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         NOAA Technical Report NMFS 106                                               February 1992


                           Marine Ranching

                           Proceedings of the Eighteenth
                           U.S. Japan Meeting on Aquaculture
                           Port Ludlow, Washington
                           18-19 September 1989

                           Ralph S. Svrjcek (editor)
























                           U.S. Department of Commerce
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      A44672

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      o. 106








                                                      NOAA Technical Report NMFS

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                                                                      NOAA Technical Report NMFS 106


                                                                      Marine Ranching

                                                                      Proceedings of the Eighteenth
                                                                      U.S. Japan Meeting on Aquaculture
                                                                      Port Ludlow, Washington
                                                                      18-19 September 1989


                                                                      Ralph S. Svrjcek
                                                                      Publications Unit
                                                                      Northwest and Alaska Fisheries Science Centers




                                                                      Panel Chairmen:
                                                                      Conrad Mahnken, United States
                                                                      Hisashi Kan-no, Japan


                                                                      Under the U, S.:fapan Cooperative Program
                                                                      in Natural Resources (UfiVR)


                                                                      February 1992



                                                  * '&VJ OF CoMr,     U.S. DEPARTMENT OF COMMERCE
                                                 0            +
                                                                      Robert Mosbacher, Secretary
                                                                 %    National Oceanic and Atmospheric Administration
                                                                      John A. Knauss, Under Secretary for Oceans and Atmosphere
                                                                      National Marine Fisheries Service
                                                   S)Arrs' of pO      William W. Fox Jr., Assistant Administrator for Fisheries




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                                                                                                     Preface





                                                The United States and Japanese counterpart panels on aquaculture were formed in 1969 under
                                                the United States-Japan Cooperative Program in Natural Resources (UJNR). The panels
                                                currently include specialists drawn from the federal departments most concerned with
                                                aquaculture. Charged with exploring and developing bilateral cooperation, the panels have
                                                focused their efforts on exchanging information related to aquaculture which could be of benefit
                                                to both countries.
                                                      The UJNR was begun during the Third Cabinet-Level Meeting of the joint United
                                                States-Japan Committee on Trade and Economic Affairs in January 1964. In addition to aqua-
                                                culture, current subjects in the program include desalination of seawater, toxic microorganisms,
                                                air pollution, energy, forage crops, national park management, mycoplasmosis, wind and
                                                seismic effects, protein resources, forestry, and several joint panels and committees in marine
                                                resources research, development, and utilization.
                                                      Accomplishments include increased communication and cooperation among technical
                                                specialists; exchanges of information, data, and research findings; annual meetings of the panels,
                                                a policy-coordinative body; administrative staff meetings; exchanges of equipment, materials,
                                                and samples; several major technical conferences; and beneficial effects on international
                                                relations.




                                                                                                                             Conrad Mahnken - United States
                                                                                                                                          Hisashi Kan-no            Japan



















                                                                       The National Marine Fisheries Service (NMFS) does not approve, recom-
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                                                                       communicate preliminary results, interim reports, and similar timely
                                                                       information. It is not subject to formal peer review.



                                                                                         Text printed on recycled paper








                                                                         Contents


                                E. S. CHANG        Reproductive endocrinology of the shrimp SitYonia ingmtis.                        I
                               W. A. HERTZ         steroid, peptide, and terpenoid hormones
                          G. D. PRESTWICH


                                    1. YANO        Hormonal control of reproductive maturation in penaeid shrimp                     7

                                H. KAGAWA          Reproductive physiology and induced spawning of yellowtail                      15
                                                   (Seriola quinqueradiata)

                                 I- T. WADA        Gametogenesis of triploid b ivalves with respect to aquaculture                 19
                                A. KOMARU


                                   M. HARA         Increasing the growth rate of abalone, Haliotis discus hannai,                  21
                                 S. KIKUCHI        using selection techniques

                              S. M. RANKIN         Ovarian development in the South American white shrimp,                         27
                            J. Y BRADFIELD         Penaeus vannamez .
                                L.L.KEELEY


                               W. S. ZAUGG         Changes in hatchery rearing and release strategies resulting                    35
                                J. E. BODLE        from accelerated maturation of spring chinooksalmon by
                             J. E. MANNING         photoperiod control

                                 K HIROSE          Induced spawning ofjapanese eel with LHRH-A copolymer pellet                    43

                                 1. 1. SOLAR       Reproductive physiology of sablefish (Anoplopomafimbfia)                        49
                        E. M. DONALDSON            with particular reference to induced spawning
                                 I. J. BAKER
                                  H. M. DYE
                       A. VON DER MEDEN
                                   J. SMITH

                              J. H. BEATTIE        Geoduck culture in Washington State: reproductive development                   55
                            C. L. GOODWIN          and spawning '

                              J. TSUKIDATE         Ecology of Sargassum spp. and Sargassum forest formation                        63

                               P. SWANSON          Salmon gonadotropins                                                            73

                                     H. ITO        Breeding season ofjapanese scallop off the eastern coast                        77
                                                   of Hokkaido


                                     T. SEKI       A better method for oyster farming injapan                                      85

                                  H.LAUFER         Hormonal regulation of reproduction in female crustacea                         89
                                E. HOMOLA
                                M. LANDAU

                              G. P. MOBERG         Reproduction in cultured white sturgeon, Acipenser transmontanus                99
                           S. 1. DOROSHOV





                                                                            iii







                                         ContentS (continued)


                   S. UMEZAWA   Experimentation to improve recruitment of blood ark shell, 105
                                Scapharca broughtonii, in the Seto Inland Sea

                   H.NAKAHARA   The Marine Ranching System: the integration of biology 115
                                and engineering technology

                       K- TAYA  Economic problems of the Marine Ranching System    129
                  A. HASEGAWA
                      Y "U1








































































                                                  iv








                                       Reproductive Endocrinology of the Shrimp
                                                                Sicyonia ingentis:
                                       Steroid, Peptide, and Terpenoid Hormones



                                ERNEST S. CHANG, WILLIAM A. HERTZ, AND GLENN D. PRESTWICH*

                                                                  Bodega Marine Laboratory,
                                                                    University of California,
                                                            R 0. Box 24 7, Bodega Bay, CA 94923




                                                                      ABSTRACT


                                  Female reproduction in penaeid shrimp is carefully regulated by several different en-
                                docrine factors. Their precise modes of action have not yet been fully elucidated. Three
                                endocrine factors, each representing a different chemical class of hormones, have been
                                investigated in the penaeid shrimp Sicyonia ingentis in our laboratory: ecdysteroids, vitel-
                                logenesis-inhibiting hormone (VIH), and methyl farnesoate (MF). Ecclysteroids (the
                                steroid molting hormones of arthropods; predominantly 20-hydroxyecdysone), are ini-
                                tially present in low levels (<10 ng/mg) in shrimp embryos. As development of the
                                embryos nears time of hatch, the ecdysteroid levels increase to approximately 150 ng/
                                mg, indicating that they may be of embryonic origin and involved in embryonic develop-
                                ment. An assay was developed for shrimp VlH, which presumably is a protein. Delay of
                                onset of the next reproductive cycle was observed following injection of sinus gland
                                extracts into shrimp that had previously had their eyestalks removed. A photoaffinity
                                analog was synthesized for the putative shrimp reproductive hormone MF-a terpenoid         '
                                This analog, farnesyl diazomethyl ketone (FDK), was used to demonstrate the presence
                                of specific binding proteins for MF in shrimp hemolymph.



             Introduction                                                          We examined three areas of shrimp reproduction.
                                                                                First is the role of the arthropod molting hormones,
             The control of female reproduction in penaeid                      ecclysteroids, in ovarian and embryonic development.
             (members of the superfamily Penaeoidea) shrimp is                  Although there have been a number of studies on the
             highly complex. It appears that a number of environ-               role of ecdysteroids in crustacean molting (Chang
             mental signals can influence different hormonal                    1989), relatively little is known about the action of
             factors which in turn regulate various aspects of the              these steroid molting hormones on ovarian and em-
             reproductive process. The understanding of this                    bryonic development.
             regulation is an area of intense research (see reviews                Second, we investigated the activity of the vitello-
             by Adiyodi 1985; Charniaux-Cotton and Payen 1988).                 genesis-inhibiting hormone (VIH). The initial
             In addition to the basic biological studies in com-                observations that provided a basis for the existence of
             parative endocrinology, there is great interest in the             the VIH were made by Panouse (1943, 1944). He ob-
             applied aspects of this research.                                  served that, depending, upon the molt stage, removal
               Penaeid shrimp comprise one of the most eco-                     of the eyestalks from the shrimp Palaemon serratus
             nomically important marine products both                           resulted in accelerated ovarian development and
             domestically and worldwide (Rosenberry 1990).                      spawning. Similar observations have been made in a
             While natural fisheries for shrimp have declined,                  number of natantians (see Chang 1992).
             there has been a concomitant surge in aquatic cul-
             ture of penaeids. One of the major problems
             preventing optimization of the commercial culture of                 Permanent address: Department of Chemistry, State University of
             shrimp is control of female reproduction.                            New York, Stony Brook, NY 11794-3400.







              2       NOAA Technical Report NNM 106


                                                   0
                                                       OCH,          juvenile hormone III



                                                       OCH,          methyl farnesoate


                                                                                                                           Figure 1
                                                                                                                    Chemical structures of
                                                       CHN,          farnesyl diazomethyl ketone                    juvenile hormone III,
                                                                                                                    methyl farnesoate, and
                 H                                                                                                  tritiated farnesyl dia-
                                                                                                                    zomethyl ketone.


                Finally, we examined the presence of binding pro-                 Concentrations of ecdysteroids in whole animal ex-
              teins for the putative gonadotropin methyl                        tracts of S. ingentis from spawning to early nauplii
              farnesoate (MF). Based upon the similarities between              were also determined by RIA. At least 100 embryos or
              crustaceans and insects in terms of the endocrine                 larvae were collected on a filter disc at various times
              regulation of molting, it was hypothesized that an                and vacuum dried for one min to remove excess wa-
              analog to the insect juvenile hormone UH; Fig. 1)                 ter. They were homogenized in a Dounce
              may be present in crustaceans. Recent work indicates              homogenizer, extracted with methanol, and centri-
              that a sesquiterpenoid other than JH may be the                   fuged (5,000 X g, 10 min). The supernatants were
              modulator of crustacean development. The related                  dried and then - solubilized in water. The extracts
              compound, methyl farnesoate (Fig. 1), was isolated                were loaded onto CH Sep-Pak (Waters Assoc.) car-
              from the hemolymph of the crab Libinia emarginata                 tridges and the ecclysteroids were eluted with 60%
              (Laufer et al. 1987). Further evidence for an endo-               methanol and then assayed by RIA.
              crine role of MF in crustaceans would be the
              demonstration of a hemolymph binding protein,
              similar to that found in insects (see Goodman and                 Vitellogenesis-Inhibiting Hormone
              Chang 1985).
                                                                                Females with well-developed ovaries were held in a
                                                                                tank with running seawater until they initiated
              Materials and Methods                                             prespawning swimming behavior. They were removed
                                                                                to a smaller tank for spawning and then placed into a
              Experimental Animals                                              separate holding tank following spawning. Bilateral
                                                                                eyestalk ablations were performed within 24 hours
              Sicyonia ingentis (ca. 24 g wet weight) were collected            with the use of iris scissors. Shrimp were allowed to
              off Southern California and maintained in seawater                recover for 24 hours before receiving their first injec-
              tanks (15 ï¿½ I' Q under an ambient photoperiod at                  tion.
              the Bodega Marine Laboratory.                                       The injections consisted of 1.0 sinus gland equiva-
                                                                                lent or, for the controls, an equivalent amount of
                                                                                non-sinus gland neural tissue. The source of both tis-
              Embryonic Ecdysteroids                                            sues were eyestalks of reproductively quiescent
                                                                                females obtained during February and March (winter
              To measure the circulating levels of ecdysteroids,                animals). The glands or control tissue were homog-
              25 IiL samples of hemolymph were removed and ex-                  enized with a Dounce homogenizer in 4 liL of
              tracted with 75% methanol at various times during                 distilled water per gland or equivalent. The
              the course of the molt cycle. The extracts were centri-           homogenate was stored at -75' C until needed. Prior
              fuged (5000 X g, 10 min) and the pellets washed                   to injection, the homogenate was thawed, spun (5000
              with 75% methanol. The supernatants were com-                     X g, 5 min) and the supernatant was removed. A
              bined and analyzed by radioimmunoassay (RIA)                      10 1iL syringe (Hamilton) with a 28 gauge needle was
              according to the method of Chang and O'Connor                     used to inject 4 jiL of the supernatant. The animal
              (1979).                                                           was immobilized and the needle was inserted from






                                                                                  Chang et al: Reproductive Endocrinology of the Shrimp               3
                one side of the ventral midline of the fourth abdomi-                give a final protein concentration between 0.5 and
                nal segment to the first segment where the sample                    2.5 mg/mL.
                was injected. The needle was withdrawn after 10 sec-                 Photoaffinity labeling-Glass plates with depression-
                onds to minimize sample leakage.                                     wells (ca. 250 liL volume) were coated with 1%
                Injections of sinus gland or control tissue extracts                 polyethylene glycol (PEG) MW 20,000 and then
                were administered every 48 hours until 30 animals in                 rinsed with water. Each experiment consisted of com-
                each group had received four injections. The females                 peted and uncompeted samples for each tissue. Thus,
                were monitored daily and the subsequent time to                      for each tissue, two wells were loaded with 100 jLL of
                spawn was noted.                                                     a pH 8.3 buffer (20 mM Tris HCI). Four I.LL of either
                                                                                     ethanol or the competitor (MF) in ethanol (1.6 X 10-2
                                                                                     M) were added and the solutions were gently mixed
                Methyl Farnesoate Photoaffinity Analog                               on an orbital shaker for 20 minutes. The plates w             *ere
                                                                                     then cooled on an ice pack while 100 VL of tissue
                Chemicals-The unlabeled and tritium-labeled hor-                     homogenate was added to each well. The chilled
                mones farnesyl diazomethyl ketone (FDK) and                          samples were mixed for 30 minutes, and then 2.5 pL
                methyl farnesoate (MF) were synthesized as de-                       of a 5 X 10-' M solution of [IHJ-FDK in ethanol was
                scribed by UjvAry and Prestwich (1990) and purified                  added to each well. Mixing continued for 45 minutes
                by silica gel chromatography. The ['HI-FDK had a                     at 4' C, and then the wells in the chilled plate were
                specific activity of 6.6 Ci/mmol.                                    irradiated from ca. 3 to 4 cm above with an 8-watt,
                Dissection and tissue preparation-Dissected tissues                  254-nm germicidal lamp for times ranging from 15
                were homogenized (Dounce) in TM buffer (10 mM                        seconds to 4 minutes
                Tris HCl, 5 mM MgCl     2' pH 6.9) and centrifuged for               Electrophoresis and autoradiograiphy@A 70 [tL ali-
                20 min at  10,000 X g (4' Q to remove cellular de-                   quot of each well was transferred into a 0.5 mL
                bris. Each supernatant was diluted with TM buffer to                 plastic tube containing 60 RL of 2X SDS-sample



                E  200--


                    150--


                0   100--

                LIJ
                >0   50--                                                                                          Figure 2
                                                                                           Ecdysteroid titers (mean ï¿½ S.D.) of Sicyonia ingentis at
                LLJ    0--                                                                 various times before (negative days) and after molt
                           -40           -20            0           20            40       (positive days). Molt occurred on day 0. Hemolymph
                                             TIME (days)                                   (25 liL) was removed and extracted with 75% metha-
                                                                                           nol and analyzed by radioimmunoassay (n          3 to 16).


                E
                N,    200-


                Z
                0     150-




                Z
                W     100-
                0                                                                                                  Figure 3
                Z
                0                                                                          Concentrations (mean ï¿½ S.D.) of ecdysteroids in
                0      50-                                                                 whole animal extracts of Sicyonia ingentis from spawn-.
                2                                                                          ing (= fertilization; 0 h) to early nauplii. Hatching
                0                                                                          occurred at approximately 20 hours. At least 100 em-
                LU                                                                         bryos or larvae were collected on a filter disc,
                         01
                                                                                           homogenized, and assayed by radioimmunoassay as
                >_         6              1'0            20              So
                a                                                                          described in Materials and Methods section. Sample
                0                   TIME AFTER SPAWN (hours)                               numbers at 4, 8, 12, 16, 20, and 35 hours were 7, 4, 4,
                Ui
                                                                                       1   3, 2, and 1, respectively.







                  4       NOAA Technical Report NXIFS 106

                  buffer, mixed, and boiled 4 minutes. Samples (45                              ply that there is relatively little maternal investment
                  liL) were loaded onto a 0.75 X 150 X 150 mm dena-                             of ecdysteroids in the eggs and that the increasing
                  turing polyacrylamide gel (SDS-PAGE, 12%                                      levels of hormone observed in the extracts is likely
                  acrylamide), stacked at 13 mA per gel, and separated                          due to endogenous synthesis.
                  at 10' C at 18 mA per gel using a Tris-glycine pH 8.3                            These data are in apparent contrast to observations
                  running buffer. Gels were stained (4 h) with                                  that our laboratory has made in the crab Cancer
                  Coomassie Blue R250 and then destained (95%                                   anthonyi (Okazaki and Chang 1991). These crab em-
                  ethanol: H,O:ace tic acid, 50:40:10).                                         bryos have relatively high levels of ecdysteroids at the
                    The destained gel was rinsed for 5 minutes with                             time of spawn (ca. 9 ng/mg wet weight). that decrease
                  glacial acetic acid and then impregnated with                                 during embryonic development. This implies an em-
                  diphenyloxazole (15% PPO in glacial acetic acid) for                          bryonic utilization of the hormones during
                  25 minutes. The acetic acid was poured off and the                            development. These differences may be due to either
                  get was treated with 50% PEG 2000 at 50-75' C for 30                          the dramatically different rates of development (S.
                  to 40 minutes. The miniaturized gels (Mohamed et                              ingentis hatches after ca. 30 hours, C. anthonyi after
                  al. 1989) were dried and exposed to pre-flashed                               ca. 35 days) or' to differences in the chemical forms
                  Kodak XAR-5 x-ray film for 5-15 days at -75' C.                               of the molting hormones. For example, conjugated
                                                                                                metabolites of ecdysteroids have a much lower
                                                                                                affinity for the ecdysteroid antiserum and hence, if
                  Results and Discussion                                                        present, would give the overall appearance of less
                                                                                                total ecdystc@roid RIA activity. However, we fa-
                  Embryonic Ecdysteroids                                                        vor the former explanation (differential rates of
                                                                                                development).
                  The concentration profile           of ecdysteroids in       the he-             Recently, data were presented that implicate a role
                  molymph of Sicyonia ingentis is similar to those of                           for ecdysteroids in the mediation of embryogenesis
                  most other crustaceans that have been examined                                in the shrimp Palaemon serratus (Spindler et al. 1987).
                  (Fig. 2; see Chang 1989). There are low levels during                         In that species, although much lower concentrations
                  postmolt and intermolt with a dramatic increase just                          were measured (peaks of about 80 ng/g), a similar
                  prior to ecdysis (premolt). This peak falls back to                           hormone pattern was observed of low levels of
                  basal levels just prior to ecdysis. Approximately simi-                       ecdysteroids at egg extrusion followed by increasing
                  lar values were obtained in the shrimp Palaemon                               levels near hatch.
                  serratus (Baldaia et al. 1984; Van Wormhoudt et al.
                  1986).
                    Since ecdysteroids play such an important role in                           Vitellogenesis-Inhibiting Hormone
                  larval, juvenile, and adult molting (Chang 1989), we
                  assayed extracts of developing embryos to determine                              S. ingentis is a useful species for the assay of VIH
                  if these steroid hormones were also involved in em-                           because it undergoes several cycles of reproduction
                  bryonic development. Figure 3 shows the total RIA                             without intervening molt cycles in the summer
                  activity of extracts of embryos at various times after                        months. Following a spawn, shrimp were injected
                  spawning (fertilization). Negligible levels of                                with extracts of sinus glands obtained from winter
                  ecdysteroids were present in the embryos at spawn-                            (nonreproductive) female shrimp. A significant inhi-
                  ing, but the concentrations increased significantly as                        bition (P<0.01) of ovarian development and
                  embryonic development proceeded. These data im-                               spawning resulted. This effect was not observed in





                                                                                         Table I
                                                         Effect of shrimp sinus gland extracts on spawning duration.


                                                                                         Spawning Duration
                                              Extract                                         (days  S.D.)                             N

                                              nonsinus gland neural tissue                    16.31   1.31                             16
                                              sinus glands                                    19.93   2.08a                            15


                                           a p < 0.01 (Student's t-test) .






                                                                           Chang et al: Reproductive Endocrinology of the Shrimp          5

            control shrimp that were injected with nonsinus                   oocyte diameters. Antisera were also raised against
            gland neural tissue (Table 1) or summer shrimp that               this lobster VIH which cross-reacted with sinus gland
            were injected with sinus gland extracts obtained from             extracts from a number of different clecapods (Meusy
            summer donor females (Chang and Hertz, unpubl.                    et al. 1987). A 3300 dalton factor was characterized
            data).                                                            from eyestalks of Penaeus setiferus as assayed in vitro
              We have observed differences in the pepticle pro-               using fiddler crab ovaries. This assay utilized precipi-
            files from sinus glands of winter (quiescent) and                 tation of radiolabeled leucine by antibodies that had
            .summer (active) shrimp following separation using                been generated against fiddler crab vitellogenin
            high-perf6rmance liquid chromatography (Chang                     (Quackenbush and Keeley 1988).
            and Hertz, unpubl. data). We have purified these
            shrimp sinus gland pepticles and are currently assay-
            ing them for VIE activity.                                        Farnesyl Diazomethyl Ketone
              There are reports that provide some chemical in-
            formation on VIH. Most of these previous                          Using the radiolabeled photoaffinity analog of
            experiments utilized a heterologous assay system.                 methyl farnesoate, ['H]-FDK, we examined a number
            Bomirski et al. (1981) extracted a factor with a mo-              of different tissues from several different species for
            lecular weight of approximately 2000 from eyestalks               specific hormone binding. We examined both the cy-
            of the crab Cancer magister Their assay consisted of              tosol and membrane fractions of these tissues.
            measuring ovarian growth in the shrimp Crangon                    Although we were unable to demonstrate cellular
            crangon. A 5000 dalton factor was extracted from eye-             binding proteins for ['H]-FDK from any tissue, we
            stalks of the spiny lobster Panuhrus argus. It was                consistently observed specific binding in the hemo-
            assayed by measuring ovarian growth in the fiddler                lymph. Figure 4 shows the effects of increasing
            crab Uca pugilator (Quackenbush and Herrnkind                     amounts of unlabeled MF in the presence of a con-
            1983). A 7500 dalton peptide was isolated from sinus              stant amount of ['H]-FDK. The radiolabeled analog
            glands of the American lobster Homarus americanus                 forms a covalent bond with a binding protein of ca.
            and assayed in vivo in the shrimp Palaemonetes varians            36,000 daltons and is effectively competed with a 250-
            (Soyez et al. 1987). The assay consisted of measuring             fold excess of the unlabeled hormone. In addition,



                                                                                  M     M
                                              lox         50x       250x        500      0
                 1511 30" 1'        21 41           25x        I 0ox      500x
                                                                                                                      Figure 4
                                                                                             -*--w 2 00     Comparison of photolysis
                                                                                                            times (15 s, 30 s, I min, 2
                                                                                                            min, and 4 min), and the
                                                              M,
                                                                                             -o-66          quantity (fold excess) of
                                                                                                            unlabeled methyl farnesoate
                                                                                                            (MF) used to displace the la-
                        n-
                                                                                                  4 5
                                                                                                            beling of female shrimp
                                                                                             4-36           hcmolymph       with     [3H]-
                                                                                                            farnesyl diazomethyl ketone
                                                                                                            (photolysis time was 4 min).
                                                                                   J:@--     4-29
                                                                                                            The band at ca. 36,000
                                                                                             4- 24
                                                                                Me,
                                                                                                            daltons appears to be specifi
                                         WO      P    @4 -                                                  cally labeled. The lanes
                                                                                                   20       marked M500 and MO repre7
                                           -k
                                                                                                            sent hemolymph from male
                                                                                                            shrimp labeled with [-H]-
                                                                                                            FDK with and without a
                                                                       mvl,                  4-14
                                                                                                            500-fold excess of unlabeled
                                                                                                            MF, respectively. Molecular
                                                                                                            weight markers (X 10-') are
                                                                                                            given on the right of the au-
                                                                     A
                                                                                                            toradiograph. No labeling is
                                                                                                            observed at zero photolysis
                                                                                                            time.







                  6      NOAA Technical Report NNUS 106

                  the effects of increasing the length of time of U.V.                     Charniaux-Cotton, H., and G. Payen.
                  irradiation is demonstrated. A high degree of attach-                         1988. Crustacean reproduction. In Endocrinology of Se-
                  ment of ['H]-FDK to the hemolymph binding protein                               lected Invertebrate Types (H. Laufer and R.G.H. Downer
                                                                                                  eds.), p. 279-303. Alan R. Liss, Inc., NY
                  is observed after only 15 sec (Fig. 4). The 36,000                       Goodman, W.G., and E.S. Chang.
                  dalton protein appears to be present in both female                           1985. juvenile hormone cellular and hemolymph binding
                  and male shrimp (Fig. 4).                                                       proteins. In Comprehensive insect physiology, biochemis-
                   We have also recently utilized ['H]-FDK to charac-                             try and pharmacology, vol. 7 (G.A. Kerkut and L.I. Gilbert,
                  terize an analogous binding protein for MF in the                               eds.), p. 491-510. Pergamon Press, Ltd., Oxford.
                                                                                           Laufer, H., D. Borst, F.C. Baker, C. Carrasco, M. Sinkus, C.C.
                  hemolymph of adult female American lobsters                                Reuter, L.W. Tsai, and D.A. Schooley.
                  (Prestwich et al..1990). The lobster MF binding pro-                          1987. Identification of ajuvenile hormone-like compound in
                  tein has an approximate molecular weight of 42,000.                             a crustacean. Science 235:202-205.
                   Although a definitive hormonal role for MF has                          MeusyJ.-J., G. Martin, D. Soyez,J.E. van Deijnen, andj.-M. Gallo.
                  not yet been established, these data suggest that MF                          1987. Immunochemical and im        -munocytochernical studies
                                                                                                  of the crustacean vitellogenesis-inhibiting hormone
                  has a specific binding protein that may prevent its                             (VIH). Gen. Com@' Endocrinol. 67:333-341.
                  rapid degradation and may facilitate its cellular ac-                    Mohamed, M.A., K.A. Lerro, and G.D. Prestwich.
                  tion. The role of the crustacean MF binding protein                           1989. Polyacrylamide gel miniaturization improves protein
                  may be analogous to the insect juvenile hormone                                 visualization and autoradiographic detection. Anal.
                  binding protein.                                                                Biochem. 177:287-290.
                                                                                           Okazaki, R.K., and E.S. Chang.
                                                                                                1991. Ecdysteroids in the embryos and sera of the crabs,
                                                                                                  Cancer magister and C. anihonyi. Gen. Comp. Endocrinol.
                  Acknowledgments                                                                 81:174-186.
                                                                                           Panousej
                   We thank M. J. Bruce for technical assistance. Fi_                           1943. Influence de I'ablation du p6doncule oculaire sur la
                                                                                                  croissance de l'ovaire chez la Crevette Leander serratus.
                  nancial support was provided by NOAA, National Sea                              Comptes Renclus Acad@mie des Sciences, Paris 217:553-555.
                  Grant College Program, Department of Commerce,                                1944. L'action de la glande du sinus sur Fovaire chez la
                  under grant number NA85AA-D-SG140, project num-                                 Crevette Leander. Comptes Renclus Acad@mie des Sciences,
                  ber R/A-68, through the California Sea Grant                                    Paris 218:293-294.
                  College Program (to E.S.C.). We also acknowledge                         Prestwich, G.D., MJ. Bruce, 1. Ujvdry, and E.S. Chang.
                                                                                                1990. Binding proteins for methyl farnescoate in lobster tis-
                  Prof. J. Clegg and the University of California,                                sues: detection by photoaffinity labeling. Gen. Comp.
                  Bodega Marine Laboratory for Distinguished Re-                                  Endocrinol. 80:232-237.
                  search Fellow support to G.D. Prestwich. The NSF is                      Quackenbush, L.S., and W.F. Herrnkind.
                  acknowledged for support of the ligand synthesis                              1983. Partial characterization of eyestalk hormones control-
                  (Grants CHE-8809588, DCB-8509629, and DCB-                                      ling molt and gonadal development in the spiny lobster
                                                                                                  Panuhrus argus. J. Crustacean Biol. 3:34-44.
                  8812322 to G.D.P.).                                                      Quackenbush, L.S., and L.L. Keeley.
                                                                                                1988. Regulation of vitellogenesis in the fiddler crab, Uca
                                                                                                  pugilator Biol. Bull. (Woods Hole) 175:321-33 1.
                  Citations                                                                Rosenberry, B.
                                                                                                1990. World shrimp farming 1989. Aquaculture Digest, San
                                                                                                  Diego, 28 p.
                  Adiyodi, R.G.                                                            Soyez, D.,J.E. Van Deijnen, and M. Martin.
                     1985. Reproduction and its control. In The biology of crus-                1987. Isolation and characterization of a vitellogenesis-inhib-
                        tacea, vol. 9 (D.E. Bliss and L.H. Mantel, eds.), p. 147-                 iting factor from sinus glands of the lobster, Homarus
                        215. Acad. Press, Inc., Orlando.                                          americanus. J. Exp. Zool. 244:479-484.
                  Balclaia, L., P. Porcheronj Coimbra, and P. Cassier.                     Spindler, K.-D., A. Van Wormhoudt, D. Sellos, and M. Spindler-
                     1984. Ecdysteroids in the shrimp Palaemon serratus: relations           Barth.
                        with molt cycle. Gen. Comp. Endocrinol. 55:437-443.                     1987. Ecclysteroid levels during embryogenesis in the
                  Bomirski, A., M. Arendarczyk, E. Kawinska, and L.H. Kleinholz.                  shrimp, Palaemon serratus (Crustacea Decapoda): quantita-
                     1981. Partial characterization of crustacean gonad-inhibiting                tive and qualitative changes. Gen. Comp. Endocrinol.
                        hormone. Int.j.lnvertebr.Reprod.3:213-219.                                66:116-122.
                  Chang, E.S.                                                              UjvAry, I., and G.D. Prestwich.
                     1989. Endocrine regulation of molting in Crustacea. Rev.                   1990. An efficient synthesis of the crustacean hormone
                        Aquatic Sci. 1:131-157.                                                   [12-'H]-methyl farnesoate and its photolabile analog
                     1992. Endocrinology of shrimp and other crustaceans. In                      [13-H]-farnesyl diazornethyl ketone. J. Labelled Compd.
                        Culture of marine shrimp: principles and practices (AW.                   & Radiopharm. 28:167-174.
                        Fast and LJ. Lester, eds.). Elsevier Science Publ., NY. In         Van Wormhoudt, A., P. Porcheron, and L. Le Roux.
                        press.                                                                  1986. Ecdyst6roids et synthese proteique dans I'hepatopan-
                  Chang, E.S., andj.D. O'Connor.                                                  cr6as de Palaemon serratus (Crustacea, Decapoda) au cours
                     1979. Arthropod molting      hormones. In Methods of hor-                    du cycle d'intermue. Bull. Soc. Zool. Fr. 110:191-204.
                        mone radio immun oassay, 2nd ed. (B.M. Jaffe and H.R.
                        Behrman eds.), p. 797-814. Acad. Press, Inc., NY.






                Hormonal Control of Reproductive Maturation in Penaeid Shrimp



                                                                    ISAO YANO

                                                        National Research Institute of Aquaculture
                                                                Nakatsuhama Nansei-cho,
                                                                   Mie 516@01, Japan





                                                                   ABSTRACT


                                Control of reproductive maturation is a major problem to the development of com-
                              mercial aquaculture programs for penacid shrimp. Although hormonal control of
                              maturation is well documented in oviparous vertebrates and insects, similar knowledge
                              for crustaceans is fragmentary. It has long been suspected that reproductive maturation
                              may be controlled by two antagonistic hormones, one that stimulates and the other that
                              inhibits. Ovarian maturation in penaeid shrimp has been induced and accelerated by
                              implantation of thoracic ganglion prepared from maturing female lobsters. This indi-
                              cates that ovarian maturation may be induced by a gonad-stimulating hormone (GSH)
                              secreted by the thoracic ganglion of maturing females. Many workers have reported that
                              ovarian maturation is regulated by a gonad-inhibiting hormone (GIH) from the X organ-
                              sinus gland complex. This GIH probably has an antagonistic relationship with GSH
                              secreted by the thoracic ganglion. This paper further details our present understanding
                              of the endocrine systems of penaeid shrimp and how they control reproductive matura-
                              tion. It is suggested that the brain, mandibular organ, or ovary has a close relationship
                              with the X organ-sinus gland complex or thoracic ganglion, which secrete GIH or GSH,
                              in regulating ovarian maturation in penaeid shrimp.



            Introduction                                                     functions, which are closely related with the release
                                                                             of gonad-stimulating factors or hormone (s) in crusta-
              Control of reproductive maturation is a major                  ceans. This paper summarizes- our present knowledge
            problem to the development of commercial aquacul-                of the endocrine systems of-penaeid shrimp and how
            ture programs for penaeid shrimp. Controlling                    they control reproductive maturation.
            reproduction in captivity could help to provide a reli-
            able year-round supply of juveniles, serve in
            developing selective breeding programs, and be gen-              Eyestalk Hormone Effects
            erally useful for obtaining disease-free spawners.               on Maturation
            Eyestalk ablation has been used to mature female
            shrimp in captivity in conjunction with the manage-                 Eyestalk ablation stimulates ovarian maturation in
            ment of water temperature, photoperiod, light                    penaeid shrimp, palinurid lobsters, and other deca-
            intensity, areal density, sex ratio, and nutrition               pod crustaceans (Caillouet 1973; Yano 1984; Yano,
            (Caillouet 1973; Lumare 1979; Yano 1984; Primavera               unpubl. data). This treatment reduces the produc-
            1985; Crocos and Kerr 1986). Knowledge of, hor-                  tion of a gonad-inhibiting hormone (GIH), and thus
            monal control of reproductive maturation in                      permits maturation of the ovaries in females. Many
            crustaceans, however, is fragmentary. Although many              workers suggest that reproductive maturation in
            observations of endocrine systems have been con-                 penaeid shrimp is regulated by a GIH from the X
            ducted on the inhibition of reproductive maturation              organ-sinus gland complex in the eyestalks. In this
            by eyestalk hormone (s) since the pioneering work of             complex, the acidophilic sinus gland is connected
            the 1940s (Panouse 1943, 1944), recent research has              with the axons from the neurosecretory cell of the X
            focused mostly on organs (e.g., brain, thoracic gan-             organ, which is located in the medulla terminalis of
            glion, ovary, and mandibular organ) and their                    penaeid shrimp (Yano, unpubl. data) and the other

                                                                                                                                         7






            8        NOAA Technical Report NNOFS 106









                                                                                      7





                                                                                                      93,




                                                                                                                            Figure I
                                                                                                                 Transverse section of the eye-
                                                                       W                                         stalk of   Penaeus japonicus,
                                                           V
               -M VM@4-'
                 V
                                                                                                 "MA
                                                                                                                 showing the sinus gland (ar-
                                "Y
                                                                                                  @u             row). Sinus gland contained
                                                                                                                                             Hae-
                                              J
                                                                                                                 acidophilic granules.
                                             jr                                                                  matoxylin and eosin        stain,
                                                                                                                 X 130 (Yano and Chinzei 1990)


                                                                                  Bomirski et al. (1981), Quackenbush and Herrnkind
                                                Eyestalks intact                  (1983), Charniaux-Cotton (1985), and Meusy et al.
                                                                                  (1987) partially purified this hormone and character-
                                                                                  ized the bioactive factor as a peptide of three
                                                                                  different molecular sizes: 2000, 5000, and 7000 Da.


                                                 Eyestalk ablation
                                                                                  Thoracic Ganglion Hormone
                                                                                  Effects on Maturation
                                       Figure 2
            Rocket immunoelectrophoresis of sera, two weeks after uni-            Smaller size P vannamei did not mature even after
            lateral eyestalk ablation of immature female Penaeus                  eyestalk ablation (Yano, unpubl. data). Thus, ovarian
            japonicus. Immunoprecipitate by anti-vitellin-serum shows             maturation is controlled by two factors, one which
            Vg synthesis and secretion into the blood after eyestalk ab-          inhibits and the other which stimulates. In immature
            lation (Yano et al. 1988).                                            females the ovary-stimulating principle is absent or
                                                                                  not yet functioning.
                                                                                    Otsu (1960) reported that the accumulation of
            crustaceans (Carlisle and Bart 1959). In general, the                 yolk granules in oocytes was stimulated by repeated
            sinus gland is easily recognizable on the neurilema,                  implantation of pieces of the thoracic ganglion in the
            surrounding the optic ganglia (Carlisle and Bart                      immature female crab Potamon dehaani. Oyama
            1959). However, in the penaeid shrimp, kuruma                         (1968), Hinsch and Bennett (1979), Eastman-Reks
            prawn Penaeusjaponicus and white prawn P vannamei,                    and Fingerman (1984), and Takayanagi et al. (1986)
            which are members of the most primitive family in                     reported similar effects of the thoracic ganglion on
            the macrura, the sinus gland (Fig. 1) is present be-                  ovarian maturation with in vivo and in vitro experi-
            tween the medulla externa and the medulla interna                     ments. In addition to these findings, Yano et al.
            (Bell and Lightner 1988; Yano, unpubl. data). Unilat-                 (1988) demonstrated that ovarian maturation of
            eral eyestalk ablation stimulates vitellogenin (Vg)                   Penaeus vannamei can be induced and accelerated by
            synthesis and secretion into the blood in immature                    implantation of pieces of thoracic ganglion tissue
            P japonicus (Yano and Chinzei 1990, Fig. 2). Eyestalk                 prepared from female lobsters, Homarus ameficanus,
            ablation also induces a rapid increase in yolk protein                with developing ovaries (Figs. 3 and 4). These results
            synthesis in P vannamei (Quackenbush 1989). These                     indicate that ovarian maturation may be induced by a
            findings suggest that GIH, secreted by the X organ-                   gonad-stimulating hormone (GSH) secreted by the
            sinus gland complex, inhibits Vg synthesis and                        neurosecretory cells of the thoracic ganglion (Fig. 5)
            secretion into the blood in female penaeid shrimp.                    of maturing females and that this GSH is not species-






                                                                 Yano: Hormonal Control of Reproductive Maturation in Shrimp               9
                                                                            specific in activity between this shrimp and lobster.
                                                                            Injection of thoracic ganglion extract, prepared from
                                                                            maturing females, is effective in increasing serum Vg
                                                                            in P japonicus (Fig. 6; Yano, unpub]. data). This
                                                                            means that GSH also stimulates Vg synthesis and/or
                                                                            secretion, or both, into the blood in penaeid shrimp.
                                                                            Thoracic ganglion extract, prepared from vitel-
                                              .-W__                         logenic kuruma prawn females, was fractionated by
                                                                            gel filtration high-performance liquid chromatogra-
                                                   hk _L@                   phy; high Vg-stimulating activity was detected in the
                                                                            fraction corresponding to a molecular weight of
                                                                            10,000 (Yano, unpubl. data). This fraction was inacti-
                                                                            vated by trypsin; therefore, this bioactive factor, GSH,
                                                                            may be characterized as a peptide hormone.

        X,
                                                                            Vitellogenesis and
                                                                            Its Control by Hormones

                                                                            Vitellogenin, a precursor of egg yolk, is a necessary
                                                                            prerequisite for ovarian oocytes to reach full matura-
                                                                            tion in female penaeid shrimp. Hormonal regulation
                                                                            of Vg synthesis is well documented in oviparous verte-
                                                                            brates (Tata 1978) and in insects (Hagedorn and
                                                                            Kunkel 1979). Knowledge is fragmentary, however,
                                                                            on the hormonal induction of Vg synthesis or secre-
                                 04                                         tion into the blood in crustaceans. As with ovarian
                                                                            maturation, it has long been suspected that vitello-
                                  Figure 3                                  genesis in crustaceans is controlled by two
       Induced ovarian maturation in Penaeus vannamei, 18 days              antagonistic hormones; in penaeid shrimp, GIH
       after the implantation of thoracic ganglion, prepared from           is secreted from the X organ-sinus gland complex
       a female lobster, Homarus americanus, with developing                and inhibits vitellogenesis, and GSH is secreted from
       ovary (Yano et a]. 1988).                                            the thoracic ganglion and stimulates vitellogene-
                                                                            sis. Gonad-stimulating hormone probably has an

                                      4@_













                               4,
            ;V.                         7;    k

                                                                                                  W
                                                                                                                   Figure 4
                                                                                                      Nearly ripe ovary of Penaeus
                                                                                                      vannamei, 18 days after implanta-
                                                            4'J                                       tion of thoracic ganglion, pre-
                                                                                                      pared from a female lobster,
                                                                                 ',W                  Homarus americanus, with develop-
                                                                            i,                        ing ovary (Yano et al. 1988).
                                                                                                      Haernatoxylin and eosin stain,
                                                                                                      X 260.







               10      NOAA Technical Report NMEFS 106


                         _p                                    r


















                                                                            A
                                                                                                                         Figure 5
                                                                                                             Thoracic ganglion, showing
                                                                                                             neurosecretory cells in Penaeus
                                                                                                             japonicus. NeUrosecretory cells
                                             V,                                                              contained basophilic granules
                                                                                                             dispersed throughout the cyto
                                                                                                             plasm (Yano, unpubl. data).
                                                                                                             Haernatoxylin and eosin stain,
                                                                                                             X 65.

                 (Va/Vi)                                                         antagonistic relationship with GIH in regulating vitel-
                      5                                                          logenesis (Fig. 7). Unilateral eyestalk ablation was
                    5                                                            not effective in increasing serum Vg in maturing fe-
                    F                                                            male R japonicus (Fig. 8, Yano, unpubl. data). This
                    (4)4                                                         suggests that GIH may decrease quick]       y immediately
                    n
                    S                                                            before the initiation of vitellogenesis and then stay at
                                                                                 a low level until after the vitellogenesis is completed.
                    Z 3
                                                                                 Therefore, eyestalk ablation may no longer be effec-
                                                                                 tive in regulating the production of GIH after
                    > 2                                                          vitellogenesis has been initiated. On the other hand,
                    4-                                                           injection of thoracic ganglion extract, prepared from
                    0
                                                                                 vitellogenic females, was effective in increasing se-
                                                                                 rum Vg, even in maturing females (Fig. 6). This
                                                                                 indicates that after initiation of vitellogenesis, higher
                      0                                                          amounts of GSH, which are increased by injection of
                                RS            AG             TG                  thoracic ganglion extract, accelerate Vg synthesis and
                                                                                 its release into the blood. This implies the possibility
                                        Figure 6                                 that in penaeid shrimp, GSH levels may increase fur-
               Rate of Vg increase in sera of maturing female Penaeus            ther with advancement of vitellogenesis, parallel to a
               japonicus, 48 hours after injection of abdominal (AG) and         decrease in the level of GIH (Fig. 7).
               thoracic ganglion (TG) extracts. Vi: initial Vg concentra-          It is well known that biogenic amines release
               tion; Va: Vg concentration 48 hours after injection; RS:          peptide neurohormones from neuroendocrine struc-
               Ringer solution (Yano, unpubl. data).


                                     Vitellogenesis                Vitellogenesis
                 a)
                 >  High
                 .2
                 0                       GSH
                 C                                                                                                  Figure 7
                 0
                 E                                                                                 Model for the control of vitellogenesis in
                 I-
                 0                                                                                 penaeid shrimp. Gonad-stimulating-
                    Low                                                 GIH                        hormone (GSH) probably has an an-
                                                                                                   tagonistic relationship with gonad-
                                                 Previte[ logenesi s                               inhibiting-hormone (GIH) in regulating
                                                                                                   vitellogenesis.






                                                                   Yano: Hormonal Control of Reproductive Maturation in Shrimp            I I

                                                                                of GIH from the X organ-sinus gland complex in
                (Va/V0                                                          crustaceans.
                        3                                                         Vitellogenin has been identified electrophoreti-
                                                                                cally and immunologically in the haemolymph of
                                                                                vitellogenic female crustaceans (Horn and Kerr
                  U     2                                                       1969; Fielder et al. 1971; Wolin et al. 1973; Fyffe and
                  E                                                             O'Connor 1974; Yano 1987a; Yashiro 1989). There-
                  >                                                             fore, extra-ovarian tissue has been suspected for a
                      E
                  0                                                             long time as the site of Vg synthesis in crustaceans.      In
                      (D
                                                                                fact, evidence has been presented to show that Vg is
                  M
                  W                                                             synthesized by the fat body or adipose tissue in am-
                        0     Eyestalk        Eyestalks                         phipods and isopods (Picaud 1980; Croisille and
                              ablation        intact                            Junera 1980; Souty and Picaud 1981). Recently, sev-
                                                                                eral workers demonstrated that Vg is synthesized on a
                                                                                large scale by the ovaries of crayfish (Lui et al. 1974),
                                   Figure 8                                     fiddler crabs, Uca pugilator (Eastman-Reks and
            Rate of Vg increase in sera      of maturing female                 Fingerman 1985), kuruma prawns (Yano and Chinzei
            Penaeus japonicus, 48 hours after unilateral eyestalk               1987), and white prawns (Quackenbush 1989; Rankin
            ablation. Vi: Initial Vg concentration; Va; Vg concen-              et al. 1989). Considering these observations, the site
            tration 48 hours after injection (Yano, unpubl. data).              of Vg synthesis in decapods is different from that in
                                                                                isopods and amphipods. Vitellogenin is secreted into
                (Va/Vi)                                                         the haemolymph after its synthesis and then accumu-
                        25                                                      lated in the developing oocytes as vitellin (Yano
                      E                                                         1988). It is known that ecdysone, which is produced
                                                                                in the Y-organ, stimulates vitellogenesis in the isopod
                      W                                                         Procellio dilatatus and amphipod Orchestia gammarella
                      .E 20                                                     (Souty et al. 1982; Blanchet-Tournier 1982;
                                                                                Charniaux-Cotton 1985). Recently, evidence has
                      M
                                                                                been presented to show that 17ot-hydroxy-progester-
                      U 15                                                      one stimulates Vg synthesis or release, or both, into
                      .E
                      al                                                        the blood in kuruma prawns (Fig. 9; Yano 1987a),
                      >                                                         fresh water prawn, Macrobrachium lanchesteri (George
                      4..
                      0 10                                                      and Khoo 1989), and black tiger shrimp, P monodon
                                                                                (Yashiro 1989). The hormone 17 et-hydroxy-progest-
                                                                                erone is generally distributed in the ovary of
                        5                                                       crustaceans (Kanazawa and Teshima 1971). Junera et
                                                                                al. (1977) deduced the existence of a Vg-stimulating
                                                                                ovarian hormone           (VSOH) which controls
                                                                                vitellogenin synthesis in females of 0. gammarella. It is
                        0
                                     ET                  17P                    probable that 17 ot-hydroxy-progesterone stimulates
                                                                                Vg synthesis and release into haemolymph as a VSOH
                                      Figure 9                                  in penaeid shrimp and other shrimp (Fig. 10). By
            Rate of Vg increase in sera of control (0.1 Rg pure etha-           immunofluorescence, Vg was found to occur for the
            nol/g body weight) and treated female Penaeus japonicus,            first time in the follicle cells of the oil globule stage-I
            48 hours after 17 a--hydroxy-progesterone (0.1 Rg/g body            oocytes in early developing ovaries, which actively
            weight) injection. ET: Ethanol; 17P: 17 a-hydroxy-proges-           synthesize Vg (Yano and Chinzei 1987). The follicle
            terone; Vi: Initial Vg concentration; Va: Vg concentration          cells greatly expanded on the oil globule stage-I
            48 hours after injection (Yano 1987a).                              oocytes (Yano and Chinzei 1987; Yano 1988). There-
                                                                                fore, the follicle cells are nominated as a possible cell
                                                                                type responsible for ovarian Vg synthesis in kuruma
            tures in several crustaceans (Fingerman 1985). Sero-                prawns. It is suggested that 17 ot-hydroxy-progester-
            tonin has been found to induce the release of                       one, probably secreted from the ovary as a VSOH,
            molting-inhibiting hormone from isolated eyestalks                  stimulates Vg synthesis in follicle cells and Vg release
            (Mattson and Spaziani 1985). This implies the possi-                into the haernolymph in penaeid shrimp (Fig. 10).
            bility that biogenic arnines may stimulate the release              On the other hand, brain extracts, prepared from







                12        NOAA Technical Report NMEFS 106
                                                                                         tion in R japonicus (Laubier-Bonichon 1978; Yano
                                     Temperature                                         1984). High temperature (25' Q and long daylength
                                                    An                                   (15 hours) induce Vg synthesis and secretion into the
                                                                                         blood in kuruma prawn (Yano 1987a). Lumare
                                                                                         (198 1) demonstrated that even in ablated P japonicus
                                                               Light                     full sexual maturation did not occur below 17' C. In-
                                                         A00                             creasing ovarian development and rapid maturation
                                                                                         were observed as water temperature rose above 18' C
                           Xo-Sg                        Br                               (Yano 1987b). The change from 12 to 13 hours of
                                                                                         daylight was also observed to accelerate the ovarian
                                                  BH                                     maturation (Yano 1987b). In unablated R monodon, a
                                        Ile                                              longer photoperiod of 19 hours (light) did not stimu-
                                      Th                                                 late ovarian maturation (Beard and Wickins 1980).
                                                                                         These findings indicate that the effect of GSH and
                            GIH                       GSH                                GIH on induction and inhibition of ovarian matura-
                                                                                         tion or vitellogenesis is affected by water temperature
                                                                                         and photoperiod in female shrimp. It is probable
                                                                Fc                       that the release of GSH and GIH from the thoracic
                     00      VAR&                                                        ganglion and the X organ-sinus gland complex may
                                                                                         be induced directly or indirectly by water tempera-
                                                                                         ture and light stimuli, via respectively the antermule
                    Ov                                       17P                         (Barber 1961) and eye (Waterman 1961) in female
                      \1                                                                 penaeid shrimp, as shown in Figure 10.


                                    Vitellogenesis
                                                                                         Citations
                                        Figure 10
                Scheme of the factors affecting vitellogenesis and as-                   Barber, S.B.
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                Oocyte; Ov: Ovary; BH: Brain hormone; GIH: Go-                           Beard, T.W., and J.F. Wickins.
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                hormone; 17P: 17 a-hydroxy-progesterone.                                       recirculation systems. Aquaculture 20:79-89.
                                                                                         Bell, T. A., and D. V. Lightner.
                                                                                             1988. Compound eye and associated organs. In A hand-
                                                                                               book of normal penaeid shrimp histology, p. 26-33. World
                maturing females induced Vg synthesis in R japonicus                           Aquaculture Society, Baton Rouge, LA.
                (Yano, unpubl. data). This suggests the presence of a                    Blanche t-Tourn ier, M.F.
                brain hormone that stimulates the release of GSH in                          1982. Quelques aspects des interactions hormonales entre la
                                                                                               mue et la vitellogenesis chez le Crustace Amphipode
                penaeid shrimp (Fig. 10). Also, mandibular organ                               Orchestia gammarella (Pallas). Reprod. Nutr. Dev. 22:325-
                implantation could stimulate vitellogenesis in the spi-                        344.
                der crab Libinia emarginata (Hinsch 1980). Laufer ei                     Bomirski, A.M., A.E. Kawinska, and L. H. Kleinholz.
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                unepoxidated analog of the insect juvenile hormone                             hormone. Int.J. Invertebr. Reprod. 3:213-220.
                                                                                         Caillouet, C.W.
                111, was synthesized by the mandibular organ of spi-                         1973. Ovarian maturation by eyestalk ablation in pink
                der crabs, L. emarginata. However, the function of                             shrimp Penaeus duorarum Burkenroad. Proc. World
                methyl farnesoate is not known to control maturation                           Maricul. Soc. 3:205-225.
                in crustaceans.                                                          Carlisle, D.B., and F.K. Bart.
                                                                                             1959. The neurosecretory system of the head and thorax. In
                                                                                               Endocrine control in crustaceans (M. Abercrombie, P. B.
                                                                                               Medawar, and G. Sal, eds.), p. 13-39. The Univ. Press,
                Temperature and Light Effects                                                  Cambridge.
                on Maturation                                                            Charniaux-Cotton, H.
                                                 17S





































                                                                                             1985. Vitellogenesis and its control in malacostracan
                It is well known that ovarian maturation is affected by                        Crustacea. Am. Zool. 25:197-206.
                                                                                         Crocos, P.J., andj.D. Kerr.
                water temperature and photoperiod in female                                  1986. Factors affecting induction of maturation and spawn-
                shrimp. A photoperiod of 14-16 hours (light) and                               ing of the tiger prawn, Penaeus escuLenius (Haswell), under
                temperature of 24-26' C stimulate ovarian matura-                              laboratory conditions. Aquaculture 58:203-214.







                                                                              Yano: Hormonal Control of Reproductive Maturation in Shrimp                         13

              Croisille, Y, and H.Junera.                                                        1981. Artificial reproduction of Penaeusjaponicus Bate as ba-
                   1980. Recherche du lieu de synthese de la vitellogenine chez                     sis for commercial production of eggs and larvae. J. World
                     le Crustace amphipode Orchestia gammarella (Pallas). Dem-                      Maricul. Sec. 12:335-344.
                     onstration, a I'aide d'anticorpos specifiques, cle la presence         Mattson, M. P., and E. Spaziani.
                     de vitellogenine dans le tissu adipeux sous-epidermique de                  1985. 5-hydroxytryptamine mediated release of molt-inhibit-
                     fernaelles en vitellogenese seconclaire. C. R. Acad. Sci.,                     ing h6rmone activity from isolated crab eyestalk
                     Paris 290:1487-1490.                                                           ganglia. Biol. Bull. (Woods Hole) 169:246-255.
              Eastman-Reks, S., and M. Fingerman.                                           MeusyJ.-J., G. Martin, D. Soyez,J.E. van Deijnen, andj.-M. Gallo.
                   1984. Effects of neuroendocrine tissue and cyclic-AMP on                      1987. Immunochemical and immunocytochernical studies of
                     ovarian growth in vivo and in vitro in the fiddler crab, Uca                   the crustacean vitellogenesis-inhibiting hormone (VIH).
                     pugilator Comp. Biochem. Physiol. 79A:679-684.                                 Gen. Comp. Endocrinol. 67:333-341.
                   1985. In Vitro synthesis of vitellin by the ovary of the fiddler         Otsu, T.
                     crab, Uca pugilator. J. Exp. Zool. 233:111-116.                             1960. Precocious development of the ovaries in the crab,
              Fielder, D.F., K.K. Rao, and M. Fingerman.                                            Potamon dehanni, following implantation of the thoracic
                   1971. A female-limited lipoprotein and the diversity of                          ganglion. Annot. ZooI.Jpn. 33:90-96.
                     hemosyanin components in the dimorphic variants of the                 Oyama, S.N.
                     fiddler crab, Uca pugilalor as revealed by disc electro-                    1968. Neuroendocrine effects on ovarian development in
                     phoresis. Comp. Biochem. Physiol. 3911:291-297.                                the crab Thalamita crenata Latreille studied in vitro. Ph. D.
              Fihgerman, M.                                                                         diss., Univ. Hawaii, Honolulu, Hawaii.
                   1985. The physiology and pharmacology of crustacean                      Panouse,J. B.
                     chromatophores. Am. Zool. 25:233-252.                                       1943. Influence cle I'ablation du predoncule oculaire sur la
              Fyffe, W.E., andJ.D. O'Connor.                                                        croissance de Povaire chez la crevette Leander serratus.
                   1974. Characterization of quantification of a crustacean                         C. R. Acad. Sci., Paris 217:553-555.
                     lipovitellin. Comp. Biochem. Physiol. 47B:851-867.                          1944. L'action de la glande du sinus sur Povaire chez la
              George, S., and H.W. Khoo.                                                            crevette Leander C. R. Acad. Sci., Paris 218:293-294.
                   1989. Effects of estrogens and progesterones on vitellogenin             Picaud, J.-L.
                     levels in the haernolymph of the shrimp, Macrobrachium                      1980. Vitellogenin synthesis by the fat body of Porcelio
                     lanchesteri. In Abstracts of the Fifth International Congress                  dilatalus Brandt (Crustacea, Isopoda). Int. J. Invertebr.
                     of Invertebrate Reproduction; 23-28 July 1989, Nagoya, Ja-                     Reprod. 2:341-349.
                     pan, p. 57. Publ. by Int. Soc. Invert. Reprod., Mainz, Ger-            Primavera,J.H.
                     many.                                                                       1985. A review of maturation and reproduction in closed
              Hagedorn, H.H., andJ.G. Kunkel.                                                       thelycum penaeids. In Proceedings of the first international
                   1979. Vitellogenin and vitellin in insects. Annu. Rev.                           conference on the culture of penaeid prawns/shrimps; 4-7
                     Entomol. 24:475-505.                                                           December 1984, Iloilo City, The Philippines (Y Taki, J.H.
              Hinsch, G.W.                                                                          Primavera, and J.A. Llobrera, eds.), p 47-64. Japan Int.
                   1980. Effect of mandibular organ implants upon the spider                        Cooper. Agency, Tokyo, Japan.
                     crab ovary. Trans. Am. Microsc. Soc. 99:317-322.                       Quackenbush, L.S.
              Hinsch, G.W., and D.C. Bennett.                                                    1989. Yolk protein production in the marine shrimp Penaeus
                   1979. Vitellogenesis stimulated by thoracic ganglion im-                         vannamei. J. Crustacean Biol. 9:509-516
                     plants into clestalked immature spider crab, Libinia                   Quackenbush, L. S., and W. F. Herrnkind.
                     emarginata. Tissue & Cell 11:345-351.                                       1983. Partial characterization of eyestalk hormone controlling
              Horn, E.C., and M.S. Kerr.                                                            molt and gonadal development in the spiny lobster,
                   1969. The haemolymph protein of the         blue crab, Callinectes               Panulirus argus. J. Crustacean Biol. 3:34-44
                     sapidus. 1. Hemocyanins and certain       other major protein          Rankin, S.M.,J.Y. Bradfield, and L.L. Keeley.
                     constituents. Comp. Biochem. Physiol. 29:493-508.                           1989. Ovarian protein synthesis in the South American white
              Juncra, H.,C. Zerbib, M. Martin, andJJ. Muesy.                                        shrimp, Penaeus vannamei, during the reproductive
                   1977. Evidence for control of vitellogenin synthesis by an                       cycle. Int.j. Invertebr. Reprod. 15:27-33.
                     ovarian hormone in Orchestia gammarella (Pallas), Crustacea,           Souty, C., andJ.L. Picaud.
                     Amphipoda. Gen. Comp. Endocrinol. 31:457-462.                               1981. Vitellogenin synthesis in the fat body of the marine
              Kanazawa, A., and S. Teshima.                                                         crustacea isopoda, Idotea ballhica basteri, during
                   1971. In vivo conversion of cholesterol to steroid hormones                      vitellogenesis. Reprod. Nutr. Dev. 21:95-102.
                     in the spiny lobster, Panuhrus japonicus. Bull. Jpn. Soc.              Souty, C., G. Besse, andj.-L. Picaud.
                     Fish. 37:891-898.                                                           1982. Ecclysone stimulates the rate of vitellogenin release in
              Laubier-Bonichon, A.                                                                  haemolymph of the terrestrial crustacean Isopoda Porcellio
                   1978. Ecophisioloie cle la reproduction chez la crevett                          dilatatus Brandt. C.R. Acad. Sci., Paris, 294:1057-1060.
                     Penaeus japonicus trois annees d'experience en milieu                  Takayanagi, H., Y. Yamamoto, and N. Takeda.
                     controle. Oceanol. Acta. 1:135-150.                                         1986. An ovary stimulating factor in the shrimp, Paraiya
              Laufer, H., D. Borst, F.C. Baker, C. Carrasco, M. Sinkus, C. Reuter,                  compressa. J. Exp. Zool. 240:203-209.
                L.W. Tsai, and D.A. Schooley.                                               Tata, J. R.
                   1987. The identification of a juvenile hormone-like com-                      1978. Induction and regulation of vitellogenin synthesis by
                     pound in a crustacean. Science 235:202-205.                                    estrogen. In Biochemical actions of hormones, Vol. 5
              Lui, CW., B.A. Sage, andJ.D. O'Connor.
                   1974. Biosynthesis of lipovitellin by the crustacean ovary.                      (G. Litwack, ed.), p. 397-431 Acad. Press, NY.
                     J. Exp. Zool. 188:289-296.                                             Waterman, T.H.
              Lumare, F.                                                                         1961. Light sensitivity and vision. In The physiology of
                   1979. Reproduction of Penaeus kerathurus using eyestalk                          Crustacea, Vol. 2 (T.H. Waterman, ed.), p. 1-64. Acad.
                     ablation. Aquaculture 18:203-214.                                              Press, NY.







                14        NOAA Technical Report NMIFS 106

                Wolin, E.H., H. Laufer, and D.F. Albertini.                               Yano, L, and Y. Chinzei.
                     1973. Uptake of the yolk protein lipovitelliu by developing              1987. Ovary is the site of vitellogenin synthesis in kuruma
                       crayfish oocytes. Dev. Biol. 35:160-170.                                  prawn, Penaeus japonicus. Comp. Biochem. Physiol.
                Yano, 1.                                                                         86B:213-218.
                     1984. Induction of rapid spawning in kuruma prawn, Penaeus               1990. Effects of human chorionic gonadotropin and unilat-
                       japonicus, through unilateral eyestalk enucleation. Aqua-                 eral eyestalk enucleation on vitellogenin induction and
                       culture 40:265-268.                                                       ovary development in kuruma prawn, Penaeusjaponicus. In
                     1987a. Effect of 17   a-hydroxy-progesterone on vitellogenin                Shrimp culture in the world (C.C. Justo, ed.), p. 365-
                       secretion in kuruma prawn, Penaeusjaponicus. Aquaculture                  370. Midori Shohou, Tokyo, Japan
                       61:49-57.                                                          Yano, L, B. Tsukimura, J.N. Sweeney, and J.A. Wyban.
                     1987b. Maturation of kuruma prawns Penae-us japonicus cul-               1988. Induced ovarian maturation of Penaeus vannamei by
                       tured in earthen ponds. In Reproduction, maturation and                   implantation of lobster ganglion. J. World Aquacult. Soc.
                       seed production of cultured species: proceedings of the                   19:204-209.
                       twelfth U.S.-Japan meeting on aquaculture; 25-29 October           Yashiro, R.
                       1983, Baton Rouge, LA (Cj. Sinderman, ed.), p. 3-                      1989. Biochemical, immunological and histological studies
                       7. NOAA Tech. Rep. NMFS 47.                                               on ovarian maturation and rematuration of Penaeus monodon
                     1988. Oocyte development in the kuruma prawn Penaeus                        Fabricius. Ph.D. diss., Univ. Philippines System. Quezon
                       japonicus. Mar. Biol. (Berlin) 99:547-553.                                City, Philippines.






                       Reproductive Physiology and Induced Spawning of Yellowtail
                                                          (Setiola quinqueradiata)



                                                                  HIROHIKO KAGAWA

                                                            National Research institute of Aquaculture
                                                                             Nansei
                                                                       Mie 516-01, Japan




                                                                        ABSTRACT


                                  For the large-scale production of the yellowtail Seriola quinqueradiata an understanding
                                of its reproductive physiology and the ability to artificially control its reproduction are
                                necessary. This article briefly reviews recent studies on the reproductive physiology and
                                hormonally induced spawning of the yellowtail. Plasma steroid hormone levels associated
                                with ovarian development and the results of spawning induced by using human chorionic
                                gonadotropin and gonaclotropin releasing hormone are discussed.




             Introduction                                                         (April and May). Since yolky oocytes still remain in
                                                                                  the ovary after ovulation, yellowtail appear to be ca-
             In Japan, the yellowtail Seriola quinqueradiata is one of            pable of multiple spawnings during a spawning
             the most highly regarded fish species for fresh con-                 season.
             sumption. In addition, its suitability for marine                       It is well known that estradiol-17P induces
             aquaculture is good. The annual production of cul-                   vitellogenin synthesis in the teleost liver. The in-
             tured yellowtail currently amounts to about 165,000                  crease in plasma estradiol-17-P levels correlates well
             metric tons (approximately 70% of total production                   with increase of gonadal weights (Kagawa et al.
             of marine finfish aquaculture; DSI 1988). Currently,                 1983). Changes in plasma estradiol-17P levels in yel-
             wildjuvenile fish are caught as seed for aquaculture.                lowtail (Fig. 1) also correlate with the development
             For the large-scale production of the yellowtail and                 of the ovary; estradiol-170 levels are constantly low
             stable production of seed, an understanding of its                   (below 1 ng/mL) in the previtellogenic stage from
             reproductive physiology and the ability to control its               December to February and increase rapidly during
             reproduction are necessary. This article briefly re-                 the vitellogenic period reaching a maximum level in
             views recent studies on the reproductive physiology                  the spawning season at a concentration of about 9
             and hormonally induced spawning of yellowtail.                       ng/mL (Kagawa et al., unpubl. data). These values
                                                                                  found in female yellowtail are of the same order as
                                                                                  those reported for other fish that have asynchronous-
             Reproductive Physiology                                              type ovaries (goldfish, Carassius auratus, Kagawa et al.
                                                                                  1983; common carp, Cyprinus carpio, Santos et al.
             After the fry stage, female yellowtail normally attain               1986). The compound 17a, 20P-dihydroxy-4-
             sexual maturity in floating net pens in about three                  pregnen-3-one (17ot, 20P-diOHprog) was first
             years. Oocyte diameter begins increasing in February.                identified as a maturation-inducing steroid in amago
             The most advanced oocytes rapidly increase th           'eir di-     salmon Oncorhynchus rhodurus (Nagahama 1987) and
             ameter after vitellogenesis starts in March and reach                has been known to induce final oocyte maturation in
             about 700 @Lrn at the tertiary yolk globule stage in                 many other fish. Plasma levels of this steroid dramati-
             April (Fig. 1). The yellowtail has an asynchronous-                  cally increased at the time of oocyte maturation in
             type ovary which contains oocytes at various                         many te'leost species. In yellowtail, an increase of 17ot,
             developmental stages ranging from the perinucleolus                  20P-diOHprog (Fig. 2) was also observed during the
             to tertiary yolk globule stage in the spawning season                final oocyte maturation that was induced using hu-


                                                                                                                                                15


16	NOAA Technical Report NMFS 106

Figure 1

Changes in plasma estradiol-17B levels and oocyte diameter during the oocyte
development in the yellowtail (Kagawa et al., unpubl. data).


Figure 2

Changes in plasma 17a, 20B-dihydroxy-4-pregnen-3-one (17a, 20B-diOHprog)
levels and oocyte diameter in the yellowtail after injection of human
chorionic gonadotropin (Kagawa et. al., unpubl. data).


man chorionic gonadotropin (HCG) injection (Kagawa et. al.,unpubl. data).
Values for plasma 17a, 20B-diOHprog obtained from yellowtail are on the
same order as those reported for goldfish (Kagawa et. al. 1983), but very
low compared with those reported in salmonids (Young et. al. 1983).  The
difference between plasma 17a,20B-diOHprog levels of yellowtail and
salmonids may be due to the lower capacity of the yellowtail ovarian
follicle to produce this steroid as was suggested in goldfish (Kagawa et.
al. 1983).  It is still uncertain whether 17a,20B-diOHprog is the maturation
inducing steroid in yellowtail.  Further studies on in vitro effect of this
steroid on oocyte maturation and its production in the ovarian follicles
are necessary.

	There is little information on the reproductive physiology of male
yellowtail.  Males mature very early; sperm formation can be observed in
the testis of fish only 1 year old.  Little attention has been paid to the
collection of milt for fertilization because sperm formation occurs naturally
even under net-culture conditions.  The only attempt to control male





                                                                                    Kagawa: Reproductive Physiology and Spawning of Yellowtail                        17


                                                                                       Table I
                  Number of ovulated eggs, fertilization rates, and hatching rates in the yellowtail treated with single intramuscular
                  injection of human chorionic gonadotropin.


                              Experiment            No. of               No. of fish           No. of eggs                   % of                  % of
                               number              fish used             ovulated              ovulated                      fert.              hatching

                                    1                   3                     3                1,710,000                     49.1                  14.6
                                    2                 15                    13                 9,906,000                     69.6                  49.8
                                    3                 17                    14                 4,460,000                     72.6                  51.1
                                    4                 15                    11                 7,290,000                     62.5                  39.2
                                    5                 11                    lo                 8,210,000                     32.5                  21.2




                                                                                       Table 2
                  Number of spawned eggs, fertilization rates, and hatching rates in the yellowtail implanted with cholesterol pellet
                  containing the gonadotropin releasing hormone analog.


                                    Spawning                       No. of spawned
                                       date                              eggs                             % fertilized                     % hatched

                                     May 1                               217,000                              71.4                             71.4
                                           2                             630,000                              41.6                             41.6
                                           3                             882,000                              25.9                             25.9
                                           4                             623,000                              52.1                             27.3
                                           5                           1,477,000                              16.1                              8.8
                                           6                           2,212,000                              12.2                              4.9
                                           8                             949,000                              19.7                              8.7
                                           9                           2,562,000                              13.5                              7.5
                                           11                            98,000                                0                                0





              reproductive physiology is the use of a single injec-                            Eggs were fertilized using milt from male fish treated
              tion of HCG two days before fertilization, to ensure                             with 6,000 IU of HCG two days before fertilization.
              that a sufficient volume of milt is collected.                                   Fertilized eggs were then incubated in a I-ton tank
                                                                                               for 2 days at 20' C. The mean fertilization rate was
                                                                                               about 60% and the hatch rate was about 40%. Ovula-
              Induced Spawning                                                                 tion, fertilization, and hatch rates varied with the fish
                                                                                               used because ovarian maturity at the time of injection
              Although female yellowtail have ovaries that contain                             was often different between fish. Their ovaries often
              oocytes at the tertiary yolk stage Oust prior to final                           contained many degenerated oocytes and only a few
              oocyte maturation) during the spawning season, they                              oocytes at the tertiary yolk stage. Assessment of ovar-
              do not ovulate and spawn in the floating net pens                                ian maturity by cannulating oocytes from the fish
              naturally. It has been known that HCG induces spawn-                             before hormone treatment will improve these results.
              ing in many fish including marine species, such as sea                             As mentioned earlier, yellowtail have a potential to
              bream Sparus aurata (Gordin and Zohar 1978). In cul-                             spawn several times during a spawning season; how-
              tured female yellowtail (mean body weight                                        ever, they do not spawn again if ovulation is induced
              8.6 kg), ovulation is induced by a single intramuscular                          by HCG injection. One of the most important rea-
              injection of HCG at a dose of 600-700 IU/kg body                                 sons for this phenomenon is the stress incurred by
              weight (- 6,000 IU/fish). After HCG treatment, ovu-                              handling while they are injected with hormone and
              lation occurred within 24 to 48 hours. For practical                             their abdomens stripped to obtain ovulated eggs.
              purposes eggs were collected between 50 and 60                                   These stressful treatments probably induce the de-
              hours after HCG injection by manually stripping their                            generation of yolky oocytes. Gonadotropin releasing
              abdomen. The number of eggs obtained by HCG                                      hormone (GnRH) and its analog are used success-
              treatment (Table 1) averaged about 500,000/fish.                                 fully to induce spawning in a variety of fish (Marte et







                18       NOAA Technical Report NMFS 106

                al. 1988). Recently in our laboratory, attempts to use                 Branchs of the Japan Sea-Farming Association for
                an implanted GnRH analog pellet to reduce the                          their kind collaboration and technical assistance in
                amount of handling and induce multiple natural                         performing this work. A part of the data in this paper
                spawning have been started.                                            has already been published in the Annual working
                  Seven female yellowtail (mean body weight 8.0 kg)                    reports of the Japan Sea-Farming Association
                were implanted with a cholesterol pellet of gonado-                    (Shinko Bldg., 2-1, Kanda Ogawa-cho, Chiyoda-ku,
                tropin-releasing-hormone analog, des Gly" [D-Alal]                     Tokyo 101 1987). Unpublished data from this labora-
                LHRH ethylamide (Sigma) 1,000 jig/fish on 29                           tory is a result of collaboration with K. Hirose and S.
                April. Cholesterol pellets (2 X 5 mm in size) which                    Arai who are gratefully acknowledged.
                were composed of cholesterol powder, cocoa butter,
                and GnRH-analog were intramuscularly implanted.
                Females were reared with seven males in a 60 ml tank                   Citations
                and the spawned eggs were collected by net every
                morning. Collected eggs were incubated in a 1-ton                      Department of Statistical Information (DSI)
                tank for 2 days at 20' C.                                                  1988. Fisheries Statistics of Japan, 142 p. DSI, Ministry of
                  Preliminary results (Table 2) show that implanta-                          Agriculture, Forestry, and Fisheries, Government of Japan.
                                                                                       Gordin, H., and V Zohar.
                tion of a cholesterol pellet containing GnRH analog                        1978. Induced spawning of Sparus aurata by means of hor-
                induced natural spawning. Yellowtail spawned eggs                            monal treatment. Ann. Biol. Anim. Biochem. Biophys.
                over I I days; fertilization and hatch rates were rela-                      18:985-990.
                tively high during the first 4 days but decreased                      Kagawa, H., G. Young, and Y. Nagahama.
                rapidly from day 5-even though the number of                               1983. Changes in plasma steroid. hormone levels during go-
                spawned eggs increased. In this experiment, we could                         nadal maturation in female goldfish Carassius auratus.
                                                                                             Nippon Suisan Gakkaishi 49:1783-1787.
                not know how many times one fish spawned during                        Matte, C.L., N. Sherwood, L. Crim, andj. Tan.
                the experiment. Total number of spawned eggs by                            1988. Induced spawning of maturing milkfish (Chanos
                the GnRH-analog treated fish (1,378,000 eggs/fish)                           chanos) using human chorionic gonadotropin and mamma-
                was more than twice that of HCG-injected fish                                lian and salmon gonaclotropin releasing hormone
                (500,000 eggs/fish). Thus, it is possible that some                          analog. Aquaculture 73:333-340.
                fish probably spawned more than twice during the                       Nagahama, Y
                                                                                           1987. 17ot, 200-Dihydroxy-4-pregnen-3-one: A teleost matura-
                experiment. Because fertilization and hatching rates                         tion-inducing hormone. Dev. Growth & Differ. 29:1-12.
                rapidly decreased during the experiment, further                       Santos, A.J.G., K. Furukawa, K. Bando, K. Aida, and 1. Hanyu.
                studies are necessary to improve the techinques for                        1986. Photoperiodic determination of preovulatory gonado-
                obtaining eggs of high quality.                                              tropin surge on set time in the carp Cyprinus carpio.
                                                                                             Nippon Suisan Gakkaishi 57:1167-1172.
                                                                                       Young, G., L. Crim, H. Kagawa, A. Kambegawa, and Y Nagahama.
                                                                                           1983. Plasma 17a,20P-dihyroxy-4-pregnen-3-one levels dur-
                Acknowledgment                                                               ing sexual maturation of amago salmon (Oncorhynchus
                                                                                             rhodurus): Correlation with plasma gonadotropin and in
                Thanks is extended to the staffs of the Komame                               vitro production by ovarian follicles. Gen. Comp.
                (Kochi Prefecture) and Goto (Nagasaki Prefecture)                            Endocrinol. 51:96-105.






                      Gametogenesis of Triploid Bivalves with Respect to Aquaculture



                                                      KATSUHIKO T. WADA AND AKIRA KOMARU

                                                                 National Research Institute of Aquaculture
                                                                                    Nansei
                                                                              Mie 516-01, Japan






                                                                              ABSTRACT


                                     Triploid bivalves have been induced in many species and are expected to be better in
                                   quality than diploid animals in aquaculture in relation to retarded gametogenesis
                                   (Stanley et al. 1981; Allen et al. 1982, 1986). Recently however, triploids of some bivalve
                                   species have been reported to mature and to produce active sperm and ripe eggs. This
                                   paper deals with the gametogenesis in triploid bivalves with respect to resolving problems
                                   associated with their release when they are introduced into intensive culture in the sea.
                                   We have been studying triploids of the Japanese pearl oyster Pinctada jucata martensii
                                   (Uchimura et     al. 1989; Wada et al. 1989; Komaru and Wada 1990) and the scallop
                                   Chlamys nobilis   (Komaru et al. 1988). Gametogenesis proceeds very differently in these
                                   two species as   seen during the seasonal histological observation of gonads. Retardation
                                   of maturation,   was observed'in both sexes of the triploid scallop: neither sperm, nor
                                   mature eggs were detected (Komaru and Wada 1989). However, some triploid pearl
                                   oysters produced sperm and ripe eggs in gonads that were less developed than those of
                                   diploids (Komaru and Wada 1990). Sperm dissected from some of the triploid pearl
                                   oysters were active during microscopic examination. The eggs dissected from triploids
                                   showed germinal vesicle breakdown after treatment with ammonium sea water as seen in
                                   diploid eggs. Sperm from triploid pearl oysters had about 1.3 times higher mean values
                                   and a wider distribution range of relative DNA contents than those from diploids. We
                                   have not observed the spawning of triploid pearl oysters in the histological observation
                                   of animals cultured under natural conditions (Wada and Komaru unpubl. data). How-
                                   ever, in the oyster Crassostrea gigas, abnormal DNA-content values of sperm dissected
                                   from triploids have been reported and the possibility of genetic abnormality was sug-
                                   gested in the D-shaped larvae. This genetic abnormality may have resulted from the
                                   insemination of normal eggs from a diploid female With sperm dissected from a triploid
                                   male (Allen 1987; Allen and Downing 1990; Akashige 1990). In the natural area occu-
                                   pied by the intensive cultures, it is necessary to avoid genetic abnormality in the zygotes
                                   from the triploids because such a zygote might affect the native stocks. If triploid animals
                                   release functional gametes to the sea, they should be cultured in a restricted and closed
                                   area to avoid damage to natural reproduction. Another proposal may be the introduction
                                   of triploids of exotic species into areas of the sea that they do not currently inhabit and
                                   where growth of the species could be expected after the introduction.



              Citations                                                                          Proc. world symp. on selection, hybridization and genetic
                                                                                                 engineering in aquaculture, Vol. 11; (K. Tiews ed.),
              Akashige, S.                                                                       p. 208-217. Bordeaux, 27-30 May 1986 Heeneman
                   1990. Growth and reproduction of triploid Japanese oyster in                  Verlaggesellschaft, Berlin.
                     Hiroshima Bay. In Proceedings of the fifth international con-        Allen, S.K. Jr., P.S. Gagnon, and H. Hidu
                     gress of invertebrate reproduction; 23-28 July 1989, Nagoya              1982. Induced triploidy in the soft-shell clams: cytogenetic
                     (M. Hoshi and 0. Yamashita eds.), p. 461-468. Elsevier,                     and allozymic confirmation. J. Hered. 73:421-428.
                     Amsterdam.                                                           Allen, S.K. Jr., H. Hidu, and J. G. Stanley
              Allen, SX, Jr.                                                                  1986. Abnormal gametogenesis and sex ratio in triploid soft-
                   1987. Gametogenesis in three species of triploid shellfish:                   shell clams (Mya arenaria). Biol. Bull.(Woods Hole)
                     Mya arenaria, Crassostrea gigas and Crassostrea virginica. In               170:198-210.
                                                                                                                                                             19







                  20         NOAA Technical Report NMB 106

                  Allen, S. K. Jr., and S. L. Downing                                              StanleyJ.G., S. K. Allenjr. and H. Hidu
                       1990. Performance of triploid Pacific oysters, Crassostrea                       1981. Polyploidy induced in the American oyster, Crassostrea
                          gigas:gametogenesis. Can. J. Fish. Aquat. Sci. 47:1213-1222.                    virginica, with cytochalasin B. Aquaculture 23:1-10.
                  Komaru, A. and K I Wada                                                          Uchimura, Y, A. Komaru, K T. Wada, H. leyama, M. Yamaki. and
                       1989. Gametogenesis       and growth of induced triploid scal-                H. Furuta
                          lops Chlamys nobilis.  Nippon Suisan Gakkaishi 55:447-452.                    1989. Detection of induced triploidy at the different ages of
                       1990. Gametogenesis of triploid Japanese pearl oyster, Pinctada                    larvae of the Japanese pearl oyster, Pinclada fucata martensii
                          fucata martensii. In Proceedings of the fifth international con-                by microfluorometry with DAPI staining. Aquaculture
                          gress of invertebate reproduction; 23-28 July 1989, Nagoya                      76:1-9.
                          (M. Hoshi and 0. Yamashita eds.), p. 469-474. Elsevier,                  Wada, YL T, A. Komaru and Y. Uchimura
                          Amsterdam.                                                                    1989. Triploid production in the Japanese pearl oyster,
                  Komaru, A., Y. Uchimura, H. leyama and K. I Wada                                        Pinctadafucata martensii. Aquaculture 76:11-19.
                       1988. Detection of induced triploid scallop, Chlamys nobilis,
                          by DNA microfluorometry with DAPI staining.
                          Aquaculture 69:201-209.






                      Increasing the Growth Rate of Abalone, Haliotis discus hannai,
                                                     using Selection Techniques





                                                  MOTUYUKI HARA AND SHOGO KIKUCHI

                                                         Tohoku Regional Fishery Research Laboratory
                                                                 3-27-5 Shinhama, Shiogama
                                                                     Miyagi 985, Japan




                                                                      ABSTRACT


                                 Recently, a large number of abalone, Haliotis discus hannai, seeds have been produced
                               by artificial fertilization and cultured in Japan. Most of the seeds have been released into
                               the sea to increase abalone resources. However, some people have tried to continue the
                               cultivation of the abalone seeds to commercial size in tanks. These efforts have been
                               economically unsuccessful because of the typically low growth rate of this species. There-
                               fore, it is necessary to improve their growth genetically. The genetic variation among lots
                               of the seeds produced from different parents were examined. Differences in growth
                               among the lots were observed for 190 days during rearing in the same tank. Moreover, to
                               test the association between growth and isozyme genes, the Gallele frequency at the
                               P9__1 locus was compared between the large and small shell size classes. A higher fre-
                               quency in the large shell size class was observed in 8 of 11 lots. This result suggests that
                               growth in abalone is closely related to genetic factors. We also attempted to genetically
                               improve abalone. By selecting and reculturing the fastest growing individuals for two
                               generations in small scale culture in our laboratory. The results suggest a possibility of
                               improving the growth rate of cultured abalone using selection techniques.



            Introduction                                                        studied at all. It is known that variations in the shell
                                                                                size exist within and between lots of abalone seed.
            Presently, many marine species are produced by arti-                Such a phenomenon could be caused by both genetic
            ficial fertilization and are cultured in Japan. One of              differences and environmental differences such as
            the most impor tant species is abalone. Twenty to                   food, temperature, and density of individuals.
            thirty million abalone seeds are produced every year
            by over fifty farming centers (DSI 1990). Most of the
            seeds are cultured to the size of 10-30 mm in shell                 Estimation of the
            length then released into the sea in order to increase              Parental Effects on Growth
            the abalone resource. Some people have tried to con-
            tinue the cultivation of abalone seeds to commercial
            size in tanks; however these efforts have not been                  We reared several lots of abalone (Haliotis discus
            economically successful to date because the seeds ex-               hannai) seed in the same tank and compared the
            hibited low growth rates. Therefore, it is necessary to             growth of individuals. Plastic film tags (Hara. 1989)
            improve growth genetically. Artificial spawning tech-               were used to identify individuals under         the culture
            niques, diets for juveniles, and rearing equipment                  conditions. The influence of the tagging on growth
            needed for abalone culture have been researched for                 was tested. At the beginning of this study (day 0), the
            more than 20 years (Kikuchi and Uki 1974;                           mean shell length of tagged and nontagged individu-
            Takahashi and Koganezawa 1988 ; Uki 1989), but the                  als was 32.4 and 32.8 mm, respectively (Table 1).
            genetic characters of artificial seed have been not                 After 50 days, the mean shell lengths were 35.8 and


                                                                                                                                            21







              22       NOAA Technical Report NMEFS 106



                                                                         Table I
                                      Size of tagged and non-tagged seeds after 50 days rearing in the same tank.

                                                                                Days in culture

                                                                         0                          50


                                   Tagged                           32.4 ï¿½ 2.3                   35.8-- 2.2
                                   Nontagged                        32.8 _-2.1                   35.7  2.0



                                            9,

                                         _9r *




                        Cultivation for one year in each        tank






                 Measured with day 0        -       Collect about 40 individuals

                                                      from each lot and mix

                 Measured with day 50       -         in same tank after tagging



                 Measured with day 90       -       Experimental conditions

                                                      Water temp. : 20 ï¿½ VC

                 Measured with day 140      -         Food : Diatoms , Laminaria
                                                              and Eisenia                                       Figure I
                                                                                                Experimental procedure used to culture
                 Measured with day 190 -              Tank size : 158x11Ox6Ocm.1 ton            abalone and timctable used in assessing
                                                                                                their growth.


              35.7 mm. Thus, no difference in growth was observed             than that of lot A (t-test, P<0.05). This difference in-
              after 50 days rearing in the same tank.                         dicates parental effects in cultured abalone and
                To examine parental effects, we compared the                  suggests genetic deficiencies.
              growth among three lots of offspring,     each produced           In order to clarify these results, differences in
              from different sets of parents. Figure    I shows the ex-       growth of offsprings were estimated among three pa-
              perimental procedure. Each lot was produced by four             rental pairs (A, B, and Q each divided into and three
              females and three males in separate 1-ton tanks (158            different size classes (small, medium, and large). Fig-
              X 110 X 60 cm). After I year of cultivation, about 40           ure 3 shows the frequency distribution of their
              individuals were collected from each lot, tagged, and           offsprings' shell lengths. Average daily growth and
              then mixed in the same tank. Shell lengths were mea-            the coefficient of variance for each set of offspring is
              sured at about 50 days intervals. Seawater                      shown in Table 2 by size class. In the offspring of pair
              temperature was about 20' C, and food was supplied              A, the average daily growth of the small class was 58.6
              first by diatoms, then by Laminaiia and Eisenia spp.            pm/day, that of the medium class was 82.3 lim/day,
                Figure 2 shows the growth curve (by shell length)             and the growth of the large class was 111.4 jim/day.
              of each lot. At day 0 the mean shell length of lots A,          In offspring B, the average daily growth for small,
              B, and C were nearly identical at 23.6, 23.3, and 23.8          medium, and large classes were 143.0, 134.1, and
              mm, respectively. As the abalone grew, differences in           127.7 lim/day, respectively. In the offspring of C, the
              shell length became large among the three lots. After           small, medium, and large classes were 127.8, 120.5
              190 days from the beginning of the experiment, the              and 134.6 lim/day, respectively. Thus, daily growth
              mean shell length of lot B was significantly larger             means of the offspring from pairs B and C were re-








                                                                                         Hara and Kikuchi: Growth Rate of Abalone           23
                  50-                                                           these results, one could surmise that the growth was
                                                                  B-87          related to genotypes of parents.

                                                                     A
               1540-                                                C-87        Association between Pgm4 AHele
               M                                                                in Isozyme and Growth
               4J                                                    0
                                                                  A-87
                                                                                To test the association between genotypes and
                                             A @@@
                  30                         0                                  growth, the enzyme polymorphism of phosphoglu-
                                                                                comutase (PGM) isozyme was used. Electrophoresis
                                                                                was carried out using an 11% starch gel in 15.5 mM
                                                                                tris and 4.5mM citrate acid buffer. The electrode
                  20-                                                           buffer was 0.155M tris and 0.045M citrate acid (pH
                                                                                7.0). A voltage of 14.25 V/crn was applied for 6 hours
                        0          50        90        140         190          at 4' C. The detection of phosphoglucomutase on
                                           Days                                 the gel was done by the staining procedure in Show
                                                                                and Prasad (1970). Figure 4 shows electrophoretic
                                       Figure 2                                 pattern of PGM isozyme in abalone, and the pheno-
            Growth of offspring from different parental stocks. Age of          typic variation observed. There are two loci, namely
            abalone at day 0 was 1 year old.                                    Pgm-1 and Pgm-2. Appearance of Pgm-2 is not stable;
                                                                                therefore, Pgm-1 was used as the genetic marker in
            markably higher than that of the siblings from pair A               this study. A, B, C, and D indicate names of alleles.
            in all size classes. These results support the hypoth-              The B and C alleles at the Pgm-1 locus were predomi-
            esis that there are parental effects on growth in all               nate in all the seeds from the 11 lots examined. The
            size classes. Differences were also noted in the daily              C allele of the Pg-m-l locus was selected as the marker
            growth among the size classes within the pair B and                 gene in the present experiment.
            the pair C offspring. The coefficient of variance in                  To test the association between genotype and
            the offspring of pair B ranged from 19.9 to 27.1%.                  growth, the C allele frequency among 11 lots was ex-
            The coefficient of variance in those from pair C                    amined. After cultivation for about one year, the
            ranged from 11.2 to 28.0%. Pair A offspring, which                  seeds were divided into three classes: large, medium
            showed the greatest differences in daily growth                     and small. The Gallele frequency at the Pgm-1 locus
            means between the small and large size classes, also                was compared between the large and small classes. A
            showed larger coefficients of variance than pair B                  higher frequency (8 lots out of 11) was observed in
            and pair C offspring in all size classes. The small size            the large class (Table 3). If growth is controlled by
            class of the pair A offspring had the lowest'daily                  the C allele at the Pg-m-l locus, the Gallele frequency
            growth and largest coefficient of variance. This obser-             would be universally high in the large class. If growth
            vation might be caused by the existence of a large                  is independent of the allele at the Pg-?n-I locus, the C
            number of individuals with low growth rates. From                   allele's appearance is likely to be equal in both the


                    40-           A-88                     B-88                        C-88


                     30-

                >1
                U
                ',u  20-
                Cr
                CU
                S_
                U_

                     10-
                                                         S                                                               Figure 3
                      0         S                              M      L              S     M     L           Frequency distribution of shell
                         18        24      2B    32 18      22      26    30    16      22     26     30     length in offspring of parental
                          ri IS                                                ;S
                                                                                                             pairs A-88, B-88, and C-88. S
                                                      Shell length (mm)                                      small size class; M = medium
                                                                                                             size class; L =,large size class.







                  24        NOAA Technical Report NXEM 106



                                                                                      Table 2
                                        Daily growth (@tm/day) and coefficient of variance in three size class of offspring from
                                                         three parental pairs after 190 days of laboratory culturing.

                                             Parental                                                     Offspring size class
                                                pair                     Small                     Medium                        Large

                                                A-88                    58.6                        82.3                       111.4
                                                                       (79.0%)                     (57.1%)                     (41.7%)
                                                B-88                   143.0                       134.1                       127.7
                                                                       (19.9%)                     (23.8%)                     (27.1%)
                                                C-88                   127.8                       120.5                       134.6
                                                                       (28.0%)                     (30.7%)                     (11.2%)




                                                                                               A
                                                                                               B
                                                                                               C   Pgm-i
                                                                                               D





                                                                                               A
                                                                                               B   Pgm-2
                                                                                               C




                             8/8       C/C      A/B     A/C       B/D      B/C      C/o
                                                                                                                                     Fig"e 4
                                                Genotype of Pgm-1                                               Phenotypic variations of PGM isozyme ob-
                                                                                                                served in abalone.




                                                                                        Table 3
                                                        C-allele frequencies at the Pg7n locus of large and small classes.

                                          Lot                                  Large class                                  Small class

                                          A-85                                 0.330    (427)-               >              0.216   (162)
                                          B-85                                 0.315    ( 62)                >              0.235   (249)
                                          C-85                                 0.465    (187)                >              0.365   (230)
                                          A-87                                 0.919    (301)                >              0.872   ( 82)
                                          B-87                                 0.466    (326)                <              0.525   (100)
                                          C-87                                 0.497    (301)                <              0.505   (107)
                                          D-87                                 0.662    (339)                >              0.587   (104)
                                          E-87                                 0.787    (338)                >              0.720   (134)
                                          A-88                                 0.216    (117)                >              0.201   ( 67)
                                          B-88                                 0.497    (149)                >              0.485   (132)
                                          C-88                                 0.500    ( 93)                <              0.564   (110)


                                          a Number of individuals examined.




                  classes. It could be tentatively concluded that the as-                      Growth of Selected Abalone
                  sociation between the Gallele frequency and growth
                  is related to the linkage of the phosphoglucornutase                         The results above suggest that growth in abalone is
                  isozyme gene with the genes related to growth.                               closely related to genetic factors. If this is true, an







                                                                                     Hara and K&uchi: Growth Rate of Abalone          25



               1st Generation
                                                                                          80
                                                                                               195 rm/day

                                              Selection of                                60.
                                                                                      tj
                                              large sized seeds                                240
               2nd Generation

                                                                                          40-


                                                                                      V)

                                              Selection of                                20
               3rd Generation                 large sized seeds
                                                                                          0   L
                                                                                                8    10         14        18
                                              Large sized seeds                                          Months
                                              used in this experiment                                 Figure 6
                                                                                    Growth of abalone offspring selected for su-
                                                                                    perior growth rate.
                    Process of selecting experimental seeds.


                                     Figure 5
            Process used to select abalone geeds for experimentation.





                                                                       Table 4
                     Average daily growth (l.Lm/day) of seeds from parents selected for high growth rate and commercial seeds.

                                  Shell size                                            Seeda
                                    (mm)                               Selected                           Control


                                    20-30                               188                               < 155
                                    30-50                               -                                 < 141
                                    30-70                               240                                   -
                                    50-70                               -                                 <  147
                                     >70                                195                                   -


               a Maximum values of commercial seed growth reported in the past (Uki et al. 1981).




            effect should be expected when selecting for the                 culture and the large-size class offspring were simi-
            maximum growth rate in abalone. The effect of selec-             larly obtained. These offspring were also cultured for
            tion for shell length was thus tested. Figure 5 shows            8 months. The large-size class from this lot was used
            the process of selecting abalone for this experiment.            in this experiment.
            The original lot (first generation) were large-sized               The results are shown by the growth curve in Fig-
            seed taken from a fishermen's cooperative farming                ure 6. The average shell length increased from 21 to
            center in Japan. The first generation was grown in               84 mm over a duration of 10 months (from age 8 to
            culture and produced offspring. The large-size class             age 10 months). While abalone were from 20 to 30
            of these offspring (second generation) was.grown in              mrn in shell length, the average daily growth was 188







                 26        NOAA Technical Report NMFS 106
                 VLm/day. From 30 to 70 mm in size, the daily growth                       Citations
                 averaged 240 jim/day and those over 70 mm more
                 over grew about 195 @Lrn/day.                                             Department of Statistical Information (DSI)
                   These daily growth rates were compared to those                              1990. Fisheries Statistics of Japan. DSI, Ministry of Agricul-
                                                                                                  ture, Forestry, and Fisheries, Government of Japan. (In En-
                 of commercial seed (Table 4). The average daily                                  glish.)
                 growth from 20 to 30 mm in shell length was 188                           Hara, M.
                 @Lrn/day in this experiment, while the maximum daily                           1989. Effect of parents on growth in the abalone seeds,
                 growth in commercial seed was less than 155 @tm/day                              Suisanikushu 14:39-42. (Injapanese.)
                 as reported by Uki et al. (1981). The daily growth                        Kikuchi, S. and N. Uki
                 rate of the selected seed from 30 to 70 mm in size was                         1974. Technical study on the artificial spawning of abalone,
                                                                                                  genus Haliotis. Part 11: effect of irradiated sea water with
                 240 @tm/day, while commercial seed ranging from 30                               ultraviolet rays on inducing to spawn. Bull. Tohoku Reg.
                 to 50 mm and 50 to 70 mm in shell length grew at a                               Fish. Res. 33:79-86.
                 rate of only 141 and 147 jim/day, respectively.                           Takahashi, K. and A. Koganezawa.
                 Clearly, the daily growth in the present experiment                            1988. Mass culture of Ulvella lens as a feed for abalone,
                 was larger than that reported in the past.                                       Haliolis discus hannai. In New and innovative advances in
                                                                                                  biology/ engineering with potential for use in aquaculture:
                   This result indicates that selective breeding is use-                          proceedings of the fourteenth U.S.-Japan meeting on aqua-
                 ful for improving the growth rate of cultured                                    culture (AX Sparks, ed.) p. 29-36. NOAA Tech. Rep.
                 abalone. It is difficult to obtain stable conditions in                          NMFS 70.
                 rearing experiments. Therefore, it is necessary to re-                    Uki, N.
                 peat this or a similar rearing experiment in order to                          1989. Abalone seedling production and its theory (3). Int.
                                                                                                  J. Aquat. Fish. Technol. 1:224-231.
                 get a @7eneral conclusion.                                                Uki, N.,J.F. Grant and S. Kikuchi
                                                                                                1981. juvenile growth of the abalone, Haliotis discus hannai,
                                                                                                  fed certain benthic micro algal related to temperature.
                 Acknowledgments                                                                  Bull. Tohoku Reg. Fish. Res. Lab., 43:59-64.
                                                                                           Show, C.R. and R. Prasad
                                                                                                1970 Starch gel elecLrophoresis of enzymes-a compilation
                   The authors would          like to thank Y Fujio and A.                        of recipes. Biochem. Genet. 4:297-320.
                 Kijima for critically reading this manuscript. This
                 study was supported by grants to the Agriculture, For-
                 estry, and Fisheries Research Council Secretariat.







                       Ovarian Development in the South American White Shrimp,
                                                           Penaeus vannamei



                                  SUSAN M. RANKIN, JAMES Y BRADFIELD and LARRY L. KEELEY
                                                   Laboratoriesfor Invertebrate Neuroendocrine Research
                                                               Department ofEntomology
                                                                 Texas A &M University
                                                             College Station, TX 77843-24 75




                                                                     Abstract


                                Ovarian development is characteriz ed in part by an accumulation of several polypep-
                              tides (protein subunits). These polypeptides become the most abundant components of
                              the mature ovaries. Thus, the factors that regulate synthesis of these polypeptides are
                              major factors that control egg development. In the South American white shrimp,
                              Penaeus vannamei, these polypeptides are synthesized by the ovaries, presumably under
                              the direction of one or more hormones. We are using contemporary techniques of
                              molecular biology and peptide chemistry to identify the factors which inhibit or promote
                              synthesis of these important reproductive polypeptides. Our characterization of these
                              factors will lead to the development of analogs to promote reproduction under maricul-
                              ture conditions. In this paper we describe ovarian maturation in shrimp and review our
                              progress in (1) promoting an understanding of the intricate biological activities that
                              culminate in spawning and (2) exploiting this knowledge to regulate reproduction in
                              intact broodstock females under mariculture conditions.




            Introduction: the Problem                                          Thus, a fundamental problem in the shrimp mari-
                                                                             culture industry is the lack of predictable, abundant
            Achievement of the full economic potential of the                supplies of offspring of known heritage. The resolu-
            shrimp mariculture industry depends on the success-              tion of this problem lies in a multidisciplinary,
            ful domestication of the shellfish, along with genetic           multidimensional approach that involves the coop-
            selection for desired traits such as rapid growth, or            eration of physiologists, chemists, molecular
            high tolerance for changes in temperature, salinity,             biologists, and producers. We have an obligation
            or water quality. The key to domestication lies in en-           to combine our efforts to relieve this problem by de-
            hanced, controlled reproduction by the broodstock                veloping biotechnology and by establishing rear-
            animals.                                                         ing conditions (appropriate temperatures, light in-
              To date, efforts to domesticate shrimp have been               tensities, and diets) that induce reproductive
            hampered, at least in many of the U.S. firms, by the             development.
            practice of harvesting wild stock and promoting re-                Here we describe ovarian maturation in shrimp
            production by eyestalk ablation ( i.e., surgical                 and our progress in establishing the tools. necessary
            removal of an eyestalk). This practice has several               for identification of the hormones that affect repro-
            drawbacks: 1) continual introduction of wild stock as            duction. Furthermore, we describe our efforts in
            the basis for the next generation does not allow ge-             establishing the basis for bioengineering these organ-
            netic selection and risks introduction of shrimp                 isms to suit the needs of mariculture. We represent
            predators and pathogens; 2) many eyestalk-ablated                several disciplines working together to solve a com-
            animals fail to reproduce, which reduces total yield             mon problem: understanding shrimp reproduction
            and predictability of numbers of progeny; and 3) eye-            and exploiting that understanding to improve shrimp
            stalk ablation also reduces the longevity and total              production.
            fecundity of the broodstock.


                                                                                                                                      27







               28       NOAA Technical Report NAM 106


                             y   41.998 + 341.23x + 1942.8XA2 RA   2  0.756
                        500
                  CL


                        400
                  >
                  0
                        300



                        200
                  E
                  C     100             0                                                                   Figure I
                  ZD                                                                  The total amount of protein in the ovaries as a func-
                                                                                      tion of oocyte diameter in P vannamei. Each point
                  IL       0                                                          represents the measurement for a pair of ovaries
                           0.0         0.1          0.2          0.3          0.4     from a single broodstock female. (Rankin et al.
                                      Oocyte diameter (mm)                            1989.) Diameters below the resolution of the dissect-
                                                                                      ing microscope are graphed at "0.0".


               Morphology: Identification of the Players                         Biochemical Analysis of Ovaries

               Animals. Broodstock Penaeus vannamei, the species                 Oocyte diameter increased from below the resolution
               most favored for commercial mariculture in the                    of the dissecting microscope ("0", ie., <0.01 mm) up
               United States, were obtained from Laguna Madre                    to about 0.3 rum. Total protein in pairs of ovaries
               Shrimp Farm (Los Fresnos, TX) and Sea Critters                    increased from < 5 mg to about 400 mg (Fig. 1;
               (Tavernier, FL). Most of the broodstock females were              Rankin et al. 1989). The protein measurements
               40-60 g and unilaterally eyestalk ablated to promote              shown in Figure I are the sum of a number of differ-
               reproductive development (Rankin et al. 1989).                    ent kinds of proteins. Fortunately, ovarian growth is
                  Eyestalk ablation is presumed to promote repro-                marked by the accumulation of a few specific pro-
               ductive development by removing a gonad inhibiting                teins that we designate as the major ovarian proteins.
               hormone (see Char niaux-Cotton and Payen 1988).                   Factors that regulate the synthesis of these particular
               The eyestalks synthesize, store, and release a number             proteins are therefore major elements that control
               of hormones, with one or more of these presumably                 ovarian development.
               suppressing reproduction. The bluish-white sinus                    To identify these major ovarian proteins, we used
               gland is the neurohemal organ that stores and                     as our initial criteria that they should be absent from
               releases neurohormones in the eyestalk (Charniaux-                previtellogenic (immature) ovaries, and become in-
               Cotton and Payen 1988).                                           creasingly prominent, to strikingly abundant in
                  The mature ovary runs the length of the abdomen,               nearly mature ovaries. We used sodium dodecyl Sul-
               surrounds the hepatopancreas, and extends into the                fate polyacrylamide gel electrophoresis (SDS PAGE)
               head region (Bell and Lightner 1988). During matu-                to separate polypeptides, subunits of proteins. This
               ration, the ovaries progress from a clear, empty                  approach is unorthodox in that procedures for iden-
               appearance, to white, then usually to a creamy yellow             tifying major ovarian protein usually include salt
               as they near maturity. There is some variation, how-              precipitation and column chromatography prior to
               ever, in the color of vitellogenic ovaries; they may              or instead of SDS PAGE. We intentionally omitted
               appear greenish or orange instead of yellow.                      precipitation and chromatography to avoid potential
                  The hepatopancreas is analogous to the liver of a              loss of major protein candidates. The potential for
               vertebrate, serving as a general center of intermedi-             loss of major proteins was great in Penaeus vannamei,
               ary metabolism and as a site for storage of reserves.             because solubility in some buffers was low (Rankin et
               Compared to the mature ovary, it is a relatively dis-             al. 1989).
               crete and small organ, comprised interiorly of a vast               The results of SDS PAGE of ovaries in varying
               number of tubules that are surrounded and held in                 stages of reproductive maturity are shown in Figure 2
               place by the highly pigmented sheath (Bell and                    (Rankin et al. 1989). Ovaries in each stage had many
               Lightner 1988).                                                   different polypeptides. Most of the polypeptides were






                                                                             __ Rankin et al.: Shrimp Ovarian Development                       29
                                                                                   weight polypeptides that we designated the major
                  M
                       x 10-3                                                      ovarian polypepticles. Yano and Chinzei (1987) have
                    r                2 3            123                            demonstrated that the ovary of Penaeus japonicus is a
                       205-                                                        site of synthesis of ovarian proteins, whereas Tom et
                                                                                   al. (1987b) suggest an extraovarian source in
                                                                                   Parapenaeus longirostris.
                                                                                     Not shown are hepatopancreas tissue samples;
                       11.6-                                                       those appeared to lack the high molecular weight
                                                                                   polypeptides. We also tested whether the hepatopan-
                       66-                                                         creas made and secreted these polypeptides for
                                                                                   transport to the ovary by similarly incubating the
                                                                                   hepatopancreas and then monitoring the culture me-
                                                                                   dium for the presence of secreted polypepticles.
                       45 -                                                        Using this strategy, we could not detect synthesis of
                                                                                   the high molecular weight polypeptides in the me-
                                                                                   dium after incubation (Fig. 2C). Thus, the hepato-


                       29-                                                                                  1       2      3
                                                                                                   kb 3
                                     A                                                              9.5-1

                                        Figure 2
             SDS-PAGE of P vannamei ovarian and hepatopancreatic
                                                                                                   4.4
             polypeptides during the gonadotrophic cycle. (A)
             Coomassie blue-stained ovarian samples: Lane 1,
             previtellogenic ovary; lane 2, ovary at the onset of vitello-
             genesis; lane 3, ovary in mid to late vitellogenesis. (B)
             Autoradiograph of the same samples incubated with ["S]-
             methionine. (C) Autoradiograph             of [15S]
                                                                   -labelled
             polypeptides in the culture medium after incubation of
             hepatopancreas from shrimp in mid-late vitellogenesis.
             Bracket indicates the major polypeptides (-175-200 kD)
                                                                                                         4
             synthesized and'accumulated by ovaries during yolk forma-
             tion. (Rankin et al. 1989.)                                                            1.4-@..


             found in ovaries of each of the three stages exam-
             ined. However, several polypepticles were prominent
             in developed ovaries and absent from immature
             ones. These polypeptides were 175-200 kDa, and met
             our criteria for major ovarian polypeptides.
                                                                                                   0.3-1
               The relative insolubility of the major ovarian
             polypepticles suggested that in P vannamei, either the
             ovary was making the polypepticles itself, or it was                                            Figure 3
             modifying them after they were taken up. We investi-                                Total RNA samples (5 j.Lg/lane)
             gated the origin of these polypeptides using                                        from mid-reproductive cycle R
             autoradiography (Rankin et al. 1989). Tissues were                                  vannamei tissues were dena-
             incubated in a culture medium with "S-methionine,                                   tured, separated by electro-
             then the protein was extracted and separated by SDS-                                phoresis in agarose, transferred
             PAGE.                                                                               to nylon membrane, and hybrid-
               The autoradiographic visualization of the polypep-                                ized with the cloned and
                                                                                                 labelled 3 kb ovarian cDNA.
             tides synthesized by the ovary is shown in Fig. 2B. It                              Lane 1, ovary; lane 2, hepato-
             appears that most of the polypeptides that were                                     pancreas; lane 3, muscle. RNA
             present in the ovary (Fig. 2A) could also be synthe-                                size markers are indicated at
             sized there. It clearly synthesized the high molecular                              left. (Bradfield et al. 1989.)







                30         NOAA Technical Report NNITS 106


                                               Construction of Expression Plasmid



                                                                             Insertion site
                                                 13-gal promoter
                                   pUR 291                                                         pUR 291
                                                                      6-gal code,,
                                                                                11     1,                                                  Figure 4
                                                                                                                                   Expression vector pUR
                                                                                                                                   291 was linearized near
                                                                                                                                   the T-terminus of the
                                                                                                                                   P-galactosidase coding
                                  13-gal promoter                                                                                  region, and ligated in
                   pU R 291                                                                       '@@@pUR 291                      phase with the 3 kb
                                                       13-agal co@de',                                                             shrimp ovarian cDNA
                                                                     Shrimp polypeptide code
                                                                                                                                   for production of a fu-
                                                                                                                                   sion protein.
                                                               2                           pancreas does not appear to synthesize these poly-
                              M
                                       0-3                                                 peptides. Similarly, in P japonicus, yolk protein is not
                                 rxl                                                       immunciprecipitated from the hepatopancreas (Yano
                                     205-                                                  and Chinzei 1987). These results do not preclude
                                                                                           other major contributions by the hepatopancreas to
                                                                                           ovarian development, such as lipid synthesis neces-
                                                                                           sary for yolk, as has been suggested by Castille and
                                    116- "0010"                                            Lawrence (1989).

                                       97-
                                                                                           Beginning Genetic Analysis of Ovaries
                                      66-'
                                               40400      tow                              The future contribution of genetic engineering to
                                                                                           aquaculture lies in the production of genetically al-
                                                                                           tered individuals with phenotypes that are well suited
                                                                                           to aquacultural conditions. Realization of the ben-
                                                                                           efits of genetic engineering depends on the
                                                                                           generation of complementary DNA (cDNA) libraries,
                                      45-                                                  identification of genes and gene products, and on
                                                                                           judicious use of molecular technology. We have be-
                                                                                           gun this long process in P vannamei and have used
                                                                                           modern recombinant genetic techniques to explore
                                                                                           the processes of ovarian development, again, with the
                                                                                           goal of promoting the understanding and eventual
                                              Figure 5                                     manipulation of those events.
                           E. colijM101 was transformed with plasmid                         Our genetic analysis began with the construction
                           pUR 291, and plasmid-encoded P-galactosi-                       of an ovarian cDNA library (Bradfield et al. 1989). To
                           dase was induced by addition of                                 accomplish this, mRNA was purified from total RNA
                           isopropyl-R-D-thiogalactopyranoside. Cell                       that was isolated from the ovaries. The mRNA was
                           extracts were separated by SDS-PAGE                             then used to make the cDNA library. The cDNA for
                           (7.5%) and stained with Coomassie Blue.                         one of the major ovarian polypeptides was isolated,
                           Lane 1, native pUR 291; lane 2, recombi-                        cloned, and used as a probe to identify tissues active
                           nant pUR 291 containing the 3 kb ovarian                        in synthesis of that polypeptide. This is a very sensi-
                           cDNA. The heavy band at 116 kDa in lane                         tive assay for determining tissue sources of particular
                           I is unfused P-galactosidase. The band at                       polypeptides.
                           205 kDa in lane 2 is a fusion consisting of
                           P-galactosidase linked to a polypeptide en-                       The tissue source and the size of this highly ex-
                           coded by the ovarian cDNA. (Bradfield et                        pressed transcript were made visible using northern
                           al. 1989.)                                                      hybridization. In this procedure, RNA was extracted






                                                                                   Rankin et a].: Shrimp Ovarian Development        31








                                                                    4



                                           4':


                           M X







                                                                                                         "A'












                                                                                         01





                                                                     Figure 6
            Localization of the 200 kDa ovarian polypeptide using immunocytochemistry.    5  Rm ovarian sections were incubated with
            (Panel A) preimmune rabbit Inununoglobin G (IgG) and (Panel B) IgG from the rabbit inoculated with the gel-purified
            fusion polypeptide (see Fig. 2). Immunoreaction was visualized with an alkaline phosphatase-linked second antibody. Arrows
            indicate cortical granules. Scale bars = 100 @tm. (Bradfield et al. 1989.)


            from various tissues (muscle, hepatopancreas, and               product (Bradfield et al. 1989).
            ovaries) from vitellogenic females, separated on an               Figure 4 shows diagrammatically how this was
            agarose gel, transferred to nitrocellulose (which im-           done, illustrating the promoter region, the R-gal
            mobilizes the RNA), then hybridized with the                    code and the insertion site. The recombinant plas-
            radioactive DNA probe representing a major ovarian              mid was then inserted into E. coli. The genetically
            mRNA in P vannamei. The result was made visible by              engineered fusion polypeptide was  205 kDa: -115
            autoradiography.                                                kDa P-gal and -90 kDa R vannamei ovarian polypep-
              Only the ovary had detectable levels of this major            tide. Figure 5 shows that fusion polypeptide as it
            ovarian polypeptide RNA (Fig. 3). The size of the               appeared on an SDS gel. It is only by coincidence
            transcript was  6.5 kb. This is very large, but of             that the fusion polypeptide was about the same size
            course the major ovarian polypepticle is also very              as the ovarian polypeptide from which it was in part
            large. The mRNA for this polypeptide was not de-                derived.
            tected in muscle or hepatopancreas of either                      In summary, we established a cDNA expression li-
            previtellogenic or vitellogenic females. Thus, we had           brary from shrimp ovary, and from that, isolated an
            confirmed by genetic analysis some of our previous              ovarian polypeptide cDNA. That gene Was inserted
            results from in vitro experiments.                              into E. coli, for production of the genetically engi-
              We then made a genetically engineered polypep-                neered fusion product. The genetically engineered
            tide consisting in part of shrimp ovarian polypeptide           protein was then injected into rabbits for production
            and in part, bacterial P-galactosidase (P-gal). To do           of polyclonal antibodies.
            this, the 3 kb cloned cDNA representing a portion of              Immunocytochemistry on 5 @Lrn paraffin sections
            the 6.5 kb ovarian transcript was inserted into the             was used to determine the cellular localization of this
            plasmid pUR 291, in order to get high level expres-             major ovarian polypepticle (Bradfield et al. 1989).
            sion of a fusion polypeptide consisting of                      The clarkly@staining regions, which indicated immu-
            plasmid-encoded P-gal, linked to the cDNA-encoded               noreactivity, were located in the cortical special-







               32      NOAA Technical Report NMFS 106

                                                                                are extraordinarily prominent. Because these cortical
                                                                                specializations are highly abundant and large, shrimp
                                                      X                         provide a beautiful model system for studying regula-
                                                                                tion of synthesis of major cellular organetles.
                                                                                  In contrast to the 200 kDa ovarian polypeptide, a
                                                                                175 kDa polypeptide, appears to be a major compo-
                                                                                nent of the yolk (Fig. 7). In this case, gel-purified
                                                                                polypeptide, rather than genetically engineered
                                                                                polypeptide was used to generate polyclonal antibod-
                                                                                ies in rabbits. Yolk polypeptides serve as food for the
                                                                                developing embryos. This polypeptide is larger than
                                                                                yolk polypeptides described for other decapods (Lui
                                                                                and O'Connor 1977, 1976; Zagalsky 1985; Eastman-
                                                                                Reks and Fingerman 1985; Tom et al. 1987a). In
                                                                                P longiroshis, yolk polypeptides are 45 and 66 kDa,
                          inw                                                   repectively (Tom et al. 1987a).
                                                                                  In summary, we have demonstrated that two major
                                                                                high molecular weight polypeptides in vitellogenic
                                                                                shrimp ovaries are immunologically distinct, and
                                    _"J                                         serve quite different functions for the developing
                                                                                embryos. We now have probes for these two major,
                                                                                distinct ovarian polypeptides which may or may not
                                                                                be regulated by the same factors. Both of these
                                                                                polypeptides are highly abundant and both are essen-
                                        Figure 7                                tial to egg development.
               Localization of a 175 kDa ovarian polypeptide using immu-
               nocytochemistry. 5 @Lrn ovarian   sections were incubated
               with IgG from the rabbit inoculated with gel-purified ovar-      Conclusions
               ian polypeptide. No immunoreactivity was detected in
               control samples (see Fig. 6, panel A). Immunoreaction was          Now that we have identified several important
               visualized with an alkaline phosphatase-linked second anti-      polypepticles that accrue in the ovary during the
               body. Scale bars = 100 pm.                                       cycle of egg development and have demonstrated
                                                                                that the ovary synthesizes them,. we are in a position
               izations (Fig. 6). Thus,   the cortical specializations at       to pursue factors that regulate the production of
               the periphery of the oocyte contained the 200 kDa                those important polypepticles. One goal is to identify
               major ovarian polypeptide.                                       and characterize the hormones that affect reproduc-
                 Cortical specializations, also called cortical rods or         tion and then to determine the mechanisms by which
               cortical bodies, are membrane-bound organelles that              the hormones act. We anticipate that this work, in
               are assembled during oocyte development and be-                  turn, will lead to the development of stable hormone
               come associated with the cell membrane in mature                 analogs which will promote controlled, predictable
               eggs. In response to one or more stimuli (such as                reproduction by intact females of known bloodlines.
               contact with water at spawning, or fertilization), the           We have an obligation to bring people together to
               cortical specializations are rapidly extruded to form a          solve our common problem-understanding repro-
               layer of material that encompasses the egg. In                   cluction-and to exploit that understanding to help
               shrimp, the layer is rapidly dissipated into the sur-            industry improve both production and profits. This
               rounding seawater (Clark and Lynn 1977; Clark et al.             requires not only physiologists, but also chemists and
               1980). Cortical-specialization composition, function             molecular biologists as well-all working with pro-
               and regulation are largely unknown. It is speculated             ducers to achieve our common goal.
               that they prevent polyspermy or serve as an environ-
               mental protectant.
                 The club-shaped cortical specializations of P                  Acknowledgments
               vannamei (Figs. 6 and 7) and other shrimp
               (Duronslet et al. 1975; Clark and Lynn 1977; Clark et            This work was supported by institutional grant NA
               al. 1980; Anderson et al. 1984; Tom et al. 1987b; Bell           85AA-D-SDI28 to Texas A&M University by the Na-
               and Lightner 1988;, Tan-Fermin and Pudadera 1989)                tional Oceanic and Atmospheric Administration's






                                                                                                    Rankin et al.- Shrimp Ovarian Development                   33

              Sea Grant Program, and by the Texas Advanced Tech-                           Eastman-Reks, S.B., and M. Fingerman.
              nology Research Program. The research was                                         1985. In vitro synthesis of vitellin by the ovary of the fiddler
              conducted at the Texas Agricultural Experiment Sta-                                  crab, Uca pugilator J. Exp. Zool. 233:111-116.
                                                                                           Liu, C.H., and O'ConnorJ.D.
              tion.                                                                             1976. Biosynthesis of lipovitellin by the crustacean ovary.
                                                                                                   Part II: Characterization of and in vitro incorporation of
                                                                                                   amino acids into the purified subunits. J. Exp. Zool.
              Citations                                                                            195:41-52.
                                                                                                1977. Biosynthesis of crustacean lipovitellin. Part III: the in-
              Anderson, S.L., E.S. Chang and W.H. Clarkjr.                                         corporation of labeled amino acids into the purified
                   1984. Timing of postvitellogenic ovarian changes in the                         lipovitellin of the crab Pachygrapsus crassipes. J..Exp. Zool.
                                                                                                   199:105-108.
                     ridgeback prawn Sicyonia ingentis (Penaeidae) determined              Rankin, S,M.,J.Y. Bradfield, and L.L. Keeley.
                     by ovarian biopsy. Aquaculture 42: 257-271.                                1989. Ovarian protein synthesis in the South American white
              Bell, T.A., and D.V, Lightner.                                                       shrimp, Penaeus vannamei, during the reproductive
                   1988. In A handbook of normal penaeid shrimp                                    cycle. Invertebr. Reprod. Dev. 15:27-33.
                     histology. Allen Press, Lawrence, KS.                                 Tan-Fermin,J.D., and R.A. Pudadera.
              Bradfield, J.Y., R.L. Berlin, S.M. Rankin, and L.L. Keeley.                       1989. Ovarian maturation stages of the wild giant tiger
                   1989. Cloned cDNA and antibody for an ovarian cortical                          prawn, Penaeus monodon Fabricius. Aquaculture 77:229-
                     granule polypeptide of the shrimp, Penaeus vannamei. Biol.                    242.
                     Bull. (Woods Hole) 177:344-349.                                       Tom, M., M. Goren, and M. Ovadia.
              Castille, F.L., and A.L. Lawrence.                                                1987a. Purification and partial characterization of vitellin from
                   1989. Relationship between maturation and biochemical                           the ovaries of Parapenaeus [ongirostris (Crustacea, Decapoda,
                     composition of the gonads and digestive glands of the                         Penaeidae). Comp.Biochem.Physiol.87B:17-23.
                     shrimps Penaeus aztecus Ives and Penaeus setiferus (L.). J.                1987b. Localization of the vitellin and its possible precursors
                     Crustacean Biol. 9:202-211.                                                   in various organs of Parapenaeus longirostris (Crustacea,
              Charniaux-Cotton, H., and G. Payen.                                                  Decapoda, Penaeidae). Int. J. Invertebr. Reprod. and Dev.
                   1988. Crustacean reproduction. In Endocrinology of se-                          12:1-12.
                     lected invertebrate types, p. 279-303. Alan R, Liss, Inc, NY.         Yano, L, and Y. Chinzei.
              Clark, W.H. Jr., andj.W. Lynn.                                                    1987. Ovary is the site of vitellogenin synthesis in kuruma
                   1977. A Mg" dependent cortical reaction in the eggs of                          prawn, Penaeus japonicus. Comp. Biochem. Physiol.
                     penaeid shrimp. J. Exp. Biol. 200:177-183.                                    8611:213-218.
              Clark, W.H. Jr., JW. Lynn, A.I. Yudin, and H.O. Persyn.                      Zagalsky, P.F.
                   1980. Morphology of the cortical reaction in the eggs of                     1985. A study of the astaxanthin-lipovitellin, ovoverdin,
                     Penaeus aztecus. Biol. Bull. (Woods Hole) 158:175-186.                        islolated from the ovaries of the lobster, Homarus gammarus
              Duronslet, MJ., A.I. Yudin, R.S. Wheeler, and W.H. ClarkJr.                          (L.). Comp. Biochem. Physiol. 8011:589-597.
                   1975. Light and fine structural studies of natural and artifi-
                     cially induced egg growth of penaeid shrimp. Proc. Meet.
                     World Mari. Soc. 6:105-122.






                       Changes in Hatchery Rearing and Release Strategies Resulting
                           from Accelerated Maturation of Spring Chinook Salmon
                                                          by Photoperiod Control



                                                                     WALDO S. ZAUGG*

                                                          National Oceanic and Atmospheric Administration
                                                                  National Marine Fisheries Service
                                                                      Northwest Fisheries Center
                                                             Coastal Zone and Estuarine Studies Division
                                                         2 725 Montlake Boulevard East, Seattle, WA 98112


                                                    JACK E. BODLE AND JACK E. MANNING
                                                                  U. S. Fish and Wildlife Service
                                                            Little White Salmon National Fish Hatchery
                                                                      Cook WA 98605, USA





                                                                            Abstract


                                   Early maturation and spawning of adult spring chinook salmon (Oncorhynchus
                                 tshawytscha) was induced by controlling photoperiod at the Little White Salmon National
                                 Fish Hatchery. Tagged groups of progeny from these adults were released as subyearlings
                                 in May and June, and again at their normal release time as yearlings in April of the
                                 following year. Fish released as subyearlings were recovered as adults 1) in a ratio of 1:3.5
                                 to fish released during the same years (1984 and 1985) as yearlings; 2) at a higher ratio
                                 of males to females than yearlings; 3) with reduced numbers of precocious males Oacks)
                                 compared with yearlings; 4) predominantly as 4-year-old fish, similar to fish released as
                                 yearlings; and 5) at a larger size at the same age than adults released as yearlings. In spite
                                 of the lower return of adults from subyearling releases, this is a cost- effective method of
                                 supplementing the yearling release program and provides an excellent means of aug-
                                 menting the run.




             Introduction                                                           for the maintenance of harvestable numbers of
                                                                                    adults. Methods used in the hatcheries to rear and
             Historically, the abundant runs of chinook salmon                      release juvenile chinook salmon vary considerably,
             (Oncorhynchus tshawytscha) were used as an important                   depending to some degree upon physical facilities
             food source and for barter among Native Americans                      and available water supplies of the individual hatch-
             inhabiting the Columbia River Basin. Later, as settlers                eries, but more importantly upon the subspecies of
             immigrated into the basin, these fish became impor-                    chinook salmon being reared.
             tant to commercial harvesting and recreational                           Adult chinook salmon can be found in the Colum-
             fishing and were soon overharvested. Presently,                        bia River system, migrating to native waters or
             stocks of chinook salmon are depleted in most of the                   rearing facilities, in nearly every month of the year.
             basin because of blocked and degraded habitat; artifi-                 There are, however, three principal groups: spring,
             cial production in hatcheries has become essential                     summer, and fall chinook salmon, so designated to
                                                                                    correspond approximately to their upstream migra-
               Present address: National Marine Fisheries Service, Cook Field       tion and spawning times. These groups are described
               Station, Cook WA 98605, USA                                          on the next page:


                                                                                                                                                   35







                  36        NOAA Technical Report NMB 106

                     Salmon          Migration        Spawning         Length of                November they were transferred to outside ponds
                     group           time             time             time in river            where water temperatures ranged from 4 to 7' C. In
                     Spring          Jan-May          Jul-Sep          4-6 months               late March and early April 1983-85, groups of ap-
                        chinook                                                                 proximately 50,000 juveniles each were tagged with
                     Summer          Jun-Aug          Sep-Nov          3 months                 coded wire tags and held for release as subyearlings
                        chinook                                                                 in either May or June of the same spring or as year-
                     Fall chinook    Aug-Dec          Sep-Jan          I month                  lings in April of the following year (1984-86) as
                     Spring chinook salmon remain longer in fresh wa-                           indicated below (brood year in parenthesis):
                  ter before spawning than the other major fish groups                                                               Released as      Released as
                  and for much of this time exist in excellent condi-
                  tion. They are highly prized by recreational and                                   Year             Tagged        subyearlings        yearlings
                  commercial freshwater fishermen. However, serious                                  1983             + (82)           + (82)              -
                  problems arise when these fish move into hatchery                                  1984             + (83)           + (83)            + (82)
                                                                                                     1985             +(84)            + (84)            + (83)
                  holding ponds early in the spawning run and must be                                1986                                -               + (84)
                  held for an extended time before spawning. Such
                  confinement often results in high prespawning losses                          Comparisons of adult returns between                     groups re-
                  due to stress, physical injury, and disease. At the                           leased in the same spring (different brood years)
                  Little White Salmon National Fish Hatchery (Little                            could only be made for 1984 and 1985, as in 1983
                  White Salmon NFH) in Washington State, measures                               only tagged subyearlings were released and in 1986
                  have been taken to reduce some of the mortality asso-                         only tagged yearlings were released.
                  ciated with long-term holding by accelerating                                    Fish were fed Oregon Moist Pellets (OMP)
                  maturation with controlled photoperiods. Early                                throughout the rearing period. Groups of tagged
                                                                                                subyearlings that, for six weeks prior to release, had
                  spawning not only reduces prespawning mortality but                           received OMP to which 7% NaCl (on a dry weight
                  allows earlier hatching of eggs. The resulting prog-                          basis) had been added, were liberated with May re-
                  eny experience greater growth because of longer                               leases in 1984 and 1985. Thus, three groups of
                  rearing times and manifest physiological and behav-                           subyearlings were released in each 1984 and 1985;
                  ioral characteristics of developing smolts from 10 to                         two in May (one salt-fed) and one in June.
                  11 months prior to the demonstration of such char-                               Information on the post-release migratory perfor-
                  acteristics in progeny from adults spawned under                              mance of subyearlings released in 1983 was obtained
                  normal conditions.                                                            from captures in beach seines and mid-river purse
                     This report provides information on releases and                           seines at the upriver boundary of the estuary Uones
                  subsequent adult recoveries of tagged subyearling                             Beach, Oregon) after fish had migrated 186 kin
                  and yearling spring chinook salmon from the Little                            (Dawley et al. 1985; Zaugg et al. 1986). Seining op-
                  White Salmon NFH. Some preliminary information                                erations were not conducted after the 1983 season.
                  has been reported previously (Zaugg et al. 1986).                             Information on adult recovery was obtained from a
                                                                                                data base of the Pacific States Marine Fisheries Com-
                                                                                                mission, Portland, Oregon. Weights of recovered
                                                                                                tagged adults were estimated from lengths by com-
                  Materials and Methods                                                         parison with a length/weight relationship deter-
                                                                                                mined in 1989 on a group of 72 adults held at the
                  The Little White Salmon NFH is located on the Little                          hatchery for antibiotic injections.
                  White Salmon River (Washington) about 2 kin from
                  its confluence with the Columbia River. Adult spring
                  chinook salmon returning to holding ponds at the                              Results
                  hatchery by mid-May were moved into portions of
                  raceways covered by a metal building (9.9 X 13 in)                            Figure I compares the abbreviated adult holding and
                  equipped with six sodium vapor lamps (Lucalox G.E.,                           juvenile rearing times that result from photoperiod
                  LU 150/55) located 3.1 in above the water's surface.                          treatment of adults with times for controls that un-
                  They were then subjected to a reduced photoperiod                             dergo normal spawning and juvenile rearing. Under
                  schedule that began with 12 hours of light per day                            the accelerated spawning program, juveniles were re-
                  and decreased at a rate of 30 minutes per week until                          leased as subyearlings in both May andjune. Progeny
                  spawning occurred about mid-July (Zaugg et al.                                from the early spawn began feeding earlier and, con-
                  1986). After hatching, fry were reared inside in tanks                        sequently, were larger on any given date thereafter
                  containing water ranging from about 7 to 9' C. In                             (Table 1; Zaugg et al. 1986).






                                                                           Zaugg et al.: Hatchery Rearing and Release Strategies for Chinook Sahnon                                 .37


                      Feb   -------- May           Jun---- Aug
                     rAdult migration              At hatchery           Spawn


                      NORMAL



                                                                           Hatching and juvenile ivating


                     I Augl Sen I Oct I Novi Dec I Jan I Feb I Max I Am I MaJ Jun I Jul I Aual Seo I Oct I NA Dec I Jan I Feb I Mat I Am


                                                Hatchingand
                                                           juvenile rearing


                     ACCELERATED



                         Adult migration           At hatchery         Spawn
                        Feb   .......... May       Jun  ----- Jul

                                                                                              Figure I
                Comparison of spring chinook salmon adult handling and juvenile rearing between normal and advanced photoperiod
                schedules at the Little White Salmon National Fish Hatchery. Adults returning to this hatchery probably enter the Columbia
                River no earlier than February and are normally spawned in August. For release as yearlings, juveniles from normally
                spawned adults were reared in the hatchery until their second April. For subyearlings, juveniles from treated adults that were
                spawned injuly were released as subyearlings the following May andjune.


                                                                                               Table I
                     Historical spawning, hatching, and fry information for spring chinook salmon from the Little White Salmon National
                                                                             Fish Hatchery (Zaugg et al. 1986).


                                                                Spawning                          Mean wt (g)                      Date of                          Mean wt (g)
                     Group                                        dates                          at first feeding               first feeding                       on I October

                     Control (1976-79)                      11-17 August                              0.32                          10-28 Dec                            13.8
                     Photoperiod (1980-82)                  16-22 July                                0.27                     29 Oct-12 Nov                             24.4




                                                                                               Table 2
                     juvenile migration of subyearling spring chinook salmon from the Little White Salmon National Fish Hatchery (1983),
                                                                                      and adult recoveries.

                                                                              Number caught"
                      Release                Mean               Beach               Purse                 Migration                        Adult recoveryb
                         date               wt (g)               seine              seine              rate (km/day)              Number                   Percent

                     4 May 1985                6.7                37                   7                       12                     14                    0.03
                     24june 1983              10.3                 0                  89                       16                     23                    0.05

                     ,a Number caught adjusted for fishing effort (Dawley et al. 1985).
                     b Numbers adjusted to a release of 50,000; includes commercial and sport fishery catches and hatchery returns (source: Pacific States
                      Marine Fisheries Commission data base).







                  38.       NOAA Technical Report NMFS 106



                                                                                        Table 3
                      Adult recoveries fromjuvenile salmon released from the Little White Salmon National Fish Hatchery in 1984 and 1985.

                        Release year                              Release                                                  Adult recoveries4
                        and group                   Date                         Wt (g)                       Number                          Percent


                      1984
                        Yearling                    19 Apr                         36.6                         109                             0.22
                        Subyearliin                 7 May                           7.0                            5                            0.01
                          (salt  fed;
                        Subyearling                 7 May                           7.0                           11                            0.02
                          (controls)
                        Subyearling                 22 Jun                         11.7                           30                            0.06


                      1985
                        Yearlings                   17 Apr                         43.7                         266                             0.53
                        Subyearliin                 6  May                          7.6                         104                             0.21
                          (@@ah  fed;
                        Subyearling                 6  May                          7.1                         101                             0.20
                          (controls)
                        Subyearling                 20 Jun                         10.5                           68                            0.14


                        Based on 50,000 released; data includes commercial and sport fishery catches and hatchery returns (source: Pacific States Marine
                        Fisheries Commission data base).
                      b Fed a diet containing an additional 7% (dry wt) NaCl for 6 weeks prior to release.



                                                                                        Table 4
                      Numbers of tagged female and male adult salmon returning to the Little White Salmon National Fish Hatchery from
                                                                                 releases in 1984-85.


                                                                Females                                    Males                         Female/male


                      Yearling releases                             199                                      89                               2.24
                        lessjacks                                   199                                      75                               2.65
                      Subyearling releases                          158                                     119                               1.33
                        lessjacks                                   158                                     118                               1.34



                      During the first year of the study, juveniles released                     female to male ratio for adults returning from
                   in June were larger, migrated faster, and produced                            subyearling releases was nearer to 1:1 than the same
                   more adults than those released in May (Table 2).                             ratio for adults returning from yearling releases.
                   Fish released in June were caught exclusively in the                          Only one precocious male (jack) was observed out of
                   mid-river purse seine as they reached the lower river,                        119 males (1%) returning from subyearling releases,
                   whereas those released in May were captured prima-                            whereas 14 jacks out of 89 males (16%) returned
                   rily in the beach seine.                                                      from yearling releases.
                      More adults were recovered from yearling than                                Examination of the age distribution of tagged
                   subyearling releases made in 1984 and 1985 (Table                             adults from both the fishery and hatchery returns
                   3). Subyearlings released in June 1984 were recov-                            showed that fish released as either subyearlings or
                   ered as adults in higher numbers than those released                          yearlings were recovered predominantly as 4-year-
                   in May, whereas the opposite was true for                                     olds (Table 5). Age-4 adults from subyearling releas-
                   subyearlings released in 1985. The overall rate of re-                        es were equivalent in size to age-5 adults returning to
                   turn for all groups was much higher for fish released                         the hatchery from yearling releases (Table 6), both
                   in 1985 than for fish released in 1983 or 1984 (Tables                        age groups having spent about 3 years in the ocean.
                   2 and 3).                                                                       Table 7 presents the ratios of numbers and weights
                      Sex determinations on adults returning to the                              of adults recovered from yearling releases in 1984
                   hatchery from tagged fish released in 1983-85 indi-                           and 1985 to those of subyearlings released in the
                   cated a greater survival of females (Table 4). The                            same years. The generally lower total weight ratios






                                                                   Zaugg et a].: Hatchery Rearing and Release Strategies for Chinook Salmon                       39


                                                                                     Table 5
                  Numbers of adult salmon (% of total in parentheses) taken in the fishery and returning to the Little White Salmon
                                                           National Fish Hatchery, according to age in years.


                                                                                                    Age

                  Release year and group                       2                        3                          4                               5


                  1983
                    Subyearling (May)                          -                        -                          14(100)                         -
                    Subyearling Uune)                          -                        1 (4)                      24  (89)                     2  (7)
                  1984
                    Yearling (April)                           -                        1 (1)                      62  (63)                   35   (36)
                    Subyearling (May, salt)                    -                        -                          5 (100)                         -
                    Subyearling (May, control)                 -                        1 (9)                      10  (91)
                    Subyearling Uune)                          -                     10 (35)                       19  (65)                        -
                  1985
                    Yearling (April)                           -                     13 (5)                        173 (68)                   69 (27)
                    Subyearling (May, salt)                    -                     18 (18)                       80  (82)                        -
                    Subyearling (May, control)                                       19(19)                        79  (81)
                    Subyearling Uune)                        1   (1)                 12 (18)                       54  (81)                        -
                  Totals
                    Yearling                                   -                     14   (4)                      235 (67)                   104 (29)
                    Subyearling                              1 (03)                  61 (17)                       285 (82)                    2(0.6)



                                                                                     Table 6
                  Mean fork lengths in centimeters             of fish measured) of adult salmon returning to the Little White Salmon National
                                                                 Fish Hatchery, according to age in years.

                                                                                                  Age

                  Release year and group                       2                        3                              4                           5


                  1983
                    Subyearling (May)                          -                        -                          89  (11)                        -
                    Subyearling Uune)                          -                     66   (1)                      87  (20)                   101 (2)
                  1984
                    Yearling (April)                           -                     61   (1)                      76  (63)                   90(30)
                    Subyearling (May, salt)                    -                                                   92  (4)
                    Subyearling (May, control)                 -                     72   (1)                      90  (7)
                    Subyearling Uune)                          -                     71 (10)                       88  (19)                        -
                  1985
                    Yearling (Aprili                           -                     56(13)                        77 (156)                   89 (58)
                    Subyearling (May, Salt)                    -                     73 (18)                       89  (65)                        -
                    Subyearling (May, control)                 -                     71 (17)                       90  (61)
                    Subyearling Uune)                        46 (1)                  73 (12)                       90  (52)                        -
                  Totals
                    Yearling                                   -                     56 (14)                       77 (209)                   89   (88)
                    Subyearling                              46(l)                   72 (59)                       89 (239)                   101  (2)


              (compared to the total number ratios) reflects the                             tion and spawning in salmonids (MacQuarrie et al.
              larger average size of recovered adults from subyear-                          1978, 1979; Whitehead et al. 1978a, 1978b;, Eriksson
              ling releases (86.3 cm fork length, 7.74 kg, n = 348)                          and Lundqvist 1980; Bromage et al. 1982). Johnston
              to yearling released fish (79.7 cm fork length, 5.95                           et al. (1990) have shown that it is possible to, mature
              kg, n = 348).                                                                  Atlantic salmon (Salmo salar) at nearly any time of
                                                                                             the year by making appropriate photoperiod adjust-
              Discussion                                                                     ments., At the Little White Salmon NFH, the use of
                                                                                             advanced photoperiods on returning adult chinook
              Controlling photoperiod regimes to which adults are                            salmon accelerated maturation and spawning by 4 to
              exposed is an effective way of accelerating matura-                            5 weeks, which has resulted in the accomplishment of







                    40        NOAA Technical Report NWS 106



                                                                                             Table 7
                       Ratios of numbers and estimated weights of adults recovered from yearling releases to those of adults from sub-
                                                                                       yearling releases.

                                                               Number of                Relative number             Estimated             Relative weight        Average
                          Release year                          recovered              of recovered adults         total wt (k         of recovered adults          adult
                          and group                               adults             (yearling/subyearling)         of adults         (yearling/ subyearling) wt (kg)

                       1984
                          Yearling                                  109                         -                       677                      -                  6.2
                          Subyearling                                  5                       21.8                       48                    14.1                9.6
                            (May, salt)
                          Subyearling                                11                         9.9                       91                     7.4                8.3
                            (May, control)
                          Subyearling                                30                         3.6                     198                      3.4                6.6
                            Uune)
                          Subyearling average                        15                         -                       112                      -                  7.5
                       1985
                          Yearling                                  266                         -                      1,552                     -                  5.8
                          Subyearling                               104                         2.6                     796                      2.0                7.7
                            (May, salt)
                          Subyearling                               101                         2.6                     782                      2.0                7.7
                            (May, control)
                          Subyearling                                68                         3.9                     532                      2.9                7.8
                            Uune)
                          Subyearling average                        91                         -                       703                      -                  7.7
                       Totalsc
                          Yearling (1984+1985)                      375                         -                      2,229                     -                  5.9
                          Subyearling                               106                         3.5                     816                      2.7                7.7


                          Based on release of 50,000; numbers include commercial and sport fishery catches and hatchery returns (Pacific States Marine
                          Fisheries Commission data base).
                       b Weights estimated from length/weight relationships determined in 1989 from 72 returning adults.
                          Averages of the three 50,000-fish release groups for each year are totaled and compared with the sum of values obtained from the
                          two yearling release groups.



                    two original goals: 1) to reduce prespawning mortal-                              (1983) were low (0.03-0.05%). However, no tagged
                    ity by decreasing the adult holding time and 2) to                                yearlings were released that year, so a comparison of
                    produce larger yearling smolts because of a longer                                relative recoveries from the two year classes was im-
                    rearing time.                                                                     possible. Adult recoveries from subyearling releases
                       However, an unanticipated benefit resulted from                                made in the second year (1984) were also low (0.01-
                    the incorporation of photoperiod-accelerated spawn-                               0.06%), but these could be compared to adult
                    ing into the hatchery production scheme-that of                                   recoveries from yearlings released at their normal
                    smolt development in progeny during their first                                   time in April of the same year (0-22%). Adult recov-
                    spring of rearing. Development of smolt-like charac-                              eries from both subyearlings and yearlings that were
                    teristics, such as increased silver coloration, fin                               released in 1985 were much higher (0.14-0.21 and
                    clarification, a dark band in caudal fins, crowding at                            0.53%, respectively).
                    the outlet screens, increased gill Na+-K+ ATPase ac-                                 Although numbers of recovered adults were small,
                    tivities, and active seaward migration, suggested that                            it is nevertheless apparent that the release of
                    these fish could possibly be released 10 to 11 months                             subyearlings resulted in a ratio of adult females to
                    earlier than normal and survive to adulthood (Zaugg                               males more closely approximating 1 (1.33) than did
                    et al. 1986). This study was designed to test whether                             the release of yearlings (2.24). In addition, very few
                    such early releases of subyearlings could be used to                              jacks were observed in hatchery returns from sub-
                    effectively augment current production and release                                yearling releases (1 of 119 returning males ), whereas
                    of yearling juveniles. Adult recoveries from tagged                               there were 14 jacks of 89 males that returned from
                    subyearlings released in the first year of the study                              yearling releases. This greater proportion of adult






                                                              Zaugg et al.: Hatchery Rearing and Release Strategies for Chinook Salmon                 41
             males, and the lack of jacks, results in a greater op-                    Citations
             portunity to spawn individual pairs of fully matured
             adults, a practice presently being used to control dis-                   Bromage, N.R., C. Whitehead, J. Elliot, B. Broton, and A. Matty.
             ease.                                                                         1982. Investigations into the importance of daylength on the
                Eighty-two percent of adults recovered from                                  photoperiod control of reproduction in the female rainbow
                                                                                             trout. In Proceedings of the international symposium on
             subyearling releases were age 4, whereas 67% of re-                             reproductive physiology of fish; 2-6 August 1982,
             coveries from yearling releases fell into this age                              Wageningen, The Netherlands, (CJJ. Richter and HJ. Th.
             group. This is a majority, however, for each release                            Goos, eds.), p. 233-237. Pudoc Press, Wageningen.
             group. Age-4 adults from subyearling releases aver-                       Dawley, E.M., R.D. Ledgerwood, and A.L. Jensen.
             aged 12 cm longer than age-4 adults from yearling                             1985. Beach and purse seine sampling of juvenile salmonids
                                                                                             in the Columbia River estuary and ocean plume, 1977-83.
             releases, and were estimated to be about 1.75 kg                                Vol. 11: Data on marked fish recoveries, 397 p. NOAA
             heavier. This size differential, which was a character-                         Tech. Memo. NMFS F/NWC-75. Northwest Fisheries Cen-
             istic of the adults in each age category, translated                            ter, 2725 Montlake Blvd. E., Seattle, WA 98112. NTIS PB85-
             into an estimated ratio of 2.7 for the combined                                 187722.
             weight of adults recovered from yearling releases to                      Eriksson, L.O., and H. Lundqvist.
                                                                                           1980. Photoperiod entrains ripening by its differential effect
             the combined weight of adults recovered from                                    in salmon. Naturwissenschaften 67(4):202.
             subyearling releases made in 1984-85. The ratio of                        Johnston, C.E., S.R. Farmer, R.W. Gray, and M. Hambrook.
             total numbers of recovered adults from these same                             1990. Reconditioning and reproductive responses of Atlantic
             two groups was 3.5.                                                             salmon kelts (Salmo salar) to photoperiod and temperature
                Although the ratios of adults returning from year-                           manipulation. Can. J. Fish. Aquat. Sci. 47:701-710.
                                                                                       MacQuarrie, D.W., J.R. Markert, and W.E. Vanstone.
             ling/ subyearling releases varied considerably for the                        1978. Photoperiod induced off-season spawning of coho
             various groups released in the 2 years that compari-                            salmon (Oncorhynchus kisutch). Ann. Biol. Nnim. Biochim.
             sons could be made (1984 and 1985), the results                                 Biophys. 18(4):1051-1058.
             suggest that it would be necessary to release three to                    MacQuarrie, D.W., W.E. Vanstone, andj.R. Markert.
             four times as many subyearlings as yearlings to re-                           1979. Photoperiod induced off-season spawning of pink
                                                                                             salmon (Oncorhynchus gorbuscha). Aquaculture 18(4):289-
             cover an equivalent number of adults. We estimate                               302.
             that three to four times as many subyearlings as year-                    Whitehead, C., N.R. Bromage, B. Breton, and R. Billard.
             lings could be reared in the same pond space, but for                         1978a. Effects of altered photoperiod on serum gonado-
             a much shorter rearing time and with lower produc-                              tropin levels and spawning in female rainbow trout. J.
             tion costs. In addition, the larger size of adults                              Endocrinol. 79(2):29-35.
                                                                                       Whitehead, C., N. R. Bromage, J.R. M. Forster, and AJ. Matty.
             returning from subyearling releases and the reduced                           1978b. The effects of alterations in photoperiod on ovarian
             number of jacks makes this strategy even more eco-                              development and spawning time in the rainbow trout (Salmo
             nomically b@neficial. The release of subyearling                                gairdneri). Ann. Biol. Anim. Biochim. Biophys. 18(4)1035-
             progeny of adult spring chinook salmon matured                                  1043.
             early through the use of photoperiod control is an                        Zaugg, W.S., J.E. Bodle, J.E. Manning, and E. Wold.
                                                                                           1986. Smolt transformation and seaward migration in O-age
             effective hatchery practice at the Little White Salmon                          progeny of adult spring chinook salmon (Oncorhynchus
             NFH. A similar program might be used at other                                   tshauytscha) matured early with photoperiod control. Can.
             hatcheries to provide an effective method of main-                              J. Fish. Aquat. Sci. 43:885-888.
             taining and augmenting spring chinook salmon runs
             in the Columbia River Basin or elsewhere.






                Induced Spawning of Japanese Eel with LHRH-A Copolymer Pellet


                                                                   KEIJI HIROSE
                                                         National Research Institute of Aquaculture
                                                                         Nansei
                                                                   Mie 516-01, Japan




                                                                      Abstract


                                A new hormone therapy was tested for inducing maturation and ovulation in the
                              Japanese eel Anguilla japonica. A luteinizing hormone-releasing hormone analogue
                              (LHRH-A) copolymer pellet, which shows a slow and constant release of hormone over
                              75 days, was used in this study. The fish were treated with eight weekly injections of
                              salmon pituitary and human chorionic gonadotropin (HCG), after which time the
                              LHRH-A pellets were implanted in the fish. Final maturation and ovulation was induced
                              in the mature fish at 15-29 weeks by injection with 17ot-hydroxyprogesterone (17ot-
                              OHprog) and HCG; the treated fish spawned naturally for 2 days. This hormone
                              treatment schedule could induce a high success of spawning because it eliminates the
                              stresses incurred by repeated injections of hormones. The best result was obtained in fish
                              kept at a low water temperature of 15' C.


            Introduction                                                      However, repeated administration of exogenous hor-
                                                                              mones results in gonadal atresia, which may be
            Recently, eel consumption has increased, reaching 70              caused by stress and the production of an undesir-
            thousand metric tons (t) per. year in Japan. However,             able antibody. The repeated treatments of exogenous
            the production of eel elvers in natural waters has                hormones may be related to the failure to induce
            been decreasing for the past 20 years (Statistics and             ovulation, low spawning rates, and also problems in
            Information Department, Ministry of Agriculture,                  egg quality. To avoid stress and undesirable results
            Forestry, and Fisheries 1990). Presently, there is a              from the repeated treatments, we developed a co-
            shortage of seed which is barely covered by imports.              polymer pellet containing luteinizing hor-
            The development of artificially induced breeding                  mone-releasing hormone analogue (LHRH-A), which
            techniques of eel have long been needed in Japan.                 provides a slow and constant release of hormone over
            Thus, the controlled reproduction of the Japanese                 a long period of time and which succeeded in induc-
            eel Anguilla japonica has been attempted for a long               ing final maturation and ovulation when implanted
            time. Ten to fifteen years ago, several research groups           in ayu (Plecoglossus altivelis) (Yoshida et al. 1986;
            succeeded in inducing spawning of the eel by treat-               Hirose et al. 1990). In this study, the author tested
            ment with fish pituitary (Yamamoto et al. 1976;                   the potency of this pellet for inducing maturation
            Motonobu et al. 1976; Satoh 1979). Since then, there              and spawning in Japanese eels.
            have been almost no excellent results on the induced
            spawning of eels. In those previous experiments, the
            spawning rate is very low, 5-10%. Final maturation                Materials and Methods
            and ovulation are not well controlled and the larvae
            do not survive over 10-15 days. In general, catadro-              The cultured eels used for this study were over 10
            mous type eels (silver eel A. japonica) are most widely           years old and were raised at the Inland station of the
            used for induced spawning. The initial state of their             National Research Institute of Aquaculture. Before
            ovaries is immature just before or shortly after initia-          beginning the experiments, fish were transferred to
            tion of yolk formation. If we wish to induce ovarian              the main station of the institute and held in a I-t
            maturation and spawning in immature eels, a rela-                 flow-through seawater aquarium with temperature
            tively long-term hormone therapy is necessary.                    constant at 15 or 20' C.



                                                                                                                                        43







               44       NOAA Technical Report NMIFS 106

                                                                                  Treatment schedule A-Weekly treatment with chum
                                                                                  salmon pituitary (30 mg/fish) and HCG (300 IU/
                                                                                  fish) for the first 8 weeks. Only chum salmon pitu-
                   N2                                                             itary was administered thereafter until ovulation was
                                                                                  induced artificially.
                                        -78*C
                               60                                                 Treatment schedule B-Weekly treatment with chum
                                Co T -ray        5X10    rad
                                                                                  salmon pituitary (30 mg/fish) and HCG (300 IU/
                                                                                  fish) for 8 weeks followed by implantation of the
                          Molecular sieve 3A           25mg                       LHRH-A polymer pellet. The fish were not treated
                 ..............                                                   with pituitary and HCG after implantation of the pel-
                          LHRH-A                       5GJJ9
                                                                                   t until ovulation was induced.
                                                                                  le
                   .....  polymer(2G: 1 4G)
                                                                                  Treatment schedule C-Weekly treatment with chum
                                         Figure I                                 salmon pituitary (30 mg/fish) and HCG (300 IU/
               Schematic diagram for preparation of LHRH-A copolymer              fish) for 8 weeks followed by chronic treatment with
               pellet using the methods of Yoshida et al. 1986. Luteinizing       the LHRH-A polymer pellet, which was implanted in
               hormone-releasing hormone analog (LHRH-A, 50 Vg) dis-              the 8th week. Chum salmon pituitary was adminis-
               solved in phosphate buffer was absorbed to 25 mg of                tered at a level of 30 mg/fish for the first 8 weeks: 20
               molecular sieve 3A. Following evaporation, molecular sieve         mg/fish thereafter. No HCG was given after 8 weeks
               3A containing hormone was charged into a glass ampoule.            until ovulation was induced.
               A hydrophobic diethylene glycol dimethacrylate (2G) and              Both experiments were conducted in the 1-t tank
               hydrophilic polyethylene glycol #600 dimethacylate (14G)           using groups of three female fish per treatment. Ex-
               monomer mixture was also charged into the ampoule. The             periment I was held at 20' C and involved treatments
               ampoule was sealed off under nitrogen gas and irradiated           A and B only. Experiment 11 was held at 15' C and
               with gamma-rays from a Cc' source at 5 X 10' rad/hr, at            involved treatments A, B, and C.
               -78' C. The co-monomer in the supercooled state was com-             Body weight was measured weekly and changed
               pletely polymerized by the irradiation. The product was            little until final maturation was induced. A some-
               then removed from the ampoule mold.                                what rapid increase in body weight occurs during
                                                                                  final maturation and ovulation due to hydration of
               Hormones                                                           oocytes. Therefore, 17a-hydroxyprogesterone (17a-
                                                                                  OHprog I mg/fish) and HCG (1000 IU/fish) were
               Luteinizing   hormone-releasing hormone analogue,                  administered to the mature eel when it showed a
               des Gly"@[D-Ala'] LHRH ethylamide (Sigma), human                   slight increase in body weightjust prior to the rapid
               chorionic gonadotropin, (HCG, Teikoku Hormone                      increase in body weight. Ovulation and spawning
               MGF. Co. LTD., Tokyo) and chum salmon                              were induced within 2-3 days following the treatment
               Oncorhynchus keta pituitary (caught in Iwate Prefec-               with 17a-OHprog and HCG. For males, six or seven
               ture) were used. The schema for the preparation of a               weekly injections with HCG (300 IU/fish) were
               dry-copolymer composite in rod form (1.6 mm in di-                 enough to induce spermiation.
               ameter and 10 mm long) is shown in Figure 1.
               Detailed preparation methods of the copolymer pel-
               let by radiation-induced polymerization are described              Results and Discussion
               by Yoshida et al. (1986). In vitro experiments showed
               that the copolymer pellet released LHRH at a rela-                 At the initiation of the experiments the cultured eels
               tively constant rate over 75 days (Hirose et al. 1990).            had not yet attained the primary yolk globule stage
               Iii this study, a copolymer pellet containing 50 @Lg               and their gonadal somatic indexes were below 2.0.
               LHRH-A was used. The pellet was implanted intra-                   After eight weekly injections of chum salmon pitu-
               muscularly. Chum salmon pituitary and HCG were                     itary and HCG, the fish ovaries had oocytes in the
               also given weekly to induce maturation and spawning                primary yolk globule stage. At this time, LHRH-A
               of the eels.                                                       polymer pellet was implanted to the eel.
                A
                U

































               Treatment Schedules                                                Experiment I

               Two experiments involving the following three treat-               In the treatment schedule A group (Table 1), one
               ment schedules were undertaken (see Fig. 2).                       (D-3) of three fish spawned after 15 of the weekly







                                                                                                     Hirose: Induced Spawning of Japanese Eel                 45


                                                        Hormone treatment schedule


                                                                                                                                    symbol

                                                                                                               16    spawning        SP
                         A                                                                                 -J    ------
                                      salmon pituitary 30mg                                  salmon pituitary 30mg                  HCG
                                            + HCG 300 1U


                                                                LHRH-A polymer
                          B                                                                                                              polymer
                                     salmon pituitary 30mg

                                           + HCG 300 lU


                                                               LHRH-A polymer
                                                                                                              16

                                     salmon pituitary 30mg
                                                                                             salmon pituitary 20mg
                                          + HCG 300 lU


                                    17o(-hydroxyprogesterone(lmg/tish)and HCG(10001U/tish)were used for inducing
                                    ovulation and spawning.



                                                                                  Figure 2
              Hormone treatment schedules for inducing maturation and spawning in Japanese eel in Experiments I and IL Experiment I
              was held at 20' C and involved treatments A and B only. Experiment II was held at 15' C and involved treatments A, B, and
              C. HCG = Human chorionic gonadotropin; SP = Chum salmon pituitary. Time on line marked in weeks. 17ot-
              hydroxyprogesterone (I mg/fish) and HCG (1000 IU/fish) were used to induce ovulation and spawning.





                                                                                  Table I
                 Experimental results from experiment I which was performed at 20' C. See text for descriptions of treatment schedules.

                                                Body            % increase            Gonadal                Time of
                    Fish                      weight              ofbody              somatic                injection
                    no.                         (g)                weight              index                  (weeks)                     Remarks


                 Treatment schedule A
                    D-1                         606                    8.7              33.4                     16                   over-mature

                        2                       627                  30.5               35.2                     16                   over-mature
                        3                       656                   -a                19.7+                    15                   spawned, unfertilized


                 Treabnent schedule B
                    E-1                         545,                    0               19.7                     20                   dead, final
                                                                                                                                      maturation
                        2                       561                  24.5                 -                      16                   spawned, larvae
                                                                                                                                      survived 7 days
                        3                       633                  26.3                                        20                   spawned, larvae
                                                                                                                                      survived 7 days


                 a      data not collected.







                  46        NOAA Technical Report NNOS 106



                                                                                     Table 2
                      Experimental results from experiment H, which was performed at 15' C. See text for description of treatment schedules.

                                                    Body            % increase           Gonadal                Time of
                         Fish                     weight              ofbody              somatic               injection
                         no.                        (g)               weight                index               (weeks)                Remarks


                      Treatment schedule A
                         F-1                        691                 13.2                42.3                   16                dead, final
                                                                                                                                     maturation

                            2                       1019                30.0                _a                     26                over-mature
                            3                       1713                -7.5                20.5                   31                yolf deposition

                      Treatment schedule B
                         G-1                        1492                13.3                37.8                   24                ovulated, artificial
                                                                                                                                     fertilization
                            2                       988                 33.3                -                      29                spawned, larvae
                                                                                                                                     survived 8 days
                            3                       1250                12.9                -                      27                spawned, larvae
                                                                                                                                     survived 12 days


                      Treatment schedule C
                         H-1                        1600                12.8                25.5                   22                dead, mature
                            2                       1400                11.4                34.5                   18                dead,larvae
                            3                       1579                16.0                -                      27                spawned, larvae
                                                                                                                                     survived 8 days


                      a     data not collected.




                  injections, although the eggs were not fertilized. The                      A combination of 17a-OHprog and HCG was given to
                  others quickly developed to an overly-mature state                          the mature fish to induce final maturation and ovula-
                  and could not spawn. In the treatment schedule B                            tion and, thereafter, they were expected to spawn
                  group, two (E-2 -and 3) of -three fish spawned natu-                        naturally within 48 hours. The processes of final
                  rally and the resulting larvae survived for 1 week.                         maturation and ovulation proceeded very quickly at a
                  Female fish developed to a mature or spawning state                         water temperature of 20' C. At that temperature the
                  after 16-20 weeks of injections.                                            fish showed a very rapid increase in body weight and
                                                                                              high gonadal somatic index, changing into an overly
                                                                                              mature state.
                  Experiment H                                                                  Figure 3 shows the frequency of egg diameter from
                                                                                              mature eels kept at 20' C (treatment schedule C in a
                  In the treatment C group (Table 2), one fish (H-3)                          preliminary experiment). The most developed oo-
                  spawned at 27 weeks, while the other fish died when                         cytes, which were in the migratory nucleus stage and
                  they became completely mature. In the schedule B                            contained many small oil droplets, were 0.7-0.8 mm
                  group, two of the three fish spawned naturally; the                         in diameter. Following ovulation, the oocytes quickly
                  other one ovulated earlier at 24 weeks at which time                        changed into an overly mature state, as seen by the
                  artificial fertilization was performed. Some of the                         presence of eggs that now contained only a few large
                  schedule B larvae survived for 8-12 days but they                           oil droplets and were not fertilized (Hirose, unpubli.
                  could not ingest any live organisms (rotifers or fertil-                    data). Moreover, ovulation was not always preceded
                  ized oyster eggs). Treatment schedule A fish could                          by final oocyte maturation when the mature fish were
                  not spawn. The lower temperature used for experi-                           kept in water temperatures over 20' C. Thus, high
                  ment 11 (15' C) lengthened the duration of the                              ternperature conditions may cause the production of
                  weekly injections to 24-29 weeks.                                           poor quality eggs in a very short time. Mature fish
                      Based on the results from two experiments, treat-                       should therefore be kept in somewhat cool water
                  ment schedule B performed at 15' C is the. best                             (15' Q. If gravid eels are cultured under the lower
                  method for inducing maturation in the Japanese eel.                         temperature condition, body weight does not in-






                                                                                     Hirose: Induced Spawning Of Japanese Eel        47



                          20-
                                 Dec . 22 , 1988                                                     symbol
                                 MK-OHprog

                                 + HCG
                          15
                     C                                                                                      W.T. 20*C


                         10-
                     LL


                           5






                                Dec . 24   1988
                     21"  10-                                                                        ")GV
                                                                                                  .00
                                                                                         ..........
                     C                                                                              0

                                                                                             ov r-maturation
                         5

                    L16

                                            dz      @t       Ot       0-@      0.9     '09       1.0      1             (mm)
                                                            Egg diameter


                                                                     Figure 3
           Frequency distribution of oocytes in the mature eel treated with 17ot-hydroxyprogesterone (17ot-OHprog) and HCG at
           20' C.(Hirose, unpubl. data). Fish were observed to mature using treatment schedule C in a preliminary experiment. Oocyte
           samples were obtained using a polyethylene carmula inserted into the urogenital pores of the eels. Thereafter, the oocytes
           were observed under a microscope. The mature oocytes (0.7-0.8 mm in diameter) had germinal vesicles (GV) and many
           small oil droplets. After hormone treatment, the oocytes rapidly changed into an overly mature state (shaded portion) in 2
           days; germinal vesicle breakdown (dotted circle) and a few large oil droplets were also present-a typical feature in overly
           mature eggs.


           crease as rapidly during final maturation, as shown in           tance. In this study, a high frequency of maturation
           previous reports (Sugimoto et al. 1976; Yamauchi                 and spawning was induced using hormone treatment
           and Yamamoto 1982). Consequently, a final injection              schedule B in experiment Il (15' Q followed by an
           of HCG and 17u,-OHprog for inducing ovulation is                 injection of 17ot-OHprog and HCG. If we can in-
           timely when given to mature fish, and a high fre-                crease the spawning frequency in the hormone-
           quency of natural spawning can be anticipated.                   treated fish, the artificial propagation of eels will be
             In general, it is said that the stress of handling             achieved in the future.
           during hormone injection decreases the efficiency of
                           m
           hormone treat      ent and may cause rapid atresia of
           the gonads (Hirose 1980; Billard et al. 1981). This              Acknowledgment
           LHRH-A polymer pellet has a long hormone delivery
           time and is especially useful for inducing maturation
           in fish requiring a series of hormone treatments over            I am deeply grateful to Dr. M. Yoshida of Japan
           a long time period. The ability to induce a high per-            Atomic Energy Research Institute for kindly supply-
           centage of treated eels to spawn is of great impor-              ing the LHRH-A copolymer pellet.







                   48        NOAA Technical Report NWN 106

                   Citations                                                                        Sugimoto, Y, Y. Takeuchi, K. Yamauchi, and H. Takahashi.
                                                                                                          1976. Induced maturation of female Japanese eels (Anguilla
                   Billard, R., C. Bry, and C. Billet.                                                      japonica) by administration of salmon pituitaries, with notes
                        1981. Stress, environment, and reproduction in teleost fish.                        on changes of oil droplets in eggs of matured eels. Bull.
                          (A.D. Pickering, ed.), p.185-208. Acad. Press, NY.                                Fac. Fish. Hokkaido Univ. 27:107-120.
                   Hirose, K.                                                                       Yamamoto, K_ M. Nakamura, H. Takahashi, and K. Takano.
                        1980. Effects of repeated injections of human chorionic go-                       1976. Cultivation of larvae ofJapanese eel. Nature 263:412.
                          nadotropin (HCG) on ovulation and egg qualities in the                    Yamauchi, K. and K. Yamamoto.
                          ayu, PLecoglossus altivelis. Nippon Suisan Gakkaishi 46:813-                    1982. Experiments on artificial maturation and fertilization
                          818.                                                                              of the Japanese eel (Anguilla japonica). In Proceedings of
                   Hirose, K., H. Kagawa, M. Yoshida, M. Kumakura, and H.                                   the international symposium on reproductive physiology
                     Yamanaka.                                                                              of fish (CJJ. Richter and H.J. T. Goos, eds.), p. 185-
                        1990. Application of LHRH copolymer pellet for induction                            189. Center for Agricultural Publishing and Documenta-
                          of final oocyte maturation and ovulation in ayu PL-coglossus                      tion, Wageningen, the Netherlands.
                          altivelis. Nippon Suisan Gakkaishi 56:1731-1734.                          Yoshida, M., M. Asano, 1. Kaetu, IL Imai, H. Yaasa, H. Nakayama,
                   Motonobu, T., K Yamashita, and H. Oka.                                              I- Shida, K. Suzuki, K_ Wakabayashi, and 1. Yamazaki.
                        1976. Memoir on the hatched larva of the Japanese eel                             1986. Pharmacological response in male rats with controlled
                          Anguilla japonica, from the adults matured artificially. Bull                     release formulations of luteinizing hormone-releasing hor-
                          Shizuoka Pref. Fish. Exp. Station 10:87-90. (In Japanese.)                        mone agonist. Polymerjour. 18:287-296.
                   Satoh,H.
                        1979. Towards the complete cultivation of Japanese eel. Iden
                          (Genetics) 33:23-30 (injapanese).
                   Statistics and Information Department, Ministry of Agriculture,
                     Forestry, and Fisheries.
                        1990. Gyogyo-yoshokugyo-seisan-toukei-nenpou. Tentative
                          English title: Fisheries statistics ofJapan, p.181 and 268. (In
                          Japanese.)







                   Reproductive Physiology of Sablefish (Anoplopomafimbria) with
                                    Particular Reference to Induced Spawning



                                      IGOR 1. SOLAR, EDWARD M. DONALDSON, IANJ. BAKER,
                                 HELEN M. DYE, ALEXANDRA VON DER MEDEN, and JACK SMITH
                                                           Department ofFisheries and Oceans
                                                               Biological Sciences Branch
                                                               West Vancouver Laboratory
                                                                  4160 Marine Drive
                                                         West Vancouver, B. C. V7V IN6 Canada




                                                                     Abstract


                               This paper summarizes information on the reproductive physiology of sablefish
                              (Anoplopoma fimbria) obtained during five years of studies on the induced ovulation of
                              captive female sablefish. The ability to induce ovulation and spawning in captive
                              broodstock is considered critical for the development, expansion, and diversification of
                              aquacultural operations on the Pacific coast of Canada into indigenous marine species.
                              Results are discussed on the use of partially purified salmon gonadotropin and several
                              mammalian luteinizing-hormone releasing hormone analogues (LHRHa); the ovarian
                              hydration response; the effect of administering LHRHa by different routes on plasma
                              estradiol levels; and the use of a dopamine antagonist. Other preliminary and unpub-
                              lished data are also presented.




            Introduction                                                       The Pacific Biological Station (PBS), Nanaimo,
                                                                            B.C., initiated studies on sablefish mariculture in the
            Sablefish (Anoplopoma fimbria), also known as                   early 1970's (Kennedy 1972). These studies have con-
            blackcod (Fig. 1), range along the North American               tinued using a multidisciplinary approach both at the
            coast from the northeastern Bering Sea to Baja Cali-            PBS and at the West Vancouver Laboratory since
            fornia. Its depth distribution ranges from the                  1985. The present review summarizes our findings to
            continental shelf to about 1500 m (McFarlane and                date in the area of induced spawning.
            Beamish 1983). Spawning, which takes place at
            depths of 300-700 m, lasts from January to March
            and peaks in February (Mason et al. 1983, Fujiwara              Maturation of Captive Fish
            and Hankin 1988).
              The sablefish is considered a prime candidate for             Sablefish do not usually undergo final maturation in
            commercial aquaculture because of its adaptability to           captivity. Since 1985,. only I of over 150 females rip-
            confinement and its indiscriminate feeding behavior.            ened spontaneously (fish in stock group, without
            The development of methods for spawning sablefish               treatment) at the Pacific Biological Station. Although
            in captivity and for rearing the larvae are expected to         sablefish are known to be yearly spawners in the wild,
            facilitate the future establishment of mariculture op-          only one individual maintained in captivity from one
            erations for this species on the west coast of Canada.          spawning season to the next responded to hormone
            Early studies on the culture of sablefish were depen-           therapy a second time. The inability of females to
            dent on the impoundment of wild juveniles, and                  undergo ovarian maturation in captivity has been at-
            studies of larval rearing have relied upon the capture          tributed to enviromental and nutritional factors.
            of wild maturing broodstock and the stripping of                Males, on the other hand, frequently mature and
            their gametes at sea (McFarlane and Nagata 1988).               spermiate without endocrine treatment.
            Neither of these practices would be feasible or desir-             Hormonal treatments applied to maturing captive
            able as a basis for commercial culture..                        fish in our laboratories from 1985 to 1989 resulted in

                                                                                                                                     49







               50       NOAA Technical Report NAM 106



















                          10















                                                                         Figure I
                                                        Adult female sablefish, Anoplopomafimbria.



               an ovulatory response in 59.4% of the experimental                several unresponsive females at the end of the latter
               animals while no fish in the control groups ovulated.             experiment revealed that most had immature ovaries.
                                                                                  In 1987, the effect of desGly" [D-Alal) LH-RH
                                                                                 ethylamide alone or in combination with dom-
               Hormonal treatments                                               peridone, a dopamine antagonist, was investigated
                                                                                 (Solar et al. 1988). Results suggested that dopaminer-
               Several compounds have been assayed for their abil-               gic control of gonadotropin release in this species is
               ity to induce spawning in sablefish. Our study                    less apparent than in certain other teleosts (Cyprin-
               conducted in 1985 (Solar et al. 1987) demonstrated                ids, Peter et al. 1986; catfish, van Oordt and Goos
               for the first time the feasibility of inducing ovarian            1987). A second experiment in the same year investi-
               hydration and ovulation in sablefish (65% ovulation               gated the effect of injections of D-Ala     6 LH-RH or
               response). In this first study, we utilized a single intra-       microencapsulated D-Trp6 LHRHa (Solar et al.
               peritoneal (ip) injection of 1.0 mg/kg body weight                1988). Prior to the experimental work conducted in
               (bw) of partially purified salmon gonadotropin (SG-               1987, fish sex was tentatively identified by measuring
               G100) or 0.2 mg/kg bw of Gly" [D-Ala']LH-RH                       plasma estradiol-170. levels. These preliminary deter-
               ethylamide luteinizing hormone releasing hormone                  minations were later confirmed by ovarian
               analogue (LHRHa), a potent synthetic analogue of                  catheteriza tion.
               mammalian LHRH. Limited success (19.0% ovula-                      A preliminary study conducted in 1988 investi-
               tiop response) was achieved the following year using              gated the influence of certain environmental
               two doses of SG-G100 (1.0 and 0.5 mg/kg bw), SG-                  parameters (water temperature and light) on the in-
               G100 in combination with LHRHa (0.5 ï¿½ 0.2 mg/kg                   duction of ovulation. Maintenance of the fish in
               bw), or LHRHa as a primer followed by a second                    flowing chilled seawater (3-4' Q and darkness for
               dose (0.1, 0.2 mg/kg bw) three days later. Autopsy of             about three months prior to hormonal treatment did






                                                                                     Solar et al: Reproductive Physiology of Sablefish        51


                 E   25-
                 S
                 T
                 R   20-
                 A
                 D
                 1   15 -
                 0
                 L
                     10-

                 n   5
                 9

                        ..................... ......... ...... ...  N7
                 M   Oz
                 1      0        4           8        12         16         20         24                         Figure 2
                                                    DAYS                                      Plasma estradiol levels in female sablefish in re-
                                                                                              sponse to intraperitoneal injection or choles-
                           LHRH INJECTION         E) LHRH PELLET        -V-CONTROL            terol pellet implantation of des-Gly10[D-AW]LH-
                                                                                              RH ethylamide. (Solar et al., unpubl. data 1989.)
                               [-Al
                 %   25-
                 W        --)K- TREATED (N-13) -9- CONTROL (N-7:)]
                 I=  20-
                 1
                 G
                 H   Is-
                 T


                     IO_
                 N
                 C
                 R
                 E   6-
                 A
                 S
                 E   0-
                     28        24      20        16       12                 4        0
                                    DAYS PRIOR TO OVULATION







                                                                                   (13)
                     100-        B@
                                F


                 %   80-

                 0
                 V   60-                               (7)
                 U                                                                                                Figure 3
                 L                                                                            (A) Mean percent body Weight changes in fe-
                 A   40-                                                                      male sablefish treated with desGly" [D-Ala6]
                 T                            (4)
                 1                                                                            LH-RH ethylamide to induce ovulation, relative
                     20-               (2)                                                    to body weight at the time of injection. (B) Cu-
                 N                                                                            mulative percent ovulation response in
                                                                                              sablefish shown in A. Numbers in brackets indi-
                     0-                                                                       cate actual number of fish ovulated and
                        0        5          10        is        20        26         30       spawned to the date. Arrow indicates the tim-
                        f               DAYS POST-INJECTION                                   ing of treatment. (Solar et al., unpubl. data
                                                                                              1989.)







              52       NOAA Technical Report NMTS 106



                   OAYS

                      30-










                      20-








                                                                                                                      Figure 4
                                                                                                          Relationship between egg diam-
                                                                                                          eter (mm) at the time of
                                                                                                          injection and the duration of
                                                                                                          the latency period (days to ovu-
                       0-                                                                                 lation) in hormonally induced
                                                                                                          female sablefish (r = 0.8). Dot-
                        1.00     1.05     1.10    1. 15    1.20     1 .25   1.30     1.35     1.40        ted lines represent 95% confi-
                                                  INITIAL EGG SIZE                                        dence limits for mean predicted
                                                                                                          values. (Solar et al., unpubl.
                                                                                                          data 1989.)


              not improve the ovulation response relative to fish               biologically active nonapeptide absorption by the fish
              held on a natural photoperiod at 9-10' C (I. Solar et             gut (Solar et al. 1990).
              al. unpubl. data 1988).
                 In 1989, changes in plasma estradiol levels during
              the latency period (time span between drug adminis-               Ovarian Hydration and Latency Period-
              tration and ovulation) were investigated following
              treatment with D-Ala6LHRHa by ip injection and                    The female fish responding to hormonal treatment
              slow-release cholesterol pellet implantation. Both in-            have typically shown protrusion of the uro-genital pa-
              jection and implantation of the 5% D-Ala'LHRHa                    pilla, gradual distention of the abdominal region,
              pellet at the same dose (0.1 mg/Kg bw) induced ovu-               and an increase in total body weight of about 25% of
              lation in 100% of the experimental animals (n = 13),              the weight at the time of injection. The increase in
              while none of the control fish (n = 7) ovulated.                  body weight is normally slow during the days immedi-
              Changes in plasma estradiol levels measured by ra-                ately following D-Ala' LH-RHa injection and very
              dioimmunoassay were followed at 4-day intervals                   rapid during the three days prior to ovulation (Fig.
              during the 24 days postinjection. In both treatments              3). The increase in weight is due to oocyte hydration.
              there was an increase of the estrogen level which                 The eggs, which at the time of injection are small and
              peaked at day 4 and decreased to levels similar to                opaque (mean diameter 1.25 ï¿½ 0.1 mm), became
              controls on day 8. The increase in plasma estradiol               larger and transparent (2.2 ï¿½ 0.1 mm) at the time of
              was more pronounced in the injected group than in                 ovulation (Sol Iar et al. unpubl. data 1988).
              those that had received the slow-release pellet (Fig.               Latency period has been variable ranging from six
              2) (Solar et al. unpubl. data 1989). In a second ex-              days to close to a month (mean 15.8 ï¿½ 8 days). Statis-
              periment, during the same year, the oral                          tical analysis showed that egg diameter at the time of
              administration of 1.5 mg/kg bw D-Ala' LH-RHa in                   inje.ction and the number of days to spawn (latency
              two doses (1.0 and 0.5 mg/kg bw, respectively, 11                 period) are negatively correlated (r = 0.8) (Fig. 4)
              days apart) resulted in 75% of the fish ovulating                 (Solar et al. unpubl. data 1989).
              within 18 days from the first treatment. The time
              course of uptake and net plasma presence of LHRHa                 Artificial Fertilization and Incubation
              during an 8-hour period following delivery was also
              determined thereby providing the first evidence for               The fecundity of female sablefish ranges from about
                                                                                60,000 to close to one million eggs, depending on






                                                                                             Solar et al: Reproductive Physiology of Sablefish              53
              fish size (Mason et al. 1983). The average female pro-                     Fujiwara, S., and D.G. Hankin.
              duces close to 200,000 eggs (G.A. McFarlane and                                  1988. Sex ratio, spawning period, size and age at maturity of
              M.W. Saunders, PBS unpubl. data). Success in fertiliz-                            sablefish (Anoplopoma fimbria) off northern California.
                                                                                                Nippon Suisan Gakkaishi 54:1333-1338.
              ing the eggs obtained from induced sablefish has                           Kennedy, W.A.
              been variable and generally lower than the fertiliza-                            1972. Preliminary study of sablefish culture, a potential new
              tion rate of gametes obtained from mature sablefish                               industry. J. Fish. Res. Board Can. 29:207-210.
              collected from the wild during the peak spawning                           McFarlane, G.A. and RJ. Beamish.
              season. During the 1989 season, the best fertilization                           1983. Biology of adult sablefish (Anoplopoma fimbria) in wa-
              rates (those which were also followed by normal de-                               ters off western Canada. In Proceedings of the Interna-
                                                                                                tional Sablefish Symposium. Alaska Sea Grant Rep. 83-
              velopment to the 8-cell stage) were 62.4 ï¿½ 7.1 % for                              8:59-80.
              eggs from captive induced females and 52.6 ï¿½ 3.7%                          McFarlane, G.A., and W.D. Nagata.
              for those from wild noninduced female. Average fer-                              1988. Overview of sablefish mariculture and its potential for
              tilization rates, however, were 25.0 ï¿½ 17.6% (wild)                               industry. In Proceedings of the 4th Alaska Aquac. Conf.;
              and 14.5 ï¿½ 17.4% (induced) U. Jensen, PBS, pers.                                  18-21 Nov., Sitka, Alaska (S. Keller, ed.), p. 105-120.
                                                                                                Alaska Sea Grant Report 88-4.
              commun. June 1989).                                                        Mason,J.C., RJ. Beamish, and G.A. McFarlane.
                 Sablefish eggs are very fragile and easily injured                            1983. Sexual maturity, fecundity, spawning, and early life his-
              during culture and therefore require special incuba-                              tory of sablefish (Anoplopomafimbria) off the Pacific coast of
              tion techniques (Alderdice et al. 1988a, 1988b). The                              Canada. Can. J. Fish. Aquat. Sci. 40:2126-2134.
              mature fertilized eggs are clear and normally remain                       Peter, R.E., J.P. Chang, C.S. Nahorniak, R.J. Ome1janiuk,
                                                                                            M. Sokolowska, S.H. Shish, and R. Billard.
              suspended in the water column. The incubation pe-                                1986. Interactions of catecholamines and GnRH in regula-
              riod from fertilization to hatch lasts about 12 days at                           tion of gonadotropin secretion in teleost fish. Recent Prog.
              6' C (McFarlane and Nagata 1988).                                                 Horm. Res. 42:513-548.
                                                                                         Solar, I.I., IJ. Baker, and E.M. Donaldson.
                                                                                               1987. Effect of salmon gonatropin and a gonadotropin re-
                                                                                                leasing hormone analogue on ovarian hydration and ovula-
              Acknowledgments                                                                   tion in captive sablefish (Anoplopomafimbria). Aquaculture
                                                                                                62:319-325.
              Thanks are due to Dr. R.J. Beamish and Mr. G.A.                            Solar, I.I., 1J. Baker, and E.M. Donaldson.
              McFarlane for making the fish used in these studies                              1988. Induced ovulation in sablefish (Anoplopomafimbria) us-
              available to us and for continued support. We also                                ing gonadoLropin releasing hormone analogues. In Pro-
                                                                                                ceedings of the 3rd. Int. Symp. on Reprod. Physiol. of Fish;
              thank J.O.T. Jensen and E. Groot for allowing us to                               2-7 Aug. 1987, D.R.Idler, L.W. Crim and J.M. Walsh (edi-
              quote unpublished fertilization and incubation data.                              tors) St. John's, Newfoundland. Mem. Univ., Newfound-
                                                                                                land.
                                                                                         Solar  I.I., E. McLean, I.J. Baker, N.N. Sherwood and E. M.
                                                                                            Donaldson.
              Citations                                                                        1990. Induced ovulation of sablefish (Anoplopomafimbria) fol-
                                                                                                lowing oral administration of desGly"(D-Ala')LH-RH
              Alderdice D.F.,J.O.T Jensen, and F.P.J. Velsen.                                   ethylamide. Fish Physiol. Biochem. 8:497-499.
                  1988a. Preliminary trials on incubation of sablefish eggs              van Oordt, P.G.W.J., and HJ.Th. Goos.
                     (Anoploppmafimb,ria). Aquaculture 69:271-290,                             1987. The African catfish, Clarias gariepinus, a model for
                  1988b. Incubation of sablefish (Anoplopoma fimbTia) in a sys-                 the study of reproductive endocrinology in teleosts.
                     tem designed for culture of fragile teleost eggs.                          Aquaculture 63:15-26.
                    Aquaculture 71:271-283.







                                          Geoduck Culture in Washington State:
                                     Reproductive Development and Spawning


                                              J. HAROLD BEATTIE and C. LYNN GOODWIN
                                                         Washington State Department ofFisheries
                                                                Point Whitney Laboratory
                                                                  Brinnon, WA 98320





                                                                     Abstract


                                The Washington State Department of Fisheries operates and maintains a hatchery for
                              the geoduck clam (Panopea abrupta). The purposes of this hatchery are to provide clam
                              seed to plant subtidal areas which commercial harvesters have depleted, and to eventu-
                              ally double the present annual harvest from 2,000 to 4,000 metric tons. Hatchery
                              operations occur from March through July, centering on the peak of natural spawning in
                              late spring. We induce spawning by elevating seawater temperatures to 15' C and increas-
                              ing algal concentrations. In our hatchery, females release an average of 2 to 10 million
                              eggs per spawning.




            Introduction                                                     allow commercial subtida     I harvest of these clams.
                                                                             This act not only created the commercial fishery for
            The geoduck, Panopea abrupta, is the largest burrow-             this resource but designated the WI)F and the Wash-
            ing clam in the world. These clams live about a meter            ington State Department of Natural Resources
            deep in the sediment and have long siphons which                 (DNR) as cooperative managers. The state sells the
            they can extend up their burrows for feeding, respi-             right to harvest subti 'dal tracts of geoducks to compa-
            ration, and excretion (Fig. 1). Extensive populations            nies whose divers excavate the clams with water jets.
            of geoducks occur intertidally and subtidally in Wash-           A portion of the proceeds from these lease fees are
            ington State throughout Puget Sound and Hood                     used to fund compliance, management, patrol, and
            Canal. The average weight for adults taken from                  enhancement efforts by the DNR and the WDF.
            Washington waters is about 0.8 kg; however, records                In 1972, the VV`DF began conducting subtidal sur-
            show specimens in excess of 5 kg (Anderson 1971).                veys of harvested tracts. By 1975, the results of these
            The Washington State Department of Fisheries                     surveys showed that, although in many areas recruit-
            (WDF) has established a maximum sustainable yield                ment was sufficient to maintain healthy population
            estimate for geoducks in Washington State at 2,000               levels, in some areas there was little or no recruit-
            metric tons (t) per year. Geoducks are very long                 ment. Thus, in 1982, the DNR and WDF decided to
            lived; Shaul and Goodwin (1982) recorded ages of                 implement an enhancement program which would
            adults in excess of 100 years. Buried beyond the                 replenish poorly recruiting areas..
            reach of natural predators, having no known dis-                   Central to this enhancement eff6-rt was the estab-
            eases, and exhibiting low recruitment, geoducks tend             lishment of a hatchery and nursery program. Using
            to form stable, uniformly sized populations.                     data from Point Whitney Laboratory research
              The commercial harvest of subtidal geoducks in                 (Goodwin 1973, Goodwin et al. 1979), this program
            Washington State began in 1970. Prior to that year,              adapted shellfish hatchery and nursery techniques
            state regulations permitted only personal use of a               developed for oysters and hard shell clams. Moving
            harvest from hand digging. During the late 1960's,               through experimental, pilot, and commercial scale
            surveys by the WDF documented extensive subtidal                 phases, the enhancement project targeted a goal of
            beAs of geoducks in Puget Sound. In 1970, the Wash-              doubling the annual harvest of geoducks from 2,000
            ington State legislature changed the regulations to              to 4,000 t.


                                                                                                                                      55







              56      NOAA Tecbnical Report NWS 106
                                                                            Reproductive Development

                                                                            Conducting a successful hatchery program depends
                                                                            on understanding the physiology and timing of re-
                                                                            production. Like most other mollusks, geoducks have
                                                                            no external indicators of gender. They are dioecious
                                                                            and sexually intransmutable; hermaphroditic indi-
                                                                            viduals are rare. The gonadal ducts are intertwined
                                                                            with the intestinal mass, and together they form two
                                                                            large spheroids which lie on each side of a medial
                                                                            line (Fig. 2).
                                                                              When they are approximately 3 years old, male
                                                                            geoducks become sexually mature; female geoducks
                                                                            begin egg production when they are about 4 years
                                                                            old (Anderson 1971). When mature, the gonadal tis-
                                                                            sues begin a cycle of development and maturation
                                                                            which leads to spawning and to tissue resorption or
                                                                            both. They continue this cycle for most of their lives.
                                                                            Sloan and Robinson (1984) found reproductively ac-
                                                                            tive specimens that were 105 years of age. Anderson
                                                                            (1971) and Goodwin (1976) found ripe geoducks in
                                       Figure I                             at least eight months of the year from November
                       A cut away drawing of a  geoduck in its              through June.
                       natural habitat. Shell height is 16 cm.
                       (From King 1986.)



                                                                                         Anterior



                  Right Valve





                    Gonad -



                   Foot





                                                                                                                        Mantle




                                                          A..

                                                                                               V



                                                                                                            -Ctenidia







                                                                      Figure 2
              The gross morphology and location of the gonads in geoducks. Shell length is 12 cm. Drawing by Don Velasquez, Univ. Washington.






                                                                        Beattie and Goodwin: Geoduck Culture in Washington State            57







                                                                                                      4



                                             -IM      0





                                                                                     %



                                                                  Vs
                                                 0 -                                                                    Figure 3
                                              4"7-0  ;.4,
                                                                                                            Histological section showing the
                                                                  f                                         early active phase of male gonad
                                                                                                            with only a few pockets of sperm
                                                                                                            showing. (Scale bar = 100 pm;
                                                                                _r                  4  7    stain = Harris' hernatoxylin and
                                                                                                            eosin.)






















                                                                                                                        Figure 4
                 Or"
                                                                                                            Histological section of a ripe
                                                                                   _rw_                     male, follicles packed with
                                                                                                            sperm. (Scale bar = 100 @tm;
                                                                                                            stain = Harris' hematoxylin and
                                                                                                            eosin.)


            Histology: Spermatogenesis                                          Histology: Oogenesis

            In. the early stage (September through December),                     In the early stages (September through Novem-
            the reproductive follicles are small and possess only               ber), ovacytes are just beginning to form in the
            pockets of sperm (Fig. 3). When the animals reach                   ovarian follicles (Fig. 6). During the late active stage
            maturation (November through June), the follicles                   (September through January), ovacytes begin to fill
            are packed with sperm (Fig. 4). Once spawning com-                  the follicles, clinging to the ovarian walls by a nutri-
            mences (December through July), the follicles                       tive stalk (Fig. 7). When females are ripe (November
            partially empty (Fig. 5); and when spent (March                     through June), the eggs detach and float free in the
            through August), the follicles partially collapse and               lumen (Fig. 8). By the partially spent stage Uanuary
            connective tissue proliferates in the gonads.                       through July), fewer eggs exist and there are very few,







                 58       NOAA Technical Report NMFS 106


                                                                r,

                                                                      4%.



                                                                                                Vf
                                % *iO @V                                                AN)
                                               V
                      %

                         i'4 J"
                                                                                   A
                                                                                   A
                                                                                   A
                                    it




                                    _4;_

                                                                         Alt

                                                              -4



                                                                                                                                 Figure 5
                                                                                                                    Histological section of a partially
                                                 r;
                                                                                                                    spawned male. (Scale bar = 100
                                                                                                                    vm; stain = Harris' hematoxylin
                                                                                                                    and eosin.)





                                                                                                         AA,


                                                                                                      A      41











                                                                   v_A
                                                                         14  A
                                                                                                                                 Figure 6
                                                                                                  :A
                                                                                       v                            Histological section of a female
                                                                                                                        duck gonad. A few ovacytes
                                                                                                                    geo
                     N
                       _01
                      ,           J
                        AW                                                                                          are just showing. (Scale bar
                                                                                                                    100 @xm; stain     Harris' hema
                                                                                                                    toxylin and cosin


                 if any,  developing ova. Figure       9 illustrates a   nearly         animals. By August, they are entirely spawned and
                 spent female in which the ovarian follicles are empty                  the spawning cycle begins anew (Fig. 10). Females
                 and leucocytes may be present (May through Au-                         with ripe gonads occur from fall until early summer;
                 gust).                                                                 however, mass spawning only begins in April (again
                                                                                        as evidenced by the occurrence of spent animals),
                                                                                        and is completed by August (Fig. 11). The temporal
                 Reproductive Cycle                                                     window for spawning in the animals examined by
                                                                                        Anderson (1971) began from the time of first spawn-
                   Male geoducks begin gonadal development during                       ing in January and continued until August and that
                 September. By November some are ripe. Though                           the majority of spawning occurred in May and June.
                 some spawn earlier, they start mass spawning in                        In obtaining and spawning geoducks for our hatch-
                 March as indicated by the first occurrence of spent                    ery work, we have found that animals from south






                                                                           Beattie and Goodwin: Geoduck Culture in Washington State               59

                                                        0, 4,@_
                                               lAt
                                                QV
                   A,@


                       f




                                                  jq





                                                                                                                              Figure 7
                                                                                                                 Histological  section of the late
                                                                                                                 active stage of female geoduck.
                                                                                                                 (Scale bar = 100 @Lrn; stain = Har-
                                                                                                                 ris', hematoxylin and eosin.)






                                   1k
                            10


                                                -14
                        ja&                                         @:2                               -i

                                                                                                    k;

                                                                                                   A     ."L,




                                                                                                                              Figure 8
                                                                                    0
                                                                    IA                                           Histological section from a ripe
                                                                                                                 female geoduck gonad. Eggs are
                                            4M                                                                   fl ating free in the lumen.
                                                                                    _k
                                                                                                                           r     100 @Lm; stain
                                                                                                                 (Scale ba
                                                                             AWL-
             I                                                                                                   Harris' hematoxylin and cosin.)

             Puget Sound ripen two to          three weeks    earlier    than       these tanks can hold 160 geoducks. The conditions in
             those from north Puget Sound. We attribute this phe-                   the tank were the following: flow-through ambient
             nomenon to the warmer waters and high productivity                     (10' Q, unfiltered sea water and a constant density
             characteristic of south Puget Sound.                                   of about 5,000 cells/mL of cultured diatoms
                                                                                    (Chaetoceros mulle7i). After two to four weeks in these
                                                                                    conditions, geoducks will usually spawn. The males
             Spawning in the Hatchery                                               are slightly precocious: even though a 1:1 proportion
                                                                                    exists between the sexes among the geoducks, we of-
             To obtain mass spawnings earlier than they occur in,                   ten found that a disproportionate number of males
             nature (March and April), we accelerated gonadal                       spawned in the early season. In March 1990, for ex-
             development in February. We ripened the                                ample, of the 684 animals under culture 302 males
             broodstock in five tanks (1.2 X 2.2 X 0.6 m). Each of                  spawned, but only 178 females released eggs. By






                 60    NOAA Technical Report NWN 106

                                                                            "'A
                                      j


                     1@7
                               NZ
                 7,1'1@1@114*-@V 1,

                 -.4
                       .1 A@,




                                           Zi


                 TJ*,V                                                                                                Figure 9
                                                                                                          Histological section from a
                                                                                                          n
                                                                                                            arly spent female geoduck. A
                                                                                                           e
                                                                                                          few residential ova are evident.
                                                                                                          (Scale bar = 100 @tm; stain
                                                                                                          Harris' hernatoxylin and eosin.)



                                            SPAWNING CYCLE - MALE GEODUCK


                     100

                     90

                     80

                     70

                     60

                 %   50

                     40-
                     30                                                                                             Figure 10
                     20                                                                                 Area graph   depicting the annual
                     10                                                                                 reproductive cycle of male geo-
                       0                                                                                ducks as represented by the
                       sep    Oct    nov     dec  jan   feb    mar    apr    may   jun   july   aug     percentage of animals in each
                                                                                                        gonadogenic stage at the time of
                    go spent            partially   M ripe          El late active 0 early active       sampling. The preponderance of
                                        spawned                                                         spawning in nature occurs be-
                                                                                                        tween the first and last occur-
                                                                                                        rences of spent animals. (Data
                                                                                                        from Anderson 1971.)


                 April, naturally occurring gonadogenesis precludes               To induce geoducks to spawn, we stopped the wa-
                 the need for a ripening period. During this time, we          ter flow and increased algal density in the spawning
                 could bring animals directly to the hatchery for              tank to a range of 100,000 to 200,000 cells/mL. Since
                 spawning, and males and females would spawn in ap-            algae in culture live at 18 to 20' C, this influx of algae
                 proximately equal numbers. We generally attempted             causes a rise in water temperature in the spawning
                 to spawn a group of at least 100 animals at a time. On        tank. After the temperature reaches 15' C, we begin a
                          on     20 to 50
                 average,    ly              percent of the females            flow of 14-15' C seawater.
                 spawned well. Each female will spawn from 2 to 10                When spawning commenced, we determined the
                 million eggs. Thus, an average spawn will result in 60        sex of each animal by observing its spawn: In a
                 million eggs.                                                 flashlight beam, sperm give the appearance of milk






                                                                             Beattie and Goodwin: Geoduck CiAture in Washington State            61



                                           SPAWNING CYCLE - FEMALE GEODUCK


                   100

                   90

                   80

                   70

                   60

               %   50

                   40
                   30                                                                                                      Figure I I
                                                                                                              Area graph depicting the annual
                   20                                                                                         reproductive cycle of female geo-
                   10  -                                                                                      ducks as represented by the
                     0 1                                                                                      percentage of animals in each
                      sep    oct    nov     dec   jan     feb    mar     apr    may    jun    july   aug      gonadogcnic stage at the time of
                                                                                                              sampling. The preponderance of
                  Im spent             partially     E ripe           El late active 0 early active           spawning in nature occurs be-
                                       spawned                                                                tween the first and last occur-
                                                                                                              rences of spent animals. (Data
                                                                                                              from Anderson 1971.)


             mixed with water, whereas the eggs look like dust                     niles to a sand substrate nursery. Over the next 90
             suspended in the air, We removed the spawning ani-                    days, they grew t   'o seed size: an average of 9 mm shell
             mals from the tank and placed them into several                       height. During the past four years, the WDF geoduck
             spawning trays (30 X 46 X 15 cm) filled with 14-                      hatchery has produced and planted 18 million seed.
             15' C seawater keeping males separate from females.                   In a good growing area, geoducks will grow to a 0.8-
                Once they had completely spawned, we removed                       kg mean harvest weight in five to six years. Thus, with
             the females and fertilized the ova with a few millili-                an expected field survival of between 0.5 and 1.0%,
             ters of sperm suspension from the male spawning                       we anticipate that we will have enhanced the re-
             tray(s). For cleaning the fertilized eggs we used two                 source by between 70 and 140 t by the time these
             screens. Each of these screens is constructed by                      animals reach harvest size.
             stretching and attaching precision woven nylon fabric
             (NyteO') across     one end of a 38-cm diameter PVC
             tube. Using 14' C seawater, we gently washed the zy-                  Acknowledgments
             gotes through a 130-micron screen onto a 35-micron
             screen, thus removing larger and smaller particles                    The authors thank Alex Bradbury, of the Washington
             and excess sperm. One of the problems we encoun-                      Department of Fisheries for his assistance with the
             tered with using high densities of phytoplankton to                   manuscript.
             induce spawning was that, while spawning, the well
             fed geoducks produced excessive amounts of feces
             and pseuclofeces. This material mixed with the eggs,                  Citations
             and made washing through the 35-micron screens a
             tedious process.                                                      Anderson, A.M., Jr.
               After they were washed, we placed the fertilized                        1971. Spawning, growth and spatial distribution of the geo-
             eggs into a 20-liter container of 15' C seawater. To                         duck clam, Panope generasa (Gould) in Hood Canal,
             monitor development, we microscopically examined                             Washington. Ph.D. diss., Univ. Washington, Seattle,
             samples for polar bodies and first cleavage. When we                         133 p.
             were assured that normal development was occur-                       Goodwin, C.L.
             ring, we placed the zygotes in a 20,000 L tank of                         1973. Effects of salinity and temperature on embryos of the
                                                                                          geoduck clam (Panope generosa). Proc. Nat. Shellfisheries
             14' C seawater.                                                              Assoc. 63:93-95.
               At 14' C, embryonic development was completed                       Goodwin, C. L.
             in 72 hours and ended at the first veliger form. The                      1976. Observations on spawning and growth of subtidal geo-
             veliger larval period lasts 25 days at 17' C. After they                     ducks (Panope generosa, Gould). Proc. Nat. Shellfisheries
             had metamorphosed, we moved the geoduck juve-                                Assoc. 65:49-58.







                 62        NOAA Technical Report NNOFS 106

                 Goodwin, C.L., W. Shaul, and C. Budd                                       Shaul, W., and C.L. Goodwin
                      1979. Larval development of the geoduck clam (Panope                      1982. Geoduck (Panope generosa: Bivalvia) age as determined
                        generosa, Gould). Proc. Nat. Shellfisheries Assoc. 69:73-76.              by internal growth lines in the shell. Can. J. Fish Aquat.
                 King,jj.                                                                         Sci. 39:632-636.
                      1986. juvenile feeding ontogeny of the geoduck Panope                 Sloan, N.A. and S.M.C. Robinson
                        abrupta (Bivalvia: Saxicavacea), and comparative ontogeny               1984. Age and gonad development in the geoduck clam
                        and evolution of feeding. Ph.D. diss., University of                      Panope abrupta (Conrad) from southern British Columbia,
                        Victoria, Victoria, BC, 281 p.                                            Canada.      Shellfish Res. 4(2):131-137.






                        Ecology of Sargassum spp. and Sargassum Forest Formation



                                                              JUN-ICHI TSUKIDATE
                                                         Nansei National Fisheries Research Institute
                                                                Maruishi 2-17-5, Ohno Saeki
                                                                 Hiroshima 739-04, Japan




                                                                       Abstract


                                 Sargassum spp. are distributed in the coastal waters of Japan from the northernmost
                              island of Hokkaido down to the southern island of Kyushu. The forests they create in the
                              sublittoral zone of the sea are important spawning and rearing grounds for fish and
                              shellfish. A study of Sargassum spp. was conducted to support a project of creating Sargas-
                              sum forests. Methods for producing these forests are described. The majority of species
                              started growth in autumn as the water temperature began to fall. The release of oospores
                              was also temperature dependent and varied between species. Many species matured
                              between May and June when the rising water temperature approaches 18' C. Contrary
                              to previous findings, oospores were released irrespective of the tide. Both mono- And
                              multi-species forests were created and maintained for two years in Hiroshima before
                              being invaded by other seaweed species.



            Introduction                                                       Prefecture, Cystophyllum sisymbrioides was the first to
                                                                               release oospores at 14' C in March. Sargassum horneri,
            Sargassum spp.    (Fig. 1) are members of the brown                S. micracanthum, S. lortile, and S. muticum released
            algae family and are among the most highly devel-                  oospores at around 15' C in April (Okuda 1981).
            oped seaweeds in both their morphology and life                    Many species matured frorn May to June when the
            cycle. Morphologically, they have a holdfast, stipe,               water temperature rises to 18' C and higher.
            and lamina and thus resemble a terrestrial tree. Hav-                It is generally thought that oospores are dis-
            ing no asexual stage in their life history (Fig. 2), each          charged at high tide but we found oospore were
            plant is a gametophyte producing both female (oo-                  released irrespective of the tide (Okuda 1.981, 1982)
            spores) and male gametes (spermatozoids). They are                 (Fig. 3). S. micracanthum liberated oospores every sev-
            distributed throughoutjapan from the northernmost                  enth or eighth day, S. patens at 3-day intervals, and
            island of Hokkaido down to the southern island of                  most other species every fourth day.
            Kyushu (Arasaki 1984). Most species grow in a wide
            range of water temperatures and form forests in the
            sublittoral zone of the sea that are known to be                   Sargassum Forest Formation
            spawning, nursery, and feeding areas for many kinds
            of fish, shellfish, and other marine organisms.                    Most Sargassum spp. start to grow in autumn when
              This paper describes the growth and maturation of                the water temperature begins to fall. Figure 4 shows
            Sargassum spp. and the propagation of Sargassum for-               the growth of several species on a wet weight basis at
            ests with artificial reefs and seeded string.                      one of the research areas near Hiroshima, where a
                                                                               forest spreads for about 1.1-1.4 ha (Takaba and
                                                                               Mizokami 1982). S. horneri and S. tortile grows pro-
            Oospore Liberation and                                             fusely here from November to the following January.
            Growth of Sargassu                                                 The largest standing crop for the four species was
                                                                               15 kg wet weight/M2     in 1981, and production is esti-
            The optimum time for oospore liberation varied                     mated to be 15-21 kg wet weight/          (M2   9 yr). The
            among species with respect to season and water tem-                growth of S. ringgoldianum in another area located off
            perature (Table 1). At a research area in Fukuoka                  Tokushima Prefecture is shown in Figure 5 (Nakahisa


                                                                                                                                          63







              64      NOAA Technical Report NMFS 106











                                                Lamina



























                                                              Stem






                                                         Holdfast




                                                                                                            Figure 1
                                                                                                    Morphology of Sargassum.



              and Kojima 1982). This species matures in autumn at           oxygen demand ranged from 0.15 to 0.85 ppm. Nu-
              around 22' C with the largest growth observed in Oc-          trient concentrations fluctuated during the year.
              tober just before maturation. The standing crop at            Dissolved inorganic nitrogen was 1-2 [Lg-atoms/L in
              this time was 5-6 kg wet weight/M2.                           August and 4-5 Rg-atoms/L in November.
                 The environmental characteristics of the water in
              the research field off Yashiro Island are summarized
              in Table 2. (Takemoto et al. 1984). The temperature           Mono-Species Culture
              at the bottom is lower than that at the surface in
              August, although no difference in temperature was             Figure 6 shows an outline of the artificial reefs placed
              found in November. Salinity was observed to be a              in the sea for the various Sargassum forest formations
              little low in August. Dissolved oxygen concentrations         (Komoto et al. 1987). Fifty concrete blocks were
              were around 5 mg/L and saturated in both August               placed in both the eastern (E) and western (W)
              and November. Turbidity changed from 0.2 to 1.6               zones of the study area. The E-zone was close to a
              ppm depending on the tide and current. Chemical               natural Sargassum forest, while the W-zone was about






                                                                                                          Tsukidate: Ecology of Sargassum Forest Formation                           65




                                                                     Fertilized egg


                                                     f
                                                                                      Young gametophyte

                                                              Fusion


                     Spermatozoid                       Oospore


                                                                                                Gametophyte
                  Conceptacle







                              Reductio
                                division


                                                                                  Receptacle


                                                      Conceptacle


                                                                                                                                                 Figure 2
                                                                                                                                    Life Cycle of Sargassum spp.


                                                                                                       50 m from it. The natural forest was dominated by
                                                  Table I                                              several species of Sargassum and Ecklonia cava. The
                   Maturing season of Sargassum spp. and coastal water                                 seasonal variation in the number of individuals of
                   temperature (Okuda 1982)                                                            S. patens is shown in Figure 7 (Yoshikawa and
                                                                   Month and                           Tsukidate 1984, 1985). Mortality was very high dur-
                        Species                                    Temperature                         ing the first three months; afterwards the number of
                                                                                                       plants stayed at the level seen in the natural forests.
                        Cystophyllum sisymbrioides                 Late March                          The seasonal variation in the total length of S. patens
                                                                   141 C                               is shown in Figure 8 (Yoshikawa and Tsukidate 1986).
                        Sargassum horneyi                          Middle and                          The total length increased toward the following
                        S. micracanthum                            late April                          spring, reaching an average of about 70 cm. Length
                        S. toride                                  15-16' C                            declined reaching its smallest size of 20 cm in Octo-
                        S. Imuticum                                                                    ber, followed by another increase to between 50 and
                        S. patens                                  Early and                           60 cm in February. Thus the maximum growth stage
                        S. hemiphyllum                             middle May                          was found between winter and spring. Figure 9 shows
                                                                   181 C                               the seasonal variation in wet weight of S patens
                                                                                                       (Yoshikawa and Tsukidate 1986). The growth pattern
                        S. thunbergii                              Middle and                          was identical to that of total length. These forests
                                                                   late May                            diminished in the summer months but enlarged
                                                                   19-201 C                            again during this winter to spring period. However,
                        Hijikia fusiforme                          late May and                        in the third year, they did not enlarge their biomass
                                                                   earlyjune                           because of the invasion of other seaweeds such as
                                                                   211 C                               Ecklonia cava and Padina arborescens. Thus, Sargassum
                                                                                                       forests were created and maintained in a roughly
                        S.ringgoldianum                            Early and middle                    1,000 M2 area with artificially prepared reefs by using
                                                                   October
                                                                   221 C                               seeded string for at least two years.







                66        NOAA Technical Report NMFS 106



                          50
                          25                                                    Sargassum thunbergii
                          571          a
                          25
                          1001                 m                                SargaSsum patens
                             I             A
                    -0    100t             I                                    Hizikia fusiforme
                                           A
                    (QU)
                    LA
                    m                      I                                    Sargassum confusum
                          70
                    LA    25
                    sy
                    0                                                           Sargassum hemiphyllum
                    0.
                    0     25
                    0                                                           Sargassum muticum

                          25
                                                                  gM            Sargassum homed
                          25                                                    Sargassum micracanthum
                          25                                                    Cystophyllum shwmbfioides
                          25                              m 0, am               Safgassum tordle
                                1 2 3 4 5 6 7 8 91011                    12
                                                                                                                              Figure 3
                                             Interval period (days)                                              Interval of oospore liberation in
                                                                                                                 Sargassum spp. (Okuda 1982).




                             10            0 Sargassum homeri
                                           x cystophyRim skymbribides
                                           * Saigassm patens
                                           * Sargassm tordle



                      -C
                      .0i:71  2
                          E


                                                                                                           x



                                                                                  x


                                           x


                              0

                                          May         July          Sept.        Nov.        Jan.         Mar.                    Figure 4
                                                                                                                         Seasonal variations in wet
                                                        1981                                     1982                    weight of Sargassum spp.
                                                                                                                         (Takaba and Mizogarni
                                                                                                                         1982).






                                                                                                      Tsukidate: Ecology of Sargassum Forest Formation                      67


                             5                                                 0    Satgasmm firxigodianum
                                                                               0    sargass- h--d




                             3
                      E





                          0.1





                             0
                                       May           July         Sept.         Nov.         Jan.     Mar.                                  Figure 5
                                                                                                                  Seasonal variations       in wet weight of 'Sargassum
                                                          1981                                   1982              ringeggoldianum      (Nakahisa and Kojima 1982;
                                                                                                                  Nakahisa et al. 1983).



                                                                                             Table 2
                    Environmental characteristics of the research field in Yashiro Island. (Takemoto et al. 1984.) S = surface; B = bottom.
                    DO = dissolved oxygen; COD              chemical oxygen demand; DIN = dissolved inorganic nitrogen. tr = trace.

                                                    Water           Water
                    Survey      Survey      Survey  depth Layer     temp.     Salinity  DO               Turbidity  COD      PO,-P      DIN      NHN      N02-N     NON
                    season      station     time     (m)    (-)     (*C)      (%o)     (mg/L)       pH     (pprn)   (ppm)   (l.Lg-at/L) (ltg-at/L) (lLg@at/L) (l.Lg-at/L) (lLg-at/L)


                    1983        LA*4        10:39           S       25.4      32-12     5.57        8.12   0.18     0.64      0.22      1.75     1.22      tr       U3
                    Aug. 30                 10:37    3.5    B       24.8      32.18     5.60        8.15   1.12     0.37        tr      1.85     1.27      tr       U8
                                L-4*8       10:56           S       25.6      32.12     5.48        8.10   0.10     0.85      0.12      1.69     0.55      tr       1.14
                                            10:54    7.0    B       24.4      32.28     5.22        8.10   0.85     0.24      0.10      1.73     0.70     0.13      0.90
                                L-12*4      11:02           S       25.4      32.18     5.80        8.12   0.15     0.14        tr      1.11     0.75      tr       0.36
                                            11:00    7.0    B       23.9      32.48     5.58        8.15   7.20     0.24      0.17      2.04     0.82     0.37      0.85
                                L-12*8      11:11           S       25.4      32.12     6.07        8.12   0.23     0.32      0.11      0.95     0.42      tr       0.53
                                            11:09    4.0    B       24.7      32.30     5.55        8.15   0.65     0.16      0.11      0.95     0.43      tr       0.52

                    1983        L-4e2                       S       16.6      32.88     5.62        8.15   0.20     0.43      0.42      3.67     1.80     0.83      1.04
                    Nov. 30                 11:52    2.5    B       16.8                5.72        8.15   1.00     0.48      0.42      4.41     2.25     0.88      1.28
                                L-4*4                       S       16.4      32.88     5.58        8.20   0.45     0.53      0.48      6.47     2.21     0.84      3.42
                                            11:55    4.0    B       16.8      32.90     5.59        8.19   0.95     0.77      0.38      3.54     1.53     0.87      1.14
                                L,4*8                       S       17.0      32.94     5.56        8.20   0.55     0.51      0.42      4.83     2.40     1.00      1.43
                                            12:00   12.0    B       16.8      32.91     5.58        8.15   1.65     0.46      0.42      4.70     2.43     0.97      1.30
                                L-12*4                      S       16.4      32.91     5.56        8.20   0.85     0.58      0.39      3.94     2.08     0.83      1.03
                                            12:05    3.5    B       16.7      32.86     5.50        8.20   0.60     0.45      0.42      3.76     1.72     0.84      1.20
                                L-12-8                      S       17.0      32.94     5.87        8.20   0.40     0.56      0.39      3.56     1.38     0.95      1.23
                                            12:08    9.0    B       16.8      32.92     5.37        8.20   0.45     0.42      0.40      3.73     1.41     1.00      1.32




                Multi-Species Culture                                                                 The ratios in wet weight of S. piluliferum to S. pa-
                                                                                                    tens are seen in Figure I I (Komoto et al. 1988).
                Two species of Sargassum out of three, namely S. pa-                                S. piluliferum dominated the artificial reefs in the first
                tens, S. piluliferum, and S. ringgoldianum were                                     year and also demonstrated a superior regenerative
                combined and planted on the concrete blocks at the                                  ability in the second and third years. In the first year,
                same time. Seeded strings with embryos were                                         S. piluliferum accounted for 74.7% of the combined
                wrapped around the blocks alternately. Thus, as                                     wet weight of the two species, 86.5% in the second
                shown in Figure 10, eight strings with S. patens and                                year and 87.7% in the third year. Figure 12 shows the
                eight with S. piluliferum resulted (Komoto et al. 1987).                            ratios in wet weight per single concrete block of







                    68           NOAA Technical Report NMFS 106


                                                                                             0 rn          20                  so



                                 B8

                                                A


                                                                                         Ur  ------
                                             Z                                           1CM
                                 013                j
                                 am                            Mono-species
                                                                   W

                                                                 QT --- i                    ----- ---
                                 U13                            'A a     1           @13 1         B
                                 U0                                13                U
                                                                                                            E
                                                                                     HE'     L      J
                                                                                                       Mono-species
                                                             131

                                                      Mul&spedes

                                                                                                                                                                      Figure 6
                                                                                                                                                    An outline of the artificial reef for
                                                                    a                                                                               seaweeds off Yashiro Island
                                                                                                                                                    (Komoto et al. 1987). W = west-
                                                                                                                                                    ern zone of the study area; E
                                                                     )@Iy. hi Is.                                                                   eastern zone of the study areajn
                                                                                                                                                    the Seto Inland Sea, off Yashiro
                                                                                                                                                    Island.









                           300






                                                                                                             W

                           200                                                                         0     E







                            100




                                                                                                                                                                  Figure 7
                                                                                                                                            Seasonal variations in number of indi-
                                                                                                                                            viduals per 75 cm string of Sargassum
                                      Aug.       Oct.     Dec.      Feb.     Apr.       June    Aug,       Oct.     Dec.                    patens (Yoshikawa and Tsukidate 1984,
                                                                                                                                            1985). 0 = western zone of the study
                                     1983                          1984                                                                     area; 0          eastern zone of the study
                                                                                                                 D a

                                                                                                          @10'u
                                                                                     ro


















































                                                                                                                                            area.







                                                                                                              Tsukidate: Ecology of Sargassum Forest Formation                               69





                                80



                                70


                                                                                                                                                                 0 W
                                60
                                                                                                                                                                 0 E

                                                                          0
                                50


                       0        40                                                 0


                                30



                                20



                                10
                                                                                                                                                             o-
                                  0                                                                                                                          *_@_

                                        Aug.      Oct.      Dec.      Feb.      Apr.      June      Aug.      Oct.     Dec.      Feb.      Apr.      June      Aug.      Oct.      Dec.

                                       1983                          1984                                                        1985


                                                                                                  Figure 8
                  Seasonal variations in total length of Sargassum patens (Yoshikawa and Tsukidate 1986).                                         western zone of the study area;
                                                                               0 = eastern zone of the study area.



                 S. piluliferum and S. patens to the invading seaweeds                                      Conclusion
                 (Komoto et al. 1987, 1988). S. piluliferum accounted
                 for 77.6%-86.2% of all seaweeds in the first and sec-                                      Sargassum forests were created and maintained in an
                 ond years and then dropped to 47.9% in the third                                           experimental area using artificially prepared reefs
                 year with the invasion of other seaweeds. Therefore,                                       with seeded string for at least two years using both
                 the forests were not solely composed of Sargassum but                                      mono- and multi-species cultures. However, in the
                 also of other seaweeds by the third year. Among the                                        third year the forests diminished and were overcome
                 other seaweeds, Ecklonia cava first appeared in the                                        by the invasion of other seaweeds. Therefore, more
                 second year, joined by Padina arborescens and Hypnea                                       seedlings must be added in the third year and other
                 charoides, all of which grew profusely during the third                                    seaweeds eliminated in order to maintain Sargassum
                 year.                                                                                      forests for a longer time. Only a small amount of
                    The natural vegetation of Sargassum spp. on the                                         grazing by herbivores was observed in the Seto In-
                 artificial reefs was surveyed during culture of the two                                    land Sea. This is thought to be one of the reasons
                 species on the nonseeded control blocks (Fig. 10). As                                      why we succeeded in forming Sargassum forests.
                 seen in Figure 13, S. horneyi was the prevailing species
                 on the control reefs the first year, probably because it
                 matures earlier than any other species and grows fast                                      Citations
                 (Komoto et al. 1987, 1988). The following year,
                 S. tortile grew vigorously and became dominant.                                            Arasaki, S.
                 S. micracanthum also grew markedly in the second and                                             1984. Seaweed forests in Japan. In Moba and Marine or-
                 third years. It is assumed that the annual Sargassum                                               ganisms (T Hisamune, ed.), p. 1-11. Report of Japan Fish-
                                                                                                                    eries Resources Conservation Association, Zenkoku Choson
                 spp. settle first followed by the perennial ones.                                                  Kaikan, 11-35, Nagatacho 1, Chiyoda-Ku, Tokyo 100.







                     70          NOAA Technical Report NMFS 106






                                 200



                                                                                                                                                0 W

                                                                                                                                                0 E



                           Q; E




                                 10  0







                                                                                                                            '10
                                                                                                                                                                                    0




                                            Aug.      Oct.     Dec.      Feb.     Apr.      June     Aug.      Oct.     Dec.      Feb.      Apr.     June     Aug.       Oct.     Dec.
                                           1983                         1984                                                     1985

                                                                                                   Figure 9
                       Seasonal variations in wet weight of Sargassum patens (Yoshikawa and Tsukidate 1986). 0                                  western zone of the study area;
                                                                                 0     eastern zone of the study area.


                         A











                                                                                                                     75  cm
                                                                                                                                   28    a


                                                                                                                                         75 c.                               75 cm

                        75 C.



                                     75 cm                         S. tinggoldianurn Control
                                  S. patens
                                                  S. pilufferum


                                                                                                  Figure 10
                     A schematic diagram of artificial reefs for growing Sargassum spp. off Yashiro Island, Seto Inland Sea (Komoto et al. 1987).
                     Blocks were planted with seeded strings as follows: Y= S. Patens; M = S. piluliferum; 0 = S. ringg-oldianum; C = no seeded string.
                     Blocks with mixed species were arranged within the reef as shown in (A) and wrapped with alternating strings of each species
                     as shown in (B).






                                                                                                                Tsukidate: Ecology of Sargassum Forest Formation                               71


                                           First year                                          Second year

                                                                 S. patens
                                                                 (25.3%)                               Mph, S. patens
                                                                                                                     (13.5%)





                                                                        S. pfluliferum
                    S. pituliterurn                                        (86.5%)
                       (74.7%)

                                                                 Third year


                                                                                       S. patens
                                                                                       (12.3%)




                                          S. Piluriferum
                                             (87.7%)
                                                                                                                                                              Figure 11
                                                                                                                                        Comparison of Sargassum patens to S.
                                                                                                                                        piluliferum (in percent wet weight) on
                                                                                                                                        an artificial reef (Komoto et al. 1988).





                                     First year                                                     Second year

                                                S. horneri
                                                   (0.3%)
                   S. Micracan;thurn                                               Other seaweeds
                                                                                                                       S. patens
                        (12%                               S. patens                   (6.8%)
                                                            (18.9%)           S. micracanthum                            (6.4%)
                                                                                   (0.4%)

                                                                               S. tortile
                                                                                (0.1%)

                                                                                 Unknown
                                                                                   (0.1%)


                                 S. piluliferum                                                      S. piluliferum
                                    (77.6%)                                                             (86.2%)
                                                              Third year
                                                S. patens
                                                   (2.8%)

                                  Other seaweeds
                                      (48.2%)
                                                                                     S. pilufferum
                                                                                       (47.9%)



                                          S. tortile
                                                                                                                                                                Figure 12
                                            (0.1%)
                                                                                                                                            Comparison         of Sargassum patens to
                                           S. micracanthwn                                                                                  S. piluliferum to other Sargassum spp.
                                                (1.0%)                                                                                      (in percent wet weight) on an artifi-
                                                                                                                                            cial reef (Komoto et al. 1987, 1988).
                                                   N,






                    72         NOAA Technical Report NWS 106


                                       First year                                        Second year


                                                   S. pilutiferum
                            Other seaweeds            (3.1%)                  Other seaweeds               S. piluliferum
                                (1.2%)              S. rnkracanthum                                              1.9%)
                        Unknown                          (2!.2%)           Un,1.nown
                                                                                %I
                         (0.3%)                                                5 )


                                                                        S. tortile                          S. miaacanthum
                                                                          (49.5%)                                2 .1%)


                                          S. homeri
                                           (93.2%)
                                                                Third year

                                                                             S. Piluriferurn
                                                       Other seaweeds


                                                                                      miaracanthurn
                                                                                       (25.4%)


                                                  S. tordle                           5. homefi                                                         Figure 13
                                                  (61.7%)                               (0.1%)                                         Comparison among Sargassum spp.
                                                                                                                                       (in percent wet weight) on an arti-
                                                                                                                                       ficial reef. (Komoto et al. 1987,
                                                                                                                                       1988).



                    Komoto, Y, T. Iwamoto, and H. Matsuura.                                                      saceae. Report of Marine Ranching Program, Nansei Nat].
                         1987. Studies on the control of Sargassum forests. Report of                            Fish. Res. Inst., Hiroshima, p. 10 1 - I 10. (In japanesc.)
                            Marine Ranching Program, Nansei Natl. Fish. Res. Inst.,                     Takaba, M., and A. Mizogami
                            Hiroshima, p. 17-33. (In Japanese.)                                               1982. Seasonal fluctuation of Sargassum communities and
                         1988. Studies on the control of Sargassum forests. Report of                            vertical distribution of Sargassaceae at Kuroshima Island
                            Marine Ranching Program, Nansei Nad. Fish. Inst.,                                    in the western Akinada. J. Hiroshima-Ken Fish. Exp. Sta-
                            Hiroshima, p. 23-42. (Injapanesc.)                                                   tion. 12:33-44.
                    Nakahisa, Y, and H. Kojima.                                                         Takernoto, K, S. Takayama, S. Yoshioka, Y Komoto, and T. Miyago.
                         1982. Ecology of Sargassum ringgoldianum and S.                                      1984. Sargassum forest formation by artificially prepared
                            giganteifolium. Report of Marine Ranching Program, Nansei                            seedlings. Report of Marine Ranching Program, Nansei
                            Natl. Fish. Res. Inst., Hiroshima, p. 71-85. (In Japanese.)                          Natl. Fish. Res. Inst., Hiroshima, p. 51-74. (In Japanese.)
                    Nakahisa, Y., H. Kojima, and N. Tanimoto                                            Yoshikawa, K, andj. Tsukidate.
                         1983. Ecology of Sargassum ringgoldianum and S.                                      1984. Sargassum forest formation by setting artificial
                            giganteifolium. Report of Marine Ranching Program,                                   reefs. Report of Marine Ranching Program, Nansei Nad.
                            Nansci Natl. Fish. Res. Inst., Hiroshima, p. 99-115. (In Japa-                       Fish. Res. Inst., Hiroshima, p. 75-89. (In Japanese.)
                            ncsc.)                                                                            1985. Sargassum forest formation by setting artificial
                    Okuda, T                                                                                     reefs. Report of Marine Ranching          Program, Nansei Nat].
                         1981. Settlement mechanisms on the embryo of Sargas-                                    Fish. Res. Inst., Hiroshima, p. 69-86. (In Japanese.)
                            saceae. Report of Marine Ranching Program, Nansei Natl.                           1986. Sargassum forest formation by settling artificial
                            Fish. Res. Inst., Hiroshima, p. 105-117. (In Japanese.)                              reefs. Report of Marine Ranching          Program, Nansei Natl.
                         1982. Settlement mechanisms on the embryo of Sargas-                                    Fish. Res. Inst., Hiroshima, p. 55-70. (In Japanese.)
                                                                                (3.5%






                                                        Salmon Gonadotropins



                                                                 PENNYSWANSON

                                                                     School ofFisheries
                                                                  University of Washington
                                                                    Seattle, WA 981951




                                                                      ABSTRACT


                                 Control of gonadal function by pituitary gonadotropins (GTHs) is a general feature of
                               vertebrate reproduction. In most tetrapods, gonadal function has long been known to be
                               regulated by two GTHs: follicle-stimulating hormone (FSH) and luteinizing hormone
                               (LH) (Licht et al. 1977). Luteinizing hormone, follicle-stimulating hormone, and a third
                               pituitary hormone, thyroid-stimulating hormone (TSH), are chemically related. All three
                               consist of an ot and P subunit, which interact noncovalently and are both glycosylated. In
                               mammals it has been found that the ot subunits of TSH, LH, and FSH are identical within
                               a species, whereas the P subunits are hormone specific and structurally conserved be-
                               tween species (Pierce and Parsons 1981).
                                 The question of whether fish reproduction is regulated by one or two pituitary GTHs
                               has been controversial for nearly two decades. A single GTH, sometimes referred to as
                               maturational GTH, has been isolated from several teleost species: chinook Salmon,
                               Oncorhynchus tschawytscha (Breton et al. 1978); common carp, Cyprinus carpio (Burzawa-
                               Gerard 1971); silver carp, Hypophthalmichthys molitrix (Chang et al. 1988a, 1990); pike eel,
                               Muraenesox cinereus (Huang et al. 1981; Liu et al. 1989); tilapia, Orechromis mossambica
                               (Farmer and Papkoff 1977); African catfish, Clarias gariepinus (Goos et al. 1986); and
                               Atlantic croaker, Micropogonias undulatus (Copeland and Thomas 1989). Maturational
                               GTH has been thought to regulate all aspects of gametogenesis (see Burzawa-Gerard
                               1982; Fontaine and Dufour 1987). The single type of GTH which has been consistently
                               isolated from the teleost species so far examined, appears to be chemically related to
                               tetrapod FSH and LH because of its glycoproteic and subunit nature. In contrast, Idler
                               and colleagues (see Idler and Ng 1983) prepared two GTH fractions from pituitaries of
                               four teleost species; one adsorbed to Concanavalin-A Sepharose and stimulated gonadal
                               steroidogenesis (Con A 11) while the other did not adsorb to Concanavalin-A Sepharose
                               and stimulated in vivo vitellogenin uptake by ovarian follicles (Con A 1). Con A 11 shows
                               some chemical similarity to classic tetrapod pituitary GTHs, whereas Con A I does not ;
                               Con A I is low in carbohydrate content, and its subunit nature has not been demon-
                               strated.
                                 More recently two pituitary GTHs, GTH I and       GTH 11, which are distinctly different
                               from each other in chemical characteristics and structurally homologous to tetrapod FSH
                               and LH, have been isolated from chum salmon (Oncorhynchus keta) (Kawauchi et al. 1989;
                               Suzuki et al. 1988 a,b; Itoh et al. 1988) and coho salmon (0. kisutch) (Swanson et al.
                               1991). Chum salmon GTH 10 and GTH 11 P subunits have only about 31% amino acid
                               sequence identity to each other. Amino acid sequence comparisons of the chum salmon
                               GTH P subunits to those of bovine FSH (bFSH) and bovine LH (bLH) revealed that the
                               GTH I P subunit has slightly greater sequence identity to the bFSH P subunit (41%) than
                               to the bLH 0 subunit (35%), whereas the GTH 110 subunit has greater identity to the
                               bLH P subunit (42%) than to the bFSH P subunit (38%). Comparisons of the cDNA
                               sequences of the chum salmon GTH P subunits to the cDNAs of the bLH P and bFSH P
                               subunits demonstrate the same structural relatedness (Sekine et al. 1989). The amino




             Present address: School of Fisheries, University of Washington, Seattle, WA 98195 and Northwest Fisheries Science Center, National Marine
             Fisheries Service, 2725 Mondake Blvd. E., Seattle, WA 98112.


                                                                                                                                          73







                74       NOAA Technical Report NMB 106

                                  acid sequences of chinook salmon (Trinh et al. 1986), common carp (Chang et al.
                                  1988b), silver carp (Chang et al. 1990), and pike eel GTH        subunits (Liu et al. 1989)
                                  show about 40% sequence homology to mammalian LH                 subunits, and about 75%
                                  sequence identity to the chum salmon GTH II P subunit. Therefore, it is apparent that
                                  both GTH I and GTH 11 have structural homology to tetrapod FSH and LH, and the
                                  single GTH molecule previously isolated from several teleost species is structurally similar
                                  to chum salmon GTH Il. GTH I had not been identified in previous studies.
                                    The physiological distinction between GTH I and GTH II is not as clear as the chemi-
                                  cal distinction. Studies done to date so far indicate that GTH I and GTH 11 can stimulate
                                  gonadal steroidogenesis. Suzuki et al. (1988c) found that chum salmon GTH I and GTH
                                  II were equally potent in stimulating in vitro estradiol 17-P production by vitellogenic
                                  amago salmon (0. rhodurus) ovarian follicles. However, chum salmon GTH Il was found
                                  to be two times more potent than chum salmon GTH I in stimulating 17ot-
                                  hydroxyprogesterone production by ovarian thecal layers and 170t,20p-dihydroxy-
                                  4-pregnen-3-one production by granulosa layers in the presence of 17of-
                                  hydroxyprogesterone (Suzuki et al. 1988c). Coho salmon GTH I and GTH 11 stimulated
                                  in vitro estradiol 17-P and total androgen production by juvenile coho salmon ovarian
                                  and testicular tissue, respectively, in a similar dose-dependent manner (Swanson et al.
                                  1989). In recent studies using in vitro incubations of coho salmon testicular fragments, it
                                  was found that coho salmon GTH I         and GTH 11 were equipotent in stimulating 11-
                                  ketotestosterone secretion (I. Planas, School of Fisheries, Univ. of Washington, Seattle,
                                  WA, pers. commun. September 1989). The only clear difference between the biological
                                  activities of GTH I and GTH 11 was the GTH I stimulation of vitellogenin uptake by
                                  rainbow trout (0. mykiss) oocytes both in vivo and in vitro (Tyler et al. 1991). In the in
                                  vitro studies by Tyler and colleagues, GTH I was roughly 100-times more potent than
                                  GTH II whereas in vivo GT    'H 11 was not active compared to GTH L which doubled the
                                  rate of vitellogenin uptake. Distinctly different actions of GTH I and GTH 11 in male
                                  salmon have not been found and should be the subject of future investigations.
                                    Although salmon GTH I and GTH 11 appear to have similar steroidogenic activities
                                  when tested in vitro, blood and pituitary levels of these two GTHs vary significantly
                                  during reproductive development. GTH I was the predominate GTH in the plasma and
                                  pituitary of vitellogenic/spermatogenic rainbow trout, whereas GTH Il was the predomi-
                                  nant GTH at the time of final reproductive maturation (Suzuki et al. 1988d). Swanson et
                                  al. (1989) found that in prespermatogenic and previtellogenic (prepubertal) coho
                                  salmon, GTH I was the only GTH detectable in the plasma. Recent analysis of plasma
                                  levels of GTH I and GTH Il in coho salmon during the final year of reproductive matura-
                                  tion have shown an extended increase in GTH I from May through October during
                                  vitellogenesis/spermatogenesis, and a decline at the time of spawning (late November or
                                  early December). On the other hand, plasma GTH 11 levels were consistently at low or
                                  nondetectable levels throughout vitellogenesis/spermatogenesis, and increased dramati-
                                  cally at the time of spawning (Swanson et al. unpublished). Therefore, the physiological
                                  relevance of the steroidogenic activity of GTH II in prepubertal or,even vitellogenic/
                                  spermatogenic coho salmon is questionable since it does not appear to be present in
                                  significant levels in the plasma during this time.
                                     Immunocytochernical studies of salmonid pituitary gonadotrophs have revealed a lack
                                  a co-localization of the two GTHs; this is contrary to what has been found for LH and
                                  FSH in tetrapods. Nozaki et al. (1990a) demonstrated that antisera directed against coho
                                  salmon GTH I P and GTH II P subunits stain two distinctly different gonadotroph cell-
                                  types in the pars distalis of salmonids and do not stain thyrotrophs, sommatotrophs,
                                  lactotrophs, corticotrophs, or melanotrophs. Moreover, in an ontogenetic study of rain-
                                  bow trout pituitary gonadotrophs, Nozaki et al. (1990b) showed that GTH I-producing
                                  cells are present prior to puberty and increase in number during vitellogenesis; GTH II-
                                  producing cells do not appear until after the onset of spermatogenesis/ vitellogenesis
                                  and are greater in number than GTH I-producing cells at the time of final reproductive
                                  maturation. These data, in addition to data on blood levels of GTH I and GTH II, suggest
                                  relationships of GTH I with gonadal growth and GTH 11 with final maturation of the
                                  gonads similar to those of FSH and Lli in mammals. More detailed investigations of the
                                  specific roles of GTH I and GTH 11 in salmonid reproductive development are necessary
                                  to determine the physiological importance of the dual gonadotropin system in fish.






                                                                                                                      Swanson: Salmon Gonadotiropins                   75

               Citations                                                                        Kawauchi H., K_ Suzuki, H. Itoh, P. Swanson, M. Nozaki, N. Naito,
                                                                                                  Y. Nagahama
               Breton B., P. Purnet, P. Reinaud                                                      1989. Duality of salmon pituitary gonadotropins. Fish
                    1978. Sexual differences in salmon gonaclotropin. Ann.                              Physiol. Biochem. 7:29-38.
                      Biol. Anim. Biochim. Biophys. 18:739-765.                                 Licht P., H. Papkoff, S.W. Farmer, C.H. Muller, H.K Tsui, D. Crews
               Burzawa-Gerard E.                                                                     1977. Evolution of gonadotropins structure and func-
                    1971. Purification d'une hormone gonadotrope hypo-                                  tion. Recent Prog. Horm. Res. 33:169-248.
                      physaire de poisson oel6ost4@en, la Carpe (Cyptinus carpio                Liu C.S., F.L. Huang, Y.S. Chang, T.B. Lo
                      L.). Biochimie (Paris) 53:545-552.                                             1989. Pike eel (Muraenesox cinereus) gonaclotropin. Amino
                    1982. Chemical data on the pituitary gonaclotropins and                             acid sequences of both a and P subunits. Eur. J. Biochem.
                      their implication to evolution. Can. J. Fish. Aquat. Sci.                         186:105-114.
                      39:80-91.                                                                 Nozaki M., N. Naito, P. Swanson, K Miyata, Y. Nakai, Y. Oota, K.
               Chang YS., F.L. Huang, C.T. Chen, T.B. Lo                                          Suzuki, H. Kawauchi
                    1988a. Isolation and properties of the pituitary gonado-                         1990a. Salmonid pituitary gonadotrophs 1. Distinct cellular
                      tropin from silver carp (Hypophthalmichthys molitrix). Int. J.                    distributions of two gonadotropins, GTH I and GTH
                      Pept. Protein Res. 31:150-156.                                                    11. Gen. Comp. Endocrinol. 77:348-357.
               Chang Y.S., CJ. Huang, F.L. Huang, T.B. Lo                                       Nozaki M., N. Naito, P. Swanson, W.W. Dickhoff, Y. Nakai, K.
                    1988b. The primary structures of carp gonaclotropin sub-                      Suzuki, H. Kawauchi
                      units deduced from cDNA nucleoticle sequences. Int. J.                         1990b. Salmonid pituitary gonaclotrophs IL Ontogeny of
                      Pept. Protein Res. 32:556-564.                                                    GTH I and GTH 11 cells in the rainbow trout (Salmo
               Chang Y.S., CJ. Huang, F.L. Huang, C.S. Liu, T.B. Lo                                     gairdneri irideus). Gen. Comp. Endocrinol. 77:358-367.
                    1990. Purification, characterization, and molecular cloning                 PierceJ.G., T.F. Parsons
                      of gonadotropin subunits of silver carp (Hypophthalmichthys                    1981. Glycoprotein hormones: structure and func-
                      molitfix). Gen Comp. Enclocrinol. 78:23-33.                                       tion. Ann. Rev. Biochem. 50:465-495.
               Copeland P.A., P. Thomas                                                         Sekine S., A. Saito, H. Itoh, H. Kawauchi, S. Itoh
                    1989. Purification of maturational gonadotropin from Atlan-                      1989. Molecular cloning and sequence analysis of chum
                      tic croaker (Micropogonias undulatus) and development of a                        salmon gonadotropin' cDNAs. Proc. Nat. Acad. Sci.
                      homologous radioimmunoassay. Gen. Comp. Endocrinol.                               86:8645-8649.
                      73:425-441.                                                               Suzuki K., H. Kawauchi, Y. Nagahama
               Farmer S.W., H. Papkoff                                                               1988a. Isolation and characterization of two distinct gonado-
                    1977. A teleost (Tilapia mossambica) gonadotropin that re-                          tropins from chum salmon pituitary glands. Gen. Comp.
                      sembles luteinizing hormone. Life Sci. 20:1227-1232.                              Enclocrinol. 71:292-301.
               Fontaine YA., S. Dufour                                                               1988b. Isolation and characterization of subunits of two
                                                                                                        distinct gonadotropins from chum salmon pituitary
                    1987. Current status of LH-FSH-like gonaclotropin in                                glands. Gen. Comp. Endocrinol. 71:302-306.
                      fish. In Proceedings of the third international symposium                      1988c. Steroidogenic activities of two distinct salmon gonad-
                      on reproductive physiology of fish       (D.R. Idler, L.W. Crim,                  otropins. Gen. Comp. Endocrinol. 71:452-458.
                      andJW. Walsh, eds.) p. 48-56. Memorial Univ. Newfound-                    Suzuki K., A. Kanamori, H. Kawauchi, Y. Nagahama
                      land, St. John's Newfoundland, Canada.                                         1988d. Development of salmon GTH I and GTH 11
               Goos HJ.T., R. De Leeuw, E. Burzawa-Gerard, M. Terlou, C.J.J.                            radioimmunoassays. Gen. Comp. Endocrinol. 71:459-467.
                 Richter
                    1986. Purification of gonadotropic hormone from the pitu-                   Swanson P., M.G. Bernard, M. Nozaki, K. Suzuki, H. Kawauchi,
                      itary of the African catfish, Clarias gwiepinus (Burchell), and             W.W. Dickhoff
                      development of a homologous radioimmunoassay. Gen.                             1989. Gonadotropins I and 11 in juvenile. coho salmon. Fish
                      Comp. Endocrinol. 63:162-170.                                                     Physiol. Biochem. 7:169-176.
               Huang, F.L., CJ. Huang, S.H. Lin, T.B. Lo, H. Papkoff                            Swanson P., K. Suzuki, H. Kawauchi, W.W. Dickhoff
                    1981. Isolation and characterization of gonadotropin                             1991. Isolation and characterization of two coho salmon go-
                      isohormones from the pituitary gland of pike eel                                  naclotropins, GTH I and GTH 11. Biol. Reprod. 44:29-38.
                      (Muraenesox cinereus). Int. J. Pept. Protein Res. 18:69-78.               Trinh KY., N.C. Wang, C.L. Hew, L.W. Crim
               Idler D.R., T.B. Ng                                                                   1986. Molecular cloning and sequencing of salmon gonado-
                    1983. Teleost gonadotropins: Isolation, biochemistry and                            tropin beta subunit. Eur. J. Biochem. 159:619-624.
                      function. In Fish physiology, Vol. 9A (W.S. Hoar, D.J.                    Tyler C., J.P. Sumpter, H. Kawauchi, P. Swanson
                      Randall and E.M. Donaldson, eds.), p. 187-221. Acad.                           1991. Involvement of gonadotropin in the uptake of
                      'Press. NY.                                                                       vitellogenin into the vitellogenic oocytes of the rainbow trout,
               Itoh H., K. Suzuki, H. Kawauchi                                                          Oncorhynchus mykiss. Gen. Comp. Endocrinol. 84:291-299.
                    1988. The complete amino acid sequences of P subunits of
                      two distinct chum salmon GTHs. Gen. Comp. Enclocrinol.
                      71:438-451.







              78      NOAA Technical Report NMTS 106


                              1400                                                         1450'E
                                                    SOYA ST@R@.
                                APAN                                               1__@OKHOTSK
                                               REBUN     S
                                 SEA             Is.                                            SEA
                                                           F
                           5ON                 RIS JR1
                                               IS.



                                               TEURI
                                               IS.                            SAROMA     NOTORO
                                                  0.    ._- @       ,  i"     LAKE        LAKE
                                                  YAGI
                                                  SH11PV                  Ij


                                                                                              RO
                                                                            2-@-VOK

                                                                                            Li W.54     SHIRI Is
                                                                     K A     I D 0          -ONN-K           V
                                                                                                       URO
                                                                                             1@ %K
                                                                                               _EKK
                                                              N



                                                                               is
                                          ;;@@ -ka:_

                        OKU-
                        S
                                                                    iS


                                         INKA BAY)




                                                           PACIFIC                                               4n
                       410
                                    Mt.                       CEAN
                                    AO



                                                                                                    1F n



                                                                  Figure I
                   Main mariculture areas in Japan and natural distribution in east Asia of the Japanese scallop, Patinopecten
                                                           (Mizuhopecten) yessoensis.



              every survey time was observed from surface to' bot-        Results and Discussion
              tom at intervals of 2 m with the use of a
              the'rmometric electrode. This field study began in          Changes in the gonado-somatic indexes of males and
              mid- to late April 1982 at every site and finished in       females were nearly synchronized, although details
              May/July 1982 as breeding was completed at each             showed they were slightly different (Fig. 7). The in-
              site. Gonads and soft body tissues were measured by         dex values at each site were lower in April and
              wet weight. The gonado-somatic index was estimated          increased in May. When the index decreased sud-
              with the following equation.                                denly after reaching a maximum,, we estimated that
                                                                          breeding occurred. Using this index, we also esti-
                    gonado-somatic index = gonad weight X                 mated that the time of breeding varied by locality. It
                              100/soft body weight                        began in earlyjune off Rausu in the north (Nemuro






                                                                                                                                                              Ito: Breeding Season of Japanese Scallop                                             79



                                                                                                                                                                    * TOTAL:JAPAN
                          PRODUCTION: METRIC TONS                                                                                                                   o HOKKAIDO
                            350,000


                                                        1910-23:Temporary                        high        Level. catch period
                                                        1924-32:StabLe high Level. catch period                                                               ...... -------      ------------
                                                        1933-44:Extreme high.LeveL catch Period
                                                        1945-68:StabLe Low Level. catch Period
                                                        1969-76:Hangins cuLture deveLopment Period                                                                                ------------
                                                        1977-88:Sowing and hanging cuLture mass
                                                                               Production Period                                                             ------ -- ---- ---   ------------



                                                    ----------- ------------   ----------- ------------      ----------- ------------         ------- ------------       -------- ------------



                                                    ----------- ------------   -----------    ----------- ------------   J------------  L-----------  I------       - ----------  ------------


                                                                ------------- --------      ---- -- --- - --------       -------------  I-----------     --- -----  i------------
                                                                                                                                        I                           I             I             I                        Figure 2
                                                                                                                                                                                                              Annual production
                                          0                                                                                                                                                                   of the Japanese scal-
                                             1900                                                                     1950                                                                  2000              lop, Patinopecten (Mizu-
                                                                                                          Y        E        A         R                                                                       hopecten) yessoensis, in
                                                                                                                                                                                                              Japan,1910-1988.




                     Straits), in mid-June off Shibetsu in the middle area,                                                                Citations
                     and in mid-May off Bekkai in the southern portion of
                     the Nemuro region. Although the breeding season                                                                       Ito, H.
                     differs by locality, it begins when the bottom tem-                                                                          1988.       1Sowing culture of scallop in Japan. InNewandinno-
                     perature reaches about 5' C in every bed. Therefore,                                                                             vative advances in biology/engineering with potential
                                                                                                                                                      for use in aquaculture: proceedings of the fourteenth
                     the bottom temperature seems to indicate the begin-                                                                              U.S.-Japan meeting on aquaculture (A.K. Sparks, ed.),
                     ning of scallop breeding. This is new information on                                                                             p. 63-69. NOAA Tech. Rep. NMFS 70.
                     the Japanese scallop. In the past, scallop researchers                                                                       1989. Concept in mariculture of Japanese scallop. Scallop
                     focused on the midpoint of the breeding season. In                                                                               (2), Hotatekai (Kushiro), p. 89-97. (In Japanese.)
                     this paper the author is proposing a new idea, that                                                                          1990. Some aspects of offshore spat collection ofJapanese
                                                                                                                                                      scallop. In Marine farming and enhancement: proceed-
                     earlier generation spat can be of great advantage to                                                                             ings of the fifteenth U.S.-Japan meeting on aquaculture
                     the scallop mariculture industry. Earlier generation                                                                             (A.K. Sparks, ed.), p. 35-48. NOAA Tech. Rep. NMFS 85.
                     spat grow to a larger size than those collected later.                                                                Kawamata, K., Y Tamaoki and A. Fuji.
                     The larger scallop also market at a better price. So,                                                                        1981. Gonad development of the cultured scallops in Funka
                     knowledge of the beginning of the breeding season is                                                                             Bay. J. Hokkaido Fish. Exp. Stn. (Hokusuisi Geppo)
                                                                                                                                                      38:132-146. (Injapanese.)
                     beneficial information. In addition, the author sug-                                                                  Kinoshita, T.
                     gests that research focus on new concepts (Ito 1989)                                                                         1934. Relation between breeding of the Japanese scallop and
                     for scallop mariculture which includes an examina-                                                                               water temperature. Report of the Hokkaido Fish. Res.
                     tion of the biology of breeding.                                                                                                 Stn. (Hokusuisijunpo) 233:3-8. (Injapanese.)
                                                                                                                                           Maru, I-L
                                                                                                                                                  1972. Morphological observations on the veliger larvae of a
                                                                                                                                                      scallop, Patinopecten yessoensis (Jay). Sci, Rep. Hokkaido
                     Acknowledgments                                                                                                                  Fish. Exp. Stn. 14:55-62. (InJapanese, English abstr.)
                                                                                                                                                  1976. Studies on the reproduction of a scallop, Patinopecten
                     The author wishes to express sincere thanks to the                                                                               Yessoensis (Jay)-l. Reproductive cycle of the cultured
                     following co-workers of the research team: Toshikazu                                                                             scallop. Sci. Rep. Hokkaido Fish. Exp. Stn. 18:9-26. (In
                                                                                                                                                      Japanese, English abstr.)
                     Fujimoto, Takao Sasaki, Haruo Moriya, Tohru Ikeda,                                                                           1978. 5tudies on the reproduction of a scallop, Patinopecten
                     Tadashi Abe, Yukiyasu Nakata, and Yoshiji Kobayashi                                                                              yessoensis (Jay)-2. Gonad development in I-year-old
                     for their expertise and friendship.                                                                                              scallops. Sci. Rep. Hokkaido Fish. Exp. Stn. 20:13-26. (In
                                                                                                                                                      Japanese, English abstr.)







                    80          NOAA Technical Report NNM 106


                                                         (55 A                          head 3-5,U                                                 Gonad index
                                                         Egg                   Sperm
                                                             Spawning     j              ail 51,111,
                                                           or
                                                             Breeding                                                                              precursor to
                                                                                                                                                   monitoripg th
                                                            (demersal egg)                                                                         @lanktonic
                                                                                                                                                     arva
                                                         Unfertilized egg
                                                         I80,@ (70-100 14 )    Fertilization
                                                         Fertilized egg
                                                         D1.
                                                         ev loping embryo          Polar lobe stage                                                                  Larval
                                                                                   First cleavage                                                                   monitoring

                                                                                   Second cleavage

                                                                                   Third cleavage

                                                                                   Fourth cleavage
                                Age                                                Blastula,
                                0 yr                                               Gastrula                       Prodissoconch stage
                                      4 days             Trochophore larva
                                   after fertilizationi
                                      5-7 days           Early veliger larva       I D-sbaped larva               <150.a
                                                                                   Early umbo-stage larva         150-190                            Plankton        Spatfall
                                                                                   Urebo-stage larva              190-240                            analysis         prediction
                                      30-35 days         Late veliger larva        Early full-grown    larva
                                      40 days            (Pedivellger)             lFull-grown larva              240<
                                      70-80 days         Alached spat                                             250-280 A<                         Spat
                                      4-5 months         Bittom inhabiting spat                                   1 cm<                              collection

                                                         i entl

                                                                    Early grown stage
                                                                                                                                                     Intermediate
                                                                    La  ite growing stage                                                            culture
                                                                    Delgenerating stage
                                Age                                 Ulifferentlated stage
                                lyr                                 Dillerentisted stage                                           Dissoconch
                                                                    (Sex differentiated stage)                                     stage
                                                                    R   Iting stage
                                                                        as
                                                                    Early growing stage                Early growing stage
                                                                    Jte growing      stage             Late growing stage
                                                                    Me  ituring stage                  Wluring stage
                                                                                                                                                     Hanging culture
                                                                    -@l                                   I                                                or
                                                         Adult      Breeding stage                     Breeding stage                                Sowing culture
                                                                    Spent stage                        Spent stage
                                Age                                 114ting stage                      Resting stage
                                2 yr                                Ea  irly growing stage             Early growing stage
                                and                                 La  4te growing stage              Late growing stage
                                older                               biat.ing stage                     buturing stage



                                                                  Life cycle of scallop                                                              Culture method


                                                                                                 Figure 3
                      Life cycle of the Japanese scallop, Patinopecten (Mizuhopecten) yessoensis, with the note of culture methods (modified from
                                                    Yamamoto 1964; Maru 1972, 1976, 1978; Osanai 1975; Kawamata et al. 1981).






                                                                                          Ito: Breeding Season of Japanese Scallop         81




                                                                                          JAPAN

                                                                                          SEA

                                                                                          COAST




                        SARUFUTSU
                                         0 000

                                                       00





                        00MU




                                                                                          OKHOTSK


                                                                                          SEA

                        SARURU                                                            COAST








                        MONBETSU



                                           U       U





                        RAUSU


                                                                   U U




                        SHIBETSU                                                          NEMURO
                                                                   t                      STRA1TS
                                                                I  Tip                    COAST
                  AO -                                                                                                   Figure 4
                  30    NOTSUKE                                                                              Changes in gonado-somatic in-
                                                                                                             dex (gonad weight X 100/soft
                  20                                                                                         body weight, %) of the Jap-
                                                                                                             anese scallop, Patinopecten
                  10.
               0                                                                                             (Mizuhopecten) yessoensis, off
                                   1     0
                   0                                                                                         the coasts of Hokkaido, 1982.
                          MAR.           APR.        MAY          JUN.         JUL.                          Specimens were mainly 3- and
                                                                                                             4-year-olds (Ito 1990).







                   82         NOAA Technical Report NMIFS 106

                                           JAPANI               WATER TEMPERATURE , OC
                                            SEA  IMASHIKE              2          4      6 8@ 10 12 14 16 18 20 22                20 1816 14 12 10 8 6
                                           c                                                         I i        M            I     N i I I I
                                                 Mow     E




                                         OKHOTSK;oomu
                                            SEA IMONBETSU-
                                           COASTI



                                                 UrORO
                                                 @AUSIJ

                                           NEW
                                               @7@EMURO     2 0               0                                                                        4
                                           C7   T1
                                                             JAN.' FEB. 'MAR.' APR. 1MAY'JUN.'JUL.AUG.'SEP.'OCT. 1 NOTDEC.

                                                                                 T I ME OF YEAR                      1982


                                                                                          Figure 5
                             Changes in surface temperature of the coastal water related with the locality of Hokkaido in 1982 (Ito 1990).



                                           I45*'E
                                                                                  5

                                    0 K H 0 1 S K    S E A                        10






                                     2.00
                                                                      4@4









                                                                                                       20-\@
                                                         U_

                                                       DAII



                                           OKHOTSK SEA

                               JAPAN SEA                              EMURO         AY
                                                          HONWO,
                                                             KAI-
                                                                                                         P,
                                            K'A 1 0 0                                 ORO


                                                                  UREN-l(W
                                       PACIFIC OCEAN                                                                                      Figure 6
                                                                                        ACIFIC OCEAN.             Survey sites (A, B, C) in Nemuro Straits, east
                                HONSHU                                                                            Hokkaido. A = site off Rausu. B = site off
                                                                                                       /1111jp
                                                                                                                  Shibetsu. C = site off Bekkai.






                                                                                                        Ito: Breeding Season of Japanese ScaHop                 83

                          RAUSU                                        SHIBETSU                                     BEKKAI
                        40-
                        30-
                        20-
                 ><     lo-
                 w
                        O_
                        40-
                        30 -
                                          POP
                        20-                           P
                 S      10.     01005"
                 M

                          APR.        MAY            JUN.    JUL.      A            M                                 A            M
                                   T1% OF       YEAR               0        41
                   WATER TEMPYATUR                                                               \9              0
                        0                                                                           8                                  \8
                                                                                                    7
                                                          9       10.      2                        6
                                                                                                    5                                   7
                                                                                                               10
                        lor     Imm
                                                                                                                     2 3           56
                 L"               2    3 4    4 567      7       20
                                                                                    0


                                                                                    Figure 7
             Changes in the gonado-somatic index of Japanese scallops, Patinopecten (Mizuhopecten) yessoensis, and the water temperature
                                                                   in Nemuro Straits, 1982 (Ito 1990).




             Osanai, K.                                                                    Yamamoto, G.
                   1975. Seasonal gonad development and sex alteration in the                   1951. Ecological study on the spawning of the scallop, Pecten
                     scallop, Patinopecten yessoensis. Bull. Mar. Biol. Stn.                        (Patinopecten) yessoensis in Mutsu Bay. Bull. Japan. Soc. Sci.
                     Asamushi. Tohoku Univ. 15(2):81-88.                                            Fish. 17(2):53-56. (Injapanese, English synop.)
             Tsubata, F.                                                                        1964. Scallop culture in Mutsu Bay. Suisan Zoyoshoku
                   1982. A history of the fisheries researches of the scallop,                      Sosho (Aquaculture series in fisheries) 6 (Tokyo), 77
                     Patinopecten yessoensis, in Mutsu Bay. Published by Aomori                     p. (Injapanese.)
                     Prcfccturc (Aomori Prcfecture govcrrunent publication),
                     120 p. (In Japanese.)
                                                                                                                      F3 4-5







                                 A Better Method for Oyster Farming in Japan



                                                                TETSUO SEKI

                                                               Oyster Research Institute
                                                            211 Higashi Mohne Karakuwa
                                                               Miyagi 988-05, Japan




                                                                     Abstract


                               TheJapanese oyster farming industry experienced a remarkable change with the devel-
                             opment of the raft culturing method. Recently, northern Japanese oyster farmers have
                             been facing difficulty in maintaining the quality of oysters. It was concluded that this
                             problem originated with certain methods utilized by the present oyster farmers of the
                             raft culture system. In order to resolve the problems, new farming practices were devel-
                             oped in which the introduced flat oyster Ostrea edulis was used at the Oyster Research
                             Institute. A new cultch-based seed collecting method for single oyster seed production
                             and a multilayered, compact hanging method were devised to better utilize the. coastal
                             water column for oyster farming. The recent progress of this strategy is described and
                             discussed.




            Introduction                                                       New methods of collecting and culturing oyster
                                                                            seed were introduced in 1989; this paper describes
            The Oyster Research Institute (ORI), a private non-             these developments and discusses the relevant back-
            profit foundation, was established in 1961 at Mohne             ground issues.
            Bay in Miyagi Prefecture. Funding for the Institute
            was initially derived from private company sources in
            Sendai. These companies hoped to redevelop the tra-             Recent Problems with Oyster Farming
            ditionally important oyster industry in Miyagi                  in Northern Japan
            Prefecture and to encourage the related studies of
            Dr. Takeo Imai, a professor at Tohoku University, by            Since 1950, substantial developments in oyster farm-
            establishing an industry-based research laboratory.             ing in Hiroshima and northeastern Japan have been
              The Oyster Research Institute pioneered seed pro-             experienced. These developments are due primarily
            duction and farming of theJapanese abalone species              to the popularity of raft culture and the introduction
            Haliotis discus hannai (Seki 1980; Seki and                     of long-line systems (Fujiya 1970; Imai 1971; Kafuku
            Cuthbertson 1988) and the French oyster Ostrea                  and Ikenoue 1983). Despite such developments, the
            edulis (Imai 1967; Imai 1971). The initial aim of in-           industry now faces a number of problems. The in-
            troducing French oysters was to establish an exotic             creasing popularity of scallop farming due to its
            oyster industry in Miyagi Prefecture. Although large            greater returns has stagnated oyster production in
            amounts of French oyster seed were produced, the                many areas. A gradual increase in the average age of
            oysters were not popular with the Japanese consumer.            oyster farmers together with increasing water pollu-
            However, recent changes in Japanese eating habits               tion in growing areas has further contributed to the
            show an increased demand for luxury seafood.                    decline of the industry.
              The aim of French oyster research at ORI was tar-                The introduction of styrofoam flotation to oyster
            geted at providing innovative alternatives for oyster           lines gave much greater buoyancy to the system. As a
            farmers in northern Japan such as improved techno-              result, farmers tended not to remove fouling organ-
            logical sophistication and ways to better utilize               isms such as mussels from the support lines. Rather
            associated coastal waters.                                      than removing long lines from the water and drying



                                                                                                                                      85







                     Hormonal Regulation of Reproduction in Female Crustacea



                                                    HANS LAUFER and ELLEN HOMOLA
                                                                University of Connecticut
                                                       Department of Molecular and Cell Biology
                                                                Storrs, CT 06269-3125
                                                                           and
                                                           The Marine Biological Laboratory
                                                            Woods Hok MA 02543 U.S.A.



                                                                MATTHEW LANDAU
                                                              Department of Marine Science
                                                                   Stockton State College
                                                                   Pomona, N.J. 08240




                                                                        Abstract


                                Basic knowledge of female reproduction is of primary importance to crustacean aqua-
                              culture. Our approach has been to investigate crustacean reproduction utilizing the
                              approach of comparative endocrinology. This paper reviews the hormonal regulation of
                              reproduction, focusing on eyestalk factors and the mandibular organ, and the target
                              tissues affected by their secretions. This approach has lead to the finding of methyl
                              farnesoate (MF), an unepoxiclated juvenile hormone, in the circulatory system. MF is
                              produced by the mandibular organ (MO), a homologue of the insect corpus allatum,
                              and appears to be a crustacean juvenile hormone. Eyestalk ablation enhances crustacean
                              reproduction by removal of a gonad inhibitory hormone (GIH). Eyestalk removal in the
                              spider crab, Libinia emarginata, stimulates egg production and vitellogenesis in
                              nonreproductive females and increases MF production by the MO. Addition of sinus
                              gland extracts to MOs inhibits MF synthesis in vitro. Thus, there is a mandibular organ
                              inhibitory hormone in the eyestalk which may be similar or identical to GlH. The action
                              of GIH then may be on target tissues such as the hepatopancreas and ovary or may also
                              be on the MO. Whether this is the major action of GlH or one of its actions remains the
                              subject of current investigations. Additional interactions influencing the female repro-
                              ductive system such as stimulatory factors from the brain and thoracic ganglion as well as
                              the role of biogenic amines are considered briefly. Other possible interactions men-
                              tioned in the literature are also indicated in this brief review of crustacean reproduction.




                                                                               become viable endeavors in the future. In some
           Introduction                                                        cases, such as the culture of penaeid shrimp, the pro-
                                                                               duction of sufficient healthy seed organisms has
           Decapod crustaceans represent a large, diverse bio-                 hindered farming. A lack of understanding of the
           logical group with significant potential as an                      reproductive process has forestalled selective breed-
           aquacultural resource. Large-scale penaeid shrimp                   ing. We feel that a better understanding of the
           culture industries currently exist in Asia and Central              hormonal mechanisms that regulate the reproduc-
           America. Crayfish are grown in the United States, Eu-               tion of these valuable resources is fundamental to
           rope, and Australia, and prawns of the genus-                       successful aquaculture.
           Macrobrachium are cultivated in many tropical envi-                    During the past two decades our understanding of
           rons. The culture of lobsters and other species may                 crustacean reproductive endocrinology, especially


                                                                                                                                            89







                90       NOAA Technical Report N@M 106



                                     brain                            sinus gland
                   commissural ganglion                                  medulla terminalis
                              Y organ               Z                              X organ
                                                    /0
                    postcommissural                                          mandibular organ
                           organ                                        subesophageal ganglion
                    pericardial    organs                                 thoracic second roots
                     thoracic ganglion                                       neural sheath
                                                                             surrounding ganglia
                                                                               & connectives
















                                                                                                                          Figure I
                                                                                                             Major endocrine and neuro-en-
                                                                                                             docrine structures of generalized
                                                                                                             female Crustacea. Included are
                                                                                                             the organs important for female
                                                                                                             reproduction, the eyestalk sinus
                                                                                                             gland x-organ, the mandibular
                                                                                                             organ, Y-organ, and thoracic
                                                                                                             ganglion.


                that of the female, has grown steadily. This is in part           tion. The JHs are produced in a pair of small endo-
                a result of the use of the comparative approach,                  crine glands, the corpora allata (CA), usually
                whereby our understanding of other crustacean                     posterior and ventral to the brain. The JHs appear to
                groups and their close relatives, the insects, is applied         play a major part not only in the development of the
                to the decapods. Major sites of endocrine activity are            insect larval stages (hence the name 'Juvenile") but
                shown in Figure 1. In this report we concentrate on               also in the regulation of reproduction (Downer and
                eyestalk factors and the mandibular organ, and the                Laufer 1983), first by stimulating the ovary to mature
                target tissues affected by their secretions. Additional           and then by stimulating the fat body to make yolk
                endocrine interactions will be discussed as they relate           proteins. FurtherJH seems to alter the oocyte mem-
                to the reproductive process.                                      brane so that the large yolk proteins synthesized by
                                                                                  the fat body can be taken up by the developing egg.
                                                                                     Since both arthropod subphyla, the Insecta and
                A Crustacean juvenile Hormone                                     Crustacea, are already known to regulate molting
                                                                                  with identical hormones, 20-hydroxyecdysone
                The role of the terpenoid hormones, collectively                  (Karlson 1956; Hampshire and Horn 1966), we (cf.
                known as the 'Juvenile hormones" UHs) orjuvenoids                 Laufer et al. 1987c) speculated that the crustacea
                (Fig. 2), has been well established in insect reproduc-           might also have a functioning JH. This speculation






                                                       Laufer et al.: Hormonal Regulation of Reproduction in Female Crustacea       91





                                                                 0


                    METHYL (2E,6E)-FARNESOATE


                                                             0
                                                               @OR
                   0
                                                                                                          Figure 2
                                                                                       The structural formula of insect juvenile hor-
                (10R)JUVENILE HORMONE III (R=CH                                        mone (JHIII) and its metabolic breakdown
                                                                           3           product, JH acid, are compared with a similar
                (10R)JUVENILE HORMONE III ACID (R=H)                                   molecule, methyl farnesoate (MF), present in
                                                                                       crustacean hemolymph and synthesised by the
                                                                                       mandibular organs.



            was supported by a considerable literature. Gomez et            and Paulus (1984) showed that when methoprene
            al. (1973) found that the cyprid to spat metamorpho-            was injected into intact and ablated female crabs,
            sis of the barnacle, Balanus galeatus, could be                 Carcinus maenas, the ovaries became enlarged.
            inhibited by two structural analogs of the JHs,                   While these experiments demonstrated that Crus-
            methoprene and hydroprene. Similar studies were                 tacea were sensitive to JH, it was not established that
            carried out by Cheung and Nigrelli (1973) and                   crustaceans possessed endogenous juvenoids. Using
            Tighe-Ford (1977). Landau and'Finney (1977)                     an insect bioassay, Schneiderman and Gilbert (1958)
            showed thatJH analogs are active, but that rnevalonic           detected some JH activity in the eyestalks of Crusta-
            acid, a precursor of JH, had no effect. Furthermore,            cea, but the chemical nature of the extract was not
            precocene 11, a compound that destroys the CA in                investigated. When thoracic ganglia or mandibular
            insects and thereby halts the production of JH, was             organs (MOs) were implanted into immature L.
            shown to' strongly inhibit the hatching of barnacle             emarginata, there was a stimulation of vitellogenesis
            embryos treated in the early stages of development              (Hinsch and Bennett 1979; Hinsch 1980). Based on.
            (Landau and Rao 1980). Such effects were revealed               morphological studies, the MO was suggested as a
            in other crustaceans as well. JH analogs were demon-            possible homolog to the insect CA (Chaudonneret
            strated to cause morphological abnormalities in                 1956; Le Roux 1968; Byard et al. 1975). If the MO
            megalopa larvae of the crab Rhithropanapeus harrisii            was a structural homolog of the CA, it was reasoned
            (Costlow 1977), and to alter the morphology and                 (Laufer et al. 1987c) that it might produce one of the
            length of development of the American lobster                   juvenile hormones or a similar compound.
            Homarus americanus (Hertz and Chang 1986). Injec-                 Methyl farnesoate (MF), the unepoxidated form of
            tion of a juvenile hormone into larval H. americanus            JHIII (Fig. 2), was detected in the hemolymph of L.
            results in delayed metamorphosis and morphometric               emarginata using gas chromatography/ mass spectrom-
            variability in newly metamorphosed animals                      etry and selected ion monitoring (Laufer et al.
            (Charmantier et al. 1988).                                      1987c) and in the hemolymph of crayfish Orconectes
              Beside affecting development, JHs and their ana-              vifilis and H. americanus as well (Tsukimura et al.
            logs were also shown to affect reproduction.                    1989). When the MOs of crustaceans were incubated
            Inhibition of reproduction in the cladoceran Daphnia            in physiological saline supplemented with a labelled
            magna was shown by Templeton and Laufer (1983),                 precursor, [methyljH] methionine, it was identified as
            and inhibition of oogenesis and spermatogenesis                 the source of the MF (Laufer et al. 1986, 1987c; Borst
            were demonstrated in the mud crab R. harfisi (Payen             et al. 1987). Mandibular organs from 12 species of
            and Costlow 1977), and the spider crab             Libinia      Crustacea are known to produce MF, most are
                   n











































            emarginata (Hinsch 1981). Paulus and Laufer (1982)              Decapoda, but also a barnacle, Balanus nubilus, pro-







               92       NOAA Technical Report NMFS 106



                    --o-Positive effect

                                             effect
                    --+-4--Effect of
                              eyestalk removal


                    Y-organ                                      Sinus Gland
                   (ecdysone)                                    X-organ complex

                                                 MIH         GIH
                                                                                                                  Figure 3
                         BRAI                                                                Major endocrine glands and their target tissues
                                                                                             involved in crustacean female reproduction. In-
                                                                                             dicated with solid lines and arrows are
                                                                                             stimulatory effects. Inhibitory interactions are
                                                                                             indicated with dashed lines and arrows. Note
                                       M            F                                        that the inhibitory interactions from the eyestalk
                                                      HP   c   Ils                           sinus gland x-organ complex can be removed by
                                T h G.".           F                                         eyestalk ablation. These include both the inhibi-
                                                                                             tory effects of the molt inhibitory hormone
                                                                                                             -organ which produces ecdysones
                  biogenic                GSH                                                (MIH) on the Y
                                                                                             and the gonad inhibiting hormone (GIH),
                    amines
                                                                                             which according to the literature may be similar
                                                                                             or the same as the vitellogenin inhibitory hor-
                                                                                             mone (VIH) and which may also inhibit the
                                                                                             mandibular organ (MO-IH) as well as other tar-
                                                                                             get tissues such as the ovary and hepatopancreas
                         Induces secondary sex characters                                    (H 'P). The brain and thoracic ganglion (ThG),
                                                                                             according to some reports, may also stimulate or
                                                                                             inhibit reproduction, or do both. Biogenic
                                                                                             amines such as serotonin and octoparnine have
                                                                                             been shown to inhibit the MO.



               duces ME Based on a similar set of in vitro studies,               with the reproductive cycle; during the reproductive
               Tobe et al. (1989) has suggested that farnesoic acid               season, they produce eggs in a regular cycle every
               rather than MF is the major product of the MO in                   19-20 days. The MO is least active immediately after
               the mud crab Scylla serrata although only MF was de-               the eggs are layed and most active during vitellogen-
               tected in the hemolymph.                                           esis; it continues its activity until just before the
                  In the insects, JH produced by the CA is reversibly             embryos hatch and oviposition of a new brood com-
               bound to a "carrier protein" (Downer and Laufer                    mences (Laufer et al. 1987c). Hinsch (1980)
               1983), which functions to protect the hormone from                 stimulated ovarian development in juvenile female
               enzymatic degradation and to increase its solubility               spider crabs by implanting adult MOs, so the MO
               in the hemolymph. Likewise, it appears that a similar              seems to be a stimulus for the vitellogenic cycle in L.
               protein, of about 40 kd, exists in the hemolymph of                emarginata. Vogel and Borst (1989) injected MF into
               the lobster (Prestwich et al. 1990) and perhaps in                 eyestalkless female L. emarginata, which according to
               other crustaceans. In L. emarginata, MF does not ap-               the findings of Panouse 1943 would enlarge their
               pear to be converted to JHIII in the hemolymph or                  ovaries, and measured the change in hemolymph
               by a variety of assayed tissues; it is, however, catabo-           vitellogenins by enzyme-linked immunosorbent assay
               lized to farnesoic acid by a number of tissues,                    (ELISA); a single injection (0.5 to 2.0 ng of MF)
               especially the hepatopancreas, but not by the hemo-                caused a modest rise in the hemolymph protein titer.
               lymph (Laufer and Albrecht 1990).                                  Similarly, Paulson and Skinner (1988) suggested that
                  Like JH in insects, the production of MF seems to               MF increased the in vitro synthesis of specific integu-
               be related to reproduction. In female L. emarginata,               mentary tissue proteins of the land crab Gecarcinus
               the rate of MF secretion by MOs in vitro is correlated             lateralis. The proteins stimulated were different from







                                                       Laufer et al.: Hormonal Regulation of Reproduction in Female Crustacea       93

           those stimulated by 20-hydroxyecdysone, while JH                 could inhibit crab Uca pugilator ovarian synthesis of
           stimulated fewer proteins than ME                                vitellogenin in vitro, while Eastman-Reks and
                                                                            Fingerman (1984) also found that U. pugilator eye-
                                                                            stalk extracts inhibit protein synthesis in cultured
           Relationship of the Mandibular Organ and                         ovaries of the crab.
           Eyestalk to Reproduction                                           Alternatively, GIH may have non-ovarian targets, or
                                                                            in fact there may be more than one eyestalk factor
           The observation by Panouse (1943) that eyestalk ab-              which inhibits ovarian growth. In the insects, the pro-
           lation enhances ovarian growth, vitellogenesis, and              duction of JH is regulated by neuropeptides:
           oviposition has already been mentioned here. Thus,               allatotropins which stimulate the synthesis or release,
           reproduction in crustaceans appears to be under the              or both, of hormones (Kataoka et al. 1989), and
           inhibitory control of an eyestalk factor known as the            allatostatins and allatohibins which inhibit hormone
           "gonad-inhibiting hormone" (GIH), which may. be                  syn thesis/re lease (Girardie 1983; Woodhead et al.
           the same as the "vitellogenin-inhibiting hormone"                1989). If there is synthesis of one of the inhibitors,
           (VIH) (Brown and Jones 1949; Quackenbush and                     thus preventing JH production and release, there will
           Herrnkind 1981; Soyez et al. 1987). The eyestalk is a            be no ovarian development; that is, the allatohibins
           source of many other hormones, including the "molt-              and allatostatins are functional insect analogs of crus-
           inhibiting hormone" (MIH) which directly inhibits                tacean GIH. Laufer et al. (1986 and 1987a,b) found
           ecdysterone production by the Y-organs in culture                that a water-soluble, heat-stable eyestalk factor(s) in-
           (Mattson and Spaziani 1985, a and b; Sch'oettker and             hibits the in vitro synthesis of MF. (Fig. 3). Based on
           Gist 1990) (Fig. 3). k@ecently a molt-inhibiting hor-            its extraction properties, it seems likely that this fac-
           mone has been purified and its amino acids                       tor is a small peptide. We have termed this peptide
           sequenced from the lobster H. ameficanus. The pep-               the "mandibular organ-inhibiting hormone" (MO-
           tide has 61% sequence identity with crustacean                   IH). Laufer et al. (1986, 1987a, b, and c), showed
           hyperglycemic hormone, (CHH) from Carcinus                       that the levels of MF in the hemolymph of the spider
           maenas, and shows significant CHH activity when in-              crab increased dramatically after eyestalk-ablation,
           jected into H. ameficanus (Chang et al. 1990). It                which was also observed in H.. americanus and 0.
           appears that crustaceans alternate between produc-               virilis (Tsukimura et al. 1989).
           tion of MIH and GIH (Anilkumar and Adiyodi 1980;                   In addition to the eyestalk GIH/MO-IH, there is
           Chang 1984). Fyhn et al. (1977) suggested that in                another, the "gonad-stimulating hormone" (GSH)
           barnacles 20-hydroxyecdysone may be the functional               that is reported to be produced in the thoracic gan-
           GIH, and Kallen and Meusy (1989) have advanced                   glion, and which may contribute to the control of
           the theory that GIH is similar in structure, but dis-            reproduction (Fig. 3). Otsu (1960, 1963) first sug-
           tinct from CHH. Quackenbush and Herrnkind                        gested its existence because eyestalk ablation caused
           (1983) and Charniaux-Cotton (1985) demonstrated                  precocious ovarian growth in adult crabs, Potamon
           that MIH and GIH could be separated chromato-                    dehaani, but not in juveniles; he reasoned that not
           graphically.                                                     only was the absence of GIH required for ovarian
             Presumably because the eyestalk is the site of GIH             growth, but the presence of a stimulatory hormone
           production, the ablation of the eyestalk may cause               was also necessary. When adult thoracic ganglia were
           increased growth of the ovarian tissue (Panouse                  implanted in eyestalk-ablated juveniles the ovaries be-
           1943;.Brown and Jones 1949; de Leersnyder and                    gan to mature. These experiments were confirmed by
           Dhainaut 1978). From an aquacultural perspective,                Hinsch and Bennet (1979) using L. emarginata.
           eyestalk ablation may not be a desirable method of               Gomez (1965) found that the thoracic ganglion, as
           increasing the stock's reproductive potential. The re-           well as@ the brain, stimulated growth of reproductive
           sulting embryos often are of inferior             quality        tissue in Paratelphusa hydrodromous; similar results
           (Anilkumar and Adiyodi 1985; Choy 1987)           because        were reported by Takayanagi et al. (1986) in the
           either hormonal imbalances are created by         eyestalk       shrimp Paratya compressa. Extracts of the thoracic gan-
           ablation or GIH release is uncontrolled, or       both. It       glia of U. pugilator sampled during their reproductive
           has been suggested that the targets of GIH        are the        season stimulated ovarian growth in intact and eye-
           ovaries and hepatopancreas which are the          sites of       stalk-ablated crabs, but extracts of the thoracic
           yolk protein synthesis (Fig. 3) (Paulus 1984; Paulus             ganglia of crabs outside the reproductive season re-
           and Laufer 1987; Quackenbush 1989). Quackenbush                  sulted in inhibition of ovarian growth in ablated
           and Keeley (1987) showed that partially purified eye-            organisms (Eastman-Reks and Fingerman 1984).
           stalk extracts from the shrimp Penaeus vannamei                  More recently Yano et al. (1989) reported in a small







               94      NOAA Technical Report NMYS 106

               number of cases that implants of H. americanus                 migTatoria CA in vitro (Lafon-Cazal and Baehr 1988).
               thoracic ganglia into non-reproductive Penaeus                 Biogenic amines are known to affect the levels of
               vannamei stimulated ovarian maturation.                        cAMP, and to a lesser extent cGMP, in the insect cor-
                                                                              pora cardiaca, the site of AKH synthesis (Pannabecker
                                                                              and Orchard 1986; Gole et al. 1987). Tsukimura et al.
               Mechanisms of Methyl Farnesoate                                (1986) treated lobster MOs with eyestalk, brain, and
               Re0ation and Action                                            thoracic ganglion extracts; they found that the eye-
                                                                              stalk extracts significantly increased the level of
                 In an effort to understand the inhibitory action of          cGMP, but not cAMP, in the MO., while the other
               the eyestalk on the MO, we surveyed the literature on          extracts had no effects.
               the control (secretion or synthesis) of JH by insect
               neuropeptides. A peptide extracted from the insect
               corpora cardiaca (CC) was reported by Applebaum                Other Hormones Regulating
               and Moshitzky (1986) to inhibit yolk production in             Reproduction
               Locusta migrato7ioides; this peptide reacted with an an-
               tibody to adipokinetic hormone (AKH). Since AKH                The molting hormones, ecdysteroids, are known to
               is very similar in structure to crustacean "red-pig-           play a role in insect reproduction, and therefore may
               ment-concentrating hormone" (RPCH) (Fernlund                   act in a similar fashion in crustaceans. We have al-
               and Josefsson 1968; Gade 1990), and because inhibi-            ready alluded to the apparent molting-reproduction
               tion of yolk protein synthesis might be the result of a        antagonism. Blanchet et al. (1979) found 20-
               substance that acts directly on the insect CA, it was          hydroxyecdysone in the ovaries of the amphipod
               decided that the effect upon MF synthesis should be            Orchestia gammarellus and speculated that, although
               determined. Unexpectedly at 10' M RPCH signifi-                molting hormones seem to influence the growth of
               cantly stimulated, rather than inhibited, the synthesis        the oocytes, vitellogenesis itself was unaffected. It was
               of MF by the MOs of crayfish Procambarus clarkii               later demonstrated that if the Y-organ of 0.
               (Landau et al. 1989). We could mimic the effect of             gammarellus is destroyed at the beginning of the molt
               RPCH by replacing it with the Cal' ionophore                   cycle the ovary will not develop, and if it is destroyed
               A23187, and synthesis could be inhibited by culturing          during ovarian growth the synthesis of vitellogenins
               the tissue in Ca 2+-free media or including lanthanum,         stops (Meusy and Charniaux-Cotton 1984). Suzuki
               which replaces Ca   2+ on cell surfaces (Weiss 1974), in       (1986) also found that the Yorgan was required for
               the culture medium. Lambert and Fingerman (1979)               oocyte growth in the isopod Armadillium vulgare. In
               had suggested that the RPCH might act as a Cal+                the decapods, Lachaise et al. (1981) showed an in-
               ionophore, and Cal+ seems to be involved in the                crease in the levels of another molting hormone,
               regulation of JH synthesis by the CA (Aucoin et al.            ponasterone A, in ovaries of the crab Carcinus maenas
               1987; Kikukawa et al. 1987; Dale and Tobe 1988).               during ovarian maturation. We have shown an accu-
               Furthermore, we found that "pigment dispersing hor-            mulation of ecdysones in Libinia emarginata oocytes
               mone" (PDH) (Rao et al. 1985) at 10-7 M Significantly          with release of ecclysteroids during embryogenesis
               inhibited MO synthesis of MF in P clarkii (Landau et           (Laufer and Deak 1990).
               al. 1989). It is interesting to note that Mangerich et           Testosterone, progesterone, and pregnenolone,
               al. (1986) found cells in the thoracic ganglia of the          the vertebrate sex steroids, have been identified in
               crab Carcinus maenus that contained RPCH-like mol-             the gonads and hemolymph of Astacus leptodacylus
               ecules using immunocytochemical techniques.                    and H. americanus (Burns et al. 1984 and Ollevier et
                  Kravitz and collaborators (Beltz 1988) reported             al. 1986, respectively). Couch et al. (1987) found sig-
               that the biogenic arnines, serotonin and octapamine,           nificant levels of estradiol and progesterone in the
               play a significant role in determining mating behav-           MOs of H. americanus, and Yano (1985, 1987) re-
               ior in the lobster H. ameficanus. The synthesis of MF          ported that vitellogenesis in two species of shrimp,
               by crab MOs also appears to be regulated in part by            Metapenaeus ensis and Penaeus japonicus, could be
               certain biogenic amines. Disaggregated MO cells                stimulated by injections of progesterone and 17-a-
               from Libinia emarginata appear to be unaffected by             hydroxyprogesterone. Vitellogenin synthesis in the
               dopamine and are only slightly inhibited by                    isopod, Idotea balthica baste7i can be stimulated by hu-
               octopamine; however, serotonin inhibited MF synthe-            man chorionic gonadotropin (hCG) (Souty and
               sis by 20-35% at 10-l' M, suggesting that it may               Picaud 1984); in the prawn Crangon crangon, vitello-
               function as a neuroregulator (Homola et al. 1989).             genesis was also stimulated by hCG (Bomirski and
               Octopamine stimulates JH synthesis from Locusta                Klek-Kawinska 1976), and in another prawn, Caridina






                                                                        Uufer et a].: Hormonal Regulation of Reproduction in Female Crustacea                               95
               catadha,yi, it not only stimulated growth of oogonial                               Borst, D.W., H. Laufer, M. Landau, E.S. Chang, W.A. Hertz, F.C.
               and follicular cells but also increased the rate of yolk                              Baker, and D.A. Schooley.
               deposition in the growing oocytes (Sarojini and                                          1987. Methyl farnesoate and its role in crustacean reproduc-
                                                                                                          tion and development. Insect Biochem. 17:1123-1127.
               Persis 1988). A molecule very similar to hCG has                                    Brown, F.A. Jr., and G.M. Jones.
               been identified in the prawn Palaemon serratus with                                      1949. Ovarian inhibition by a sinus-gland principle in the
               the aid of a radioimmuno-assay with an hCG anti-                                           fiddler crab. Biol. Bull. (Woods Hole) 96:228-232.
               body (Toullec and Wormhoudt 1987). Follicle-stimu-                                  Burns, B.G., G.B. Sangalang, H.C. Freeman, and M. McMenemy.
                                                                                                        1984. Isolation and identification of testosterone from the
               lating hormone (FSH) and luteinizing hormone                                               serum and testes of the American lobster (Homarus
               (LH) were also shown to increase the rate of ovarian                                       americanus). Gen. Comp. Endocrinol. 54:429-432.
               development in the shrimp Crangon crangon                                           Byard, E.H., R.R. Shivers, and D.E. Aiken.
               (Zukowska-Arendarczyk 1981).                                                             1975. The mandibular organ of the lobster, Homarus
                                                                                                          americanus. Cell Tissue Res. 162:13-22.
                                                                                                   Chang, E.S.
                                                                                                        1984. Ecdysteroids in crustacea: role of reproduction, molt-
               Acknowledgments                                                                            ing, and larval development. In Advances in invertebrate
                                                                                                          reproduction 3 (W. Engels, ed.), p. 223-230. Elsevier Sci-
                                                                                                          ence Publ., NY
               The research reported here was supported in part by                                 Chang, E.S., G.D. Prestwich, and M. Bruce.
               grants from the Sea Grant College Program, NOAA,                                         1990. Amino acid sequence of a pcptide with both mott-in-
               NA-AA-D-SG101, a National Research Service Award                                           hibiting and hyperglycemic activities in the lobster, Homarus
               from NIH, and the Lady Davis Trust. We wish also to                                        americanus. Biochem. Biophys. Res. Comm., 171:818-826.
                                                                                                   Charmantier, G., M. Charmantier-Daures, and D.E. Aiken.
               acknowledge the artistic talents of MaryJane Spring                                      1988. Larval development and metamorphosis of the Ameri-
               and the advice and technical assistance of Dr. Frank                                       can lobster Homarus aniericanus (Crustacea, Decapoda): ef-
               Mauri of the University of Connecticut Biotechnol-                                         fect of eyestalk ablation and juvenile hormone in-
               ogy Center.                                                                                jection. Gen. Comp. Endocrinol. 70:319-333.
                                                                                                   Charniaux-Cotton, H.
                                                                                                        1985. Vi    'tell6genesis and its control in malacostracan
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                  96        NOAA Technical Report NNOS 106

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                  Karlson, P.                                                                        1986. Immunocytological identification of structures con-
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                                                                  Laufer et al.: Hormonal Regulation of Reproduction in Female Crustacea                       97

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                      42:185-189.                                                          Rao, K.R., J.P. Riehm, C.A. Zahnow, L.H. Kleinholz, G.E. Tarr, L.
              Mattson, M.P., and E. Spaziani.                                                Johnson, S. Norton, M. Landau, OJ. Semmes, R.M. Sattelberg,
                   1985b. 5-hydroxytryptamine mediates release of molt-inhibit-              W.H.Jorenby, and M.F. Hintz.
                      ing hormone activity from isolated crab eyestalk                          1985. Characterizat'ion of a pigment-dispersing hormone in
                      ganglia. Biol. Bull. 169:246-255.                                            the eyestalks of the fiddler crab, Uca pugilator Proc. Nat.
              Meusy,J.-J., and H. Charniaux-Cotton.                                                Acad. Sci., U.S. 82:5319-5322.
                   1984. Endocrine control of vitellogenesis in malacostraca               Srcjini, R., and B. Persis.
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              Ollevier, F., D. De Clerck, H. Diederik, and A. De Loof.                     Schoettker, P.J., and D,H, Gist
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              Otsu, T.                                                                             adipeux du crustace isopode marin Idotea balthica
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              Paulson, C.R., and D.M. Skinner.                                                  1986. An ovary stimulating factor in the shrimp, Paratya
                   1988. Molecular action of 20-hydroxyecdysone, methyl                            compressa. J. Exp. Zool. 240:203-209.
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                      Zool. 28, 83A.                                                            1983. The effects of a juvenile hormone analog (Altosid ZR-
              Paulus,J.E.                                                                          515) on the reproduction and development of Daphnia
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                      tacean (Brachyura). Ph.D. diss., Univ. Connecticut, Storrs.                  Dev. 6:99-110.
              Paulus, J.E. and H. Laufer.                                                  Tighe-Ford, DJ.
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                      Carcinus maenas. Biol. Bull. (Woods Hole) 163:375-376.                       morphosis in the barnacle Elminius modestus Darwin (Crus-
                   1987. Vitellogenocytes in the hepatopancreas of Carcinus                        tacea: Cirripedia).J. Exp. Mar. Biol. Ecol. 26:163-176.
                      maenas and Libinia emarginata. Int. J. Invertebr. Reprod.            Tobe, S.S., D.A. Young, H.W. Khoo, and F.C. Baker.
                      Dev. 11:29-44.                                                            1989. Farnesoic acid as a major product of release from crus-
              Payen, G.G., andJ.D. Costlow.                                                        tacean mandibular organs in vitro. J. Exp. Zool. 249:165-
                   1977. Effects of ajuvenile hormone mimic on male and fe-                        171.
                      male gametogenesis of the mud crab, Rhithropanopeus                  Toullec,J.-Y., and A. van Wormhoudt.
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                      152:199-208.                                                                 peptides apparentes a I'hormone de croissance humaine
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                      Endocrinol. 80:232-237.                                              Tsukimura, B., T.M. Tanji, and F.I. Kamemoto.
              Quackenbush, L.S.                                                                 1986. Sinus gland activation of cyclic GMP in the mandibu-
                   1989. Yolk proteins in the hemolymph of Penaeus                                 lar organ of Homarus americanus. Am.Zool.26,91A.
                      vannamei. Am. Zool. 29, 63A.                                         Tsukimura, B., M. Martin, M, Frinsko, and D.W. Borst.
              Quackenbush, L.S. and W.F. Herrnkind                                              1989. Measurement of methyl farnesoate (MF) in crustacean
                   1981. Regulation of molt and gonadal development in spiny                       hemolymph. Am. Zool. 29, 49A.
                      lobster Panulirus argus (Crustacea: Palinuridae): effects of         Vogel, J.M., and D.W. Borst.
                      eyestalk ablation. Comp. Biochem. Physiol. 69A:523-527.                   1989. Spider crab yolk protein: molecular characterization
                   1983. Partial characterization of eyestalk hormones control-                    and the effects of methyl farnesoate (MF) on, its hemo-
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                      Panuhrus argus. J. Crustacean Biol. 3:34-44.







                  98         NOAA Technical Report NMFS 106

                  Weiss, G.G.                                                                          1987. Effect       of    17-alpha-hydroxy-progesterone            on
                       1974. Cellular pharmacology of lanthanum. Ann. Rev.                               vitellogenin secretion in Kuruma prawn, Penaeus
                          Pharmacol. 14:343-354.                                                         japonicus. Aquaculture 61:49-57.
                  Woodhead, A.P., B. Stay, S.L. Seidel, M.A. Khan, and S.S. Tobe.                 Yano, I., B. Tsukimura, J.N. Sweeney, and J.A. Wyban.
                       1989. Primary structure of four allatostatins; Neuropepticle                    1989. Induced ovarian maturation of Penaeus vannamei by
                          inhibitors of juvenile hormone synthesis. Proc. Nat. Acad.                     implantation of lobster ganglion. J. World. Aquacult. Soc.
                          Sci. U.S. 86:5997-6001.                                                        19:204-209.
                  Yano, 1.                                                                        Zukowska-Arendarczyk, M.
                       1985. Induced ovarian maturation and spawning in greasyback                   1981. Effect of hypophyseal gonadotropins (FSH and LH) on
                          shrimp. Metape-naeus ensis, by progesterone. Aquaculture 47,            the ovaries of the sand shrimp Crangon crangon. Mar. Biol.
                          223-229.                                                                63:241-247.






                 Reproduction in Cultured White Sturgeon, Acipenser trammontanus



                                                   GARY P. MOBERG and SERGE 1. DOROSHOV

                                             Department of Animal Science and Aquaculture and Fisheries Program
                                                                University of California, Davis
                                                                      Davis, CA 95616





                                                                          Abstract


                                  The value of sturgeon flesh and the demand for caviar makes the culture of sturgeon
                                an exciting opportunity for aquaculture worldwide. However, a major problem in success-
                                fully adapting sturgeon to aquaculture has been the failure to produce mature domestic
                                broodstock for egg and milt production. Even the industrial-scale sturgeon hatcheries in
                                the U.S.S.R., which have been in operation since the late 1950s, producing fingerlings
                                for mitigation, depend upon harvesting wild broodstock for eggs and milt. Recently in
                                California, several private aquaculture ventures have begun to raise white sturgeon
                                (Acipenser transmontanus) for the commercial market. While successful in producing mar-
                                ketable fish, they have not been able to raise a reliable source of domestic female
                                broodstock and must continue to harvest wild broodstock for eggs. Females raised in
                                culture remain reproductively immature until at least 10 years of age (the age of the
                                oldest cultured females in California). Reproduction in these females is arrested just
                                prior to the initiation of vitellogenesis. In this report, we discuss our observations of the
                                reproductive maturation of captive sturgeon broodstock and review our previous efforts
                                to manipulate their endocrine system t    Io promote earlier sexual maturation. It appears
                                that the hypothalamic-pituitary-gonadal axis of the cultured females is not secreting
                                sufficient gonadotropins to induce the full maturation of the reproductive system. We
                                have attempted to induce further reproductive maturity by hormone treatment. While we
                                have been successful in inducing vitellogenesis in these immature cultured females by
                                estradiol administration, the ovarian follicles will not incorporate the vitellogenin.



             Introduction                                                        out the world, with some species approaching extinc-
                                                                                 tion. One approach to assuring the survival of these
             Because of the     commercial value of its flesh and the            species is to raise sturgeon in aquaculture operations,
             use of its roe for caviar, the demand for sturgeon has              providing sturgeon for both mitigation and as a
             been intense and has resulted in a severe exploita-                 source of flesh and caviar.
             tion of wild sturgeon stocks worldwide. The impact of
             this commercial exploitation on the sturgeon popula-
             tion has been further exacerbated by the fish's low                 History of Sturgeon Culture
             reproductive rate. The females of most of the com-
             mercially valuable sturgeon species do not reach                    By the turn of the century, commercial exploitation
             reproductive maturity until 15 to 20 years of age.                  of sturgeon in Russia had already severely diminished
             Once a sturgeon has spawned, she may not repro-                     wild stocks. Russia has had the largest resources of
             duce again for several years. Further reducing                      sturgeon species in the world, where they reach un-
             fecundity is the sensitivity of these animals' reproduc-            usually high abundance in the continental brackish
             tion to a variety of environmental disturbances,                    water of the historic Sea of Tetis, the Caspian, Aral,
             especially pollution and the damming of the rivers                  and Azov Seas. The latter two are now lost as a stur-
             where they migrate to spawn. The combined result of                 geon habitat, serving as one of the worst examples of
             fishing pressure and the low reproductive rate has                  environmental mismanagement. The largest source
             been a dramatic decline of sturgeon stocks through-                 of sturgeon, the Caspian Sea, still holds substantial


                                                                                                                                              99







               100      NOAA Technical Report NAM 106

               sturgeon resources, but their fate and future com-                by Leach 1920). These attempts were only partly suc-
               mercial exploitation are endangered by the loss of                cessful because of the difficulties with capturing
               natural reproduction and the increasing pollution of              oVulatory females and incubating the highly adhesive
               the sea.                                                          sturgeon eggs. Further development of sturgeon cul-
                 The first attempt to artificially reproduce anadro-             ture in North America apparently waned until the
               mous sturgeon was made in Russia by Borodin in                    1960's, when hormonally induced spawning was suc-
               1898 (Borodin 1898). The first experimental hatch-                cessfully used to breed paddlefish Polyodon spathula
               ery operations were established in the Caspian Sea                (Purkett 1963). Interest in sturgeon culture revived
               basin (Volga and Kura Rivers) in 1937-38. However,                in the early 1980's when lake, atlantic, and white stur-
               industrial-scale sturgeon hatcheries did not evolve               geon (A. transmontanus) were spawned and raised to
               until the late 1950's when all major spawning rivers              the fingerling stage in various university laboratories
               were darnmed and most of the spawning grounds for                 and state hatcheries (Smith et al. 1980; Doroshov et
               sturgeon were lost (Kozin 1964). Research lagged on               al. 1983; Czeskleba et al. 1985). At this time, only the
               sturgeon nutrition and development of intensive ju-               artificial propagation of the white sturgeon has been
               venile culture, however, resulting in over dependence             established as a complete technology that includes
               of Soviet sturgeon culture on unreliable methods                  induced spawning, egg incubation, and intensive
               that even today require the harvesting of wild stock              rearing of juvenile and adult fish (Doroshov 1985;
               for eggs and milt. Currently the U.S.S.R.'s sturgeon              Conte et al. 1988).
               culture system annually produces 70-100 million                     The role of sturgeon culture in the restoration and
               fingerlings, which are used to stock the Caspian and              replenishment of North American stocks is still insig-
               Azov Seas, with an estimated survival to adulthood of             nificant, in spite of the potentially high efficiency of
               1-3% (Marti 1979). It is clear that this approach to              hatcheries to produce sturgeon juveniles. Most ef-
               culture has succeeded in temporarily maintaining the              forts in wild stock management are directed at
               commercial exploitation of wild sturgeon in the                   environmental protection and the stringent control
               U.S.S.R. (approximately 20,000 metric tons annual                 of the predominant recreational fisheries. The major
               catch). However, ecological changes in the Caspian                stimulus for current attempts at developing sturgeon
               Sea, environmental pollution, and the continuation                culture originates from the development of commer-
               of the commercial "caviar" fishery may endanger ex-               cial aquaculture in the United States and Western
               isting stocks.                                                    Europe.
                 Extensive commercial fisheries for sturgeon in
               North America were established during 1880-90 in
               the Delaware River, Great Lakes, and along the Pa-                Culture of White Sturgeon
               cific Coast. The commercial catches peaked during                 in California
               1890-1900, almost approaching the levels caught in
               Russia; however, the catch rates rapidly declined to                The Pacific Northwest is the home for two stur-
               insignificance by 1910 (Ryder 1890; Bajkov 1949;                  geon species-the white sturgeon and the green
               Harkness and Dymond 1961; Semakula and Larkin                     sturgeon, A. medirostyis. The biology of green stur-
               1968; Galbreath 1985; Smith 1985). Since the early                geon is practically unknown. Although important in
               part of this century, regulations for most North                  some geographic areas, this species is believed to be
               American sturgeon stocks here prohibited or effec-                of inferior value as a food fish. In contrast, the meat
               tively limited commercial catch. Nevertheless, there              of the white sturgeon has a very high market value
               is still no single example of complete stock recovery             and this species is still harvested by a small commer-
               to previous historical levels. It is also interesting that        cial fishery in the lower Columbia River. The white
             -while the commercial fishery for sturgeon has pr.acti-             sturgeon is taken by sport-fishermen throughout the
               cally ceased in North America, the sport fishery,                 Pacific Northwest. As a result, its harvest by recre-
               especially for the white sturgeon in the Pacific North-           ational fisheries in the Columbia and Sacramento
               west, has become a major user of sturgeon resources.              Rivers has reached a rate equal to that of the earlier
                 The dramatic and rapid decline in sturgeon                      exploitation by commercial fisheries (Galbreath
               fisheries prompted early attempts to artificially repro-          1985), resulting in a significant threat to the wild
               duce sturgeon for stock replenishment. Hatchery                   population.
               work with atlantic (Acipenser oxyrhynchus), lake (A.                 White sturgeon stocks live in the estuaries of three
               fulvescens) and shortnose (A. brevirostrum) sturgeons             major rivers, Fraser, Columbia, and Sacramento. This
               was pioneered during 1890-1910 (Ryder 1890; Post                  is one of the largest sturgeon species, reaching a
               1890; Stone 1901; Carter 1904; Meehan 1909; review                maximum recorded size of 1800 pounds (Moyle and







                                                            Moberg and Doroshov: Reproduction in Cultured VAiite Sturgeon        101

           Cech 1988). Males reach first sexual maturity at 10-           retain a substantial number of fish, the oldest of
           15 years of age, while females mature at 15-20 years           which have reached 10 years of age and a body
           of age. Ripe fish migrate into the rivers and spawn            weight of 50 kg (Anonymous 1990). These fish are
           from March (Sacramento River) tojune (Columbia                 raised at low density in outdoor tanks and raceways
           and Fraser Rivers), at water temperatures of 14-               and are fed different types of salmonid diets
           16'C. Spawning grounds are now limited to rivers               (Silvercup, Rangen, Moore-Clark). In 1987, eight
           below the dams, although there are small reproduc-             farms started collaborative research with the Univer-
           ing populations in the reservoirs on the Columbia              sity of California at Davis, in an attempt   to establish
           River (Galbreath 1985).                                        artificial reproduction in captive stocks    and to im-
             Preliminary information was recently obtained on             prove sturgeon broodstock management. The major
           the reproductive cycle of white sturgeon females in            objective of this research is to decrease     the age at
           the San Francisco Bay (Chapman et al. 1987;                    which the females reach reproductive maturity.
           Chapman 1989; Doroshov et al. 1990). The most im-
           portant findings were the high individual variability
           in the age of first ovulation (12-22 years) and the            Reproductive Maturation of
           apparently biennial vitellogenic cycle which appears           Captive Broodstock
           to be strictly controlled by seasonal factors (photo-
           period and, probably, temperature). It was also noted          During the past two years we have performed biop-
           that the females undergoing vitellogenesis exhibited           sies on the gonads and collected plasma from more
           elevated plasma concentrations of estrogen.                    than 1,000 cultured sturgeon. Paraffin sections of go-
             The culture of white sturgeon in California was ini-         nadal tissue, stained by hemotoxylin and eosin
           tiated in the early 1980's by several commercial               (males) or by periodic acid-Schiff stain (fernales),
           ventures. Wild fish are captured during their spawn-           have been examined microscopically. These samples
           ing migration in the Sacramento river. Ova and                 have provided us with the first insight into the go-
           semen are obtained by injecting fish with either carp          nadal maturation of cultured sturgeon and has
           pituitary extracts or synthetic gonadotropin-releasing         enabled us to identify the major problems that are
           hormone analog (Lutes et al. 1987; Conte et al.                preventing these'animals from achieving normal re-
           1988). The fertilized eggs are incubated in jars; lar-         production in culture.
           vae and fingerlings are raised in tanks on salmonid              About 90% of the captive males mature by 3-4
           diets (Hung 1989)..                                            years of age at a body size of 5-6 kg. The stages of
             Initially, the market for these small scale culture          testicular development are shown in Figure 1, (A
           operations was the sale of fry to other growers and to         through Q. Spermatogenesis of these cultured males
           aquarium retailers. During the past three years, sev-          is synchronous, occurring during the summer and
           eral commercial ventures have established the                  early fall. By October-November, testicular cysts con-
           growout of high market-value food fish. Sturgeon are           tain ripe spermatozoa, and it is possible to induce
           raised in tanks, raceways, and earthen ponds and               spermiation by hormone injections. The quality of
           marketed (primarily to restaurants) at body weight             captive male semen does not differ from the quality
           6-8 kg as 2-3 year-old fish. The growout system is also        of the semen collected from wild males (Chapman
           well established in several Western European ven-              1989), and most farmers today use only captive males
           tures that produce highly prized smoked white                  @for their hatchery production. It appears that the tes-
           sturgeon (such as Agroittica Lombarda, Calvisano,              ticular cycle is annual and that the cultured males
           Italy). The white sturgeon is superior to many other           spermiate each year. The only reproductive problem
           species in the high quality of their meat, their accep-        that has been observed in cultured males is an appar-
           tance of artificial diets, their tolerance to high             ent negative effect of warm (above 16' C) tempe-
           density and, most importantly, their fast growth rates         rature on the final phase of the testicular cycle.
           at water temperatures in the range of 20-23' C.                These temperature conditions are often observed in
             The major problem in developing a white sturgeon             some California farms using underground water sup-
           culture has been the lack of domestic broodstocks              plies that have a constant temperature of 18-20' C.
           and dependence of fish farmers on wild broodstock              The colonies of males maintained under such condi-
           capture, which is not only unreliable but severely re-         tions appear to undergo rapid testicular regression
           stricted by government regulation. As a result, several        and will not spermiate during the following spring
           aquaculture ventures in California have initiated the          spawning season U. Michael, Sierra Aquafarms,
           rearing of captive broodstocks obtained from hatch-            Elverta, CA 95626; and Ken Beer, The Fishery, Galt,
           ery-produced fish (FI generation) and they now                 CA 95632, pers. commun. 1989).







                102      NOAA Technical Report WES 106


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                                                                           Figure I
                 Selected stages of gametogenesis in captive white sturgeon: (A),    (B) and (C) show development of testicular germ cells
                 (hematoxylin and eosin stains, microscope magnification 315x); (D) and (E) show development of the ovarian follicle
                 from previtellogenic (D) to early vitellogenic (E) stage, the lattcr observed in very few captive females (magnification
                 50x); (F) peripheral area of early vitellogenic follicle possessing differentiated granulosa layer and egg chorion (periodic
                 acid-Schiff stain, magnification 315x). All fish were sampled in November 1988, at age 3-5 years (males) and 5-7 years
                 (females).


                  In  contrast to the males, most of the captive fe-              majority of cultured females stop reproductive matu-
                males remain reproductively immature for up to 10                 ration just prior to the initiation of vitellogenesis.
                years of age, which is the age of the oldest cultured             Their ovaries complete gonial proliferation by four
                females in California. These captive females have                 years of age, but the ovarian follicles remain in a
                reached a body size (40-50 kg) which is well above                refractory condition characterized by the failure to
                the size of. wild females when they first reach repro-            differentiate granulosa cells (Fig. 1, d through f).
                ductive maturity (Chapman 1989). The great                        This reproductive block could result from a number







                                                            Moberg and Doroshov: Reproduction in Cultured white Sturgeon         103
            of environmental and husbandry factors. We are cur-           increased secretion of endogenous gonadotropins
            rently focusing on the role of seasonal fluctations in        which in turn stimulated endogenous estrogen syn-
            water temperature and nutrition of the broodstock as          thesis. Regardless, the combined estrogen and
            the two most important factors that may be affecting          GnRHa treatment still did not initiate sufficient fol-
            reproduction in these animals.                                licular differentiation (i.e., granulosa cells) to permit
                                                                          the incorporation of vitellogenin. Since this com-
                                                                          bined treatment lasted for only 73 days, it is possible
            Endocrine Manipulation                                        that a longer treatment period might have initiated
            of Vitellogenesis                                             further follicular development (Moberg et al. 1990).
                                                                             From our current understanding of the endocrine
            Since the sexual maturation of the cultured female            control of sturgeon reproduction, it seems that ma-
            sturgeon is arrested at the previtellogenic stage, we         nipulation of the endocrine system still is the most
            have attempted to induce vitellogenesis and trigger           viable approach to inducing reproductive maturation
            further ovarian maturation by hormone manipula-               in the cultured female sturgeon. However, to accom-
            tion (Moberg et al. 1990). We implanted cellulose             plish this task, it will be necessary to refine the
            pellets containing various doses of estradiol into four       hormone treatment practices.
            to seven year-old white sturgeon females that were
            part of the first generation of wild broodstock
            spawned at the University of California at Davis.             Conclusions
            These fish had been raised and maintained in out-             The value of sturgeon flesh coupled with the demand
            door freshwater tanks and fed commercial trout                for caviar makes the culture of sturgeon an exciting
            broodstock diet (Murray Elevators). The estradiol im-         opportunity for aquaculture not only in California,
            plants not only elevated plasma concentrations of             but throughout the world. The adaptation of this spe-
            estrogen in these females, but also stimulated the syn-       cies to aquaculture is not only necessary for the full
            thesis of vitellogenin, raising the plasma con-               realization of its market potential, but may be the
            centration of vitellogenin to levels that were 6 to 10        most viable strategy to insure the survival of this
            times higher than the mean values observ          'ed in      unique group of fish. While remarkable progress has
            vitellogenic wild females (Chapman et al. 1987). In           been made in developing procedures to culture stur-
            spite of these high concentrations of plasma                  geon, one major problem remains, full reproductive
            vitellogenin, there was no evidence of any uptake of          maturation of the cultured females within a time
            vitellogenin by the ovarian follicles, suggesting to us       frame that makes sturgeon culture a viable economic
            that the ovarian follicles were not sufficiently devel-       enterprise.
            oped to incorporate the vitellogenin found in the               The failure of the cultured females to reach full
            plasma.                                                       reproductive maturity appears to result from failure
              These findings suggest to us that the failure of the        of the pituitary to secrete sufficient gonadotropins to
            cultured female sturgeon to mature beyond the                 induce final ovarian follicular development. The
            previtellogenic phase reflects a failure of the hypo-         most viable approach to solving this problem seems
            thalamic-pituitary-gonadal (HPG) axis to initiate the         to be the utilization of hormone treatments to either
            next stage of reproductive maturity. Since follicular         induce the secretion of gonadotropins or to dupli-
            development as well as estrogen synthesis is depen-           cate the effects of these pituitary hormones. The
            dent upon the HPG axis to secrete sufficient                  success of such hormone therapy would not only es-
            gonadotropins to stimulate the next stage of ovarian          tablish full reproduction in culture but would
            development, the failure of both events to occur in           provide a means to induce reproduction in females
            the cultured females suggests that insufficient go-           at a much younger age than that which occurs in the
            nadotropin is being secreted in these animals. To             wild, making the culture of sturgeon a more eco-
            address this problem, we sought to stimulate gonado-          nomically feasible enterprise.
            tropin secretion by treating the animals with
            synthetic gonadotropin          releasing      hormone
            (GnRHa), either alone or in combination with estra-           Acknowledgments
            diol administration. GnRHa treatment by itself did
            not induce estrogen synthesis and as a result had no            This work is a result of research sponsored in part
            effect on vitellogenin synthesis. The combined treat-         by NOAA, National Sea Grant College Program, De-
            ment of estrogen and GnRHa did result in                      partment of Commerce, Under grant number
            significantly higher plasma concentrations of estro-          NA85AA-D-SGI40, project number R/A-73, through
            gen than did estradiol treatment alone, suggesting            the California Sea Grant College Program, and in
            that this combined treatment may have stimulated an           part by the California State Resources Agency.







                  104         NOAA Technical Report NNIEFS 106

                  Citations                                                                    Hung, S.S.O.
                                                                                                    1989. Practical feeding of white sturgeon. Aquaculture
                  Anonymous.                                                                            Magazine 15:60-62.
                       1990. Third annual report of domestic white sturgeon                    Kozin, N.I.
                         broodstock research and development program. Aqua-                         1964. Sturgeon of the USSR and their artificial propagation.
                         culture and Fisheries Program, Univ. California, Davis, Sep-                   Uses. Nauchno-issled. Inst. Morsk. Rybn. Khoz. Okeanogr.,
                         tember, 1990, 36 p.                                                            Trudy, 52, p. 21-57.
                  Bajkov, A.D.                                                                 Leach, G.C.
                       1949. A Preliminary Report on the Columbia River                             1920. Artificial propagation of sturgeon, review of sturgeon
                         Sturgeon. Oregon Fish Comm., Portland, Res. Brief                              c.ulture in the United States. Reports U.S. Fish Commis-
                         2:3-10.                                                                        sion, 1919:3-5.
                  Borodin, N.A.                                                                Lutes,   P.B., S.I. Doroshov, F. Chapman, J. Harrah, R. Fitzgerald,
                       1898. Experiments on artificial insemination of sturgeon                   and M. Fitzpatrick.
                         eggs and other biological observations conducted on the                    1987. Morpho-physiological predictors of ovulatory success
                         Ural River in Spring 1987 [in Russian]. Vestnik Rybopro-                       in white sturgeon, Acipenser transmontanus. Aquaculture
                         myschlermosti, St. Petersburg, Vol. 6-7.                                       66:43-52.
                  Carter, E.N.                                                                 Marti, YY.
                       1904. Notes on sturgeon culture in Vermont. Trans. Am.                       1979. Development of sturgeon management in the south-
                         Fish. Soc.-33: 60-75.                                                          ern seas of the USSR. In Biological resources of inland wa-
                  Chapman, F.A.                                                                         ters of the USSR, p. 73-85. (Publ. House Nauka, Moscow.
                       1989. Sexual maturation and reproductive parameters of                  Meehan, W.E.
                         wild and domestic stocks of white sturgeon, Acipenser                      1909. Experiments in sturgeon culture. Trans. Am. Fish.
                         transmontanus. Ph.D. diss., Univ. California, Davis.                           Soc. 39:85-91.
                  Chapman, F.A., R.L. Swallow, and S.I. Doroshov.                              Moberg, G.P., S.I. Doroshov, F.A. Chapman, K.J. Kroll, J. Van
                       1987. Ovarian cycle of white sturgeon (Acipenser                           Eenennaam, andj.G. Watson,
                         transmontanus). In Proc. of 3rd int. symp. on reproductive                 1991. Effects of various hormone implants on vitellogenin
                         physiology of fish. St. Johns, Newfoundland, Canada; 2-7                       synthesis and ovarian development in cultured white stur-
                         August 1987 (D.R. Idler, L.W. Crim, andj.M. Walsh, eds.),                      geon, Acipenser transmontanus. In Acipenser (P. Williot, ed.)
                         p. 196. Memorial Univ. Newfoundland.                                           p. 389-399. CEMAGREF-DICOVA cedex, France. ISBN
                  Conte, F.S., S.I. Doroshov, P.B. Lutes, and E.M. Strange.                             #2853622088.
                       1988. Hatchery manual for the white sturgeon, Acipenser                 Moyle, P.B., andjj. Cechjr.
                         transmontanus. Coop. Ext. Univ. California, Div. Agricul-                  1988. Fishes, introduction to ichthyology. Prentice Hall,
                         ture and Natural Resources, Publication 3322, 104 p.                           NJ, 559 p.
                  Czeskleba, D.G., S. AveLallemant, and T.F. Thuemler.                         Post, H. 1
                       1985. Artificial spawning and rearing of lake sturgeon,                      1890. Sturgeon experiments in hatching. Trans. Am. Fish.
                         Acipenser fulvescens, in Wild Rose State Fish Hatchery, Wis-                   Soc. 19:36-40,.
                         consin, 1982-1983. In North American sturgeons: biology               Purkett, C.A.
                         and aquaculture potential (F.P. Binkowski and S.I.                         1963. Artificial propagation of paddlefish. Trans. Am. Fish.
                         Doroshov, eds.), 79-86 p. Dr. W. junk. Publishers,                             Soc. 90:125-129.
                         Dordrecht, Netherlands                                                RyderJ.A.
                  Doroshov, S.I.                                                                    1890. The sturgeon and sturgeon industries of the eastern
                       1985. Biology and culture of sturgeon, Acipenseriformes. In                      coast of the United States, with an account of experiments
                                                                                                        bearing upon sturgeon culture. U.S. Fish Commission
                         Recent advances in aquaculture, Vol. 2 U.F. Muir and Ri.                       Bull. 8:231-328.
                         Roberts, eds.), p. 251-274. Croom Heim, London and                    Semakula, S.N., and P.A. Larkin.
                         Sydney.                                                                    1968. Age, growth, food and yield of the white sturgeon
                  Doroshov, J.N., J.P. Van Eenennaam, F.A. Chapman, and 8.1.                            Ad enser transmontanus of the Fraser Rier. J. Fish. Res.
                     Doroshov.                                                                            P
                                                                                                        Board Can. 25:2589-2602.
                       1990. Histological study of the ovarian development in wild             Smith, T.1j.
                         white sturgeon, Acipenser transmontanus. In Proc. of lst int.              1985. The fishery, biology, and management of Atlantic stur-
                         symp. on sturgeon, Bordeaux, France; 2-5 Oct., 1989. Cen-                      geon, Acipenser oxyrhynchus, in North America. In North
                         tre d'ttudes du Machinisme Agricole du G6nie Rural des                         American sturgeons: biology and aquaculture potential.
                         Eauxet For@ts, France. (In press.)                                             (F.P. Binkowski and S.I. Doroshov, cds.), p. 61-71. Dr. W.
                  Doroshov, S.I., W.H. Clark Jr., P.B. Lutes, R.L. Swallow, K.E. Beer,                  junk. Publishers, Dordrecht, Netherlands.
                    A.B. McGuire, and M.D. Cochran.                                            Smith, T.I.J., E.K. Dingley, and D.E. Marchette.
                       1983. Artificial propagation of the white sturgeon, Acipenser                1980. Induced spawning and culture of Atlantic sturgeon.
                         transmontanus. Aquaculture 32: 93-104.                                         Prog. Fish-Cult. 42:147-151.
                  Galbreath,J.L.
                       1985. Status, life history and management of Columbia River             Storie, L.
                         white sturgeon, Acipenser transmontanus. In North American                 1901. Sturgeon hatching in the Lake Champlain ba-
                         sturgeons: biology and aquaculture potential (F.P.                             sin. Trans. Am. Fish. Soc. 30:137-  .143.
                         Binkowski and S.I. Doroshov, eds.), p. 119-126. Dr. W.
                         junk. Publishers, Dordrecht, Netherlands.
                  Harkness, W.J.K., andj.R. Dymond.
                       1961. The lake sturgeon: the history of its fishery and prob-
                         lems of conservations. Publication of Fish and Wildlife
                         Branch, Ontario Dep. of Lands and Forests, Canada, 121 p.






                      Experimentation to Improve Recruitment of Blood Ark Shell,
                                     Scapharca broughtonii, in the Seto Inland Sea



                                                               SATOSHI UMEZAWA*

                                                       Japan Sea Regional Fisheries Research Institute
                                                               Suido-cho, Niigata 951, Japan




                                                                       Abstract


                                 The blood ark shell Scapharaca broughtonii is one of the most important bivalves in the
                              coastal fishery of Japan. Recently, fishery hauls of blood ark shell have been decreasing.
                              The blood ark shell research group of the Marine Ranching Project has studied tech-
                              nologies to artificially propagate spawning stocks of these species in order to increase
                              recruitment in the natural fishery grounds. This paper reviews some of our efforts to
                              understand the biology and ecology of ark shells and to develop techniques to improve
                              spat collection and prevent predation from starfish.





            Introduction                                                       about 30-mm in shell length in the following year,
                                                                               from March to July. These 30-mm seeds are used for
            The blood ark shell Scapharca broughtonii is one of the            releasing into the fishery grounds and for cage cul-
            most important shellfish of the coastal fishery of Ja-             tures. In both released and cage-cultured ark shells,
            pan. Its distribution extends from southern                        one can expect to harvest the shellfish two years after
            Hokkaido to Kyushu in shallow bays and gulfs with                  birth. Because it takes over three years for blood ark
            muddy bottoms. Recently the fishery has decreased                  shells to mature sexually, these stocks will have no
            owing to excessive fishing pressure and the fluctua-               chance to contribute to reproduction. Therefore, the
            tion of the natural level of the resource. Reduction in            spawning stock is usually very small and may not pro-
            the broodstock levels of ark shell are occurring be-               duce a significant number of offspring.
            cause of the large number of premature shells                        The blood ark shell research group of the Marine
            harvested in the wild.                                             Ranching Project (MRP), which consists of the
               Over the past decade mass production techniques                 Nansei Regional Fisheries Reseach Laboratory, the
            yielding about 10 million spats annually per hatchery              National Research Institute of Aquaculture, and the
            have been established in Yamaguchi and other pre-                  Inland Sea Fisheries Experimental Station of
            fectures. First, broodstock is collected by selecting              Yamaguchi Prefecture, has studied technologies to
            spawners from the commercial catch of small trawlers               cultivate spawning stocks of blood ark shell. By artifi-
            during late March to early April. The selected                     cially propagating seeds to an age greater than two
            broodstocks are then reared in small tanks without                 years old, this group had attempted to increase re-
            feeding for several months, becoming mature when                   cruitment in the natural fishery grounds (Fig. 1).
            the water temperature rises to 20' C. The spawners                   This paper outlines the research activities and
            discharge eggs and sperm during late June to early                 some results of the blood ark shell research group in
            July with temperature stimulation. Naturally spawned               the MRP.
            eggs are collected and maintained in a larval rearing
            tank, and newly hatched larvae are initially fed with
            Pavlova luthe7i or Cheatoceros spp. for two months. The
            seeds are harvested from the tank when they reach
            I mm in shell length in September. Subsequently, the
            spats are reared in lantern nets until they reach                   Present address: Seikai National Fisheries Research Institute,
                                                                                Kokubu, Nagasaki: 850, Japan.


                                                                                                                                        105







               106       NOAA Technical Report NWS 106


                                                                                     Hatchery

                                                                                   Larval and seedling rearing





                                                      Stock breeding                                                intermediate rearing

                                                                                                          IN




                                           `X

                                Fisheryground
                                                                                                                          X
                                                                                                                                      -x:
                                                                                                                       .xx
                                                                                           Woodstock culture
                                              Releasing                                                                       Releasing

                                                              Spat collection


                                                                                                                          Natural sea
                                                                      ..................                        ......



                                   Cage culture                         . ..
                                                                             .... ................









                                                                          Figure I
               Schematic drawing of aquaculture of the blood ark shell. Methodology of artificial broodstock culture is studied
                                                              in the Marine Ranching Project.




               Environmental Conditions for the
               Cultivation of Artificial Broodstock                               1985). Water quality and bottom conditions were also
                                                                                  surveyed at each station. Figure 3 shows the results of
               Figure 2 shows the location of the pilot farms for the             cage culturing I-year-old shell during the 5-month
               cultivation of spawning stocks in the Kasado Bay at                period lasting from May to October. High survival
               Yamaguchi Prefecture. This bay was selected as a                   rates and good shell growth are seen at Station 13 in
               farming ground because 1) this area has been used                  comparison with the other stations. Two- and 3-year-
               previously to culture the species, 2) eggs and larvae              old shells have similar survival rates.
               discharged from the broodstock are expected to re-                    The characteristics of the environmental condi-
               main in the bay because of its topographical                       tions at Station 13 are as follows:
               characteristics, and 3) the local fishermen's associa-                * Water temperature is about I' C or more higher
               tion cooperated in our survey and investigation.                   during the spring to early summer period than at the
                  The artificially propagated seeds must be reared                other stations. The water depth is shallower, about
               more than two years for use as broodstock; therefore,              10 m at the station.
               the farming ground must provide environmental
               conditions for good growth and high survival rates                    * Water transparency is high.
               during cultivation.                                                   * Grain size of bottom sediment is larger, about 10-
                  Cage culture of I- to 3-year-old shell was tried at six         20gm mainly.
               stations in Kasado Bay to investigate the growth and
               survival rates of blood ark shell (Umezawa et al.                      Organic content levels of sediment are low.







                                                                                         Umezawa: Rearuitinent of Blood Ark Shell           107



                                 A
                     Yanagwhi ii@ef."-                                          MKhmtsu city

                   Sea Of SOD

                                         i koku


                             Area enlarged

                              Tokuyam City     j         1      31


                                                   4
                                                                             74
                                         05                                           17
                                                                          80
                                                       Kasado bay      90
                                                                         log*)
                                                                   I.10


                               -------   *16                     12*  '.1;


                                             018
                                                                N


                                                IN


                             14
                             0                                                        1 Mi                               Figure 2
                                                  Usado Island                16                           Map of Kasado Bay and station
                                                                                                           numbers of the environmental
                                                                                                           survey (1-18). Pilot farms for the
                                                                                                           cultivation of blood ark shell are
                                                                                                           located at stations 9 and 10.



                 The percentage of chlorophyll-a in the phyto-pig-               was the elimination of starfish with the use of traps.
             ment (chlorophyll-a plus pheopigment [pheo-                         The other was protection of the blood ark shell with
             phytin and pheophorbide]) is large, in the bottom                   capsules until the broodstock reached a large enough
             water.                                                              size to avoid predation (Takami and Kournoto 1986).
                                                                                    Figure 4 shows the arrangement of the experimen-
               It is suggested that the proper environmental con-                tal lots for predation prevention at Station 9 in
             ditions for blood ark shell propagation includes                    Kasado Bay. Lot A shells were protected by two types
             higher water temperatures during spring to early                    of starfish traps (shown in Fig. 5), which surrounded
             summer, not exceeding 25' C continually. The re-                    the ark shell release area. Lot B, blood ark shells
             quirement for bottom conditions is sandy mud                        were maintained in the capsules shown in Figure 6
             without high organic matter concentrations.                         for protection from starfish predation. Lot C shells
                                                                                 were a control group which received no protection
                                                                                 from predation.
             Prevention of Predation by Starfish                                    Starfish are usually present in the experimental
                                                                                 area before the blood ark shells are released. A large
             High mortality occurs frequently for a few weeks after              number of starfish swarmed gradually after the re-
                                                                                 lease-the greatest concentration of them near the
             the release of the blood ark shell seeds. One of the                experimental area occurring in July. From September
             causes is predation by starfish. Therefore, prevention              to October few starfish are found. In lot A, 96.5% of
             of predation by starfish is essential for the cultivation           swarmed starfish were removed from the ark shell
             of broodstock. Two methods were examined. One                       release area; type 1I was more useful than type 1.







                   108           NOAA Technical Report NMFS 106




                                            n=37                                   n=47
                              10-




                                 0
                              40-                     St.10
                                                                                            St. 13
                              30-                                             n=50
                              20-           n=50
                              10-_    I                                      /
                                 0 1 U@=

                                                                                      n=46

                       M
                              10-
                                            n
                                            =12
                       2
                       a
                       .r-       0
                       0
                              40-                     St. 9                                 St. 12
                       0
                              30-

                              20-      f
                                            n=50                               n=50
                              10--
                                                                           UL    . . . . .
                                 0          . . .



                                            n=21
                              10-
                                                                                 n=9
                                 0                                           P:d@         I    '    '
                              40-           1          St. 7                                St.11
                              30-
                              20-           n=50                               n=50                                                        Figure 3
                              10-                                                                                Composition of total weight of blood ark shell
                                 0    -4                                  UL
                                            .  I    I                        .   I    I   .                      after five months of culture at six stations in
                                     10     20 30 40 50 60                   10 20 30 40 50                      Kasado Bay. Within the three groups of two                .  sta-
                                                            Total weight (g)                                     tions each, data for 23 May 1984 are shown in
                                                                                                                 bottom graph, data for 24 October 1984 are
                                                                                                                 shown in upper graph (Umezawa et al. 1985).
                                                                                                                 n=number of specimens.


                     Figure 7 shows the survival rate of blood ark shell                                When blood ark shells are released, those over 2
                   in these experinments from May to October. During                                 years old need no protection from predation. How-
                   May to the middle of July, 1-year-old shell mortality is                          ever, I-year-old shells are still vulnerable to predation
                   presumed to result from starfish predation because                                at release time, such that about 30% survival can be
                   the size of starfish caught in this period was large                              expected even when methods to prevent starfish pre-
                   enough to prey upon the blood ark shells. After the                               dation are used.
                   middle of July, the decrement in survival rate was not
                   dependent on starfish predation, because all of the
                   experimental lots experienced almost the same de-                                 Plan against High Mortality
                   cline in survival. Two- and 3-year-old shells have no                             in the Summer
                   need for prevention from starfish predation starfish
                   because the control experiment indicated a high sur-                              From summer to early autumn, a high mortality of
                   vival potential for older shells.                                                 farmed 1-year-old shell has occurred frequently. The








                                                                                Umezawa: Recruitment of Blood Ark Shell        109












                                                 Starfish trap

                                                    *F I

                                                     LotIAPIV
                                              (Removed starfish                                           Lot C
                            *Ulna=                by traps)                                      (Without traps and capsules)















                                                     Lot B
                                              (Protected by capsules)


                                                                   Figure 4
                   Configuration of experimental system to prevent starfish predation on blood ark shells during 14 May to 27
                   November in 1985. Each * indicates an individual starfish removed by a trap. Each * indicates an
                   uncaptured starfish in the study area. El indicates a type-l starfish trap and 0 indicates a type-II trap as
                   shown in Figure 5 (Takami and Kournoto 1986).



           cause of this mortality is estimated to be the effect of      of cage cultures of only 8-32% (Table 1). Thus, high-
           high water temperature, low dissolved oxygen or in-           density hanging is useful for avoiding high mortality
           creased sulphide levels (Ishida et al. 1977;                  in summer. This method has been repeated success-
           Hamamoto 1981; Umezawa et al. 1984). It is impor-             fully for several years (Umezawa et al. 1987).
           tant to avoid this high mortality during the
           cultivation of blood ark shell stock.
             High-density hanging cultures with lantern nets             Sexual Maturation of
           were tested to counter high mortality in the summer           Planted Broodstock
           (Umezawa et al. 1987). One hundred artificially cul-
           tured blood ark shell seeds were maintained in each           Table 2 shows histological observations of gonad de-
           of the four lantern nets, which were hung at a depth          velopment in blood ark shells cultured from planted
           of 10 in in the sea. Four cage cultures, each of which        seed in Kasado Bay for one to three years
           had 50 seeds, were placed on the sea bottom. From             (Numaguchi et al. 1985). The phases of gonadal de-
           May to October, the high density hangings achieved            velopment are divided into five stages: stage 1,
           high survival rates of 94-100% compared with those            immature or sex unknown; stage II, early develop-







                110      NOAA Technical Report NAUS 106


                       4    35 cm

                                                                  r n



                 35 cm
                                                  45                                                    CM



                                    Bait bag                    60 cm
                  15 cm                                                              i   45 cm
                   4-                                                                                                     Figure 5
                                                                                                                Structure of traps designed
                           Type I                                        Type H                                 to remove starfish in lot A
                                                                                                                (Takami and Koumoto 1986).





                                        45 cm                          Nylon net

                                              18 CM                         Vinyl pipe


                                                                                                                Figure 6
                                                                      K@otton net
                                                                                          Structure of capsule to protect ark shell from pre-
                                               Capsule                                    dation of starfish in lot B (Takami and Koumoto
                                                                                           1986).


                ment; stage III, late development; stage IV, mature or            Eight species of bivalve larvae were collected in
                spawning; and stage V, after spawning. One-year-old               Kasaso Bay, and 11 species were collected off the
                shells do not mature in the first year. All 2-year-old            Hikari coast. Figure 8 illustrates the number of ark
                shells were immature in May-some male gonads de-                  shell type larvae at Kasado Bay and Hikari. There
                veloping after June. As for females, only one                     were two species of ark shell (Scapharca broughtonii
                individual matured in October. Three-year-old shells              and S. suberenata), but neither species could be spe-
                did not mature in May. After June, the gonads devel-              cifically identified by larval examinations. In Kasado
                oped in both males and females, and from August to                Bay, 3.1 individuals/ml of ark-shell type larvae ap-
                September many individuals reached the spawning                   peared in late July, and fewer were collected in
                phase. The spawning season is estimated to range                  September and October. Off the coast of Hikari, 2.0
                from July to September and the peak season is as-                 individuals/M3   were present in July and August, ris-
                sumed to be from late August to early September.                  ing to 15.3 individuals/  M3  in late September.
                  Table 3 shows the estimated egg number for 10 3-                  Two types of seed collectors, the hanging and bot-
                year-old parents. Three-year-old shells, which range              tom types, were set at two stations in Kasado Bay and
                from 65 to 75 mm. in shell length and from 77 to 91 g             one station off the coast of Hikari (Takami and
                in total weight, hold from 0.5 to 12 million eggs, aver-          Koumoto 1987). Hanging seed collectors consisted of
                aging 5.5 million per individual.                                 a lantern net containing used fish net or nylon net.
                                                                                  Bottom seed collectors which were located on the
                                                                                  bottom hung the same lantern net by means of an
                Larvae and Spats Collection                                       iron frame (Fig. 9). Figure 9 shows the number of
                from the Natural Environment                                      blood ark shell spat deposited on the two types of
                                                                                  seed collector. In the hanging type collector, more
                In order to know the period, distribution, and num-               blood ark shell spats are deposited as water depth
                                                                     ce









































                ber of larval blood ark shells, Norpac-net tows were              increases. The number of spats deposited in the bot-
                conducted at six stations in Kasado Bay and six sta-              tom type is greater than that of the hanging type.
                tions in the coastal waters off Hikari close to the east          Spats collected in mid-December measured 1-6 mm
                side of Kasado Island (Takarni and Kournoto 1987).                in shell length, indicating that the larvae appeared








                                                                                           Urnezawa: Recruitment of Blood Ark Shell            ill



                     100 -                                        100 -

                     90   -         25 1-year-old shells/m2        90   -             50 1-year-old shells/m2

                     80   -                                        80   -


                     70   -                                        70   -


                     60   -                                        60   -


                     50   -                                        50   -


                     40   -                                        40   -


                     30   -                                        30   -


                     20   -                                        20   -


                     10   -                                        10   -

                                                             t
                       0                                             0
                          May Jun Jul Aug Sep Oct Nov                   May Jun Jul Aug Sep Oct Nov




               4)    100 -                                        100 -
              Cl-
                     90   -                                        90   -                                                     Figure 7
                     80   -                                        80   -                                          Survival rate of blood ark
                                                                                                                   shell by age and density
                     70   -                                        70   -                                          from experiments to prevent
                     60   -                                        60   -                                          starfish predation. Open
                                                                                                                   circles denote lot A where
                     50   -          20 2-year-old shells/m2       50   -       20 3-year-old shells/m2            two types of starfish traps
                                                                                                                   were used; closed stars de-
                     40   -                                        40   -                                          note lot B where shells were
                                                                                                                   shells protected by a capsule;
                     30   -                                        30   -                                          closed circles denote lot C
                     20   -                                        20   -                                          where seed were released
                                                                                                                   without the protection of
                     10                                            10                                              traps or capsules (control)
                          L                                             F                                          (Takami      and     Kournoto
                       0  May Jun Jul Aug Sep Oct Nov                0  May Jun Jul Aug Sep Oct Nov                1986). The figures in each
                     1985                                          1985                                            graph shows intial shell
                                                                                                                   density.



             and attached onto the collector from late September                  collectors were smaller in 1986, indicating that spat
             to October.                                                          collection is a major problem to be solved at the pilot
               These larval and spat collection results were ob-                  scale.
             tained in the investigation for 1986. The origin of
             these spats was probably derived from the natural                    Citafion
             population because the planted broodstock in
             Kasado Bay was still immature in 1986. The results of                Hamamoto, S.
             an identical investigation conducted in 1987                              1981. Basic studies on the mortality factor and resisting
             (Ouhashi and Kournoto 1988)@, showed marked im-                             power of an ark shell, Scapharca broughtonii
             provement in the number of the ark shell type larvae                        (SCHRENCK). Scientific Report of Kagawa Prefectual
             compared to the previous year. These findings sug-                          Fisheries Experimental Station 18:1-19. (Injapanese.)
             gest that some fraction of collected larvae were                     Ishida, M., 1. Hayashi, and H. Ushima.
             spawned from the planted blood ark shell parents.                         1977. Experiment on cage culture of blood ark shell. An-
                                                                                         nual Report of Fukuoka Prefectual Buzen Experimental Sta-
             However, the number of deposited spats in the seed                          tion 49-52. (Injapanese.)







                   112         NOAA Technical Report NMEFS 106



                                                                                             Table 1
                      Comparison of growth and survival between high-density hanging cultures in lantern nets and cage cultures on the
                                                                       sea bottom in 1986 (Umezawa et al. 1987).


                                                        High-density hanging culture                                       Cage culture at bottom

                      Date                        No. of shells/net       Mean total wt. (g)                      No. of shells/net         Mean total wt. (g)

                      May 13                            100                      6.8                                       50                       8.1
                                                        100                      6.9                                       50                       7.3
                                                        100                      6.8                                       50                       7.1
                                                        100                      6.8                                       50                       6.1
                                                        100                      7.0


                      Oct. 13                            98                      23.6                                      14                       16.4
                                                         94                      25.6                                      16                       15.6
                                                        100                      23.8                                        9                      15.4
                                                         97                      20.7                                        4                      12.1






                                                                                             Table 2
                      Frequency           of blood ark shells in each phase of gonad development in I- to 3-year-old shells in 1984 (Numaguchi
                      et al. 1985). Stages I-V are phases of gonad development: Stage I, immature or sex unknown; Stage 11, early develop-
                      ment; Stage 111, late development; Stage IV, mature or spawning; and Stage V, after spawning.


                                                            1-yr-old                                  2-yr-old                                 3-yr-old

                                                  I     II     III     IV     V              1   11     111    IV     V               1     11    111    IV      V
                        Sampling date                   6

                        May 23                    100   0 0    0  0    0  0   0  0         100   0  0   0  0   0  0   0   0         100     00    0 0   0   0  0   0
                        Jun 5                     100   0 0    0  0    0  0   0  0           85 15  0   0  0   0  0   0   0           55 25 20    0 0   0   0  0   0
                        Jun 25                    100   0 0    0  0    0  0   0  0           55 45  0   0  0   0  0   0   0           55 30  5 10   0   0   0  0   0
                        Jul 4                     100   0 0    0  0    0  0   0  0           55 15  0 15   0 15   0   0   0           20 50 10 10   10  0   0  0   0
                        Jul 24                    100   0 0    0  0    0  0   0  0           35 55  0 10   0   0  0   0   0           5 25   5 45   20  0   0  0   0
                        Aug 20                    100   0 0    0  0    0  0   0  0           90 10  0   0  0   0  0   0   0           0     00 50   30  15  5  0   0
                        Sep 17                    100   0 0    0  0    0  0   0  0           55 45  0   0  0   0  0   0   0           10    00    0 0   10  10 60 10
                        Oct 31                    100   0 0    0  0    0  0   0  0           80  0  0   0  0   0  0   10  10          90    00    0 0   0   0  5   5







                                                                                             Table 3
                                    Shell length, weight and egg number of 3-year-old blood ark shell (Numaguchi and Tanaka 1987).
                                                                Shell length                                      Weight (g)                                   No. of.eggs
                                 Sampling No.                       (mm)                              Meat                          Gonad                        (X 105)

                                          1                            71                             78                              0.36                         5.4
                                          2                            77                             87                              0.86                         7.1
                                          3                            71                             81                              1.59                        16.1
                                          4                            67                             70                              1.93                        54.0
                                          5                            70                             80                              2.89                         5.5
                                          6.                           70                             77                              .3.51                       70.6
                                          7                            66                             86                              3.72                        76.5
                                          8                            65                             88                              4.09                       115.5
                                          9                            75                             91                              5.25                        78.9
                                         10                            72                             86                              5.97                       119.3

                                     Average                           70                             82.5                            3.02                        54.9








                                                                                            Urnezawa: Recruitment of Blood Ark Shell            113


                                                        Q 15.3





                  E



                  (U

                       10
                  W
                  -C




                  0



                  0
                  W                                                                                                            Figure 8
                                              Hikari
                                                                                                                     Seasonal distribution of ark
                  -2     5
                  4--                                                                                                shell type larvae at Kasado
                  0                                                                                                  Bay (water depths are about
                                                                                   Kasado bay                        15 m) and the. coastal wa-
                  E
                                                                                                                     ters off Hikari (water
                  z                                                                                                  depths are about 20 m) as
                                                                                                                     collected with a Norpac net.
                                           '0                                                                        0 = mean tows from all
                                                                                                                     depths combined at each of
                         0                                      1             do              44iW4=!=SQ             six stations. 0 = mean for
                           Jun     Jul     Aug     Sep     Oct            Jun     Jul     Aug     Sep     Oct        the middle of water depth
                         1986                                          1986                                          to upper layer (Takami and
                                                                                                                     Koumoto 1987).




                                     Kasado             Hikari
                             - 4i-
                      Om         0





                       5m
                  6



                  0
                  -f;
                  CL
                      lom


                  M
                  r
                  (D


                  tA
                      15m



                                                                                                          Figu re 9
                                                                         Number of blood ark shell spats deposited in       seed collectors at dif-
                      20m                                                ferent depths in Kasado Bay and off the coast of Hikari. Numbers in
                                                                         the triangles predict the total spat number of 21 hanging cages.
                                                                         Those with frames predict the total spat number of 12 cages located
                                                                         on the bottom (Takami and Kournoto 1987).







                  114         NOAA Technical Report NMEFS 106

                  Numaguchi, K., and Y. Tanaka.                                                        1987. Technique for collecting ark-sheli seeds from artificial
                       1987. Acceleration technique of maturation and spawning of                        broodstock. Marine Ranching Project Progress Report,
                          the ark-shell,      Scapharca broughtonii, in farming                          Bay Scallop and Blood Ark Shell 7:61-72.
                          ground. Marine Ranching Project Progress Report, Bay                    Umezawa, S., K Nogami, and 0. Fukuhara.
                          Scallop and Blood Ark Shell 7:55-59. (In Japanese.)                          1984. Relation between high mortality and some environ-
                  Numaguchi, K., S. Funakoshi, and K Wada.                                               mental conditions for ark shell, Scapharca broughtonii in cage
                       1985. Maturation and spawning of cultured ark shell,                              culture. Bull of the Nansei Regional Fisheries Research
                          Scapharca broughtonii. Marine Ranching Project Progress                        Laboratory 16:231-244. (InJapanese.)
                          Report, Bay Scallop and Blood Ark Shell 5:71-75 (In Japa-                    1985. Environmental conditions and releasing density ofark
                          nese.).                                                                        shell in the artificial farming ground. Marine Ranching
                  Ouhashi, H., and Y Koumoto.                                                            Project Progress Report 5:57-69. (In Japanese.)
                       1988. Technique for collecting ark-shell seed from artificial              Umezawa, S., S. Arima, K Oochi, and 0. Fukuhara.
                          broodstock. Marine Ranching Project Progress Report, Bay                     1987. Farming the broodstock and environmental condi-
                          Scallop and Blood Ark Shell 9:73-83. (In Japanese.)                            tions for natural seeding of ark-shell. Marine Ranching
                  Takami, T., and Y Koumoto.                                                             Project Progress Report, Bay Scallop and Blood Ark Shell
                       1986. Studies on the artificial propagation and control of the                    7:73-81. (Injapanese.)
                          predators in the ark shell. Marine Ranching Project
                          Progress Report, Bay Scallop and Blood Ark Shell 6:97-106.
                          (Injapanese.)







                        The Marine Ranching System: The Integration of Biology
                                                  and Engineering Technology



                                                            HIROYUKI NAKAHARA
                                                          Research Institutefor Ocean Economics
                                                            Maryfuji Bldg., 3-1-10 Shinbashi
                                                              Minato-Ku, Tokyo 105, Japan




                                                                     Abstract


                               This paper discusses a variety of concepts concerning aquaculture, including fish-farm-
                             ing and marine ranching with respect to their purpose, the ownership of the fish,
                             participants, the ability to measure resource production, the technologies introduced to
                             date, and future trends. Approaches to engineering systems, which should be designed to
                             meet with the biological aspects, are also examined along with some examples. Aquacul-
                             ture, which used to be deployed in rather small, calm and protected bay areas, needs
                             improved hardware and techniques in order to be performed in more offshore areas on
                             a larger scale. The expansion of fish farming should be accompanied with new environ-
                             mental-control technologies:technologies designed to enhance primary production and
                             other related engineering capabilities. To integrate the efforts of biologists and engi-
                             neers is the best way to create a successfull marine ranching system. Some of the
                             experiences reported in this paper are based upon research and development activities.
                             For example, seabed improvement efforts have been made which make use of civil engi-
                             neering and water quality improvement to fit the needs of a target species during a
                             certain stage of its growth to enhance feeding. This was done by making use of water
                             movement control technologies and by introducing new materials and structures for the
                             attachment of food organisms.



           Introduction                                                      tion from aquaculture has risen five times during this
           Japan is the largest fishing nation in the world with a           period. Sea-farming, which refers primarily to the
           total catch in 1985 of 12.2 million tons UFA 1987;                propagation of cultured seed that are released into
           MAFF 1987). Additional fishery products valued at                 the natural environment, has been performed for
           about five billion dollars were imported while exports            more than twenty years. Typical of this effort is the
           totalled one billion dollars. Because the local off-              release of two billion chum salmon (Oncorhynchus
           shore fishery in the waters around Japan consists                 keta). The mass production of other seed is now pos-
           mainly of sardines and because growing international              sible with kuruma prawn (Penaeus japonicus), blue
           constraints are making contributions from long-dis-               crab (Portunus trituberculatus), scallop (Patinopecten
           tance fishing harder to maintain, efforts are focusing            yessoensis), abalone (Hatiotis discus hannai), and oth-
           on coastal fishery production to keep up with the                 ers.
           growing demand for high-quality fishery products.                   Based upon these cultivation activities, the new
              The coastal fishery production increased from 2.24             concept of The Marine Ranching System has been
           million tons in 1965 to 2.71 million tons in 1975, and            developed in recent years, which makes use of indus-
           to 3.36 million tons in 1985. However, within these               trial engineering technologies (RIOE 1981-88). Some
           totals, the percentage from fishing boats, the tradi-             of them are in a very conceptual stage while others
           tional style of harvest, remained constant at around 2            are being experimentally executed on-site. This pa-
           million tons during these twenty years. The increases             per described the status quo of these efforts injapan,
           were realized by the marine net-pen fishery and                   which are combining biological knowledge with engi-
           aquaculture. (Figs. I and 2; DS1 1984) The contribu-              neering technologies.


                                                                                                                                      115







                    116        NOAA Technical Report NNffS 106



                        x10  TON



                     12


                     11






                        9



                                                              LONG-
                                                              DISTANCt





                        6


                                                                      OFFSHORE



                        4



                               z
                        3


                        2                                            COASTAL


                        1
                                                                     AQUACULTURE
                        0                                                                                                                    Figure 1
                                      60           65       70         7@         so    84                          Trends of fishery production in Japan (Fisher-
                                                              INLAND & FRESHWATEn-AQUACULTURS                       ies Statistics of Japan, Ministry of Agriculture,
                                                                                                                    Forestry, and Fisheries 1984).



                            13 * (Million metric tons)
                            12 e                                                                inland water(l.6%)
                                                                                                fi sheri es- cultures
                                                                                                Marine cultures
                                                                             ..........
                                                                                                              (8.7%)
                                                                      ..............
                                                                                                Coastal fisheries
                                                                    ..........
                                      ...............................
                            10 q
                                               .................                                     (17.7%)
                                       .......................

                            9


                            8


                            7


                            6


                            5        -------- --- -
                                                                                                Offshore fisheries
                            4                                                                               (54.3%)

                            3



                                                                                                Distant water
                                                                                                           fisheries
                                                                                                            (17.8%)
                                                                                                                                                Figure 2
                                                                                                                          Total catch by type of fishery, 1974-84
                                  1974                               190                    44                            (Fisheries Statistics of Japan, Ministry of
                                                                                                                          Agriculture, Forestry, and Fisheries 1984).






                                                                                                               Nakaham: The Marine Ranching System                        117
               Definition of Aquaculture, Sea-farming,                                             Aquaculture
               and Marine Ranching
                                                                                                   The term "aquaculture" generally means the cultiva-
                   Throughout the long history of mankind, fishing                                 tion of fish in the protected sea area and is frequently
               has meant the taking of live fish from the sea in a                                 symbolized by the net-cage (Fig. 2). This process usu-
               manner analogous to hunting on land. In the past,                                   ally starts with the collection of eggs from either
               we caught more fish from the sea by sailing all over                                natural or artificial environments, then rearing the
               the world using high-tech electronic instrumenta-                                   juveniles, usually with feeding until harvest time. The
               tion. Conversely, we are now developing aquaculture                                 most important characteristics of aquaculture are 1)
               techniques that are analogous to cattle breeding. In                                that the ownership of fish is quite clear, 2) that hu-
               Japan, both freshwater and marine aquaculture have                                  man control is possible throughout the whole life of
               been developed for a long time. Carp (Cyprinus                                      the fish although conditions within the net cages are
               carpio), eel (Anguilla japonica), chum salmon (0.                                   supported by nature, and 3) that the final fruits of
               keta), rainbow trout (0. mykiss), and sweetfish or Ayu                              the production are countable (Table 1).
               (Plecoglossus altivelis) are the major species cultured
               in freshwater. Marine aquaculture mainly covers laver
               (Porphyra), oyster (Crassostrea gigas), yellowtail (Se7iola                         Sea-Farming
               quinqueradiata), sea mustard (Undaria), scallop
               (Patinopecten yessoensis), and red seabream (Pagrus ma-                               Sea-farming involves the propagation of cultured
               jor)                                                                                seeds and fry into adults and their release into the




                                                                                           Table I
                                                       Major characteristics of aquaculture and fish-farming in Japan.

                                                            Aquaculture                                              Fish-farming


                   Sector                                   Private                                                  Public


                   Major species                            Laver (Porphyra)                                         Salmon (Oncorhynchus keta)
                                                            Oyster (Crassostrea gigas)                               Red seabream (Pagraus major)
                                                            Yellow-tail (Seriola                                     Kuruma prawn (Penaeusjaponicus)
                                                            quinqueradiata)                                          Yezo abalone (Nordotis discus hannat)
                                                            Scallop (Patinopecten                                    Flounder (Paralichthys olivaceus)
                                                            yessoensis)
                                                            Red Seabream (Pagrusmajor)
                   Area                                     Protected sea                                            Open sea

                   Purpose                                  Direct production                                        Resource increase

                   Human control                            Total period                                             Until releasing
                                                               (until harvesting)

                   Ownership                                Fish producer                                            None
                                                            Net cage owner


                   Bait                                     Feedable                                                 Non-feedable
                                                                                                                     (food organism in nature)


                   Production                               Countable                                                To be included in total catch
                                                                                                                     (by traditional fishing operation)
                   Technology                               Net cage structure                                       Seeding production
                                                            Formula feed                                             Tagging
                                                                                                                     Artificial reef
                   Future trend                             Larger                                                   Survival rate improvement
                                                            Deeper                                                   Environmental control
                                                            More offshore                                            Primary production enlargement
                                                            Biotechnology                                            Biotechnology







                 118       NOAA Technical Report NMFS 106



                                                   Fish & Shellfish in Nature



                                          Spawning Ground               Catch of Adul


                                                           Egg   Collection

                                    Artificial Batch ;
                                              I       Pry & Alevin Rearing
                                          Re easing       1(       1              ---1
                                                                                  Cultivation


                                                              Monitoring
                              Artificial
                                Fishing
                                Ground                  Environmental Control
                                               I              & Management

                           Limit tion 0              Prevention of Fish Oise
                           Fishing Gear,
                           Season, Area       @-@Nutrition           Feeding
                           Production             Protection from Harmful El-=--ts
                           Control by      I
                           Total Catch
                             Fish Size     I  47E-@,cemeut of Primary Production


                           INDIRECT                                                                 RESOURCE
                           RESOURCIt        I    Fish Farming                     Aquaculture       ENHANCE-
                           MANAGEMENT                                                               MENT &
                                           I                                                        MANAGEMENTI
                    (
                         Conventional          Harvest at Sea                     Harvest from
                           Fishing                                                Fish-Preserve

                                                                                                                               Figure 3
                                                                                                                     Concept of the Marine
                                                      TOTAL     FISH    PRODUCTION                                   Ranching   System (Research
                                                                                                                     Institute  for Ocean Eco-
                                                                                                                     nomics).

                 sea. Obviously they cannot be counted except when                   in other species it is quite difficult to assess the effec-
                 they are tagged.                                                    tiveness quantitatively.
                   Released fish belong to no one individual, instead                  Both aquaculture and sea-farming are considered
                 they contribute to the increase of the fishery re-                  vital parts of the marine ranching system, but when
                 sources and can harvested by anyone. For this                       we discuss their use in this progam, we are speaking
                 reason, sea-farming is done by the public sector in                 of projects that are technically upgraded in various
                 Japan. This type of cultivation is highly dependent on              perspectives. In the case of aquaculture for example,
                 the carrying capacity of the natural sea itself. Com-               the near future may bring the use of a netless caging
                 pared with aquaculture, human measures to improve                   system, which utilizes acoustic, optical or some other
                 their growth in the natural environment have had a                  advanced technology. Moreover, large floating struc-
                 rather limited effectiveness and the artificial reef is             tures to hold net cages of required size and numbers,
                 almost the only one effective method among them.                    may be used to create offshore preserves which can
                 The final product of sea-farming is included in the                 be moved if necessary.
                                                                                                Offshore
                                                                                             Large-scale
                                                                                                Net Cage





                      :@Fi;shi-g






                 total number of fish caught by the traditional fishing                Sea-farming also needs to step into a new age with
                 operations. In the case of chum salmon, it is easy to               various environmental control technologies created
                 detect the contribution of this human effort whereas                for the system. We need multiple technologies from






                                                                                                              Nakahara: The Marine Ranching System                       119


                                   Increase in the entire quantity of marine                    Studies on an efficient integrated production
                                   resources by reducing natural enemies and                    system for each Marine product
                                   deaths.
                                   1. Increase in the            Japanese horse                       I                      Studies on new fisheries
                                   survival ratio of ftshe       mackerel                            Complex resource        systems
                                                                 i.Ae
                                   and shellfishes               16t@Z_l
                                                                                                      culture technology
                                                                 Yellow tails
                            L                                                 Bluefin tuna

                                                                              A._


                                         Masu salmon                                                      Complex production
                                   Arame                         Flounders                                technology that com-
                                   seaweed                                                                bines the properties of
                                                                                                          ecology and those of
                                                                 10 , @*
                                                                  4_@                                        waters
                                   Kajime seawee
                                   (A Igae)    Bay scallops      rt@                                                        MigT3tory species
                                   40,        Oaf                Flatfuh
                                   A                                                   PToducti 3n system
                                        Ark shells                                      technology                                  Sedentary species


                                                                                  Est2blishment of production
                                                                                  system technology for each
                                   It.                                            marine product
                                   Environment control technology                                                                   Supporting technology

                                                                                                                           Expansion in optimum
                                                                                                                           sphere of life
                                                     Seawater environment


                                                                                                           Pre rition
                                                                                                           of i ases                     Provision of a better
                                                                       Sea bottom                                                        living environment
                                                                       envuonrnent
                                                                                                      Measurint
                          Studies on the technology for                                               technology
                       Loptimizing seawater and sea
                          bottom, the places of growth
                          and life of marine creatures                                                           Monitoring for maintenance
                                                                                                                 of environment



                                                                                                Prevention or diteases of
                                                                                                fished and shellfishes



                                                                                         Figure 4
                    Flowchart illustrating the research of the Marine Ranching Program. (Research Council of Agriculture, Forestry, and
                                                                                        Fisheries).


               civil, mechanical, electric and other engineering dis-                             Marine Ranching Program
               ciplines to be developed by industrial communities
               outside of fisheries. Furthermore, to increase the                                 In the government sector, the Research Council of
               seedling survival rates in the sea, biotechnol6gy will                             Agriculture, Forestry and Fisheries, collaborating
                                   /
                                   Marne
                                   seaweed
                                   Jim       e@
                                      e
                                   A
                                               Bay 'call ps
                                                   jf                                             In 1-syste.
                                        Ark  s ell,                                               y











































               be of great help in producing healthier seed than                                  with the Fisheries Agency and its national fishery re-
               ever before. Of course the total system of marine                                  search institutes, started a 9-year research program,
               ranching should be combined within the appropriate                                 called the "Marine Ranching Program" (MRP), in
               legal and social frameworks (Fig. 3).                                              1980. During the first 3 years, the ecological study of







                        120           NOAA Technical Report NAUS 106











                                                                                                                   . .....................





                                                                                                                          ........................
                                                                                                                          ................................
                                                                                                                                               Fishery
                                                                                                                          ............. ..........
                                                                                                                                               Resources
                                                                        High


                                                         (Stage of
                                                         Nutrition)
                                                                        ...........................                                 ......................
                                                                        ..............
                                                                            ...........I.............  ............. .................
                                                                        Increase from       Increase from         Fishery          Increase of
                                                                         Aquaculture         Sea                  is=           :i;! Fishery Resources
                                                                                                 Tarming          Re urce
                                                           Low
                                                                                           ...................................     from Enhancing
                                                                    .... . .......- ................. .... ...
                                                                                                                                   Primary Production



                                                                                                                                         ...... .........................
                                                                                        Addition of               (already
                                                            Addition of                                           being used)
                                                                                        Resource
                                                                                                                                                                ....... ....... ...
                                                                                                                                         .....................................
                                                            Resource                                                                   .........................................
                                                                                        (e.g. Fry &
                                                            (e.g. net pens)
                                                                                        AJevin Releasing)
                                                                                                                                        ................... ....................
                                                                                                                  Primary Production           Enhancement of                         Primary
                                                            Surplus of Primary Production                                                                                             Production
                                                           (Bait Fish     (Utilization of Local                   (already being used%         Primary Production
                                                          Relocation)      Excess Resource)                                                    (e.g. Artificial Upwelling
                                                                                                                                               Promotion of Photosynthesis)

                                       -----------------------------------------------------------------------                       - ----------------------------------------------





                                                                                 Existing Food Chain                                                   Enlarged Food Chain
                                                                                                                                                          Naturally Generated
                                                    Increase in Harvest                                                                                   Increase in Harvest
                                                    Due to Added Resources                                        [Conventional Type]                     Due to Increase in
                                                                                                                                                          Primary Production



                                                       shoiYs increase of fishery resources. (Both sides are exaWrated for better understandiig)



                                                                                                              Figure 5
                                                                                          Structure of the marine food chain.



                        various target species and preliminary study on envi-                                             fish and algae (Fig. 4). This program has been done
                        ronmental control and disease prevention were                                                     mainly by considering the biological aspects, but
                        executed. The second 3-year period was devoted to                                                 some study to integrate it with engineering technolo-
                        the study of production systems, and the last 3 years                                             gies has also been done by a group studying the
                        to the examination of multiple-species ranching sys-                                              marine ranching system organized within the Re-
                        tems. The selected target species were masu salmon                                                search Institute for Ocean Economics (RIOE), a
                        (0. masou), horse mackerel (Trachurus japonicus),                                                 research institution which examines various ocean
                        bluefin tuna (Thunnus thynnus), flounder, bay scal-                                               uses and development and to which the author be-
                        lops, and seaweed (along with abalone and others).                                                longs. The approach used for help identify
                        Each represents a different pattern of life in the sea,                                           engineering tasks within the marine ranching system
                        thus covering the categories of highly migrating,                                                 examined by RIOE is described below.
                        open-sea migrating, nonmigrating, and sedentary






                                                                                     Nakahara: The Marine Ranching System          121






                                                               SUNLIGHT
                      NUTRIENT-CONTAINED
                      SUBSTANCE(WATER)



                               SURFACE


                                SUNLIGHT
                                                                       SUNLIGHT
                       NUTRIENTS                           NUTRIENT
                                                                       TEMPE4T
                                TEMPERA U
                                                                                     '101 .. @i-L_ 4
                                                                                              IF


                               PLANKTON






                                                                    PLANKTON








                               SEA BOTTOM
                                                                                                                Figure 6
                                                                                                    Scheme of engineering on the
                           NUTRIEN                                                                  key parameters of the food chain
                                                                                                    to enhance primary production
                                    TEMPERATURE                                                     (Research Council of Agriculture,
                                                                                                    Forestry, and Fisheries).


           Approach to the Marine Ranching                                 the nutrients and the sunlight are among the critical
           System Engineering                                              factors which create optimum environmental condi-
                                                                           tions (Fig. 6) as described in below.
                                                                             The left hand side of Figure 5 shows a direct ap-
           Food Chain Scheme Approach                                      proach to increasing resources by adding them
                                                                           directly into the sea. Aquaculture represents this ap-
           When we consider utilizing the sea's potential, or con-         proach of adding the new resource into a
           sider the addition of recoverable resources into it, a          manageable sea area so that all the added resource
           view from the perspective of the food chain offers us a         can be harvested. In the case of yellowtail, for ex-
           suggestion. Such an effort involves expanding the               ample, the feeding bait is sardine which represents
           pyramid shaped hierarchy of the food chain as shown             the fruits of primary production somewhere else in
           in Figure 5. To better understand the diagram, it               the sea. In this approach, engineers may focus on the
           should be kept in mind that both sides of the pyramid           development of large scale structures to support fish
           structure have been exaggerated somewhat.                       cages which must be strong enough to cope with the
             The right hand side shows that artificial measures            harsh environment of the deeper, more offshore sea
           should be taken in order to increase primary produc-            areas. The direct release of resources to the sea is
           tion by making use of the unused but potentially                another type of approach. In this case, we expect that
           valuable resources of the sea. One idea is to pump up           a portion of the added resource will be harvested in
           nutrient-rich deep-sea water to certain sea areas, such         the future by conventional fishing operations. This
           as serniclosed bays and thus form an artificial up-             represents sea-farming, namely, the propagation and
           welling system. Another idea is to transmit sunlight            release of juveniles. The natural environment sup-
                           .I RE
                               NTS                              I N 2TI@@
                                                          I <T
                                        T






                               PLANKTON















                               SEA BO!J






































           into the dark lower layer of the sea to activate photo-         ports this enhancement scheme if we assume that the
           synthesis. These ideas are creating great challenges            existing carrying capacity is sufficient. In this case,
           which can only be solved with new engineering tech-             less effort has been necessary from the engineering
           nologies. They are based upon the basic concept that            community"







                122      NOAA, Technical Report NMFS 106
                   (F 00 C)                    (SECURITY)                           Engineering can control the level of nutrients and
                                                                                  sunlight for baitfish. In addition, they can put im-
                      BAIT                      HARMFUL ELEMENTS                  prove security from predators by controlling
                                    SUNLIGHT                                      environmental conditions or by changing the preda-
                                        NUTRIENTS           PREDATION             tors' attention to other attractive things. Environ-
                                                                                  mental conditions such as water movement, and
                                           DISEASE          FISHING               seabottom conditions can be managed by civil and
                 BAIT SPECIES                               OPERATIONS            mechanical engineering technologies, to create favor-
                                                             IMPROVEMENT OF       able environmental conditions forjuvenile fish.
                                                             SURVIVAL RATE
                  DISSOLVED       TEMPERATURE                                     Variations of Artificial
                  OXYGEN              SEAWATE             DEPTH                   Upwelling System
                                      MOVEMENT       I
                             \SALINITY               SEABOTTOM CONDITIONS
                                           ENVIRONMENTAL                            Various artificial upwelling technologies are illus-
                                           CONDITIONS                             trated in Figure 8. Among these is a land-based
                    (HEALTH)                H 0 U S E                             system using pumped up nutrient-rich, deep seawa-
                                                                                  ter, which is already operating in Hawaii. This system
                                         Figure 7                                 is also well known as a part of OTEC (Ocean Ther-
                Critical factors for the improvement of survival of marine        mal Energy Conversion), which generates electricity
                                        resources.                                by using the temperature difference between surface
                                                                                  and deep seawater. We can therefore put the deep
                                                                                  water not only in the natural sea area but also in
                                                                                  artificially made ponds, and land-based tanks as has
                                                                                  been done in Hawaii. In Japan, a similar system ex-
                Critical Factors          roach                                   clusively for the cultivation of fish will soon start near
                                       FP                                         Cape Muroto in Kochi prefecture.
                When we turn our eyes to the vital elements, or criti-              Another unique system utilizes deep water pumped
                cal factors for fish growth and suvival, we find that             up to a surface barge. Partly intended for the direct
                several important factors can be controlled by human              use of the nearby net cages which are attached to the
                beings to some degree. In the case of chum salmon, a              barge, its primary purpose is to supply a sprinkler
                migratory species, the production increases have                  system which disperses the      .pumped up water onto
                been made by accumulating fingerling releases over                the surface to bolster primary productivity and
                many years without the use of additional specific arti-           baitfish abundance in the area. Designed to be com-
                ficial measures to control environmental factors. This            pleted within a couple of years in the Toyama Bay,
                success therefore, must also have been supported by               the system moderates the cold temperture of the
                a combination of favorable conditions like the abun-              pumped deep water taken from the depth of 300 in
                dance of food sources in the northern Pacific Ocean,              by mixing it with warm surface water.
                the rather small number of harmful elements, and                    Through this field test, the optimum ratio of cold
                the mobility resulting from the swimming capacity of              deep water to warm surface water will be surveyed
                                                                                  and its effectiveness to increase the fishery produc-
                this species.                                                     tion will be estimated. The estimation index is one of
                  However, when we consider nonmigratory and sed-                 the most important items to be examined. The bay
                entary species, the effect of predators and                       has a steep seabed, is halfway closed by Noto Penin-
                environmental factors are much more influential on                sula, and holds cold deep water rich in nutrients.
                the survival rate, especially during the juvenile stage.          This unique natural condition can only be seen in
                The critical factors for the survival of a fishery re-            Toyama Bay along the Sea of Japan coast and in
                source depends upon the species and its stage of
                growth. In addition, when we discuss engineering ap-              Suruga Bay along the Pacific coast. The Toyama Bay
                proaches, some basic considerations can be given by               project should be of great interest from both the bio-
                    SEAWATER
                    CONSTITUENTS












































                                                                                  logical  and the engineering point of view, although
                making analogies with the lives of the human beings.              many difficulties exist, including the method of the
                At the very least, we need food to eat, good health to            assessment of its effectiveness. However, this chal-
                grow, a house in which to sleep, and security to live.            lenging project is said to be the first attempt at an
                The corresponding factors for fish are bait to eat,               offshore floating-type upwelling system in the world.
                security from predators, and a comfortable chemical               Seabed civil engineering systems are approaches un-
                and physical environment (Fig. 7).






                                                                             Nakahara: The Marine Rmching System      123




                                             ON-LAND (HAWAII, KOCHI,JAPAN)



                                ARTIFICIAL_
                                POND


                                             OFFSHORE
                                             STRUCTURE


                 PIPELINE
                (PUMP up)


                                             SEMI-CLOSED
                                             SEA


                                NATURAL
                                SEA


                                            -OFFSHORE OPEN
                                             SEA           (TOYAMA,JAPAN)






                                                                            HORIZONTAL
                              -MEMBRANE -TEXTILE SCREEN (HOKKAIDO, JAPAN- FLOW

                                                                       FoRimim


                 SEA BED                                                  WNW
                 STRUCTURE
                                             ARTIFICIAL DIKE
                (FLOW CONTROL)
                                                                         H0101!

                              -GRAVITY


                                            -CONCRETE SCREEN (EHIME,JAPAN)

                                                           MARREX
                                                            Type S




                                                                          MARITEX
                                                                           Type L
                                                                                                       Figure 8
                                              PLANNING STAGE                                 Various artificial upwelling
                                                                                             systems.



           der development to control water movement. A hori-       ously unfamiliar to fishery@related people are being
           zontal method of flow control is being deployed          introduced among these efforts.
           experimentarily with a textile screen in the shallow
           water off Hokkaido. an artificial seabed dike, which
           makes use of the fly ash, is also under examination.     New Materials
           Concrete seabottorn structures which channel
           deepwater flow up to the surface have also been ex-      Materials are being introduced in the fishery-related
           perimentally employed in Aomori prefecture on a          efforts mentioned above, including fly ash, a by-prod-
           small scale. In Ehime Prefecture a greater magnitude     uct continuously produced by coal-fired power
           of scale was accomplished requiring a budget of hun-     plants, and rubber, which most people equate with
           dreds of millions of yen. A rubber dike to create an     car tires. Quite unique, new materials are also being
           artificial tideland is also being executed. Further-     used for other marine ranching engineering. Two ex-
           more, subsurface structures for the creation of          amples are described here. First is "Polyethtel," which
                                                                       X


                                                                          MAR




















           artificial seabeds that employ available sunlight are    is used to construct a man-made seaweed-like tree.
           another idea promoting the multilevel utilization of     Used in combination with a concrete artificial reef, it
           the water column (Fig. 9). Many new materials previ-     upgrades the reef's attractiveness to marine life by







           124    NOAA Technical Report MIFS 106


                                                                            Rubber Dike
                                                              Tidel d                   igh-tide
                                                                                        Ow-tide



                                                                        Rubber dike deflated











                                                                    Tideland




                                                            Shore line



                          Artificial Seabed Dike             Rubber Dike





                                                                                  7






                                                                                 Photosynthesis
                                                                       qT q      (gathering fry)
                   Sunlight collect                                   --Z Oo
                   ing b"y                                               41D Ar  Accommodation
                             ip@          Z.                                     for fish

                                 Sun ight transmission                       Accumulation of
                                       ho-se                         1j. r4 _L_I
                                                                         .L L4 4 sunken orp
                                                                                      ,anisms

                                        de   ter sunlight
                                          rradiation device


                       Sunlight Transmission System                     Ii T T
                   A
                                       gh  _s'
                                          tr
                                  e ih_ t Le
                                        s


                                        de
                                          r7a d I.







                                                              STSS (Single-point Tension-leg
                                                                  Subsurface Structure)


                                                      Figure 9
                                          Examples of marine ranching technologies.






                                                                                           Nakahara: The Marine Ranching System            125




                                                                                       41













                                          Frame of Artificial Reef                          Completion of Artificial Reef




                                                                       is
                                                                                                                 40










                                                                                                           A







                                                       Attached Artificial Seaweed made by Polyethtel
                                                              (Brand Name is "Bush-Absorber")








                                             S












                                                     . ... . .. . ... . .....
                                         Installing Operation                                Installed on the Seabed

                                                                          Figure 10
                         Artificial seaweed forest formation using new materials (Wakachiku Construction Co., Ltd., Tokyo, Japan).







              126       NOAA Technical Report NMFS 106


                                                   40-












                                     Net Type                                        Mound-Covering Type





                                                                                          4W





                                                                                ID











                                   Suspended-Board Type                          Multi-Board Type


                                                                   Figure I I
                      Artificial seaweed forest formation using new materials (Chateau Marine Survey Co., Ltd., Miyakojima,
                                                                 Osakajapan).




              providing a substitute for the attachment of algae            Engineering Measures Based
              (Fig. 10). This effort was made five to eight years ago.      upon Biological Requirements
              The other example is a composite material of poly-
              ethylene and calcium carbonate that is made into              Two examples of the examination process used to
              twine and boards. The twine is used to weave a net-           model the development of engineering technologies
                                    Y




















              type structure for seaweed attachment, while the              which will contribute to the increase of resources are
              boards are used to create an artificial seabed at any         shown in the cases of flounder and ark shells (Tables
              depth. Any number of decks can be installed in mid-           2 & 3). The charts seek to identify critical factors
              water by anchoring or pile fixing (Fig. 11). This new         which determine survival, growth, and reproductive
              material was patented recently.                               rates. During the transformation period of flounder






                                                                                                         Nakahara: The Marine Ranching System                    127


                                                                                      Table 2
                                                  Basic idea of engineering measures upon biological requirements
                                                                     Flounder (Paralichthys olivaceus).


                                                                                                                 Flounder                   Other
                                                                    Habitat                                      (Paralichthys              species
                  Life stage                          Seawater                      Seabottom                    olivaceus)

                  Larval                       Prevention of dispersion
                     (floating life)           (by controlling seawater
                                               movement)


                  juvenile                                                       Improvement of                                         Protection
                     (seabottom life)          (Same as above)                   seabottom condition             Supply of bait         from predators

                  Adult                                                          (Same as above)                 (Same as above)

                  Spawning                                                       (Same as above)                 (Same as above)







                                                                                      Table 3
                                                  Basic idea of engineering measures upon biological requirements
                                                                     ark shell (Scapharca broughtonii).


                                                                                                                 Flounder                   Other
                                                                    Habitat                                      (Paralichthys              species
                  Life stage                          Seawater                        Seabottom                  olivaccus)

                  Floating                   Prevention of dispersion
                                             (by controlling seawater
                                             movement to accumulate
                                             larvae.)                                                            Supply of bait

                  Attaching and              (Same as above)                     Formation of attaching          (Same as above)        Protection from
                  transformation to                                              structures                                             predators
                  sedentary type


                  Seabottom                                                      Improvement of                  (Same as above)
                                                                                 seabottom condition


                  Spawning                                                       (Same as above)                 (Same as above)        Protection from
                                                                                                                                        predators




               from the floating life stage to the seabottom life                             case of ark shells, similar approaches could be con-
               stage, bait species change and water currents fre-                             sidered.
               quently disperse eggs and larvae to unfavorable
               seabottom conditions. Both are critical situations that
               affect the mortality of juveniles. These requirements,                         Conclusion
               determined by biological study, suggest that engi-
               neering measures should be taken to control the                                The planning and development of engineering tech-
               seawater movement in order to prevent egg disper-                              nologies should basically be supported by biological
               sion and to improve the seabottom condition. In the                            studies, otherwise successful results can not be ex-







                128        NOAA Technical Report NXM 106

                pected nor obtained. All aspects of the engineering                      JEA Uapan Fisheries Association)
                must have a biological reason and endorsement,                                1987. Fisheries of Japan 1987. (In English.)
                Even then, the engineering side must be always                            MAFF (Ministry of Agriculture, Forestry, and Fisheries)
                                                                                              1987. Annual Report on Japan's Fisheries, Fiscal 1986,
                humble to accept the demands of nature. Biologists                               Summary. (In English.)
                must also keep themselves open- minded to the new                         Research Committee on marine ranching system (Research Insti-
                technologies in other industrial communities. Mak-                          tute for Ocean Economics)
                ing use of industrial engineering capabilities for the                        1981. Model study on Environmental Monitoring system for
                development of enhanced fishery' resources is of                                 marine ranching. (In Japanese.)
                                                                                              1982. Marine Ranching Program: enlargement method of fa-
                great significance for marine ranching systems.                                  vorable environmental conditions and monitoring system.
                                                                                                 (Injapanese.)
                                                                                              1983. Marine Ranching System: a model study. (In Japa-
                Acknowledgment                                                                   nese.)
                                                                                              1984. Marine Ranching System: a Preliminary assessment.
                                                                                                 (Injapanese)
                This paper is based        upon an intensive study being                      1985. Study on enrichment method of the marine ranching
                done by the research committee on the marine                                     ground. (Injapanese.)
                ranching system, which was organized in 1981, and                             1986. Study on ranching ground use development by cultiva-
                which is still active now. This committee has been                               tion technology (1), with 1) development of data based on
                working together under a research contract with the                              marine ranching, 2) examination on the integration of biol-
                Research Council on Agriculture, Forestry, and Fish-                             ogy and engineering technologies. (In Japanese.)
                                                                                              1987. Study on ranching ground use development by cultiva-
                eries, which consists of some thirty companies among                             tion technology (11) with examination on the integration of
                the 120 member firms of the RICIE.                                               biology and engineering technologies. (In Japanese.)
                                                                                              1988. Study on ranching ground use development by cultiva-
                                                                                                 tion technology (III) with examination on the integration
                Citations                                                                        of biology and engineering technologies. (Injapanese.)

                DS1 (Department of Statistics and Information)
                     1984. Fisheries statistics ofjapan 1984, DSI. Ministry of Ag-
                       riculture, Forestry, and Fisheries, Government of Japan. (In
                       English.)






                             Economic Problems of the Marine Ranching System


                                                                   KATSUO TAYA

                                                       National Research Institute ofFisheries Science
                                                                5-5-1 Kachidoki, Chuo-ku
                                                                    Tokyo 104, Japan

                                                                AKIRA HASEGAWA

                                                                Tokyo University of Fisheries
                                                                 4-5- 7 Konan, Minato-ku
                                                                    Tokyo 108, Japan

                                                                  YOSHIO MASUI

                                                              Tokyo University of Agriculture
                                                              1-1-1 Sakuragaoka, Setagaka-ku
                                                                    Tokyo 156, Japan



                                                                     ABSTRACT


                                 The major challenge facing the Marine Ranching Project is to increase fishery produc-
                              tion in an economically efficient manner. To accomplish this, new technology such as
                              improved seed production methods is required, along with an integrated approach that
                              includes well planned development of local economies as well as total resource manage-
                              ment and harvest regulation. This report analyzes the work of the Marine Ranching
                              Project on the production and release of masu salmon (Oncorhynchus masou) by examin-
                              ing the relationship between production and release costs and the rate of smoltification.
                              In addition, survey data show that most of the masu salmon taken in Hokkaido from
                              October to March are juveniles under 600 g, and that the economic value of the fishery
                              could be raised considerably if the target fish were allowed to reach an adult size of 1,500
                              g before harvesting. The report also notes that the masu salmon fishery plays an impor-
                              tant role in the regional economies of northern Japan, and that seed release and harvest
                              regulations must be designed to meet the economic and social needs of the various
                              regions and communities that depend heavily on the resource.



            Introduction                                                       This report also analyzes the market price mecha-
                                                                               nism for masu salmon and evaluates the economic
            The most important tasks now facing the masu                       benefits of managing the fishery through restrictions
            salmon (Oncorhynchus masou) Marine Ranching                        on the harvest of undersized immature fish. Finally,
            Project are the economically efficient production and              the report reviews the role of the masu salmon
            release of smolt seed. At present, many researchers                fishery in the northern prefectures of Hokkaido,
            are developing new seed production methods and                     Aomori, Iwate, Akita, and Niigata and discusses ways
            studying the ecology and physiology of masu salmon.                of coordinating the Marine Ranching Project with lo-
              This report analyzes three methods of masu                       cal variables such as types of fishing gear, level of
            salmon seed production and release: autumn release                 fishing effort, and fishery production costs. Data on
            of 1+ year seed obtained from wild fish (hereafter                 monthly harvest rates and average fish body weights
            referred to as autumn release); spring release of I+               were collected from the Prefectural Fisheries Experi-
            year seed obtained from wild fish (spring release);                mental Stations of these governments.
            and spring release of 0+ year seed obtained from                      The research was divided into the following seg-
            hatchery@raised fish (hatchery spring release). The                ments: 1) Comparative study of masu salmon seed
            economic merits, demerits, and other problems asso-                production costs at the Prefectural Hatchery Stations
            ciated with each method are analyzed and discussed.                at Iwate; 2) Economic comparison of autumn release,

                                                                                                                                          129







                    130          NOAA Technical Report N?M 106



                                                                                               Table I
                                            Seed production cost of masu salmon per fish (in yen).                  (Iwate Prefecture Office 1974.)

                                                                        Autumn                               Spring                      Hatchery spring
                                                                          release                            release                          release


                         Eggs                                               2.0                                2.0                               2.0

                         Feed (1.5)'                                        4.69                               9.38                              7.5
                              (2.5)'                                        7.81                              15.63                             12.5

                         Power/fuel                                         0.75                               2.0                               1.2

                         Consumable items                                   0.75                               1.5                               1.2

                         Labor                                              5.28                               6.36                              4.2

                         Transportation                                     1.25                               2.5                               2.0

                         Maintenance                                        2.0                                8.0                               3.2

                         Survival ratio                                     0.5                                0.4                               0.6

                         Total cost per fish
                           (1.5)                                            18.72                             34.74                             22.63
                           (2.5)                                            21.84                             40.99                             27.63

                         aParentheses contain feed conversion ratios of 1.5 (minimum) and 2.5 (maximum).

                           Conversion ratio = (A-B)/C X 100,

                         where A= Final weight (g/fish)
                                 B = Start weight
                                 C = Feed weight.





                                100                     AuUmn release                                     Spring release         100 -                Hatchery spring release
                                                                                 100-
                                                 Feed conversion rate
                                                        a7-1.5
                                                        b=2.5
                         4@
                         2                              c--b-a
                         U
                         8       50                                              50                                               50

                                                                                                                           b
                                                                                                                          a
                                                                                                                                                                            b
                                                                            b                                                                                               a
                                                                        a           ---------
                                                                                                                    I      c                                                C
                                    0                   so       80     100         0                  50        80    100(%)        0                 50         80     100 W

                                          Smaltification ratioW


                                                                                               Figure I
                    Ratio of seed production costs to the sinoltification ratio in masu salmon. Solid lines (a and b) represent various feed
                                                          conversion rates: a = 1.5; b @ 2.5. Line c indicates the values for b-a.


                    spring release, and hatchery spring release methods                                  regions with attention paid to fish size and the unit
                    for the production and release of masu salmon, fo-                                   weight market price; and 4) Investigation of fishing
                                                        A

                                                    ee,
                                                        u

                                                        con

                                                        a-
                                                          v
                                                          -1
                                                           er
                                                               5
                                                               re

                                                               s
                                                               lea

                                                               to"


                                                        b=2    5
                                                                  se
                                                                    rate                                  Spring    release                   ,Llatclery spring rel,
                                                          _b   a








                                                                                                       .J_
                                                                                                      .j
                                                                                                                                              ........ .

                                                                            b










                    cusing on smoltification and recapture ratios; 3)                                    gear, fishing effort and fishery production costs for
                    Collection of distribution and price data in various                                 masu salmon in various regions.






                                                                                Taya et al.: Economic Problems of the Marine Ranching System                        131
                                                                                                Analysis of Economic Efficiency

                                                                                                The approximate seed production costs expected us-
                     :::o@  7                                                                   ing each of the three methods are shown in Table I
                     0                                  (A) Autumn release                      (Iwate Prefecture Office 1974). Costs are lowest for
                     U1                                                                         the autumn release method and highest for the
                                                        (B) Spring release
                            6                                                                   spring release method. The most important consider-
                                                        (C) Hatchery spring release             ation, however, is the combined production and
                     C
                     r_                                                                         release costs per fish in relation to the smoltification
                            5
                     41                                                                         ratio which is graphed for each method in Figure 1,
                                                                                                where each curve indicates a different feed conver-
                     0                                                                          sion rate. If the smoltification ratio (x) is assumed to
                            4
                                                                                                be constant between methods and varies between 50
                                                                                                and 80%, the costs per fish are as follows:
                     U      3                                               B
                                                  r - -                                            between 18.72/x and 21.48/x yen for the autumn
                                                 J-                                                release method,
                            2
                                                                            C                   0  between 22.63/x and 27.63/x for the hatchery
                                                                            A
                                                                                                   spring release method, and
                                                                                                0  between 34.74/x and 40.99/x yen for the spring
                                                                                                   release method.

                                                                                                  In actual practice, however, the smoltification ratio
                            0                    50            80       100                     tends to be around 50% for the autumn and spring
                                        Smoltification ratio                                    release methods, and around 80% for the hatchery
                                                                                                spring release method. AVhen this is taken into con-
                                                                                                sideration, the spring hatchery release method is
                                                                                                shown to be the most economically efficient in terms
                                              Figure 2                                          of production and release costs.
                Percentage    of returning adults needed in relation            to the            Production and release costs are only one part of
               smoltification ratio. A=autumn release; B=spring release;                        the overall economic aspects of the Marine Ranching
               and C=hatchery spring release.







                                                                                       Table 2
                                                   Fry production cost and value of returning fish for chum salmon.



                                    Investment               Production                                                                        Production cost of
                                      salmon                cost (yen)           Cost of returning salmon (yen)             Market price       returning salmon
                                     ranching               of salmon                                                        (yen/kg) of         per kg/market
                     Year group    (million yen)             fry/fish                per fish             per kg          returning salmon      price X 100(%)



                     1962-65          374-540                 0.99-1.60            86.7-241.9            24.8-69.1             @360-545              5.8-13.1
                     Average                465                    1.35                  16.51                 47.2                   464                9.95

                     1966-70          476-767                 1.34-2.90            70.5-126.0            20.1-36.0               439-725              3.0-6.2
                     Average                619                    1.91                   85.7                 24.5                   586                4.29

                     1971-75         992-2489                 1.72-3.10            76.8-139.6            21.9-36.3             609-1029              2.5-5.76
                     Average              1455                     2.58                  111.6                 31.9                   891                3.72

                     1976-79        2423-5806                 4.02-6.65                                3.5 kg/fish
                     Average              3698                     5.04







                  132         NOAA Technical Report NMFS 106



                                                                                         Table 3
                                  Seed production cost and value of returning fish for masu salmon. (Iwate Prefecture Office 1974.)

                                                                                  Cost (yen) of         Cost (yen) of          Market price           Cost of return
                                          Production cost (yen) Return           returning masu       returning masu           of returning       masu salmon/Unit
                      Type                        per fish           ratio       salmon per fish       salmon per kg           fish (yen/kg)        price x 100

                      Autumn release                18.72               x             18.72/x              12.48/x                  1500                   0.83/x
                      Spring release                34.72               x             34.74/ x             23.16/ x                 1500                   1.54/x
                      Spring hatchery
                       release                      22.63               x             22.63/x              15.09/x                  1500                   1.01/x






                          30                                    (A) Autumn release

                                                                 (B) Spring release

                                                                 (C) Hatchery spring release


                      o   20







                      0
                      U
                          10




                                                                                                  B
                                                                                                  C
                                                                                                  A

                                                    10                                        30     %                                  Figure 3
                                                      Recovery ratio                                            Relationship of the recovery ratio to the cost/
                                                                                                               value ratio for masu salmon (Iwate Prefecture
                                                                                                                Office 1974.)


                  System. Recapture ratios also play a vital role. If the                           Figure 2 plots smoltification ratio versus recovery
                  market price of recaptured fish is assumed to be                                ratio for each method. From the data in Figure I it
                  2,250 yen/fish, fishery production costs are assumed                            appears that the hatchery spring release method,
                  to be negligible, the marketing commission rate is                              with an 80% smoltification ratio as compared to only
                  assumed to be 5%, and the conversion ratio assumed                              50% for the other two methods, is most efficient. In
                  to be 2.0, then the upper and lower ranges of the                               reality, however, the recovery ratio for this method
                  recovery ratio (y) for each method (as it varies with                           tends to be lower than for the other two, and thus
                  the smoltification ratio x) can be calculated as fol-                           research is required to improve the recovery ratio for
                  lows:                                                                           spring hatchery release fish.
                  ï¿½   autumn release                                                                One way of evaluating the economic efficiency of
                      method                      y   257.5/x and y = 94.9/x,                     the Marine Ranching Project is to compare produc-
                  ï¿½   spring release                                                              tion and release costs with the value of the recovered
                                                                                                  fish. Production and release costs as a percentage of
                      method                      y   480.8/ x and y = 177. 1 / x,                the unit weight value of recovered fish for chum
                  ï¿½   hatchery spring                 and                                         salmon (0. keta) are shown in Table 2. Because pro-
                      release method              y   319.2/x and y = 117.6/x                     duction costs are a small fraction of the value of a







                                                               Taya et al.: Economic Problems of the Marine Ranching System        133


                                                                                       50
                                                                                       7o  B      Toyama
              80                                                                               @\kNiigata
              % A                                                                      40
              60-                                                                                      V\@          AVfta
                                      Aomori                                           3

                                                                                                                Ishikawa

              40-                                                                      20

                                            Hokkaido




              20                                                                       10
                                                 Iwate

                             7

                                                     15    17    13    7     5
                 J           M     A     M     J     J     A     S     0     N     D      J     F     M     A     M     J      J
                                   Month                                                                Month

                                                                    Figure 4
           Monthly harvest patterns for masu salmon in 1986. (A) shows average body weight of salmon harvested in Hokkaido and
           Honshu prefectures (X 100 g). D = Aomori; + = Hokkaido; X = lwate. (B) shows average body weight of sal.mon harvested
           in Honshu prefectures; El = Niigata; 9 = Akita; X = Toyama; 7 = Ishikawa.


           returning salmon, a recovery ratio of even 2% pro-              economic value of the fishery can be obtained if the
           duces an increase in the economic value of the                  juveniles harvested in Hokkaido are allowed to ma-
           fishery. The same calculations are shown in Table 3             ture before being harvested elsewhere. Calculations
           for masu salmon for each release method; these re-              show that if the fishery were closed from November
           sults are graphed -in Figure 3. If the target figure of a       to February, an increase of 23% (from 7.6 billion yen
           15% recovery ratio were achieved for all methods,               to 9.4 billion yen) could be obtained (Fig. 6). If the
           then the cost/value ratio (cost per returning salmon,           closure were extended to include the entire period
           value is market price pe-r  -fish) would vary between 5         from October to March, the increase would rise to
           and 10%, which is a desirable result. Even if the ratio         31% (from 7.6 billion yen to 10.0 billion yen). A
           is calculated at the present recovery rate of 5%, the           problem arises in that fishermen in Hokkaido and
           cost/value ratios would vary from 15-30%, indicating            elsewhere depend heavily on income from sport
           that artificial seed production projects have the po-           fishing during this period and would certainly op-
           tential for contributing to the masu salmon fishery.            pose any move to close or restrict the fishery. Thus,
              Seasonal harvest patterns of masu salmon for se-             an integrated plan is required that maximizes the
           lected prefectures in 1986 are shown in Figure 4. In            overall economic value of the fishery while protect-
           Hokkaido, most of the fish harvested between Octo-              ing the interests of local harvest groups.
           ber and March are juveniles weighing under 600 g.
           The current market value of such undersized masu
           salmon is only 500 yen/kg (Fig. 5). The market value            Role of Masu Salmon in Local
           of adult fish over 1,500 g, however, rises to 1,500 yen/        and Regional Economies
           kg. Thus, if these undersized juveniles are allowed to
           mature before harvesting, the value per fish will rise          Our investigations sho    wed that although there is a
           from 250 yen (500 g at 500 yen/kg) to 2,250 yen                 noticeable lack of stability in catch volume, masu
           (1,500 g at 1,500 yen/kg). These figures represent a            salmon play an important role in both the local and
           nine-fold increase in value. Obviously, an increase in          regional economies of Hokkaido, Aomori, Iwate, and







                 134      NOAA Technical Report NNM 106



                       2000







                                                                                                        March

                        1500 -                                                                         February

                                                                                                        January








                        1000




                                                              Log(p)-2.484+G.625Log(BW)



                         500                                                                                                    Figure 5
                             0              500            1000            1500            2000                       Relationship between price
                                                                                                                      and weight of masu salmon
                                                               Bodyweight                                             (Kamuenai Fisheries Coop-
                                                                                                                      erative 1987).



                       1000                                                      FN Sport fishing income
                                                                                 XA
                                                                                 F@@ Commercital fishery income

                        800

                               760



                    4)

                        600-


                    E

                    4)
                    E
                    0    400-
                    U






                         200-




                                                                                                                                   Figure 6
                            0--                                                                                            Evaluation of benefits
                                     Present        Closed         Closed                                                  of fishery restrictions
                                                    Nov-Feb        Oct-Mar                                                 in (Hokkaido Salmon
                                                                                                                           Hatchery 1988).






                                                                                    Taya et al.: Economic Problems of the Marine Ranching System                             135



                                                                                            Table 4
                                                    Estimated landings of masu salmon injapan (1984) (in metric tons).

                                                        Japan Sea                     Pacific Ocean           Nernuro          Okhotsk                  Total
                                              Hokkaido            Honshu         Hokkaido        Honshu         Straits           Sea       Hokkaido          Honshu


                   Off shore
                   fishing                        187.6            744.1           418.3             -              -             -             605.9           744.1
                   Coastal
                   fishery                        562.4           1162.4           224.0          143.4             2.1           43.5          832.0         1305.8
                   Total
                   landings                       750.0           1906.5           642.5          143.4             2.1           43.5         1437.9         2049.9
                   Each
                   area                                    2656.5                           785.7                   2.1           43.5                 3487.9
                   Percentage
                   M                                       76.17                            22.53                   0.06          1.25                  100.0






                                1200                                                                                                                       18.0


                                1100 -                                                                                                                     -16.5
                                                              Aomori
                                1000 -                                                                                                                     -15.0

                                900                                                                                                                        13.5
                                                                                                                        Hokk "d
                                800                                                                                                                        12.0


                                700                                                                                                                        .10.5


                                600                                                                                                                        -9.0
                         E
                                                                                                                                                                    0
                         -a     500 -                                                                                                                      -7.5    A
                         >


                                400                                                                                                                        6.0
                                                       Akita                                                                                                       >
                                300 -                                                                                                                      4.5

                                200 -                                                                               Niigata                                -3.0
                                                      Iwate
                                100                                                                                                                        -1.5



                                           1976        77         78        79         80        81        82         83         84         5        86


                                                                                            Year


                                                                                          Figure 7
                      Masu salmon landings of the coastal fisheries by weight (metric tons) and value (value assumed at 1,500 yen/kg).



               Niigata prefectures. The total annual catch fluctuates                                  Hokkaido accounted for 41% (1,438 t) and
               between 3,500 and 4,000 metric tons (t), the average                                  Honshu for 59% (2,050 t) of the total harvest. Sev-
               price varies from 1,000 to 1,500 yen/kg, and the total                                enty-six percent (2,656 t)           of the catch was harvested
               value of the fishery ranges from 35 to 60 billion yen/                                in the Japan Sea, 22% (785 t) in the Pacific Ocean,
               year. The geographical distribution of the catch for                                  1.2% (43 t) in the Sea of Okhotsk, and 0.06% (2 t) in
               1984 is shown in Table 4.                                                             the Nemuro, Straits region. Clearly, the Japan Sea







                  136         NOAA Technical Report WEN 106



                                                                                          Table 5
                      Family fishery income 1985 (1,000 yen). t = metric tons. Ministry of Agriculture, Forestry, and Fisheries, government of
                                                                                     Japan (1985).

                      Family                                      fishing                         fishing                       fishing              net income
                      fishery                                gross income                       expenses                      net income                rate

                      Japan Sea area
                      I t (under)                                  1,557                              667                          890                     57.1
                      1-3 t                                        3,017                           1,641                        1,376                      45.6
                      3-5 t                                        7,208                           4,636                        2,572                      35.6
                      5-10 t                                      14,859                          11,786                        3,073                      20.6
                      Bag net                                      6,668                           4,410                        2,258                      33.8
                      Average                                      4,212                           2,680                        1,532                      36.3


                      Pacific Ocean area
                      I t (under)                                  1,859                              773                       1,076                      57.8
                      1-3 t                                        8,581                           1,301                        2,280                      63.6
                      3-5 t                                        7,470                           4,415                        3,055                      40.8
                      5-10 t                                      10,926                           7,219                        3,707                      33.9
                      Bag net                                      5,544                           2,611                        2,933                      52.9
                      Average                                      4,220                           2,241                        1,979                      46.8





                  fishery is by far the most important, accounting for                            masu salmon is none the less a valuable local re-
                  40 billion yen in total value as compared to 8-12 bil-                          source and an important ingredient in many
                  lion yen for the Pacific Ocean fishery.                                         traditional seafood dishes. Family income by vessel
                      At the national level, coastal fisheries harvested                          size is shown in in Table 5 for fishing families in the
                  39% (1,349 t) and offshore fisheries 61% (2,137 t) of                           northern Japan Sea and northern Pacific Ocean re-
                  the total catch. The figures for the Pacific Ocean.                             gions (MAFF 1985). Net family income from fisheries
                  fishery, were 53% (418 t) for coastal and 47% (367 t)                           varies from 0.890 to 3.073 million yen in the Japan
                  for offshore areas; while in the Japan Sea the major                            Sea, and from 1.076 to 3.707 million yen in the Pa-
                  percentages were reversed, with coastal yields 35%                              cific Ocean. Because the market price of masu
                  (931 t) and offshore harvests at 65% (1,724 t). Catch                           salmon is relatively high, this fish represents an im-
                  statistics by region and fishery are shown in Table 4,                          portant source of income for these fishing families.
                  and Figure 7 graphs the volume and value of land-                               Thus, investment in masu salmon marine ranching
                  ings by prefecture for the 10-year period from 1976                             facilities will be a crucial element in developing the
                  to 1986.                                                                        local and regional economies in these areas.
                      Fishing methods for masu salmon were found to
                  vary widely, and include gillnet, bottom trawl,
                  longline, pole and line, and setnet techniques. In the                          Citations
                  Japan Sea region of Hokkaido (e.g., Shakotan Penin-
                  sula area) sportfishing was found to be an important                            Hokkaido Salmon Hatchery.
                  source of income for about 45 vessels. This                                          1988. Annual report of the Hokkaido salmon hatchery. (In
                  sportfishing industry harvests a large percentage of                                   Japanese.)
                                                                                                  Iwate Prefecture Office.
                  undersized juveniles, and as such adversely affects the                              1974. Exploitation program of masu salmon, p. 24-25. (In
                  efficiency of the masu salmon Marine Ranching                                          Japanese.)
                  Project.                                                                        Ministry of Agriculture, Forestry, and Fisheries.
                      Although landings in prefectures such as                                         1985. Annual report on fisheries economics (family
                  Yamagata, Ishikawa, and Toyama are relatively small,                                   fishery). (Injapanese.)








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