Marine ranching proceedings of the eighteenth U.S.-Japan Meeting on Aquaculture, Port Ludlow, Washington, 18-19 September 1989

[From the U.S. Government Printing Office, www.gpo.gov]

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
SH117
A44672

n
_10
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

cim

4-
LIBRARY
77-17, 7

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e co 1990 HOBSON AVE.
LU
CHAS. SC 29408-2623

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-
mend or endorse any proprietary product or proprietary material mentioned
in this publication. No reference shall be made to NMFS, or to this publica-
tion furnished by NMFS, in any advertising or sales promotion which would
indicate or imply that NMFS approves, recommends or endorses any pro-
pfietary product or proprietary material mentioned herein, or which has
as its purpose an intent to cause directly or indirectly the advertised product
to be used or purchased because of this NMFS publication. The U.S.-
Japan subscries of NOAA Technical Reports on aquaculture is used to
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

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 � 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 � 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 � 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

8 NOAA Technical Report NNOFS 106

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.
sociated endocrine patterns in penaeid shrimp. An: 1961. Chemoreception and thermoreception. In The physi-
Antennule; Br: Brain; Xo-Sg: X organ-sinus gland ology of Crustacca Vol. 2 (T.H. Waterman, ed.), p. 109-
complex; Th: Thoracic ganglion; Fc: Follicle cell; Oo: 131. Acad. Press, NY.
Oocyte; Ov: Ovary; BH: Brain hormone; GIH: Go- Beard, T.W., and J.F. Wickins.
nad-inhibiting hormone; GSH: Gonad-stimulating 1980. Breeding of Penaeus monodon Fabricius in laboratory
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
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implantation could stimulate vitellogenesis in the spi- 344.
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17S

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Yano: Hormonal Control of Reproductive Maturation in Shrimp 13

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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-

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

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 � 2.3 35.8-- 2.2
Nontagged 32.8 _-2.1 35.7 2.0

_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 � 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-

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.

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':

M X

"A'

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.
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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:

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.

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

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

50 NOAA Technical Report NAM 106

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 � 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 � 0.1 mm), became
controls on day 8. The increase in plasma estradiol larger and transparent (2.2 � 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 � 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 � 7.1 % for 8:59-80.
eggs from captive induced females and 52.6 � 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 � 17.6% (wild) industry. In Proceedings of the 4th Alaska Aquac. Conf.;
and 14.5 � 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.

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.)

Right Valve

Gonad -

Foot

Mantle

A..

-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

-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

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,@

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

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@,

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

% 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

% 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

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

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

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

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

0 W
60
0 E

0
50

0 40 0

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.

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.

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

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

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

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

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
crustacea. Am. Zool. 25:197-206.
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96 NOAA Technical Report NNOS 106

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98 NOAA Technical Report NMFS 106

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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

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

?
*J&
oil Jor
Or

41-

7, TV

lop,

@5
_,Jp
Alk

_00@
IT

a A "*4-
A
A",

-4,

IF
Ae

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

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

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

10
W
-C

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

LONG-
DISTANCt

OFFSHORE

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%)
.......................

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

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

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

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

Frame of Artificial Reef Completion of Artificial Reef

is
40

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

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

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
........ .

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

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.
� 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-
� 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
� 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

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

600-

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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form of maps showing distribution of rainfall, chemical
and physical conditions of oceans and atmosphere, TECHNICAL MEMORANDU MS- Reports of
distribution of fishes and marine mammals, ionospheric preliminary, partial, or negative research or technology
conditions, etc. results, interim instructions, and the like.

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Information on availability ofNOAA publications can be obtainedftom:
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U.S. Department of Commerce
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
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3 6668 00003 7384
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