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



                           L ILI @TE


          NOAA Technical Repoi-t NMFS 92                                            November 1990


                           Genetics in Aquaculture

                           Proceedings of the Sixkenth
                           U.S. -Japan Meeting on Aquaculture
                           Charleston, South Carolina
                           October 20 and 21., 1987


                           Ralph S. Svrjcek (editor)


























                           U.S. Department of Commerce


    SH11
    .A44672
    no.92










                  NOAA Technical Report NMFS


                       The major responsibilities of the National Marine Fisheries Service (NMFS) are to monitor and assess the abundance and geographic distribution
                  of fishery resources, to understand and predict fluctuations in the quantity and distribution of these resources, and to establish levels for their optimum
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                  surance and vessel construction subsidies. It collects, analyzes, and publishes statistics on various phases of the industry.
                       The NOAA Technical Report NMFS series was established in 1983 to replace two subcategories of the Technical Reports series: "Special Scientific
                  Report-Fisheries" and "Circular." The series contains the following types of reports: Scientific investigations that document long-term continuing
                  programs of NMFS; intensive scientific reports on studies of restricted scope; papers on applied fishery problems; technical reports of general interest
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                       Copies of NOAA Technical Reports NMFS are available free in limited numbers to governmental agencies, both Federal and State. They are also
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                  and may be reprinted entirely, reference to source is appreciated.



                  69. Environmental quality and aquaculture systems: Proceedings of the                  Beverly M. Vinter. October 1989, 651 p.
                  thirteenth U.S.-Japan meeting on aquaculture, Mie, Japan, October 24-25,
                  1984, edited by Carl J. Sindermann. October 1988, 50 p.                                81. Catch-per-unit-effort and biological parameters from the Massachu-
                                                                                                         setts coastal lobster (Horwrus ameriranus) resource: Description and Trends,
                  70. New and innovative advances in biology/engineering with potential                  by Bruce T. Estrella and Daniel J. McKiernan. September 1989, 21 p.
                  for use in aquaculture: Proceedings of the fourteenth U.S.-Japan meeting
                  on aquaculture, Woods Hole, Massachusetts, October 16-17, 1985, edited                 82. Synopsis of biological data on the cobia R"hycentron canadum (Pisces:
                  by Albert K. Sparks. November 1988, 69 p.                                              Rachycentridae), by Rosalie Vaught Shaffer and Eugene L. Nakamura.
                                                                                                         December 1989, 21 p.
                  71. Greenland turbot Reinhardliushippoglossoidesof the eastern Bering Sea
                  and Aleutian Islands region, by Miles S. Alton, Richard G. Bakkala, Gary               83. Celaphopods from the stomachs of sperm whales taken off Califor-
                  E. Walters, and Peter T. Munro. December 1988, 31 p.                                   nia, by Clifford H. Fiscus, Dale W. Rice, and Allen A. Wolman. Decem-
                                                                                                         ber 1989, 12 p.
                  72. Age determination methods for northwest Atlantic           species, edited
                  by Judy Perittila and Louise M. Dery. December 1988,           135 p.                  84. Results of abundance surveys of juvenile Atlantic and Gulf menha-
                                                                                                         den, Brevoortia Vannus and B. patrunus, by Dean W. Ahrenholz, James F.
                  73. Marine flora and fauna of the Eastern United States. Mollusca:                     Guthrie, and Charles W. Krouse. December 1989, 14 p.
                  Cephalopoda, by Michael Vecchione, Clyde F. E. Roper, and Michael
                  J. Sweeney. February 1989, 23 p.                                                       85. Marine farming and enhancement: Proceedings of the Fifteenth
                                                                                                         U.S.-Japan Meeting on Aquaculture, Kyoto, Japan, October 22-23, 1986,
                  74. Proximate composition and fatty acid and cholesterol content of 22                 edited by Albert K. Sparks. March 1990, 127 p.
                  species of northwest Atlantic finfish, by Judith Krzynowek, jenny Mur-
                  phy, Richard S. Maney, and Laurie J. Panunzio. May 1989, 35 p.                         86. Benthic macroiatina and habitat monitoring on the continental shelf
                                                                                                         of the northeastern United States. 1. Biomass, by Frank Steimle. Febru-
                  75. Codend selection of winter flounder Pseudopleuronectes amen@anus, by               ary 1990, 28 p.
                  David G. Simpson. March 1989, 10 p.
                                                                                                         87. Life history aspects of 19 rockfish species (Scorpaenidae: Sebastes) from
                  76. Analysis of fish diversion efficiency and survivorship in the fish return          the Southern California Bight, by Milton S. Love, Pamela Morris, Mer-
                  system at San Onofte Nuclear Generating Station, by Milton S. Love,                    ritt McCrae, and Robson Collins. February 1990, 38 p.
                  Meenu Sandbu, Jeffrey Stein, Kevin T. Herbinson, Robert H. Moore,
                  Michael Mullin, and John S. Stephens Jr. April 1989, 16 p.                             88. Early-life-history profiles, seasonal abundance, and distribution of
                                                                                                         four species of clupeid larvae from the northern Gulf of Mexico, 1982 and
                  77. Illustrated key to the genera of free-living marine nematodes of the               1983, by Richard F. Shaw and David L. Drullinger. April 1990, 60 p.
                  order Enoplida, by Edwin J. Keppner and Armen C. Tarjan. July 1989,
                  26 p.                                                                                  89. Early-life-history profiles, seasonal abundance, and distribution of
                                                                                                         four species of carangid larvae off Louisiana, 1982 and 1983, by Richard
                  78. Survey of fishes and water properties of south San Francisco Bay,                  F Shaw and David L. Drullinger. April 1990, 37 p.
                  California, 1973-82, by Donald E. Pearson. August 1989, 21 p.
                                                                                                         90. Elasmobranchs as living resources: Advances in the biology, ecology,
                  79. Species composition, distribution, and relative abundance of fishes                systematics, and the status of the fisheries, edited by Harold L. Pratt Jr.,
                  in the coastal habitat off the southeastern United States, by Charles A.               Samuel H. Gruber, and Toru Taniuchi. July 1990, 518 p.
                  Wenner and George R. Sedberry. July 1989, 49 p.
                                                                                                         91. Marine flora and fauna of the northeastern United States, Echinoder-
                  80. Laboratory guide to early life history stages of northeast Pacific fishes,         mata: Crinoidea, by Charles G. Messing and John H. Dearborn. August
                  by Ann C. Matarese, Arthur W. Kendall Jr., Deborah M. Blood, and                       1990, 30 p.











                                                 NOAA Technical Report NMFS 92


                                                 Genetics in Aquaculture


                                                 Proceedings of the Sixteenth
                                                 U.S. -Japan Meeting on Aquaculture
                                                 Charleston, South Carolina
                                                 October 20 and 21, 1987


                                                 Ralph S. Svrjcek (editor),
                                                 Publications Unit
                                                 Northwest and Alaska Fisheries Science Centers



                                                 Panel Chairmen:
                                                 Conrad Mahnken, United States
                                                 Takeshi Nose, Japan



                                                 Under the U.S. -Japan Cooperative Program
                                                 in Natural Resources (UJNR)



                                                 November 1990





                                  stil OF Co.    U.S. DEPARTMENT OF COMMERCE
                                                 Robert Mosbacher, Secretary
                                                 National Oceanic and Atmospheric Administration
                                                 John A. Knauss, Under Secretary for Oceans and Atmosphere
                              14,                National Marine Fisheries Service
                                 "ArEs 0V        William W. Fox Jr., Assistant Administrator for Fisheries






              C"
                                 TaE7










                                                         LIBRARY
                                                       NOAA/CCEH
                                                   1990 HOBSON AVE.
                                                 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
                                                                                                                                             Takeshi Nose          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-
                                                                       prietary product or proprietary material mentioned herein, or which has
                                                                       as its purpose an intent to cause directly or indirectly the advertised pro-
                                                                       duct to be used or purchased because of this NMFS publication.



                                                                                      Text printed on recycled paper







              CONTENTS


                  W.K. HERSHBERGER             Assessment of inbreeding and its implications for salmon broodstock                       1
                            J.M. MYERS         development
                            R.N. IWAMOTO
                            W.C. McAULEY


                    G.H. THORGAARD             Chromosome set manipulation in salmonid fishes                                            9

                            R.T. DILLON Jr.    Outcrossed lines of the hard clam Mercenaria mercenaria                                11
                             J.J. MANZI

                                   Y. FU       A preliminary study on genetics of two types of the rotifer Brachionus plicatilis      13
                            Y. NATSUKARI
                            K. HIRAYAMA


                            K.FUKUSHO          Present status of genetic studies on marine finfish in Japan                           21

                            J.C. LEONG         Recombinant viral vaccines in aquaculture                                              27
                             R. BARRIE
                     H.M. ENGELKING
              J. FEYEREISEN-KOENER
                            R. GILMORE
                               J.HARRY
                            G.KURATH
                            D.S. MANNING
                            C.L. MASON
                               L.OBERG
                            J. WIRKKULA

                            R.S. WAPLES        Genetic monitoring of Pacific salmon hatcheries                                        33
                            G.A. WINANS
                            F.M. UTTER
                            C. MAHNKEN


                              Sj. YOON         Successful gene transfer in fish                                                       39
                                  Z. LIU
                    A.R. KAPUSCINSKI
                            P.B. HACKETT
                                A.FARAS
                             K.S. GUISE


                            T. NAKANISHI       Clonal ginbuna crucian carp as a model for the study of fish immunology                45
                            H.ONOZATO          and genetics

                            T.I.J. SMITH       Aquaculture of striped bass, Morone saxatilis, and its hybrids in North America        53

                            L.J. LESTER        Computerized image analysis for selective breeding of shrimp: a progress               63
                            K.S. LAWSON        report
                     Mj. PIOTROWSKI
                            T.-C. B. WONG


                            H. MOMMA           Breeding test on abalone                                                               71

                            L.L. BEHRENDS      Two-stage hybridization and introgression for improving production traits of           77
                            J.G. KINGSLEY      red tilapias
                            A.H. PRICE III







                                      Assessment of Inbreeding and Its Implications for
                                                       Salmon Broodstock Development*


                                                 WILLIAM K. HERSHBERGER and JAMES M. MYERS

                                                                              School of Fisheries WH-10
                                                                               University of Washington
                                                                                  Seattle, WA 98195



                                                             R.N. IWAMOTO** and W.C. McAULEY

                                                                                  Domsea Farnis, Inc.
                                                                                   5500 180th S. W.
                                                                                 Rochester, WA 98579




                                                                                    ABSTRACT


                                         Inbreeding is an important part of any selection and breeding program designed to improve
                                      aquacultural broodstock. A decrease in freshwater and saltwater growth rate was noted in a strain
                                      of coho salmon, Oncorhynchus kisutch, undergoing selection to improve these traits for commercial
                                      production. Thus, an investigation was undertaken to estimate the level of inbreeding in this
                                      strain and to assess different approaches to alleviate problematic levels of inbreeding. Estimation
                                      of inbreeding level was conducted via pedigree analysis and change in heterozygosity of
                                      elctrophoretically detected ser-um proteins variants of odd- and even-year lines of coho salmon.
                                      The two methods of analysis indicated vastly different inbreeding levels. However, pedigree
                                      analysis, the more accurate of the two methods, estimated inbreeding levels not anticipated to
                                      cause the observed depression in growth traits. Two approaches, interstock crosses and crosses
                                      between parallel -selected lines, were assessed for alleviation of inbreeding problems. Both types
                                      of crosses decrease the level of inbreeding, but the performance of the two types of crosses dif-
                                      fered greatly. Crosses between unrelated year classes of the selected stock showed positive heterotic
                                      effects, while the outcrosses with unrelated lines yielded negative heterotic effects. These results
                                      indicate that careful attention should be given to the selection of the founding populations from
                                      which broodstocks are developed and that subsequent breeding information be collected to pro-
                                      duce pedigrees for population maintenance. Furthermore, the production of parallel "in-house"
                                      lines, may provide the best method of minimizing inbreeding without diluting selection gains.



                 Introduction                                                                   result in increased inbreeding levels (Falconer 1981), where
                                                                                                the magnitude will depend on the genetic characteristics
                 Inbreeding is integral to any selection and breeding pro-                      of the population and the severity of the constraints im-
                 gram designed for the development of broodstock. Such                          posed. Consequently, the factors that influence inbreeding
                 programs generally deal with a "closed" population (i.e.,                      must be integrated into the design of any program to
                 migration into the population is eliminated) having a re-                      develop genetically improved aquacultural stocks.
                 stricted breeding population size. Both of these factors                         There has been a large amount of research concerning
                                                                                                inbreeding and its effects on various traits in fish. For
                                                                                                example, work with rainbow trout, Oncorhynchus mykiss
                  * Contribution No. 760, School of Fisheries WH-10, University of Wash-        (formerly SaIrno gairdnert), has revealed that increased levels
                   ington, Seattle, WA 98195. The Project was supported by U.S. NOAA            of inbreeding result in increased egg and fry mortality,
                   Grant NA86AA-D-SCO44 A09 to the Washington Sea Grant Program                 increased numbers of abnormal fry, decreased early
                   Project No. R/A-47.
                  * Current Address: Ocean Farms of Hawaii, P.O. Box A, Kailua-Kona,            growth, and decreased fishery recovery (Kincaid 1976,
                   HI @6745                                                                     1983; Aulstad and Kittlesen 1971). Research with brook

                                                                                                                                                                       I







                    2            NOAA Technical Report NMFS 92


                    trout, Salvelinusfontinalis, has demonstrated a negative im-                                         a broodstock with traits that are beneficial to the produc-
                    pact on weight owing to inbreeding (Cooper 1961). Ryman                                              tion of 300-350 g coho salmon for the "plate-size" salmon
                    (1970) reported a decrease in recapture frequency in Atlan-                                          market.
                    tic salmon, Salmo salar, with increased levels of inbreeding.                                            The traits that have been emphasized for selective im-
                    In general, the results of these studies suggest a negative                                          provement are 1) freshwater growth, 2) smoltification, and
                    impact on a variety of biological traits in the populations                                          3) saltwater growth to harvest size. Genetic analyses of
                    studied and, consequently, on production.                                                            these traits in the stock employed by Domsea Farms re-
                        No studies have been published on the effects of inbreed-                                        vealed adequate variability to expect progress from selec-
                    ing on Pacific salmon, Oncorhynchus spp., nor have any                                               tion (Iwamoto et al. 1982; Hershberger and Iwamoto 1984;
                    published reports dealt with the effects of inbreeding in con-                                       Saxton et al. 1984).
                    junction with a selection and breeding program designed                                                  Using estimated genetic values and considering that
                    to develop a genetically improved stock for aquacultural                                             the facilities available to the program would only allow
                    purposes. To some degree, both of these deficiencies in in-                                          raising 40 families of 600 individuals or less, a selection
                    formation are being eliminated as Pacific salmon are used                                            scheme was designed to yield maximum response and to
                    for captive culture. It is imperative that data be obtained                                          be useful in a commercial operation (Fig. 1). This scheme
                    on inbreeding in these species under defined programs to                                             involved several different types of concurrent selection
                    determine their response to selection.                                                               (e.g., family and individual) and used a selection index that
                                                                                                                         incorporated heritability estimates, relative economic
                                                                                                                         values, genetic correlations, and mean values on all the
                    Research Rationale                                                                                   traits of interest. It was recognized early in the develop-
                                                                                                                         ment of this scheme that potential inbreeding problems
                    The University of Washington, Domsea Farms, Inc., and                                                could arise from the rather severe limitation in breeding
                    the Washington Sea Grant Program have been conduct-                                                  population size (only twenty individuals contribute to
                    ing a selection and breeding program with coho salmon,                                               each generation). Consequently, breeding was conducted
                    0. k1sutch, to develop a broodstock for the marine net-pen                                           by a rotational line-crossing procedure (Fig. 2) to minimize
                    industry in the State of Washington. The major objective                                             the possibility of crossing within lines. On a theoretical
                    of this nine year cooperative program has been to develop                                            basis, these steps should limit the change in inbreeding





                                                                   3.5 MONTH SALTWATER
                                                                             SAMPLING




                                 FRESHWATER
                                   SAMPLING                                                                                      8
                                                                                                                             MONTH
                                                                                                                         SALTWATER
                                                                SALTWATER                  SALTWATER                       SAMPLING
                                                                   PHASEI                     PHASEII

                          Fish in Excess                                                                                      25 Families*
                            of 600 per
                             Family*
                                                     FRESHWATER                                                      15 Families
                                                        REARING                                     SALTWATER
                                                                                                        PHASE III



                                         40 Families
                         20 Families*
                                                                  INCUBATION           MATURATION IN
                                                                                         FRESHWATER                          14 MONTH
                                                                                                                          SALTWATER
                                                                                                    SA
                                                                                                        PH
                                                                                                          'WATER
                                                                                                          ASE






                                                                                        MATU  RATI  0N  IN
                                                                                         FRIES HWATER
                                                                                                                           S,
                            INCUBATION                                                                                       'SAMPLING
                                                                                                10 Families
                                                                                     IN
                           PERFORMANCE                                                                                                                                        Figure 1
                                                                              60 Families                                    5 Families*              Diagram of the           selection scheme used to
                                                                                                                                                      develop coho salmon stocks for marine pen-
                                                                                 SPAWNING                                                             culture. The entire cycle represents a two-
                                                                                                                                                      year generation interval.







                                                                                                     Hershberger et al.: Assessment of Salmon Broodstock Development                                                              3




                                           FAMILY                  FAMILY               FAMILY                  FAMILY                FAMILY
                                              1.                     2                      3                      4                     5




                                 FROM
                                FAMILY
                                   5
                                                                                                                                                          TO
                                                                                                                                                       FAMILY1












                                                                   FAMILY                 FAMILY                 FAMIL7                FAMIL7
                                               6                      7                      8                     9                     10




                                 FROM
                                FAMILY
                                   10
                                                                                                                                                          TO                                    Figure 2
                                                                                                                                                       FAMILY 6            Diagram of the rotational line mating
                                                                                                                                                                           system used in crossing selected indi-
                                                                                                                                                                           viduals.        The asterisk indicates that
                                                                                                                                                                           each family cross is composed of six
                                                                Q                      [9d                    lid                   E                                      single-pair matings to form six double
                                                                                                                                                                      I    first-cousin families.





                       to about I % per generation (Hershberger and Iwamoto                                                       for this growth depression would be the accumulation of
                       1984).                                                                                                     deleterious alleles through inbreeding. Even with the pre-
                           In 1983 (for the odd-year line) and 1984 (for the even-                                                cautions taken in the design of the selection and breeding
                       year line) a decrease in the growth of selected fish in                                                    program, there were two potential sources of inbreeding
                       saltwater was observed (Fig. 3). One possible explanation                                                  that could not be quantitated. First, an unknown amount
                                                                                                                                  of inbreeding may have been introduced by selection and
                                                                                                                                  breeding that had occurred prior to use of this designed
                                                                                                                                  program. Second, because of some unexpected husbandry
                                   700                                                                                            problems with raising fish to maturity there was a strong
                                                                                                                                  probability that a few families contributed disporpor-
                                   600                                                                                            tionately to the subsequent generations. Prior to the defi-
                                                                                                                                  nition of pedigrees for the two lines, the importance of these
                                   500
                           Cr                                                                                      2              factors was undeterminable.
                                   400
                                                           ................ .                                      2                  As a result of these indications, studies were initiated
                                   300                                                                                            to 1) determine the actual levels of inbreeding in the two
                                                                                                                                  lines and 2) define the best approach to eliminate inbreed-
                                   200 1977    1978    1979     1980   1981    1982    1983    1984    1985     1986              ing in the selected stocks.
                                                                        BROODYEAR

                                             -M - ODD-YEAR LINE    -0 EVEN-YEAR LINE     -C- WILD CONTROLS
                                                                                                                                  Determination of Inbreeding Level
                                                                Figure 3                                                          The level of inbreeding in each of the two selected lines
                       Average weight (grams) of selected broodstock and wild controls.
                       after 8 months rearing in marine net-pens. Weights for 1986 are                                            (Le., odd- and even-year) was determined by two differ-
                       given as unadjusted (1) and adjusted (2) for density differences                                           ent methods. First, pedigree analyses were employed to
                       that year. N = 1200-2200 for selected broodstock and N                                                     determine the coefficient of inbreeding (F) (Falconer 1981).
                       15-35 for wild controls.                                                                                   Computation of this value is accomplished by tracing the








                        4            NOAA Technical Report NMFS 92



                                                                            DOMSEA COHO SALMON
                                                                            SEAWATER BROODSTOCK
                                                                            ODD-YEAR LINE PEDIGREE                                                      BROODYEAR

                                                       K777N
                                                                                           -(R           %                                                         1977
                              LDTC          LUU                             LAUC  @AUC                AU      LD C           RAT          RUT          RDU
                                                                                                      ,6 %
                                1            14                             20       25               2         3              35          53           30



                                                                                               @rH A 1U      @L'D
                              LHUS         RHUU        'R@ PUC              RHPU,' LHPUC              C            U        LDPU           DPU        RDUU          1979
                                63            3           19                20,.,A   27               41                       31          23           25
                                                                                                                    A








                              LOPU                                          LH     RDUUC              R A
                                           LDDU                                                               LHUSC        RHDUC'@      LDAUC          DDUC        1981
                               44                                                     10              0                       23           61           25
                                              34                            2








                                   U
                                         -(L'                                                                               RHAUC       RDUUC
                                             'DD'U"', rLHUS                 RDD     RDUU              LDDUC' ,RDRUj:                                   LDUU       1983
                                             54
                                                        ,66                 59,@      16              50..,@                   18           9            3


                                     --- (L   U S@)        _S C'%           LH                                                              'U'S1                  1985
                                                                                                                     @'_LDDUC            RD            RDDU
                                                        LHU                                                  rLHUU
                              LHUD                                          S     rL Z U              LHPU %
                                                          54                52        2               11                       41          55           50
                                                                                                                                                A
                                                                                                                   @c ---

                                                                                                                                                                                                     Figure 4
                                                                                                                                                                                   Pedigree of matings between se-
                                                                                                                                                                                   lected families for the odd-year
                                                                                                                                                                   1987            broodstock line (1978-1986).
                                                                                                                                                                                   Families enclosed by a striped
                                                                                                                                                                                   box are double first cousins.




                        pedigree back to common ancestors and determining the                                                       ing coefficient from these pedigrees (Table 1) indicates that
                        probability that a pair of alleles, are identical by descent.                                               the current level of inbreeding is not too severe, although
                        Second, the change in genotype frequencies of electro-                                                      the estimate for the next generation (1987 broodyear) will
                        phoretically analyzed protein differences were determined                                                   approach 8- 10%. These levels of inbreeding would not be
                        and the difference in heterozygote frequencies equated to                                                   anticipated to cause the level of change found in the re-
                        an apparent inbreeding coefficient (Hartl 1980). Electro-                                                   sponse of growth to selection. It has been estimated that
                        phoretic analyses were conducted on serum samples from                                                      in domesticated animals selection can balance an increase
                        100-120 adult fish in each of four years (1977, 1978, 1985,                                                 in inbreeding of approximately 2 % per generation (Pirch-
                        and 1986). The electrophoretic procedures employed were                                                     ner 1969). The estimated levels of inbreeding in coho
                        those reported in Utter et al. (1970) for analysis of serum                                                 salmon lines, to the point where apparent inbreeding
                        transferrins in coho salmon.                                                                                depression was noted (1983 and 1984), are below this value.
                           Construction of the pedigrees for the two lines of coho                                                  However, the coefficients reflect only the inbreeding since
                        salmon revealed more closely related families than was                                                      the program was initiated and do not provide a measure
                        originally anticipated (Fig. 4). Calculation of an inbreed-                                                 of prior inbreeding. Further, it is difficult to determine what







                                                                             Hershberger et al.: Assessment of Salmon Broodstock Development                                5



                                                                                           Table 1
                                           Inbreeding estimates based on pedigree analysis for both odd- and even-year lines, and
                                           based solely on effective population size (Ne). The estimates are calculated assuming the
                                           initial inbreeding coefficient (F) is equal to 0.

                                                                        Pedigree estimates                                     AF = (1 /2 N + 4)'

                                                     Odd-year       Control                    Even-year      Control              Odd        Even

                                           1977        0.00             -            1978           0.00          -                0.00       0.00
                                           1979        0.00             2.50         1980           0.00        2.50               2.27       2.27
                                           1981        0.32             4.71         1982           0.63        5.75               4.49       4.41
                                           1983        2.34             8.68         1984           4.22        9.11               8.78       6.58
                                           1985        4.86             11.00        1986           5.90        12.20              10.86      8.52
                                           1987        8.34             13.79                                                      12.88


                                           'Theoretical A F  excluding sib-matings.




                 the effects of an incremental change in inbreeding may be                          much larger value than was obtained from the pedigree
                 in a species that has been recently developed from naturally                       analyses (Table 1).
                 reproducing populations (Soule 1980).                                                It is possible to rationalize the discrepancy in these values
                    The second type of inbreeding assessment employed elec-                         on two bases. First, there is evidence suggesting selective
                 trophoretic analysis of the transferrin locus, which has been                      differences among the various afleles of the transferrin locus
                 shown to have three variant alleles (Utter et al. 1970) and                        (Suzurnoto et al. 1977; Pratschner 1977). T             he results of
                 is one of the few genetically variable protein loci found in                       Pratschner's research indicated that fish with the "A" and
                 coho salmon (Utter et al. 1980). Comparison of the geno-                           "C" alleles, were more resistant to challenges by Vibrio
                 type and gene frequency values in the original adult pop-                          bacteria than those with the "B" allele, and Suzurnoto et
                 ulation with those from the fourth generation of selected                          al (1977) found that the "A" allele imparted higher sur-
                 stock (Table 2) revealed changes that would be anticipated                         vival to BKD (bacterial kidney disease) challenge. If such
                 in an inbred population (Falconer 1981); that is, there was                        selective pressures were applied to the selected coho salmon
                 a decrease in the frequency of heterozygotes and, with one                         lines, analyses based on the genotype frequencies would
                 exception, there was little change in the gene frequencies.                        tend to overestimate the inbreeding coefficient. The data
                 Calculation of apparent inbreeding coefficients based on                           from the current study support the hypothesis that fish with
                 the frequency change in heterozygotes (Fig. 5) yields a                            the "A" and "C" alleles have a selective advantage, and




                                                                                           Table 2
                                           Observed transferrin gene and genotype frequencies in the odd- and even-year lines of
                                              coho salmon and their changes over four generations of selection (N = 100- 120).

                                                                                           Odd-year broodstock line
                                                                                  Genotype                                  Gene frequency

                                              Year           AA         AB      AC         BB       BC        cc          fA       fB         fC

                                           1977              0.00       0.08      0.33     0.00     0.13      0.48        0.20     0.10       0.70
                                           1985              0.05       0.03      0.08     0.00     0.18      0.68        0.10     0.10       0.80
                                              Change         +0.05      -0.05   -0.25      +0.00    +0.05    +0.20       -0.10     0.00       +0.10


                                                                                           Even-year broodstock line
                                                                                  Genotype                                  Gene frequency

                                              Year           AA         AB      AC         BB       BC        cc          fA       fB         fC

                                           1978              0.10       0.05      0.45     0.05     0.25      0.10        0.35      0.20      0.45
                                           1986              0.12       0.00      0.42     0.00     0.04      0. 42       0.33      0.02      0.65
                                              Change         +0.01      -0.05   -0.02      -0.05    -0.21    +0.32       -0.02     +0.18      +0.20







                  6        NOAA Technical Report NMFS 92


                                                                                                                              Figure 5
                             ODD-YEAR                                                         Estimates of the apprent inbreeding coefficients for the odd- and
                                                                                              even-year lines based on the changes in observed and expected
                               ACTUAL HETEROZYGOSITY VS. EXPECTED                             genotype frequencies of the coho transferrin alleles.

                             1977        .525 -.46 /.46 = +14.1 %

                             1985        .275 -.525 /.525 = - 47.6 %
                             CHANGE IN OBSERVED HETEROZYGOSITY                                                                     Table 3
                                                                                                       The relative growth and survival of interstrain (Domsea
                                                                                                       x Univ. of WA) and intrastrain (Domsea odd- x even-
                             CHANGE 1977 to 1985:.275 - .525 /.525             47.6 %                  year) crosses after 8 months rearing in marine net-pens.
                             ESTIMATED AF             41.9%                                            The weights and survivals have been standardized against
                                                                                                       the Domsea x Domsea (2 x 2) cross = 100. The index
                                                                                                       value is the cross-product of weight and survival/100. N
                             EVEN-YEAR                                                                    8-45 for each cross.
                                                                                                                             Outcrossing schemes
                                ACTUAL HETEROZYGOSITY VS. EXPECTED                                                                       Relative     Relative
                                                                                                                                         weight       survival     Index
                             1978       .75 -.635 /.635 = + 18.1 %
                                                                                                       DOMSEA (D)                        100           100         too
                             1986 .461 - .465 /.465           0.8 %                                    D x UW (9 x 0)                    147            25           36.8
                             CHANGE IN OBSERVED HETEROZYGOSITY                                         UW x D (9 x a)                    141            25           35.3
                                                                                                       University of Wash.   (UW)        55.1           21.4          11.7
                             CHANGE 1978 to 1986:.461 -.75 /.75              38.5 %                                         DOMSEA line crosses
                             ESTIMATED AF             29.3%                                                                              Relative     Relative
                                                                                                                                         weight       survival     Index

                                                                                                       DOMSEA (2 x 2)                    100            100        too
                                                                                                       DOMSEA (2 x 3)                    116.1          150        174.5
                  vibriosis is a common problem in the marine net-pen                                  DOMSEA (3 x 2)                    101.4          225        174.1
                  culture of salmon. The directed selection practiced on the                           DOMSEA (3 x 3)                    128.7          100        128.7
                  stock may also have an epistatic effect on the transferrin
                  locus. A tacit assumption made in the use of the genotype
                  frequency relationship used to calculate an inbreeding coef-
                  ficient is the absence of selection. Such an assumption is                       coho salmon stock and the hatchery stock of the Univer-
                  clearly not valid in this situation and may result in the                        sity of Washington, and between the Domsea odd- and
                  inflation of the calculated value.                                               even-year parallel- selected lines. Progeny from these crosses
                    To summarize, it appears that pedigree analysis is the                         were reared in conjunction with the broodstock line.
                  best approach to determine inbreeding levels in coho                                 It is apparent from the data (Table 3) that the progeny
                  salmon. Thus, it would seem wise to assure that a selec-                         from the crosses derived from the Doinsea intrastock crosses
                  tion and breeding program incorporates the mechanisms                            were superior to the interstock cross at the time of harvest.
                  that define accurate pedigrees of the breeding population.                       Although both of the University of Washington x Domsea
                  Further, caution should, be exercised in the use of geno-                        hybrids were larger after eight months of saltwater rear-
                  type frequency changes to determine absolute values for                          ing, relative to the Domsea controls, the overall survival
                  inbreeding coefficients. The potential effects of direct and                     of both the hybrids and the University of Washington fish
                  indirect selection must be determined for these values to                        was extremely poor under net-pen conditions. The high
                  be considered as valid measurements of inbreeding.                               values reported reflect the survival of a few large hybrids
                                                                                                   which biased the weight measurements. The University of
                                                                                                   Washington x Domsea hybrids may not necessarily be
                  Elimination of Inbreeding                                                        indicative of all interstock crosses, but the results suggest
                                                                                                   that extensive hybrid testing may be necessary to identify
                  Although the apparent levels of inbreeding in the selected                       a complementary stock. The Domsea intrastock hybrids,
                  stocks of coho salmon were not large, two approaches to                          however, showed both good growth and greatly improved
                  elimination of accumulated inbreeding were investigated:                         survival relative to controls. Maintaining "in-house"
                  outcrossing between stocks and outcrossing between lines                         parallel selection lines may be a more efficient expenditure
                  within stocks. Test crosses were made between the Domsea                         of effort relative to testing outcrosses. The "odd x even"







                                                                                Hershberger et al.: Assessment of Salmon Broodstock Development                                  7


                   crosses would appear to be the method-of-choice for allevi-                        Citations
                   ating the inbreeding "load" while preserving selection
                   gains.                                                                             Aulstad, D., and Kittlesen.
                                                                                                            1971. Abnormal body curvatures of rainbow trout (Salmogairdnen)
                                                                                                              inbred fry. J. Fish. Res. Board Can. 28:1918-1920.
                                                                                                      Cooper, E.L.
                   Implications for                                                                         1961. Growth of wild and hatchery strains of brook trout. Trans.
                                                                                                              Am. Fish. Soc. 23:614-617.
                   Broodstock Development                                                             Falconer, D.S.
                                                                                                            1981. Introduction to quantitative genetics. Longman,Inc.New
                   The coho salmon stocks that have been developed as a                                       York, NY, 340 p.
                   result of this research program have, apparently, not yet                          Gall, G.A.E.
                                                                                                            1987. Inbreeding. In Population genetics & fishery management
                   reached a level of inbreeding which would result in a strong                               (N. Ryman and F. Utter, eds.), p. 47-87. Univ. Washington
                   negative impact on their performance. The depression in                                    Press, Seattle, WA.
                   growth observed in both lines appears to have been envi-                           Hart],  D.L.
                   ronmentally generated and subsequent generations have                                    1980. Principles of population genetics. Sinauer Assoc., Inc.
                                                                                                              Sunderland, MA, 488 p.
                   performed well (Fig. 3). However, analyses of inbreeding                           Hershberger, W.K., and R.N. Iwamoto.
                   in these lines have demonstrated several areas requiring                                 1984. Systematic genetic selection and breeding in salmonid culture
                   special consideration in the development of aquaculture                                    and enhancement programs. In Proceedings of the I lth U.S.-
                   broodstocks. Where possible, a selection and breeding pro-                                 Japan Meeting on Aquaculture, salmon enhancement; 19-20
                   gram should be initiated with a large enough population                                    October 1982, Tokyo, Japan, p. 29-32. U.S. Dep. Commer.,
                                                                                                              NOAA Tech. Rep. NMFS 27.
                   size to completely address the combined needs of a reason-                         Iwamoto, R.N., A.M. Saxton, and W.K. Hershberger.
                   able selection differential and elimination of close familial                            1982. Genetic estimates for length and weight of coho salmon
                   relationships. Otherwise, definitive steps must be taken in                                (Oncorhynchus kisutch) during freshwater rearing. J. Hered.
                   the formulation of the selection and breeding program to                                   73:187-191.
                   minimize the accumulation of inbreeding from these                                 Kincaid, H.L.
                                                                                                            1976. Effects of inbreeding on rainbow trout populations. Trans.
                   factors.                                                                                   Am  .Fish. Soc. 105:273-280.
                      Further, a broodstock program should be initiated from                                1983. Inbreeding in fish populations used for aquaculture. Aqua-
                   either an outbred population with an inbreeding coeffici-                                  culture 33:215-227.
                   ent (F) equal, or close to 0, or from a stock with a defined                       Pirchner, F.
                   and well maintained pedigree. This would insure that the                                 1969. Population Genetics in Animal Breeding. W.H.Freeman
                                                                                                              and Co., San Francisco, CA, 274 p.
                   inbreeding level could be unquestionably determined and                            Pratschner, G.A.
                   the effects of any increases could be well defined. In addi-                             1977. Relative resistance of six transferrin phenotypes of coho
                   tion, research is needed to determine the response of aqua-                                salmon to cytophagosis, furunculosis and vibriosis. M.S. Thesis,
                   cultural species recently derived from wild populations to                                 Univ. Washington, Seattle, WA, 71 p.
                   an increase in inbreeding level. While the response of                             Ryman, N.
                   domesticated animals to increases in inbreeding has been                                 1970. A genetic analysis of recapture frequencies of released young
                                                                                                              of salmon (Salmo salar L.). Hereditas 65:159-160.
                   quantitated to some degree (Pirchner 1969), there is no                            Saxton, A.M., W.K. Hershberger, and R.N. Iwamoto.
                   a priori method by which to predict the magnitude of                                     1984. Smoltification in the net-pen culture of accelerated coho
                   responses in natural populations. As indicated by Gall                                     salmon (Oncorhynchus kisutch); quantitative genetic analysis. Trans.
                   (1987), the best information will be obtained from induc-                                  Am. Fish. Sec. 113:339-347.
                                                                                                      Soule, M.E.
                   ing high levels of inbreeding in such stocks and quantify-                               1980. Thresholds for survival: maintaining fitness and evolutionary
                   ing the effects. However, inbreeding effects observed in                                   potential. In Conservation Biology (M.E. Soule and B.A. Wilcox,
                   the progeny of sib-matings are indicative of, but not highly                               eds.), p. 151-169. Sinauer Assoc., Inc., Sunderland, MA.
                   correlated with the performance of individuals with equal                          Suzumoto, B. K., C. B. Schreck, and J. D. McIntyre.
                   inbreeding levels produced through generations of matings.                               1977. Relative resistances of three transferrin genotypes of coho
                                                                                                              salmon (Oncorhynchus kistuch) and their hernatological responses to
                      Finally, it appears that using parallel selection in at least                           bacterial kidney disease. J. Fish. Res. Board Can. 34:1-8.
                   two separate lines of broodstock would be a valuable ap-                           Utter,  F.M., W.E. Ames, and H.O. Hodgins.
                   proach to incorporate into a selection and breeding pro-                                 1970. Transferrin polymorphism in coho salmon (Oncorhynchus
                   gram. This provides an additional data set with which to                                   kisulch). J. Fish. Res. Board Can. 27:2371-2373.
                   evaluate a selection program and also incorporates a                               Utter, F. M., D. Campton, S. Grant, G. Milner, J. Seeb, and L. Wishard.
                   mechanism that has the potential to eliminate inbreeding                                 1980. Population structures of indigenous salmonis species of the
                                                                                                              Pacific Northwest. In Salmonid ecosystems of the North Pacific
                   effects without the loss of advances made in the traits that                               (W.J. McNeil and D.C. Hirnsworth, eds.), p. 285-304. Oregon
                   are beneficial to aquaculture production.                                                  State Univ. Press, Corvallis, OR.








                                   Chromosome Set Manipulation in Salmonid Fishes


                                                                    GARY H. THORGAARD

                                                   Department of Zoology and Program in Genetics and Cell Biology
                                                                       Washington State University
                                                                         Pullman, WA 99164-4220





                                                                               ABSTRACT


                                       Techniques to manipulate chromosome sets and produce polyploid fishes or fishes with all the
                                     inheritance from the female or male parent have been exploited in aquaculture in recent years.
                                     Some of the principal applications of this work have been to produce sterile fish or to produce
                                     monosex populations. Three additional applications of chromosome set manipulation that we
                                     have explored in salmonids in our laboratory and in collaboration with other laboratories have
                                     been 1) increased survival in triploid hybrids; 2) the potential for gene transfer by "incomplete
                                     gynogenesis"; and 3) the generation of homozygous diploids and ultimately homozygous clones
                                     through androgenesis (all-paternal inheritance).
                                       A number of researchers have demonstrated that interspecific triploid fish hybrids survive better
                                     than the corresponding diploid hybrids. Notable examples of this phenomenon include the tiger
                                     trout (brown trout x brook trout) hybrid, the rainbow trout x coho salmon hybrid, and the
                                     chum salmon x chinook salmon hybrid. The tiger trout has considerable potential as a sport
                                     fish and may be advantageous because both the diploid and triploid hybrids are essentially sterile.
                                     The rainbow trout x coho salmon hybrid has increased resistance to IHN (infectious hernatopoietic
                                     necrosis) virus characteristic of the coho salmon parent. The chum salmon x chinook salmon
                                     hybrid has early seawater tolerance characteristic of the chum salmon parent.
                                       Gynogenesis (all-maternal inheritance) experiments have normally involved complete inactiva-
                                     tion of the paternal genome by radiation or chemical treatment of the sperm. However, we have
                                     demonstrated that if a lower than normal radiation treatment is applied to the sperm, some paternal
                                     genes may still be active in the progeny. This has been demonstrated for both pigmentation and
                                     isozyme loci. It appears that the paternal genes in this situation are located on chromosomal
                                     fragments which are lost during development. If the paternal genes can be stably inherited and
                                     if desirable paternal traits can be selected for, this "incomplete gyogenesis" might potentially
                                     be used to transfer desirable traits between species.
                                       Androgenesis is induced by fertilizing radiation-inactivated eggs with normal sperm and by
                                     applying a pressure or heat treatment to block the first cleavage division and produce homo-
                                     zygous diploids. We have successfully induced androgenesis in rainbow trout and have also pro-
                                     duced androgenetic progeny from homozygous androgenetic males. Androgenesis has a number
                                     of distinctive applications for aquaculture, including generation of homozygous clones and recovery
                                     of strains from cryogenically preserved sperm.


















                                                                                                                                                           9









                             Outcrossed Lines of the Hard Clam Mercenaria mercenaria


                                                                        ROBERT T. DILLON Jr.

                                                                                Department of Biology
                                                                                 College of Charleston
                                                                                Charleston, SC 29424



                                                                              JOHN J. MANZI

                                                                         Marine Resources Research Institute
                                                                                Charleston, SC 29412





                                                                                   ABSTRACT


                                        A large-scale breeding program has been initiated in South Carolina to achieve improved growth
                                      and survival of the hard clams, M. mercenaria. This interdisciplinary, multi-institutional program
                                      uses the facilities and personnel of the South Carolina Wildlife and Marine Resources Research
                                      Institute, the College of Charleston, the University of South Carolina, and Clemson University.
                                        Nursery stocks of hard clams that had been selected for fast growth were obtained from
                                      Aquaculture Research Corporation ("ARC" - Dennis, MA) and the Virginia Institute of Marine
                                      Science ("VIMS" - Wachapreague, VA). These stocks were compared to corresponding wild
                                      populations for allele frequencies at seven polymorphic enzyme loci. Although as few as 30-60
                                      parents were spawned at each of four generations to produce these two broodstocks, neither line
                                      exhibited any reduction in heterozygosity. Both lines, however, showed evidence of genetic drift
                                      and loss of rare alleles, suggesting that crosses between them could result in genetically distinct lines.
                                        ARC and VIMS stocks were spawned on three occasions at different times of the year for pro-
                                      duction of both reciprocal outbred and pure control lines. Growth and survival were monitored
                                      regularly over two years. Early growth was strongly influenced by time of spawning, and as such
                                      was not a reliable indicator of subsequent growth. Most significant disparities between trials
                                      decreased as the lines aged. At 21 months, outbred and purebred lines were not consistently
                                      different in their heterozygosity, mean size, or size variance.
                                        Within crosses, little relationship was detected between shell length and heterozygosity aver-
                                      aged over the seven enzyme loci. However, significant differences between the largest and smallest
                                      clams were detected at individual loci in 10 of 42 tests. Results were consistent neither with the
                                      hypothesis that the alleles themselves were affecting growth, nor with the hypothesis that these
                                      enzyme loci were tightly linked to other loci affecting growth. Rather, it appears that alleles are
                                      marking the entire genomes of their parents, and that variation in the growth rates of the off-
                                      spring from individual clams may be obscuring any relationship with overall heterozygosity.








                         A Preliminary Study on Genetics of Two Types of the Rotifer
                                                                      Brachionus plicatilis


                                       YONG FU, YUTAKA NATSUKARI, and KAZUTSUGU HIRAYAMA

                                                                                 Faculty of Fisheries
                                                                                Nagasaki University
                                                                              Bunkyomachi, Nagasaki
                                                                               Nagasaki 852, Japan




                                                                                  ABSTRACT


                                         The domesticated rotifer Brachionus plicatilis can be divided roughly into two types, called
                                       L and S, using morphological differences in the shape of anterior spines on the lorica (obtuse
                                       angled and pointed, respectively). However, differences in growth responses with respect to
                                       environmental factors make this method unreliable, We have, therefore, tried to clarify differ-
                                       ences at the genetic level between types, using starch gel electrophoresis of enzymes.
                                         Thirty-four collected strains were separated by three methods into the two types. Initially, strains
                                       were qualitatively judged with respect to differences in the shape of anterior spines. Afterwards
                                       pure strains were cultured parthenogenetically and re-evaluated using the second method (quan-
                                       titative). To accomplish this, morphological features were measured, the ratios of which created
                                       an index for comparison of the strains (cluster analysis). Both the anterior spine and cluster analysis
                                       indicated that the 34 strains were composed of two large clusters consisting of 15 L and 19 S strains.
                                         Allozyme variations of the 34 strains were then detected by horizontal starch gel electrophoresis.
                                       Nine isozyme loci were recognized. Of the 42 alleles observed, 15 alleles over 6 loci showed great
                                       differences between L- and S-types. Using genetic distances according to the allele frequencies
                                       of 42 alleles, a dendrogram was drawn. The strains separated into two groups. One group con-
                                       sisted of only S-type strains, the other group was subdivided again into 3 clusters. One of these
                                       three clusters consisted only of the S-type strains, while the other two contained only L-type strains.
                                       This result indicates the great genetic differences    between L and S strains.



                  Introduction                                                                not to genetic differences. However, Fukusho and Okauchi
                                                                                              (1982, 1983, 1984) have provided evidence that differences
                  Since the introduction of the rotifer Brachionus plicatilis to              may be genetic and that the two types can be isolated from
                  nourish larval fish, aquaculturists have increased scientific               each other. In countries outside Japan, many scientists
                  attention on this organism. In Japan a significant achieve-                 recognize the variation of rotifers which is due to poly-
                  ment in rotifer biology was the discovery that the domes-                   morphosis. Scientific approaches concerning analysis of
                  ticated rotifers can be divided roughly into two so-called                  allozyme variation have therefore been investigated (Serra
                  S and L types as shown in Figure 1 (Fukusho 1983). The                      and Miracle 1983, 1985, 1987; Snell and Carrillo 1984;
                  main morphological differences between the two types are                    Snell and Winkler 1984; Suzuki 1983, 1987; King and
                  lorica size, lorica shape, and the shape of the anterior spines             Zhao 198 7), while in Japan there have been no studies to
                  on the lorica. They also exhibit differences in growth with                 detect allozyme variation in the two types by means of elec-
                  respect to temperature. The morphological and physio-                       trophorctic procedures.
                  logical differences in the two types were summarized in a                     Using strains collected from many locations, we at-
                  previous review (Hirayama 1987). The rotifer, especially                    tempted to distinguish L and S types using morphological
                  the domesticated rotifer, exhibits cyclomorphosis (seasonal                 comparisons. In order to confirm the genetic differences
                  variation in size) and also polymorphosis (change in size                   between strains, allozyme variations were detected by
                  influenced by variations in diet) (Fukusho and Iwamoto                      horizontal starch gel elect rophores is. Then, the genetic
                  1980, 1981). So, there is a probability that observed dif-                  distances among collected strains were compared for mor-
                  ferences could be attributed to cyclo- or poly-morphosis,                   phological similarities.

                                                                                                                                                                 13








             14        NOAA Technical Report NMFS 92
                   S
                                                                                                                N. @A
                                                                                                                         *@w
                                                                                                 T 7     Ai

                                                                                                                          IN






                                       IVV








                                                                                                            4












                                                                         Figure 1
                                     The two types of rotifer Brachionus plicatilis, L and S (provided by K. Fukusho).



             Materials and Methods                                              collected those eggs into test tubes reculturing them again
                                                                                with marine Chlorella. After the offspring hatching from
             We collected many strains from all over the world. On the          those eggs grew and laid their first eggs, we performed mor-
             map (Fig. 2), the localities of 34 strains used in this study      phological measurements. We removed 20 individuals per
             are shown. Table I shows the abbreviated names and                 sample and measured seven morphological features (Fig.
             origins of the strains. In the tables and figures, L- and          3, A through G). The ratios of these measurements were
             S-type strains are shown by abbreviation with capital and          used to create indices for a cluster analysis.
             small letters, respectively.
                                                                                Allozyme Analysis
             Morphological Analysis                                             The same 34 strains were used both for electrophoretic and
             We first observed the anterior spines of each of the 34            morphological analysis. Allozyme analysis for each strain
             strains and qualitatively divided them into the two types,         was conducted with a population grown from one in-
             L and S, according to whether they had obtuse angled or            dividual and cultured with marine Chlorella and baker's
             pointed spines, respectively. We classified 15 strains into        yeast. The population was harvested with a net, washed
             the L type and 19 strains into the S type. After the initial       with clean seawater several times, blotted dry using filter
             screening, one individual from each strain was selected for        paper and frozen at      30'C until analyzed. Before har-
             culturing parthenogenetically and was regarded as one              vesting, the group was starved for one day to remove the
             genetic strain for further study. Each strain was cultured         influences of food. Immediately prior to electrophoretic
             with marine Chlorella (Nannochloropsis oculata). We collected      analysis, we thawed the sample and used a small amount
             eggs and recultured each strain in marine Chlorella suspen-        of the drip absorbed by filter paper as a crude extract of
             sions in 23'C. The first eggs were laid after 48 hours. We         enzyme for allozyme alayisis. Electrophoresis were carried







                                                                                 Fu et al.: Genetics of the Rotifer Brachionus plicatilis         15







                    F-PA
                    F-PA- I I
                    F-PA- I I I
                    F-PA-IV


                                                                                a-sal

                                                              .0    c _xM                               a-mk


                                                                            P-ilo
                                            t-son                           p-le                                                  -amp
                        is-eil              t-Pu                              -0t


                                          s-sin


                                                     i-ja




                                                                               i-kay
                                                     J-SAP
                               J-NSGT                J-SAP-86
                               J-NSGT-11                                                                              J-TKU


                               J-NSU
                               J-NSC        0@4
                               J-NSZ
                               i-nsz                                        j-ebp                       J_S01
                                    i-kgko                       i-otk
                                   j-kgko-8                     J-OTK
                                          J-KAU         J-kgs                          locality of collection


                                                                             Figure 2
                                 Map of collection localities. Capital and small letters mean L- and S-type strains, respectively.




               out in 11% starch gel with three buffer systems reported              Leucine aminopeptidase (LAP, EC 3.4.11. 1); and Glucose
               by Clayton and Tretiak (1972) with minor modifications                phosphaste isomerase (GPI, EC 5.3.1.9).
               (Table 2). Staining procedures were from Shaw and Prasad
               (1970) and Siciliano and Shaw (1976). The following 18
               enzymes were tested: a-Glycerophosphate clehydrogenase                Results
               (aGPD, EC 1. 1. 1. 8); D-Sorbitol dehydrogenase (SDH, EC
               1.1.1.14); Lactate dehydrogenase (LDH, EC 1.1.1.27;                   Morphological Analysis
               3-Hydrooxybutyrate dehydrogenase (HBDH, EC
               1.1.1.30); Malate clehydrogenase (MDH, EC 1.1.1.37);                  In Figure 3, are shown the average morphological mea-
               Malic enzyme (ME, EC 1.1.1.40); Isocitrate dehydro-                   surements and standard deviations of the 15 L and 19 S
               genase (IDH, EC 1.1.1.42); 6-Phosphogluconate                         strains. The results indicate that the strains of the rotifer
               dehydrogenase (6PGD, EC 1. 1. 1. 44); Glucose-6-phosphate             could be divided clearly into the two types by quantitating
                                                                                                                                              4


















































               dehydrogenase (G6PD, EC 1.1.1.49); Superoxide                         the shape of the anterior spine (E/D, G/F). The results of
               dismutase (SOD, EC 1. 15. 1. 1); Aspartate aminotrans-                the cluster analysis (Fig. 4A) are identical to the classifica-
               ferase (AAT, EC 2.6. 1. 1); Adenylate kinase (AK, EC                  tion judging by the anterior spine shape (15 L types, 19
               2.7.4.3); Phosphoglucomutase (PGM, EC 2.7.5. 1);                      S types). Each cluster can be divided again into 2 small
               Esterase (EST, EC 3. 1. 1. 1); Alkaline phosphatase (ALP,             clusters. These results indicate that with statistical treat-
               EC 3.1.3. 1); Acid phosphatase (ACP, EC 3.1.3.2);                     ment of the morphologica charcteristics, the varieties of the







                   16             NOAA Technical Report NMFS 92



                                                                                                    Table I
                        Abbreviated names and origins of 34 strains of Brachionas plicatilis tested for morphological and genetic differences. PE: Prefec-
                        tural Experimental Station or Hatchery; SFC: Japan Sea Farming Center; AQD SEAFDEC: Aquaculture Division of South
                        East Asian Fisheries Development Center; NICA: National Institute of Coastal Aquaculture; and CE: City Hatchery. Capital
                        and small letters mean that the strain belongs to L and S type respectively.

                        Abbreviated                                                                                      Station or                 Year of                 Wild (w) or
                                  name             Country                          Locality                             hatchery                   collection           domesticated (d)

                        j-amp                   Japan                    Aomori                                     PE                                 '87                        d
                        j-kay                   Japan                    Kagawa                                     PE                                 '87                        d
                        j-ehp                   Japan                    Ehime                                      PE                                 '87                        d
                        j-otk                   Japan                    Oita                                       SFC                                '87                        d
                        j-nsz                   Japan                    Nagasaki                                   PE                                 '86                        d
                        j-kgko                  Japan                    Kagoshima (Kai Lake)                                                          '78                        w
                        j-kgko-86               Japan                    Kagoshima (Kai Lake)                                                          '86                        w
                        j-kgs                   Japan                    Kagoshima (Shibushi                        SFC                                '87                        d
                        a-sal                   USA                      California (Salton Sea)                                                       '78                        w
                        a-mk                    USA                      Florida (Makay Bay)                                                           '80                        w
                        c_xm                    China                    Fujian Fish. Res. Inst.                                                       '87                        d
                        P-ilo                   Philippines              Panay Island                               AQD SEAFDEC                        '84                        d
                        p-le                    Philippines              Panay Island                               Leganes Stn.                       '84                        d
                                                                                                                    AQD SEAFDEC
                        P-Ot                    Philippines              Oton River (Panay Island)                                                     '84                        w
                        i-ja                    Indonesia                Java                                                                          '86                        d
                        s-sin                   Singapore                Natl. Inst. of Aquaculture                                                    '86                        d
                        t-son                   Thailand                 Sonkia                                     NICA                               '87                        d
                        t-pu                    Thailand                 Puket Marine Inst.                                                            '87                        d
                        is-eil                  Israel                   Eilat                                                                         '87                        d
                        J-Sol                   Japan                    Shizuoka                                   PE                                 '78                        d
                        J-TKU                   Japan                    Univ. Tokyo                                                                   '78                        d
                        J-OTK                   Japan                    Oita (kamiura)                             SFC                                '87                        d
                        J-SAP                   Japan                    Saga                                       PE                                 '84                        d
                        J-SAP-86                Japan                    Saga                                       PE                                 '86                        d
                        J-NSU                   Japan                    Nagasaki Univ.                                                                '69                        d
                        J-NSZ                   Japan                    Nagasaki                                   PE                                 '86                        d
                        J-NSC                   Japan                    Nagasaki                                   CE                                 '86                        d
                        J-NSGT                  Japan                    Nagasaki (Goto Island)                     SFC                                '87                        d
                        J-NSGT-II               Japan                    Nagasaki (Goto Island)                     SFC                                '87                        d
                        J-KAU                   Japan                    Kagoshima Univ.                                                               '86                        d
                        F-PA                    France                   Palavas-les-Flots                                                             '87                        d
                        F-PA-11                 France                   Palavas-les-Flots                                                             '87                        d
                        F-PA-III                France                   Palavas-les-Flots                                                             '87                        d
                        F-PA-IV                 France                   Palavas-les-Flots                                                             '87                        d





                                                                                                    Table 2
                                                                         Buffer systems used for electrophoresis of enzymes.

                                                             Electrode buffer                                            Gel buffer
                        Abbreviated
                                  name       Components                                    pH         Components                                 pH                    References

                                  C-A        0.04 m Citric acid,                           6.1        Dilute 50 mL of electrode buffer           6.1        Clayton and Tretiak (1972)
                                             adjust pH up to 6.1 with                                 to I liter (Citric acid, 0.002 M).
                                             N-(3-aminopropyl)-morpholine.
                                  C-A        0.04 m Citric acid,                           6.9        Dilute 50 mL of electrode buffer           6.9        Clay and Tretiak (1972)
                                             adjust pH up to 6.1 with                                 to 1 liter (Citric acid, 0.002 M).
                                             N-(3-aminopropyl)-morpholine,
                                             then to 6.9 with NaOH.
                                  C-T        0.04 m Citric acid,                           8.0        Dilute 50 mL of electrode buffer           8.0        Clayton and Tretiak (1972)
                                             adjust pH up to 8.0 with                                 to I liter (Citric acid, 0.002 M).
                                             Tris-(hydroxymethyl)-methylamine.


                                                    Fu et al.: Genetics of the Rotifer Brachionus plicatilis                                                              17
                                                   rotifer can be divided into two groups, and that the strains
  A   S                                             within the same type display further variation.
         206                  L
o                 277                                                                                                                                                                        Allozyme Analysis
lk         2t         2t        24-0         905    28-0 3@0[pN
L                                                       Among 18 enzymes tested, 10 enzymes showed clear band-
   0.792
  0                =1 0. 821
   Tr      0                          s                 .ng patterns (Table 3). However, bandings for 3 enzymes
                                                        (AK, EST, and IDH) were not genetically interpretable.
         a75                         0a, 5             The number of alleles of each locus are summarized in
            S                                           Table 4. On MDH, 3 isozyme loci were recognized, al-
              0.619  L  B  0: 662                       though no alleles were detected at 2 loci. In Table 5 are
    C                                                    shown the number of L- and S-type strains and the alleles
       0.5     0.6     0           0,8                 they posses at each locus. The L and S strains differ con-
                       L                                 siderably in allele profiles. For instance, at Ldh where 8
        0.755         1.095                              alleles were observed, 9 of 15 L strains possessed the A
   D               Erl              _U                  allele whereas none of S strains possessed the A allele. In
 E ER;@U"     0-7        d8 0'9    10 .'1  .2           contrast, B allele appeared only in the S strains. There were
 L                                                       considerable genetic differences between L and S strains
  0. 3M  S  F   G     0-f568                              for 15 alleles at 6 loci. Allele frequencies for each allele at
  T'  A                                                   9 loci affecting 7 enzymes were estimated for each strain
    er V\1A 0'3 Ot0.5   0.6  0'7    'G                   in which individuals were considered to be genetically iden-
                                                         tical. For MDH, however, three zones of banding patterns
                                                        appeared. Although two of those three zones were not in-
     Figure 3                                                    terpretable as showing allozyme variation, we regarded
Averages and standard deviations of 5 varieties of measurements   allele frequency as one if the strain had the bandings in
   considered for differentiating L- and S-type strains.




                                                    (A)                      morphological                                                                                                     genetical                                       (B)
                                   80                     60                   40                         20                                          0                          0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 18
                                                                                                                                                      j-P  t-son
                                                                                                                                                      t-son P- t
                                                                                                                                                      i s-e ij         kgko-86
                                                                                                                                                      i-j aa-s a I
                                                                                                                                                      Po tj -k g k
                                                                                                                                                      s-Si ni-j .
                                                                                                                                                      J-kgk. - 86 i-kay
                                                                                                                                                      tpu J-ehp
                                                                                                                                                      C_xm a-mk
                                                                                                                                                      P-1 ICX.
                                                                                                                                                      i-kay Jotk
                                                                                                                                                      j-kgs p-1 e
                                                                                                                                                      Jnsz P-i         lo
                                                                                                                                                      J-ehp - i n
                                                                                                                                                      J-0tk t-P
                                                                                                                                                     1 @j-,
                                                                                                                                                      a-m kI _!         ,s z
                                                                                                                                                      a-s a Ij-P
                                                                                                                                                      J-kgko i s-e I
                                                                                                                                                      J-TKU PP         A-11
                                                                                                                                                      F-PA_RF-PA
                                                                                                                                                      F-PA JTKU
                                                                                                                                                      J-SAP J-SAP
                                                                                                                                                      J-NSZ J-NSZ
                                                                                                                                                      J-NSGTK J N SGT-11
                                                                                                                                                      J-NSGT J_NSGT
                                                                                                                 i
                                                                                                                                                      J_KAU J-NSC
                                                                                                                                                      -Sol i-so I
                                                                                                                                                      Iii -SAP.86 J-SAP .861                                                               1
                                                                                                                                                      i_0TK J-OTK
                                                                                                                                                      F-PA-M J-KAU
                                                                                                                                                      F-PA_lV F-PA-IVi
                                                                                                                                                      J-NSC F-PA-M
                                                                                                                                                      J-NSU J-NSU
                                          PC




                                                                                                              E@                                                                 9:1



                                    80                    60                    2 @0_                     20                                                                   0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
                                                                                  fusion                  level                                                                               genetical distance


Figure 4    of similarities among 34 strains by morphological and genetic snalyses. Abbreviated names by capital and small 
letters mean that the strain belongs to L and S type, respectively.
 







                   18          NOAA Technical Report NMFS 92



                                                    Table 3                                                                              Table 5
                       The different enzyme systems of Brachionus plicatilis screened                       The number of U and S-type strains for each allele at dif-
                       with various buffers.        x      no detectable bandings; A                        ferent enzyme loci.      9 = Great difference in allele posses-
                               unclear bandings; 0       find bandings.                                     sion between U       and S-type strains.

                                                               Buffer                                                               Relative mobility       L-Type       S-Type
                                                                                                            Locus         Allele                               (15)         (19)
                                            C-A                 C-A                  C-T
                       Enzyme             (pH 6. 1)           (pH 6.9)            (pH 8.0)                  Ldh                X               100                2            0
                                                                                                                          0    A               81                 9            0
                       aGPD                     X                 X                     X                                 0    B               69                 0            5
                       SDH                      X                 X                     X                                      C               64                 4            4
                       LDH                      A                 0                     A                                 0    D               47                 1            12
                       HBDH                     X                 X                     X                                      E               39                 1            3
                       MDH                      A                 0                     A                                      0,                                 0            2
                       ME                       X                 A                     A                                      0,                                 1            0
                       IDH                      A                 A                     0                   Mdh-I              A               100                0            3
                       6PGD                     A                 0                     X                                      B               83                 13           16
                       G6PD                     X                 A                     X                                      C               62                 15           9
                       SOD                      0                 0                     A
                       AAT                      A                 0                     A                   Mdh-II        0    ?               -                  0            17
                       AK                       0                 0                     A                   Mdh-III       0    ?               -                  15           6
                       PGM                      X                 0                     A                   6Pgd               A               100                0            1
                       EST                      A                 0                     A                                 *    B               91                 10           0
                       ALP                      A                 A                     A                                      BL              75                 1            1
                       ACP                      A                 A                     A                                      C               68                 4            1
                       LAP                      X                 X                     X                                      CL              55                 2            0
                       GPI                      A                 A                                                       0    D          (-)39                   0            10
                                                                                                                               DL         (-)52                   0            1
                                                                                                                          0    E          (-)68                   0            13
                                                                                                                               F          (-)75                   0            1

                                                                                                            Sod           0    A               100                0            6
                                                    Table 4                                                               0    B               95                 10           0
                               Isozyme    loci and number of observed alleles.                                            0    C               74                 0            17
                                                                                                                          0    D               49                 5            0

                       Enzyme             Locus           Aflele         Subunit structure                  Aat-I              A               100                11           8
                                                                                                                               B               75                 15           15
                        LDH               Ldh                 8              Tetramer                       Pgm-I              A               100                4            6
                        MDH               Mdh-I               3              Dimer                                             B               93                 3            2
                                          Mdh-II              ?                                                                C               89                 1            2
                                          Mdh-III             ?                                                                D               86                 7            6
                        6PGD              6Pgd                9              Dimer                                             E               79                 3            9
                        SOD               Sod                 4              Dimer                                             F               77                 0            1
                                                                                                                               G               70                 0            2
                        AAT               Aat-I               2              Dimer                                             0                                  1            0
                        PGM               Pgm-I               8              Monomer                        Gpi           0    A               100                0            6
                        GPI               Gpi                 6              Dimer                                             B               78                 0            1
                                                                                                                               C               67                 2            6
                                                                                                                          0    D               13                 0            13
                                                                                                                          0    E               28                 13           0
                                                                                                                               F                  0               7            2


                   the zone. If not, we decided allele frequency on the zone
                   as zero. According to Nei's formula (1972), the genetic
                   distances among the 34 strains were estimated from gene
                   frequencies including estimated values for MDH. The den-                             of which consists only of S type strains, and the other two
                   drograrn expressing similarities among the 34 strains was                            clusters consist only of L-type strains. Although one of the
                   also drawn from genetic distances (Fig. 413). The 34 strains                         two large clusters includes the two types of rotifers, the
                   can therefore be divided into two major groups. One group                            classification by the genetic distances also pointed out that
                   consists only of the strains which had been identified as                            there are great genetical distances between L and S strains.
                   S type judging by the anterior spine shape. The other                                Some of the strains which are genetically identical (e.g.,
                   cluster can be divided again into 3 smaller clusters, one                            genetic distance = 0) were collected from neighboring loca-








                                                                                                      Fu et al.: Genetics of the Rotifer Brachionus plicatilis                            19


                   tions or from the same hatchery, for instances between the                                    1981. Polymorphosis in size ofrotifer, Brachionusplicatilis, cultured
                   two strains of p-ilo and p-le or between J-NSGT and                                              with various feeds. Bull. Natl. Res. Inst. Aquacult. 2:1-10.
                   J-NSGT-11. However, 'in one instance (c-xm and *-otk),                                   Fukusho, K., and M. Okauchi.
                                                                                                                 1982.   Strain and size of the rotifer, Brachionus plicatilis, being
                   the samples were geographically unrelated.                                                       cultured in southeast asian countries. Bull. Natl. Res. Inst.
                                                                                                                    Aquacult. 3:107-109.
                                                                                                                 1983.    Sympatry in natural distribution of two strains of a rotifer,
                   Discussion                                                                                       Brachionusplicatilis. Bull. Natl. Res. Ins. Aquacult. 4:135-138.
                                                                                                                 1984.    Seasonal isolation between two strains of rotifer Brachionus
                                                                                                                    plicatilis in an eel culture pond. Bull. Jap. Soc. Sci. Fish. 50:909.
                   For comparison, the two dendrograms are shown in the                                     Hirayama, K.
                   same frame (Fig. 4). The dendrogram patterns for the two                                      1987. An Approach from the physiological aspect to the problems
                   methods are very similar, especially with respect to the                                         in present mass culture technique of the rotifer. In Proceedings
                   L-type strains.                                                                                  of the 15th (1986) U.S. Japan meeting on aquaculture (Al Sparks,
                      The results indicate that the rotifer Brachlonus plicatill's                                  ed.). U.S. Dep. Commer., NOAA Tech. Rep.
                                                                                                            King, C.E., and Y. Zhao.
                   can be divided into the two types of genetic constitution.                                    1987. Coexistence ofrotifer (Brachionusplicatilis) dones in Soda Lake,
                      The results in this report were drawn from 34 strains,                                        Nevada. Hydrobiologia 147:57-64.
                   collected mainly from western Japan. In the case of the                                  Nei, M.
                   L-type, the overseas strains obtained came from only one                                      1972. Genetic distance between populations. Am. Nat. 106:
                                                                                                                    283-292.
                   locality. We are now collecting more strains from all over                               Serra, M., and M.R. Miracle.
                   the world in order to make a more unequivocal conclusion.                                     1983. Biornetric analysis ofBrachionusplicatilisecotypes from Spanish
                                                                                                                    lagoon. Hydrobiologia 104:279-29L
                                                                                                                 1985. Enzyme polymorphism in Br"hionusplicatilis populations from
                   Acknowledgments                                                                                  several Spanish lagoons. Verh. Internat. Limno. 22:2991-2996.
                                                                                                                 1987.    Biometric variation in three strains of Brachionus plicatilis
                                                                                                                    as a direct response to abiotic variables.        Hydrobiologia 147:
                   The authors wish to              express their sincere thanks to                                 83-90.
                   H. Kayano, Nagasaki University, for his kind advice on                                   Shaw, C.R., and R. Prasad.
                   the interpretation of allozyme variation, to K. Fukusho who                                   1970. Starch gel electrophoresis of enzymes - a compilation of
                   kindly provided photos of L and S strains, and also to the                                       recipes. Biochem. Genet. 4:297-320.
                   scientists who kindly sent us live samples of the rotifers.                              Siciliano, M.J., and C.R. Shaw.
                                                                                                                 1976. Separation and visualization of enzymes on gels. In
                                                                                                                    Chromatographic and electrophoretic techniques, 4th ed., Vol.
                                                                                                                    2:185-209 (1. Smith, ed,). William Heinemann Medical Books
                   Citations                                                                                        Ltd, London.
                                                                                                            Snell,  T.W., and K. Carrillo.
                   Clayton, J.W., and D.N. Tretiak.                                                              1984.    Body size variation among strains of the rotifer Brachionus
                        1972. Amine-citrate buffers for pH control in starch gel electro-                           plicalihs. Aquaculture 37:359-367.
                           phoresis. J. Fish. Res. Board Canada 29:1169-1172.                               Snell, T.W., and B.C. Winkler.
                   Fukusho, K.                                                                                   1984. Isozyme analysis of rotifer proteins. Biochem Syst Eco
                        1983.    Present status and problems in culture of the rotifer Br"hionus                    12:199-202.
                           plicatilis for fry production of marine fishes in Japan. Symp. Intl.             Suzuki, M.
                           Acuacultura Coquimbo, Chike-Septiembre:361-373.                                       1983. Taxonomical study on rotifers cultured for fry production.
                   Fukusho, K@., and 1, Iwamoto.                                                                    Zool. Mag. 91:657.
                        1980. Cyclomorphosis in         size of the cultured rotifer,    Brachionus              1987. IntraspecificvariabdityofBrachionusplicatilis. Hydrobiologia
                           plicalilis. Bull. Natl. Res. Inst. Aquacult. 1:29-37.                                    147:45-48.








                       Present Status of Genetic Studies on Marine Finfish in Japan


                                                                    KUNIHIKO FUKUSHO

                                                                 National Research Institute of Aquaculture
                                                                              Fisheries Agency
                                                               Nakatsuhama, Nansei-cho, Watarai-gun, Mie
                                                                               516-01 Japan




                                                                              ABSTRACT


                                      The present paper briefly introduces the status of genetic breeding of marine finfish in Japan.
                                   The domestication of exotic species is also described. Selection is the most successful technique
                                   of genetic breeding for marine finfish, even though limited scientific data and experimental results
                                   have been reported. Selection was conducted on red sea bream, I'agrus major, and Japanese flounder,
                                   Paralichthys olivaceus, and their selected strains were supplied for industrial culture. Experiments
                                   on hybridization leading to heterosis were conducted as well as interspecific, intergeneric, inter-
                                   family, back and reciprocal crosses. Few of these hybrids, however, have been widely used by
                                   the industry, except the P. major x crimson sea bream, Evynnisjaponicus. Chromosome manipu-
                                   lation studies such as triploid production and all female production by gynogenesis have been
                                   conducted since 1984 in Japan. These technologies are strongly expected to be adopted in in-
                                   dustrial culture, even though they are currently experimental. Exotic species or strains of marine
                                   finfish have been introduced to Japan and cultured in recent years. Most marine species are
                                   imported to supply seed where local production is inadequate, not to introduce a new industrial
                                   target species or strain, except the red sea bream. Cryopreservation of sperm is used in most
                                   hybridization studies, induction of gynogeriesis, and triploid production. This technology will
                                   no doubt be adopted in androgenesis and gene bank projects for marine finfish.




                Introduction                                                             tools for improving fish quality. Therefore, various kinds
                                                                                         of experiments on genetics, including chromosome man-
                Mariculture of finfish is well developed in Japan as reflected           ipulation have been intensively conducted in recent years
                by the total harvest in 1986 of nearly 2 x 105 tons. The                 for marine finfish, despite the short history of mariculture.
                number of cultured species is approximately 30 (Table 1)                    The objective of the present paper is to provide a brief
                (Fukusho 1981). Yellowtail, Seriola quinqueradiala (14.6 x               introduction on the status of genetic breeding of marine
                104 tons), and red sea brearn (3.4 x 104 tons), are the most             finfish, except salmonids in Japan. Introductions leading
                important species. The Japanese flounder and coho                        to domestication are considered part of the study of gene-
                salmon, Oncorhynchus kisutch, have shown great promise as                tics for the purpose of this review.
                cultured species, with their production levels increasing
                rapidly in recent years (0.3 x 104 and 1.2 x 104 tons, re-
                spectively for 1987 data). All the marine species are cul-               The Introduction of Exotic Species
                tured in net cages except the Japanese flounder which is
                usually raised in land based tanks.                                         Exotic species or strains of marine finfish, such as
                  At present, the total supply is adequate to satisfy demand             yellowtail, red sea bream, rockfish, Sebasticus spp., grouper
                for yellowtail and red sea bream, taking into considera-                 Epinephelus spp., knifejaw Oplegnathus spp., have been in-
                tion the total cornsumptiorn plus the carrying capacity of               troduced to Japan and cultured in recent years. Marine
                the culture ground. Therefore, research presently focuses                finfish have been introduced for different reasons than have
                on the improvement of fish quality (e.g., high growth rate,              freshwater fish. Most marine species are imported to supply
                high resistance to pollution, good taste and flavor, and                 seed where the local production is inadequate, not to intro-
                favorable color) based on the requirements of culturists and             duce a new industrial target species of strain. An excep-
                consumers. Genetic breeding is one of the most effective                 tion is the case of the red sea bream.

                                                                                                                                                          21








                    22            NOAA Technical Report NMFS 92



                                                                                                         Table I
                                                                           Marine finfish cultured in Japan (Fukusho 1981).

                                                       Family                      Common name                             Scientific name                  Pref. No.'

                                                  Salmonidae                 Cohn salmon                              Oncorhynchus kisutch                          I
                                                  Mugilidae                  Grey mullet                              Mugil cephalus                                I
                                                  Oplegnathidae              Japanese striped knifejaw                Oplegnathus fasciatus                      19
                                                                             Japanese spotted knifejaw                0. Punclatus                                  7
                                                  Serranidae                 Sea bass                                 Lateolabraxjaponicus                          5
                                                                             Sea bass                                 L. latus                                      I
                                                                             Groupers                                 Epinephelus spp.                              3
                                                  Girellidae                 Nibbler                                  Girella punctala                           11
                                                  Sparidae                   Red sea brearn                           Pagrus major                               25
                                                                             Porgy                                    Sparus sarba                                  2
                                                                             Crimson sea brearn                       Eoynnisjaponica                            I I
                                                                             Porgy                                    Acanthopagrus schlegeli                    17
                                                                             Porgy                                    A. latus                                      2
                                                  Pomadasyidae               Grunt                                    Parapristipoma trilinea                       I
                                                  Carrangiclae               Yellowtail                               Seriola quinqueradiata                     26
                                                                             Amberjack                                S. purpurascens                               8
                                                                             Amberjack                                S. aureovittata                               2
                                                                             Horse mackerel                           Trachurusjaponicus                         20
                                                                             Striped jack                             Longirosirum delicatissimus                   9
                                                  Scombridae                 Bluefin tuna                             Thunnus thynnus                               3
                                                  Siganiclae                 Rabbit fish                              Siganusfuscessens                             3
                                                  Alutericlae                Filefish                                 Stephanolepis cirrhifer                       2
                                                  Tetradontidae              Puffer                                   Fugu rubripes                                 9
                                                  Scorpacnidae               Rockfish                                 Sebastiscus marmoratus                        3
                                                                             Rockfish                                 Sebastes inermis
                                                                             Rockfish                                 S schlegel,                                   I
                                                  Bothiclae                  Flounder                                 Paralichthys olivaceus                     10


                                                  'Number     of prefectures where the species was         cultured.




                       A deep reddish color is highly prized in cultured red sea                                  and response to new environmental conditions should always be
                    bream by the Japanese consumers. A Korean strain, which                                       considered prior to introduction, as with freshwater fish.
                    is identical to the Japanese strain taxonomically, shows a
                    much deeper reddish color than the latter (Harada et al.
                    1988, Harada et al. 1985, Kumai et al. 1986). No difference                                   Hybridization
                    has been found between the two strains in electrophoresis
                    analysis of isozymes. The Korean strain is preferred by fish                                  Experiments on hybridization leading to heterosis have con-
                    farmers because of its deeper reddish color, even though                                      tributcd to the development of larval rearing techniques for
                    the Japanese strain is superior to the Korean strain in terms                                 marine finfish (Fujita 1961, 1967; Harada 1974, 1975, 1978).
                    of growth rate (Kumai et al. 1986). The deep reddish color                                    Interspecific, intergeneric, interfamily, back and reciprocal
                    is caused by its higher content of carotenoid and astaxan-                                    crosses have also been attempted (Harada 1978).
                    thine in the skin, which results even under the same rear-                                        Hybridization of marine finfish was initiated on puffers
                    ing and feeding conditions as the Japanese strain (Kumai                                      (1961-67) of which several species are a high prized luxury food
                    et al. 1986). Hybridization between the Japanese and                                          despite the fact that parts of these fish are highly toxic (Fujita
                    Korean strains has been conducted to provide a hybrid with                                    1967) (Table 2). The Fisheries Laboratory of Kinki University
                    a deeper reddish color and higher growth rate (Harada                                         has further promoted hybridization to improve fish quality.
                    et al. 1988).                                                                                 Several successful and promising hybrids were produced that are
                       Both fertilized eggs and juveniles of Japanese red sea                                     superior in growth rate, survival rate, body color, and meat
                    bream have been exported to foreign countries; Thus                                           quality to each parent fish (Table 3) (Harada 1974, 1975, 1978).
                    marine finfish have been introduced world-wide and mari-                                      The "kindai" (Oplegnathusfasciatus x 0. punclatus), which ab-
                    culture has expanded. In each country, they are being                                         breviates the name of Kinki University and which means sea
                    cultured as an introduced species. Precise investigation and                                  brearn and sounds like "golden fish" injapanese, is significantly
                    research of a new marine species' biological characteristics                                  superior in growth rate, survival rate, and handling to each







                                                                                                              Fukusho: Genetic Studies on Marine Finfish in Japan                                         23



                                                          Table 2                                                                                           Table 3
                                    Hybridization of puffers (Fujita 1967).                                               Hybrids of marine finfish produced at the Kinki Univer-
                                                                                      Hatching rate                                       sity (Harada 1974, 1975, 1978).
                                                  Female        Male                          (%)                                         Female       x    Male

                                       Takifugu pardalis    x   T. poecilonotus               95                                   Pagrus major        x    Acanthopagrus schulegeli                1964
                                          T. poecilonolus   x   T. pardalis                   95                                        P. major       x    Sparus sarba                              67
                                              T rubripes    x   T. xanthopterus               93                          Oplegnathusfasciatus         x    A. schlegeli                              68
                        Lagocephalus lunaris spadiceus      x   T. niphobles                    0                                   0. fasciatUS a     X    0. Punctatus                              69
                                         L. 1. spadiceus    x   Canthigaster rivulata           0                         Seriola quinqueradiata'      x    S. auroeovittata                          70
                                                                                                                              S. quinqueradiaea        X    S. purpurascens                           70
                                                                                                                                S. purpurascens'       x    S. aureovittata                           71
                                                                                                                                  S. aureovittata      X    S. purpurascens                           72
                                                                                                                                        P. majora      x    Evynnis japonicus                         73
                    parent fish (Kumai 1984; Harada et a]. 1986). Because red                                                       0. punialus'       x    0. fascialus                              73
                    sea bream. are usually cultured in protected bays with vary-                                                  Auxis thazard        x    Euthynnus affinis                         76
                    ing salinity, tolerance to low salinity is an important char-                                         'Promising hybrid.
                    acteristic. Therefore, a useful hybrid of the red sea bream,
                    and the porgy, Acanthopagrus shulegeli, was developed which'
                    showed both improved tolerance to lower salinity than the
                    maternal fish plus faster growth and better taste than the
                    paternal one (Harada 1975). The Nagasaki Prefectural                                             wild red sea bream caught in the Akashi area in the Seto
                    Institute of Fisheries has also conducted studies on the                                         Inland Sea. Production of selected strains has also been
                    hybridization of marine finfish. A hybrid of a sparid, Sparus                                    intensively conducted on the Japanese flounder and the
                    sarba, and the porgy, A. shulegell', was produced in order                                       Japanese sriped knifej aw, 0. fasnatus, at the Fisheries Lab-
                    to combine the highest growth characteristics of S. sarba                                        oratory of Kinki University (Harada 1975, 1978).
                    and the low salinity resistance of A. schulegeli (Kitajima and
                    Tsukashima 1983). However, the hybrid showed mater-
                    nal characteristics in both its morphological and physio-                                        Chromosome Manipulation
                    logical characteristics (Kitajima and Tsukashima 1983).
                    The same phenomenon occurred in the hybrid of P. maj'or                                          Since 1984, chromosome manipulation studies such as
                    and the crimson sea bream, Evynnisjaponicus (Arakawa and                                         triploid production, all female production by gynogenesis,
                    Yoshida 1986, Arakawa et al. 1988).                                                              extraction and synthesis of growth hormone, production
                        Few of these hybrids have been widely used by industry,                                      of cloned fish, and cell fusion have been conducted in
                    except the hybrid of P. maj'or x E. japonicus. The reason                                        Japan to produce new strains of marine finfish. These
                    may be due to 1) conservative consumers to whom appear-                                          technologies have been called "Fisheries Biotechnology."
                    ance is very important (red sea brearn must look like the                                        In 1985, the Ministry of Agriculture, Forestry, and
                    wild red sea bream. because theJapanese people always eat                                        Fisheries (MAFF) designed and organized a large-scale
                    the whole and raw fish for sashimi and sushi; 2) limited                                         scientific project on chromosome manipulation titled
                    attempts to show clearly the difference in quality between                                       "Development of new breeding techniques by induction
                    the hybrid and parent fish; and 3) too short a period of                                         of gynogenesis in fish." The National Research Institute
                    marine finfish culture for the industry to evaluate and                                          of Aquaculture was the leading institution to promote the
                    utilize new strains or hybrids as well as exotic species.                                        project, along with three Universities (Tokyo University
                                                                                                                     of Fisheries, Hokkaido University, and Kansei Gakuin
                                                                                                                     University), two Regional Fisheries Laboratories (Nansei
                    Selection                                                                                        Regional Fisheries Laboratory and Hokkaido Regional
                                                                                                                     Fisheries Laboratory of the Fisheries Agency-MAFF), and
                    Selection is the most successful technique of genetic breed-                                     three Prefectural Institutes of Fisheries (Saitama, Hyogo,
                    ing for marine finfish, even though limited scientific data                                      and Hokkaido). The target species included the Japanese
                    and experimental results have been reported. Selected                                            flounder, red sea bream, plaices, Verasper moseri, Limanda
                    strains of red sea bream have been supplied by the Fish-                                         shrenki, L. punctatissima, Platichthys stellatus, and filefish,
                    eries Laboratory of Kinki University. These selections are                                       Stephanolepis cirrhifer, Thamnaconus modestus, Aluterus monoceros.
                    highly desired by fish farmers because their growth rates                                        The Fisheries Agency has also organized and initiated a
                    are approximately 30-40% higher than the wild forms.                                             scientific project addressing fisheries biotechnology. Several
                    Mass selection has been conducted at the Fisheries Lab-                                          Prefectural fisheries laboratories have also joined this
                    oratory of Kinki University over several generations from                                        "Local Biotechnological Study Project" where marine








                   24           NOAA Technical Report NMFS 92


                   finfish such as the red sea brearn and flounder are being                              preservation is also used in most hybridization studies,
                   studied.                                                                               induction of gynogenesis, and triploid production.
                      Triploid and gynogenetic diploid induction techniques                                  In marine fish, cryopreservation has been conducted on
                   which use cold shock have been used to block the second                                a variety of species, such as: two species of goby, Glosso-
                   polar body extrusion for red sea brearn (Arakawa et al.                                gobius olivaceus, Acanthogobw'flavimanus; porgy; mullet, Mugil
                   1987; Arakawa and Miyahara 1988; Fukusho et al. 1987b),                                cephalus; mackerel, Scomberjaponicus; bluefin tuna, Thunnus
                   porgy (Arakawa et al. 1987), and flounder (Tabata, 1988;                               thynnus; and puffer, Takaifugu nipholbles (Doi et al. 1982;
                   Tabata and Gorie 1988a, Tabata et al. 1986). The dura-                                 Kurokura 1983). A recent study on the hybridization of
                   tion of cold shock intervals are as follows: for red sea bream,                        red sea bream and crimson sea brearn showed positive
                   15-20 min. duration of OOC, starting 3 min. after insemina-                            results with high survival rates and increased growth rates
                   tion; for porgy, 25 min. duration, starting 1.5 min. after                             observed when sperm preserved for 6 months was used
                   insemination; and for flounder, 45 min. duration, start-                               (Kurokura et al. 1986).
                   ing 3-5 min. after insemination. Suppression of the Ist                                   Thus, gamete preservation is useful for hybridization of
                   cleavage was achieved by using increased hydrostatic                                   species which spawn in different seasons, genetic breeding
                   pressure (Tabata and Gorie 1988b). UV irradiation has                                  by chromosome manipulation, transplantation (introduc-
                   been effective for genetic inactivation of sperm (e.g.,                                tion), and gene bank projects for marine finfish.
                   1000-2000 erg/mm2 for red sea brearn). Also, sperm of
                   different species have often been used as an indicator of
                   the induction of gynogenesis (Fukusho et al. 1987a; Yano                               Acknowledgments
                   and Sakai 1988). Triploids were produced in red sea brearn
                   and porgy (Arakawa et al. 1987, 1988; Fukusho et al.                                   I wish to express my sincere thanks to Dr. F. Brian Davy,
                   1987b). Thus, various conditions for induction of triploid                             IDRC, Canada and Dr. Ryo Suzuki, National Res. Inst.
                   and gynogenesis have been examined for several marine                                  Aquaculture, Japan for their critical reading of this
                   finfish, and comparisons of growth rate, survival rate, and                            manuscript.
                   other biological characteristics have been conducted
                   through larval rearing (Arakawa and Yoshida 1986; Fuku-
                   sho et al. 1987a; Tabata et al. 1986; Tabata and Gorie                                 Citations
                   1988a). Comparison of growth rate during rearing to young
                   stage and commercial size was also conducted (Tabata and                               Arakawa, T., and J. Miyahara.
                   Gorie 1988a), but there is little information to evaluate                                   1988. Induction of gynogenesis with ultra violet rays in red sea
                   gynogenetic and triploid fish in view of industrial culture                                    bream, Pagrus major. Bull. Nagasaki Pref. Inst. Fish. 14:37-42.
                   because the scientific activities have only just started.                                      (In Japanese; English summ.)
                                                                                                          Arakawa, T., and Y. Yoshida.
                   Reports    .and papers on the comparison of growth and                                      1986. Growth, survival and morphologic comparison between fry
                   maturation rates and sex ratio between chromosome ma-                                          cross bred Pagrus major with Evynnisjaponica and hatchery reared
                   nipulated fish and common diploid fish are expected to                                         Pagrusu major. Bull. Nagasaki Pref. Inst. Fish. 12:27-35. (In
                   promote these techniques in industrial mariculture of fin-                                     Japanese; English summ.)
                   fish. Techniques for examination of ploidy have been                                   Arakawa, T., M. Tanaka, K. Inoue, 1. Takami, and K. Yamashita.
                   established, but with conflicting result (e.g., appearance                                  1987. An examination of the conditions for triploid induction by
                                                                                                                  cold shocck in red sea brearn and black sea bream. Bull. Nagasaki
                   of males among gynogenetic diploids of the flounder in spite                                   pref. Inst. Fish. 132:25-30. (In Japanese; English summ,)
                   of a theoretical expectation of all female production)                                 Arakawa, T., C. Kitajima, K. Yamashita, A. Ikeda, and H. limura.
                   (Tabata 1988; Tabata and Gorie 1988b). These phenom-                                        1988. Growth and morphology of crossbred Pagrus major with Evynnis
                   ena could not be explained by the XX and XY sex                                                i.aponica. Bull. Nagasaki Pref. Inst. Fish. 14:31-3 5. (In Japanese;
                                                                                                                  English summ.)
                   chromosome theory. Further investigation on embryology                                 Doi, M., T. Hoshino, Y. Taki, and Y, Ogasawara.
                   and sexual differentiation might be required as well as                                     1982. Activity of the sperm of the bluefin tuna Thunnus thynnus under
                   genetic studies. The mechanism of sexual differentiation                                       fresh and preserved conditions. Bull. Japan. Soc. Sci. Fish. 48:
                   should be clarified to advance the technology of chromo-                                       495-498. (In Japanese; English summ.)
                   some manipulation.                                                                     Fujita, S.
                                                                                                               1961.    Studies on life history and aquaculture of important puffers
                                                                                                                  in Japan.    Special report of the Nagasaki Pref. Inst. Fisheries,
                                                                                                                  No. 2, 121 p. (In Japanese)
                   Cryopreservation of Sperm                                                                   1967. Artificial interspecific and intergeneric hybridization among
                                                                                                                  the tetradontid puffer (Prelim. rep.). Jpn. J. Michurin Biol.,
                   Experiments on androgenesis have been conducted for                                            3:5-11. (In Japanese; English summ.)
                   freshwater fish in Japan. Cryopreservation of sperm in                                 Fukusho, K.
                                                                                                               1981. Present status and view of fry production and genetic breed-
                   combination with androgenesis is useful in preserving en-                                      ing of marine finfish. Fish Genetics and Breeding 6:1-10. (In
                   dangered species, and also in all male production. Cryo-                                       Japanese.)








                                                                                                Fukusho: Genetic Studies on Marine Finfish in Japan                             25


                 Fukusho, K., M. Okauchi, H. Nanba, M. Hoshi, and H. Tsubaki.                                 Oplegnaihusfasciatus (Temminck and Schlegel). Bull. Fish. Lab.
                       1987a. Comparison in growth and survival rate among gynogenetic                        Kinki Univ. 2:1-127. (In Japanese; English summ.)
                         larvae of red sea bream, being induced by sperm of red sea bream,            Kumai, H., M. Nakamura, Y. Kubo, and Asada.
                         flounder, and striped knifejaw. Proc. MeetingJapan. Soc. Sci.                      1986. Comparison of growth and morphological characteristics
                         Fish. 1987 (Hakodate), p. 149. (In Japanese.)                                        among Japanese, Korean, and Hong Kong red sea bream.
                       1987b. An attempt of triploid induction of red sea bream, using                        Proc. Meeting Japan. Soc. Sci. Fish. 1986 (Tokyo), p. 28. (In
                         fertilized eggs by natural spawning in net cage. Proc. Meeting                       Japanese.)
                         Japan. Soc. Sci. 1987 (Hakodate), p. 149. (In Japanese.)                     Kurokura, H.
                 Harada, T.                                                                                 1983. Cryopreservation of fish sperm. Fish Genetics and Breeding
                       1974. Genetic improvement of sea bream. Yoshoku (Midori-                               Sci. 8:42-53. (In Japanese.)
                         shobow), 11:50-54. (Injapanese.)                                             Kurokura H., S. Kasahara, H. Kumai, and M. Nakamura.
                       1975. Target species of marine finfish for fry production. In                        1986. Hybridization of red sea brearn and crimson sea brearn by
                         Feeding and development of larvae and juvenile (Japan. Soc. Sci.                     cryopreservation of sperm. Proc. MeetingJapan. Soc. Sci. Fish.
                         Fish., eds.), p. 90-96. Koseisha-Koseikaku, Tokyo, Japan. (In                        1986 (Tokyo), p. 50. (In Japanese.)
                         Japanese.)                                                                   Tabata, K.
                       1978. Cross breeding of marine finfish. Yoshoku (Midori-shobow)                      1988. Review: Studies on chromosome manipulation in Hirame,
                         15:32-35. (In Japanese.)                                                             Paralichthys olivaceus. Fish Genetics and Breeding Sci. 13:9-18.
                 Harada, T., 0. Murata, S. Miyashita, S. Oda, and S. Maeda.                                   (In Japanese.)
                       1985. Incubation and larval rearing of Korean red sea bream.                   Tabata, K., and S. Gorie.
                         Proc. Meeting Japan. Soc. Sci. Fish. 1985 (Tokyo), p. 54. (In                      1988a. Comparison of the growth of gynogenetic diploids with con-
                         Japanese.)                                                                           trol diploid in Hirame Paralichthys olivaceus in the same tank.
                 Harada, T., H. Kumi, and 0. Murata.                                                          Bull. Japan. Soc. Sci. Fish. 54:1143-1147. (In Japanese; English
                       1986. Artificial hybrids between Japanese parrot fish and spotted                      summ.)
                         parrotfish. Bull. Japan. Soc. Sci. Fish. 52:613-62 1. (In Japanese;                1988b. Induction of gynogenetic diploids in Paralich1hysalivaceus by
                         English summ.)                                                                       suppression of the I st cleavage with special reference to their sur-
                 Harada, T., 0. Murata, and S. Miyashita.                                                     vival and growth. Bull. Jpn. Soc. Sci. Fish. 54:1867-1872. (In
                       1988. Artificial hybridization between Japanese red sea bream and                      Japanese; English summ.)
                         Korean red sea bream, and three years culture of the hybrids.                Tabata, K., S. Gorie, and N. Taniguchi.
                         Proc. MeetingJapan. Soc. Sci. Fish., 1988 (Tokyo), p. 276. (In                     1986. Verification by isozyme gene marker for gynogenetic
                         Japanese.)                                                                           diploidization and triploidization in Hirame, Paralighthys
                 Kitajima, C., and Y. Tsukashima.                                                             olivaceus. Fish Genetics and Breeding Sci. 11:35-41. (In Japa-
                       1983. Morphology, growth and low temperature and low salinity                          nese.)
                         tolerance of sparid hybrids. Jpn. J. Ichthyol. 30:275-283. (In               Yano,   Y., and Y. Sakai.
                         Japanese; English summ.)                                                           1988. Introduction of gynogenetic diploids in two species of flat
                 Kumai, H.                                                                                    fish. Bull. Hokkaido Reg. Fish. Res. Lab. 52:167-172. (In
                       198C Biological studies on culture of the Japanese parrot fish,                        Japanese; English summ.)







                                          Recombinant Viral Vaccines in Aquaculture*


                           JO-ANN C. LEONG, R. BARRIE, H.M. ENGELKING, J. FEYEREISEN-KOENER,
                                  R. GILMORE, J. HARRY, G. KURATH, D.S. MANNING, C.L. MASON,
                                                                  L. OBERG, and J. WIRKKULA

                                                                            Department of Microbiology
                                                                              Oregon State University
                                                                          Corvallis, Oregon 97331-3804




                                                                                  ABSTRACT


                                        Viral pathogens in aquaculture have largely been controlled by the culling and destruction
                                     of carriers and infected animals and eggs. Because most viral pathogens in aquaculture are trans-
                                     mitted via water and because sensitive animals reside in the neighboring waters, the administra-
                                     tion of attenuated viral vaccines has not been feasible. Attenuated vaccines require costly trials
                                     to assure that these modified live viruses are nonvirulent in all species and that reversion to virulence
                                     does not occur. Killed viral vaccines have been too expensive to produce for the aquaculture
                                     industry. Thus, subunit viral vaccines developed by recombinant DNA techniques are attractive
                                     alternatives for the industry. These vaccines are nonreplicating and inexpensive to produce. The
                                     molecular cloning and expression of viral genes in several host vector systems for the develop-
                                     ment of subunit viral vaccines for aquaculture has been the primary research focus of the authors'
                                     laboratory. Work on the development of such vaccines for infectious hematopoietic necrosis virus
                                     (IHNV), a fish rhabdovirus, and infectious pancreatic necrosis virus (IPNV), a fish birnavirus,
                                     is presented. Laboratory tests of both vaccines in vivo have indicated that fish develop protective
                                     immunity to live virus after vaccination.



                Introduction                                                                  earthen ponds and stable viruses, like the baculoviruses and
                                                                                              the picornaviruses, these disinfection procedures may not
                One of the major factors that will have an impact on the                      work.
                success of the aquaculture industry is the control of                           Another facet of present day controls for viral diseases
                diseases. As the industry grows and greater productivity                      in aquaculture is the requirement for certified pathogen-
                demands are madeon facilities, the incidence of disease                       free stocks and eggs and the use of specific pathogen-free
                outbreaks will increase. Thus, the need for more effective                    water in the facility. When available, these requirements
                disease controls has been receiving more attention. The                       have been very effective in preventing disease outbreaks.
                viral diseases are particularly important because there are                   However, it has not always been possible to obtain disease-
                no suitable treatments available. In the United States, there                 free stocks for highly-prized strains nor economically prac-
                are no approved antiviral drugs or vaccines that can be                       tical to use specific pathogen-free water. Thus, the aqua-
                used in the aquaculture industry today.                                       culture industry has a definite need for viral vaccines. Our
                   At the present time, the control of viral diseases is based                group reports here the successful development of two pro-
                largely on management. Current recommendations for the                        totype viral vaccines by recombinant DNA techniques.
                control of viral disease outbreak include the destruction of                    Two viruses were selected for vaccine development
                diseased stocks, drainage of ponds, disinfection of con-                      because these viruses affect economically important aqua-
                taminated areas with chlorine, treatment with sunlight or                     culture species (salmon and trout) in the United States,
                lime, and the restocking of the facility with disease-free                    Europe, and Japan. In addition, these viruses, infectious
                stock. These procedures are very expensive and instituted                     hematopoetic necrosis virus (IHNV), and infectious pan-
                with understandable reluctance by the industry. With                          creatic necrosis virus (IPNV), affect very young fish, and
                                                                                              immunization of large numbers of fish at this size by im-
                                                                                              mersion is fairly easy. IHNV is a rhabdovirus with an
                *Oregon Agricultural Experiment Station Technical Paper No. 8961.             enveloped virion and glycoprotein peplomers on the

                                                                                                                                                                  27








                28         NOAA Technical Report NMFS 92


                envelope surface; it has a viral genome of single-stranded                   pared against purified IHNV and IPNV were made as
                RNA of negative sense (      'McAllister and Wagner 1977). In                described (Engelking and Leong 1989).
                contrast, IPNV is a nonenveloped virus with a single cap-
                sid and a genorne of two segments of double-stranded RNA
                (Dobos 1975). The techniques that were involved in the                       Construction of Recombinant Plasmids
                construction of recombinant plasmids containing the genes                    The construction of a recombinant plasmid containing the
                for the major immunogenic proteins of these two viruses                      trp E promoter and the trp E gene fused to an immunogenic
                have been described (Kurath and Leong 1985; Huang                            region of the gene for IHNV glycoprotein gene or the
                et al. 1986). The expression of these proteins in bacteria                   IPNV VP2 gene is shown schematically in Figure 2. The
                and the use of these expressed proteins as vaccines will be                  isolation, cloning and sequence analysis of these genes have
                described here.                                                              been reported (Kocner et al. 1987; D. S. Manning 1988).
                                                                                             The pATH vectors were the generous gift of A. Tzagaloff
                                                                                             (Dieckmarm and Tzagoloff 1985). The constructions were
                Materials and Methods                                                        verified by DNA sequence analysis by the dideoxy method
                                                                                             (Sanger et al. 1977). The plasmid pUC 19, which served
                Cells, Virus, and Antisera                                                   as the negative control for pTA1 in Figure I was obtained
                The following viruses here used in this study: the IHNV                      from Pharmacia, Inc., Piscataway, NJ.
                isolate from Round Butte was obtained from W. Groberg
                (Oregon Department of Fisheries and Wildlife) and the                        Immunization Trials in Fish
                IPNV isolates, Sp and Buhl, were obtained from R. Hed-
                rick, University of California at Davis. The virus used for                  Bacterial crude lysates were prepared as described (Kleid
                challenge studies was prepared by infecting rainbow trout                    et al. 1981). Proteins were analyzed by sodium dodecyl
                (Oncorhynchus mykiss) fry and reisolating the virus from fish                sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
                dying of IHN disease in the case of the IHNV isolates and                    and Western immunoblotting as previously described
                IPN disease in the case of the IP@NV isolates. Subsequently,                 (Gilmore et al. 1988). The crude lysates were used to im-
                the virus was grown for two passages in chinook salmon                       munize fish by immersion. Rainbow trout fry at 0.4 g were
                embryo cells (CHSE-214 cells) (Fryer et al. 1978). The                       immunized in sets of 100 fish. Immunization was ac-
                tissue-culture supernatant fluid containing the virus was                    complished by bathing groups of 100 fry in 25 mL of the
                used as the challenge virus. The IHNV and IPNV used                          vaccine preparation (ca. 3 mg/mL total protein concen-
                as molecular weight markers in Figure I were prepared                        tration) for I minute. At that time, the immersion solu-
                as described in Kurath and Leong (1985) for IHNV and                         tion volume was increased to 250 mL with water and fish
                Huang et al. (1986) for IPNV. The            rabbit antisera     pre-        were incubated in this diluted solution for an additional





                                                               12                      LO                                          Figure I
                                                                              .2                              Analysis of bacterial production of trpE-viral gene
                                                                              I.-      tu
                                                               E              4        E                      fusion proteins by antibody reactivity on an elec-
                                  >                                 CL        CL
                                  Z
                   A.          'L
                           CL                    B.               1 5 25   1 5 25    1 2                      trophorectic transfer blot of a 10 % SDS-polyacr-y-
                           -------                                                                            lamide gel of bacterial extracts. (A) Development
                                                                                                              of the blot made with anti-IHNV sera. Lane a
                                                    110                                  _VP1                 is the trpE-G fusion protein detected in cells con-
                                                        -#                           WWI,                     taining the recombinant plasmid, p52G; Lane b
                                                     66-                                 -4- TrpE-VP2         are proteins detected in cells containing the ex-
                                                    45-                                  -VP2                 pression vector pATH3, without a viral gene in-
                                                                                                              sert; and Lane c is purified IHNV. (B) Develop-
                                                                    *".mow                                    ment of the blot made with anti-IPNV sera.
                                                     31-1                                    VP3              Lanes a and i contain the molecular weight
                                                                                             VP3A             marker proteins: phosphorylase B (110 000 Da);
                                                                                                              bovine serum albumin (66000 Da); ovalbumin
                                                                                                              (45 000 Da); and carbonic anhydrase (31000 Da).
                           a b c                              a b c    d  e f g h I                           In lanes b, c, and d are cell lysates from bacterial
                       Anti-IHNV Sera                              Anti-IPNV Sera                             cells containing the plasmid pUC 19 with no viral
                                                                                                              insert; the samples were loaded at 1, 5 and 25
                                                                                                        -     pL respectively in lanes b, c, and d. In lanes c,







                                                                                     Leong et al.: Recombinant Viral Vaccines in Aquaculture                            29


                 2 minutes. These fish were then placed in aquaria of 5                          from stained gels and Western blots of total bacterial ex-
                 gallons with a water flow rate of 0.25 gal/min in a con-                        tract. In Figure 1, the product of a trpE-IHNV glycopro-
                 stant water temperature of 10'C. The control fish were                          tein fusion gene from the plasmid p52G and the major cap-
                 exposed to saline in the same procedure or left undisturbed.                    sid protein of IPNV from the plasmid pTAl is shown in
                   Approximately one month after immunization, the ex-                           Western blots of the appropriate bacterial lysates. A deter-
                 perimental and control fish were placed in separate aquaria                     mination of the DNA sequence of p52G indicated that a
                 in groups of 25. The fish were exposed to serial log virus                      264 bp fragment of the lHNV glycoprotein gene had been
                 dilutions in I liter of water. The challenge virus was                          inserted in-frame with the trpE protein to produce a fusion
                 prepared as described by Engelking and Leong (198 1). In                        protein of 49000 daltons (49 kDa = 37 kDa [trpEl + 11
                 Figure 3, the data for fish exposed to 7.2 x 10 + 5 plaque                      kDa [glycoprotein gene fragment]). In addition, a second
                 forming units per mL (PFU/mL) is presented. The data                            fragment of the IHNV glycoprotein gene had been inserted
                 represents the mean of duplicate experiments. All dead fish                     out-of-frame adjacent to the 264 bp fragment and this ad-
                 were assayed for the presence of infectious virus in chinook                    ditional nucleic acid resulted in I kDa (84 bp extra) more
                 salmon embryo cells (CHSE-214) as described by Engel-                           of amino acid residue owing to the fusion protein (Gilmore
                 king and Leong (1981).                                                          et al. 1988).
                                                                                                    The IPNV expressing plasmid, pTAl, contained the
                                                                                                 entire coding region of the A segment of the viral
                 Results                                                                         genome for the isolate Sp. It was constructed so that
                 Antigen      Production in Bacteria                                             the viral genetic information was fused in-frame to the
                                                                                                 trpE protein (Figure 2) and all the proteins encoded by the
                 The size and quantity of virus-specific antigen produced                        A segment were synthesized in the bacteria. Thus, VP2
                 in bacteria hosting the recombinant plasmids was estimated                      (major capsid protein), and VP3 (minor capsid protein)








                                                                                                                                                            TrpE



                                                                                          PLASMID WITH                                 EXPRESSION
                                                                                          CLONED VIRAL                                   VECTOR                 Eco R1
                                                                                              GENE                               AMP  R                         Bam H1
                                                                                                                                                                Hind III
                                                                                  Pst I                      Pst I


                                                                                          VIRAL GENE

                                 Figure I (Continued)
                 f, and g are 1, 5 and 25 pL of cell lysates from
                 bacterial cells containing the plasmid pTA1. Lane                          VIRAL GENE                                          Restrict with either
                 h contains purified IPNV. The arrow indicates the                                                                                   Eco Rl
                 trpE-VP2 fusion protein found in lanes e, f, and g.                      Restrict with compatible                                   Bam Hl
                 The symbol VP1 indicates virion protein 1; VP2,                                enzyme                                               Hind III
                 virion protein 2; VP3, virion protein 3; and VP3a,
                 breakdown product of VP3.


                                                                                                                 TrpE

                                                                                                   TrpE
                                        Figure 2                                                   Promoter                         VIRAL GENE
                 Construction of the expression vectors for trp E-viral                                                                INSERT
                 gene fusions. The cDNA cloned insert of the IHNV                                                                               IMMUNOLOGICAL
                 glycoprotein gene or the A segment of the IPNV                                                                              01 SCREENING FOR
                 genome was restricted with a compatible nuclease                                                                               EXPRESSION
                 to permit the insertion of a portion of the viral gene
                 into the expression vector in the proper reading
                 frame. The resulting plasmids were used for expres-
                 sion of a trpE-viral gene fusion protein in E. coli.








                  30            NOAA Technical Report NMFS 92




                          100 -

                                                                                              CONTROL
                                80-
                       _j

                                                                                                                                        Figure 3
                       0        60
                                                                                                                 Immunization of rainbow trout with a subunit vac-
                                                                                                                 cine against IHNV. Rainbow trout fry (0.4 g) were
                       Z
                       Uj       40                                                                               immersed in a bacterial lysate (3 mg/mL, contain-
                       cc                                                                                        ing 10% expressed IrpE-G fusion protein) as de-
                       Ui
                                20                                                            VACCINE            scribed in Materials and Methods section. The
                                                                                                                 results are expressed as mean percent total mortality
                                0                                                                                on the ordinate and days after the initiation of viral
                                0         4         8           12       16         20          24               challenge on the abscissa. There were 25 fish in the
                                                                                                                 control group and 26 fish in the vaccinated group.
                                              DAYS AF7ER VIRAL CHALLENGE                                         In this particular challenge, the fish received 7.2 x
                                                                                                                 lo,5 plaque forming units/mL.





                                50-

                                                                                           CONTROL

                                40-

                                                                                                                                        Figure 4
                                30 -                                                                             Immunization of rainbow trout with a subunit vac-
                       0                                                                                         cine against IPNV. Rainbow trout fry (0.3 g) were
                                20                                                                               immersed in a bacterial lysate (3 mg/mL) contain-
                       Z                                                                                         ing the 1rpE-VP2 fusion protein as described in
                                                                                                                 Materials and Methods section. The results are
                       W        10-                                                                              expressed as percent total mortality on the ordinate
                       a.
                                                                             0 0 0 0       VACCINE               and days after the initiation of viral challenge on the
                                0                       a        6 0         1          1           1            abscissa. There were 25 fish in both control and vac-
                                0         10         20          30         40          50         60            cinated groups. The fish received 10" plaque
                                                 DAYS AFTER VIRAL CHALLENGE                                      forming units/mL of IPNV-Buhl strain for viral
                                                                                                                 challenge.




                  of IPNV-Sp were expressed by this recombinant plasmid                           Discussion
                  in bacteria.
                                                                                                  We have presented initial findings on the efficacy of bac-
                  Immunization Trials with Subunit Vaccine                                        terially expressed viral proteins as subunit vaccines for fish.
                                                                                                  Both the IHNV and the IPNV vaccines were effective in
                  Viral challenges provided data on the efficacy of the                           immunizing fish against lethal viral challenge in laboratory
                  bacterially expressed protein as vaccine&. A significant                        trials. Moreover, the vaccinations were carried out on rain-
                  level of protection (697o) was conferred on fish immu-                          bow trout fry that were 0.4 g in size. These fish were able
                  nized with p52G versus unimmunized fish when chal-                              to respond effectively to the viral vaccine. Previous studies
                  lenged with the Round Butte isolate of IHNV (Figure 3).                         of immunization in fish have indicated that the minimum
                  The glycoprotein used in constructing the fusion pro-                           size for successful immunization by immersion was 0.8 g
                                                              r


























                  tein was derived from this strain. In Figure 4, the pro-                        for chinook salmon (Fryer et al. 1978) and 1-2.5 g for rain-
                  tection that was achieved by immunization with pTAI                             bow trout Uohnson et al. 1982).
                  against the Buhl isolate of IPNV, a heterologous virus                             The use of these vaccines with different species of fish
                  strain, is shown. A decrease in virus-induced mortalities                       and against a variety of different viral strains must be
                  from 45 To to 3 To was found for the immunized group of                         tested. In addition, the duration of effective immunity must
                  fish.                                                                           be determined. However, the possibility now exists for








                                                                                            Leong ct al.: Recombinant Viral Vaccines in Aquaculturc                                 31


                  developing an inexpensive and effective vaccine for fish                                      necrosis virus. Nucl. Acid Res. 3:1903-1919.
                  using recombinant DNA technology.                                                     Engelking. H.M., and J.C. Leong.
                                                                                                             1981. IHNV persistently infects chinook salmon embryo cells.
                     The development of any vaccine must have safety as well                                    Virol. 109:47-58.
                  as efficacy as one of its primary considerations. The safety                               1989. The glycoprotein of infectious hematopoeitic necrosis virus
                  of live attenuated vaccines has been questioned for the                                       elicits neutralizing antibody and protective responses. Virus Res.
                  aquaculture industry because of the nature of the environ-                                    13:213-230.
                  ment where the vaccine would be applied. The vaccine has                              Fryer, J.F., J.S. Nelson, and R.L. Garrison.
                  to be completely safe for cultured and wild salmonid fish                                  1978. Immunization of salmonids for control of vibriosis. Mar.
                                                                                                                Fish. Rev. 1,0:20-23.
                  in the watershed. Moreover, the vaccine has to be eco-                                Gilmore, R.D. Jr., H.M. Engelking, D.S. Manning, and J.C. Leong.
                  nomical and a subunit vaccine produced in bacteria seems                                   1988. Expression in Escherichia coli of an epitope of the glycopro-
                  to be a viable alternative. The initial trials of the subunit                                 tein of infectious hematopoietic necrosis virus protects against viral
                  vaccines reported here suggest that bacterially expressed                                     challenge. Bio/Technology 6:295-300.
                  viral proteins, even in crude lysates, can be used as effec-                          Huang, M., D.S. Manning, M. Warner, E.B. Stephens, andJ.C. Leong.
                                                                                                             1986. A physical map of the viral genome for infectious pancreatic
                  tive and economical viral vaccines.                                                           necrosis virus Sp: Analysis of cell-free translation products derived
                                                                                                                from viral cDNA clones. J. Virol. 60(3):1002- 1011.
                                                                                                        Johnson, K.A., J.K. Flynn, and D.F. Amend.
                                                                                                             1982. Onset of immunity in salmonid fry vaccinated by direct im-
                  Acknowledgments                                                                               mersion in Vibrio anguillarum and Yersinia ruckeri bacterins. J. Fish.
                                                                                                                Dis. 5:197-205.
                  This publication is the result of research sponsored by                               Kleid, D.G., D. Yansura, B. Small, D, Dowbenko, D.M. Moore,
                  Bonneville Power Administration Contract DE-A179-                                        MJ. Grubman, P.D. McKercher, D.O. Morgan, B.H. Robertson,
                  84BP16479, Project 84-43 (G.R. Bouck and R. Morinaka                                     and H.L. Bachrach.
                                                                                                             1981.    Cloned viral protein vaccine for foot-and-mouth disease:
                  served as the Contracting Office Technical Representatives                                    Responses in cattle and swine. Science 214:1125-1129.
                  on the project) and, in part, by Oregon Sea Grant with                                Koener, J.F., C.W. Passavant, G. Kurath, and J.C. Leong.
                  funds from the National Oceanic and Atmospheric Ad-                                        1987. Nucleotide sequence of a cDNA clone carrying the glycopro-
                  ministration, Office of Sea Grant, Department of Com-                                         tein gene of infectious pancreatic necrosis virus, a fish rhabdo-
                  merce, under grant no. NA85AA-D-SGO95 (project no                                             virus. J. Virol. 61(5):1342-1349.
                                                                                                        Kurath, G., and J.C. Leong.
                  R/FSD-l 1) and from appropriations made by the Oregon                                      1985. Characterization of infectious hernatopoietic necrosis virus
                  State Legislature. We thank Rebecca Day for typing the                                        in mRNA species reveals a non-virion rhabdovirus protein. J.
                  manuscript.                                                                                   Virol. 53:162-468.
                                                                                                        McAllister, P.E., and R.R. Wagner.
                                                                                                             1977. Virion RNA polymerases of two salmonid rhabdoviruses. J.
                                                                                                                Virol. 22(3):839-843.
                  Citations                                                                             Manning, D.S.
                                                                                                             1988. Deletion mapping and Expression of the Large Genomic seg-
                  Dieckmann, C.L., and A. Tzagoloff.                                                            ment of Infectious Pancreatic Necrosis Virus. Doctoral Diss.,
                       1985. Assembly of the mitochondrial membrane system. J. Biol.                            Oregon State Univ., Corvallis, Oregon.
                          Chem. 260:1513-1520.                                                          Sanger, F., S. Nicklen, and A.R. Coulson.
                  Dobos, P.                                                                                  1977. DNA sequencing with chain-terminating inhibitors. Proc.
                       1975.    Size and structure of the genome of infectious pancreatic                       Natl. Acad. Sci. USA 7,t(12):5463-5467.








                                     Genetic Monitoring of Pacific Salmon Hatcheries


                                           ROBIN S. WAPLES, GARY A. WINANS, FRED M. UTTER,
                                                                    and CONRAD MAHNKEN

                                                                           Northwest Fisheries Center
                                                                   National Marine Fisheries Service, NOAA
                                                                           2725 Montlake Blvd. East
                                                                               Seattle, WA 98112




                                                                                ABSTRACT


                                       In the last few decades, and in response to substantial reductions in the abundance of wild
                                     populations of Pacific salmon, an enormous amount of resources in both Asia and North America
                                     has been devoted to artificial propagation programs. Several factors increase the possibility of
                                     rapid (often detrimental) genetic change in cultured populations, but genetic considerations are
                                     often overlooked in the effort to increase short-term productivity. Here, we discuss recent studies
                                     using electrophoretic data for chinook salmon, Oncorhynchus tshawytscha, that address three im-
                                     portant concerns for hatchery populations: levels of genetic variability, stability of allele frequen-
                                     cies, and genetic interactions (due to straying or overplanting) between hatchery and wild popula-
                                     tions. Results indicate that although there is no evidence for a general reduction in levels of genetic
                                     variability in hatchery stocks relative to wild populations from the same geographic area, allele
                                     frequencies over a period of one generation changed much more in samples from hatchery popula-
                                     tions in Oregon than in nearby wild populations. The genetic changes in the hatchery stocks
                                     appear to be due to a combination of two factors: genetic drift due to reduced effective popula-
                                     tion size, and (in some cases) the infusion of genes from other populations through straying or
                                     transfer of broodstock between hatcheries.




                 Introduction                                                               Materials and Methods


                 As a consequence of increased fishing pressure, loss of                    The electrophoretic data discussed here were collected over
                 spawning habitat, and blockage of migratory routes, re-                    the last decade at the National Marine Fisheries Service
                 turns of wild anadromous salmonids in the Pacific North-                   laboratory in Seattle. A considerable database exists for
                 west have declined substantially in this century. In part                  all the North American species of Pacific salmon, Oncorhyn-
                 to mitigate these losses, an extensive public hatchery sys-                chus, but here we consider only data for chinook salmon,
                 tem has been developed during the last several decades.                    0. tshawy1scha; for this species, data are available for pop-
                 Throughout most of this period, management practices at                    ulations from California to Alaska. Whole juvenile fish or
                 the hatcheries have been dictated primarily by production                  tissue samples (muscle, liver, eye, heart) from adult fish
                 demands, and relatively little consideration has been given                were collected in the field and stored at - 70'C until
                 to the genetic quality of released fish and their effects on               analyzed. Starch gel electrophoresis was performed as
                 wild fish. The availability of large amounts of data pro-                  described by Aebersold et al. (1987). Each sample was
                 duced by protein electrophoresis over the last decade has                  surveyed for genetic variation at up to 100 presumptive
                 made possible a critical evaluation of the genetic status of               gene loci, and genotypes inferred from the phenotypic
                 Pacific coast hatchery populations of salmonids. Here, we                  banding patterns (see Utter et al. 1987 for discussion) were
                 summarize results from several recent studies which are                    used to compute allele frequencies and a variety of stan-
                 pertinent to three important concerns: 1) levels of genetic                dard indices of genetic variability and differentiation.
                 variability found in hatchery and wild populations; 2)
                 stability of allele frequencies in hatchery and wild popula-               Levels of Genetic Variability
                 tions; and 3) genetic interactions (due to straying or over-
                 planting) between hatchery and wild populations.                           Recent policy statements (e.g., Northwest Power Planning

                                                                                                                                                               33








               34        NOAA Technical Report NMFS 92


               Council, 1987) regarding anadromous salmonids express                   One drawback to the above analysis is that average
               two major concerns: that existing levels of genetic diver-            heterozygosity is not very sensitive to the presence or
               sity be maintained, and that unique gene pools be pre-                absence of uncommon alleles. Although they contribute
               served. Loss of genetic variability is a real concern for             little to the measurement of heterozygosity, such alleles are
               managed populations because constraints on money, space,              potentially very important to a population because they
               and other resources often limit the size of the breeding              allow a greater degree of plasticity in response to changes
               population. In a closed population, approximately 1/2N,               in the environment. The presence of numerous allctes (even
               of the existing genetic variation is lost each generation, with       those at low frequency) in a population ensures that each
               N, being the effective number of breeders (Crow and                   generation, many genotypic combinations are produced
               Kimura 1970). The effective population size (N,) is less              upon which natural selection might act. Because alleles at
               than the actual number (N) if the sex ratio is uneven or              low frequency are easily lost if the effective breeding size
               if the variance in reproductive success among families is             is small, the average number of alleles per locus is a more
               large-both factors that might be influenced by hatchery               sensitive indicator than average heterozygosity of undesir-
               management procedures. Furthermore, if population size                able changes in the genetic makeup of a population.
               changes over time, long-term N, is determined primarily               According to Utter et al. (1989), the average number of
               by the effective number of breeders in the generation(s)              alleles per locus for seven hatchery and six wild popula-
               with smaller size. Therefore, a population bottleneck (re-            tions from Oregon were similar (1.74 and 1.68, respec-
               duced effective breeding size in one or a few generations)            tively). This lends additional support to our conclusion that
               can contribute appreciably to the long-term erosion of                the wholesale reduction of genetic variability reported in
               genetic variability.                                                  some hatchery populations of Salmo (e.g., Stahl 1983) has
                 To determine whether these effects are important in                 apparently not occurred in chinook salmon hatcheries in
               Pacific salmon, we examined two measures of genetic                   the Pacific Northwest.
               variability (average heterozygosity and effective number                This result is encouraging, but by no means constitutes
               of alleles per locus) in a series of hatchery and wild pop-           a clean bill of health for hatchery populations. If the genetic
               ulations of chinook salmon. The occurrence of consis-                 makeup of the source populations is to be perpetuated as
               tently lower levels of genetic variability in hatchery stocks         accurately as possible, it is important not only to conserve
               would suggest that artificial propagation has caused pop-             overall levels of genetic variability, but also to avoid large
               ulation bottlenecks. The heterozygosity data, however,                changes in frequency of the alleles present. For example,
               provide no evidence of the erosion of genetic variability             consider a locus with two alleles (A and a), sampled in a
               in cultured populations of chinook salmon in the Pacific              population at two times, with the following frequencies
               Northwest. In each case where data are available for a                observed-time 1: A = 0.8, a = 0.2; time 2: A = 0.2,
               comparison (Fig. 1), hatchery and wild populations from               a = 0.8. Hardy-Weinberg expected heterozygosity (2Aa
               the same area have very similar levels of heterozygosity.             = 0.32) remains unchanged, but allele frequencies have
               This result differs from that reported in a number of studies         shifted drastically. Clearly, it is also important to monitor
               of Atlantic salmon, Salmo salar, and rainbow, Oncorhynchus            allele frequencies over time in artificially propagated
               mykiss, cutthroat, 0. clarkil, and brown trouts, Salmo trutta         populations.
               (review, Allendorf and Ryman 1987); some cultured
               populations of these species have been found to have greatly
               reduced levels of heterozygosity relative to the ancestral            Temporal Stability
               wild stocks.                                                          of Allele Frequencies
                 Some interesting trends are apparent in the heterozy-
               gosity data for chinook salmon but these relate to geo-               To evaluate the temporal stability of allele frequencies, we
               graphic differences rather than to differences between                examined electrophoretic data for 21 coastal chinook
               hatchery and wild populations. In the Columbia River                  salmon populations from Oregon and California that were
               basin, coastal populations have higher heterozygosity than            sampled in each of two years (Waples and Teel 1990). For
               do lower river populations, which in turn retain more                 each population, allele frequencies in the two samples
               genetic variability than Snake River populations from far-            were compared at an average of 10 polymorphic loci. For
               ther upstream (Fig. 1). Populations from the Klamath and              each locus, a contingency chi-square test was used to test
               upper Fraser rivers also show reduced levels of genetic               the hypothesis that the population frequencies were un-
               variability relative to those closer to the coast (Georgia            changed. Results of these tests are very revealing (Table
               Strait, Puget Sound). Presumably, these differences reflect           1). For the three California hatchery and the nine Oregon
               the essentially independent evolutionary histories of the dif-        wild populations, the number of single locus tests show-
               ferent areas and, perhaps, the smaller population size or             ing a significant change in allele frequency (1/16 = 6%
               increased frequency of population bottlenecks in the up-              to 7/88 = 8 To) was close to the value (517o) expected to
               river populations (Winans 1989).                                      arise from sampling error, while the figure for the nine







                                                                   Waples et al.: Genetic Monitoring of Pacific Salmon Hatcheries                35


                                                       155   Upper
                                   5                   10    Fraser
                                                             River
                                15    Georgia          5
                                10   Strait
                                                       0
                                5

                                0




                                             - - - - - - - - - - - - -   T
                                                15     8     t            I
                                                       j Puge
                                                       Sound


                                                 5

                                                 0                                L




                                                 8
                                             15

                                             10
                   Oregon -     7  -6          5       Lower                     1   3
                                                       Columbia
                                   ::1.
                   Coast 10                                                            Snake
                                   XX
                                               0       River                            River
                                                                              10
                            5
                                                                               5
                            0
                                                                               0
                                                       3     2
                                                       15    Klamath
                                                       10-   River

                                                       5-
                                                       0                   - - - - - -

                                                       155
                                                             acramento
                                                       10  Ri;ver
                                                       5                         i@i:@ Wild
                                                                                i Hatchery
                                                       0





                                                                             CL
                                                                               0                                       Figure I
                   Heterozygosity in hatchery                              Heterozygosity           Comparison of average heterozygosity values for
                   and wild Chinook salmon                                                          hatchery and wild stocks of chinook salmon by
                                                                                                    geographic area.




               Oregon hatchery populations was much higher (29/81                    cients to explain such large frequency shifts in a single
               36% of all tests showing significant allele frequency                 generation. Waples and Teel also showed that the observed
               change). In addition, combined chi-square tests over all              differences can be accounted for by genetic drift only if the
               loci indicate very significant (P<0.01) or highly significarit        effective number of breeders in the Oregon hatcheries
               (P<0.001) changes in allele frequency between 1981 and                averaged about 50 or less. Examination of brood stock data
               1985 samples in eight of the nine Oregon hatcheries (Table            indicates that effective population size may indeed have
               1).                                                                   been quite low in at least some of the hatcheries (Waples
                  Possible causes of short-term allele frequency change in-          and Teel 1990).
               dude natural selection, genetic drift, and migration. In the            Another possibility is that some of the genetic changes
               present example, selection appears to be an unlikely cause,           resulted from the infusion of new genes during the transfer
               given Waples and Teel's (1990) demonstration that it is               of fish between hatcheries, or from natural straying into
               necessary to invoke unrealistically large selection coeffi-           the hatcheries. Evidence to support this hypothesis comes








                     36            NOAA Technical Report NMFS 92



                                                                                                             Table 1
                                               Twenty-one chinook salmon populations sampled in each of two years. Number of significant
                                               (a = 0.05) single locus chi square tests comparing allele frequencies in two years are shown, and
                                               significance levels are given for combined chi-square test over all loci and a test for gametic
                                               disequilibria (n.s.          not significant).

                                                                                                          Between-year cmparisons'                      Gametic disequilibriad

                                                                             Sample size                 Single locus
                                               Population'                     1981    1985'           (no. sig./total)              All loci             1981             1985,

                                                                                                 Oregon wild populations
                                               Alsea                            94      50                   0/11                    n.s.                 ii.s.            n.s.
                                               Chetco                          100      93                   1/7                     n.s.                 n.s.             n.s.
                                               Coquille                        115      50                   1/12                    n.s.                 ii.s.            n. s.
                                               Nehalern                        141      50                   1/9                     n. s.                n.s.             n. s.
                                               Nestuca                          60      50                   1/9                     n.s.                 n.s.             n.s.
                                               Siletz                           92      50                   0/11                    n.s.                 n.s.             n.s.
                                               Sixes                           100      50                   1/8                     0.01                 n.s.             n.s.
                                               Siuslaw                          82      34                   1/11                    n.s.                 n. s.            n.s.
                                               Tillamook                        88      50                   1/10                    n.s.                 n.s.             n.s.
                                                  No. sig./total                                             7/87                    1/9                  0/9              0/9
                                                                                              Oregon hatchery populations
                                               Cedar Creek                      99     100                   4/9                     0.001                0.01             0.001
                                               Cole R. (S)                     113      50                   1/9                     n.s.                 n.s.             n.s.
                                               Cole R.                          50     100                   5/13                    0.01                 n. s.            n. s.
                                               Elk R.                          100     100                   2/9                     0.001                n. s.            n. s.
                                               Fall Creek                      100     100                   2/7                     0.01                 0.001            n. s.
                                               Rock Creek (S)                  100     100                   4/9                     0.001                0.05             0.001
                                               Salmon                           99     100                   5/8                     0.001                n. s.            0.001
                                               Trask (S)                       100     100                   3/10                    0.001                n. s.            0.05
                                               Trask                           100     100                   3/7                     0.01                 0.01             0.001
                                                  No.    sig./total                                          29/81                   8/9                  4/9              5/9
                                                                                            California hatchery populations
                                               Iron Gate                        99      50                   1/8                     n. s.                n. s.            n. s.
                                               Trinity (S)                      50     100                   0/5                     n.s.                                  0.05
                                               Trinity                         100      50                   0/3                     n.s.                 n.s.             n.s.
                                                  No. sig./total                                             1/16                    0/3                  0/2              1/3


                                               'Spring run denoted by         (S); all others are fall run stocks.
                                               'Samples taken in 1983         for Oregon wild populations, 1984 for California populations.
                                               'Data from Waples and Teel (in press).
                                               'Data from Waples and Smouse (1990).





                     from gametic disequilibrium analysis, a powerful means                                            reasons. First, the transfer of eggs, fry, and brood stock
                     of detecting samples which are actually a mixture of                                              among hatcheries is a common occurrence that complicates
                     distinct gene pools. Gametic disequilibrium (the non-                                             the problem of identifying the genetic makeup of hatchery
                     random association of alleles at different gene loci)                                             populations. Second, strays of hatchery or transplanted fish
                     occurs as the result of a mixture of gene pools that dif-                                         may have an adverse effect on wild populations adapted
                     fer in allele frequency at two or more loci (Nei and Li                                           to local conditions. The genetic consequences of such
                     1973).                                                                                            admixtures are difficult to evaluate by traditional methods
                                                                                                                       (physical tags, behavioral observations) because the pres-
                                                                                                                       ence of exotic fish in a population does not ensure that they
                     Genetic Interactions Between                                                                      will interbreed with the residents and produce viable off-
                     Hatchery and Wild Populations                                                                     spring. If the potential source populations can be identified
                                                                                                                       and adequate genetic markers are available, estimates of
                     Admixtures (mixtures of fish from more than a single gene                                         the mixture fractions are possible (Campton 1987). How-
                     pool) involving hatchery populations are a concern for two                                        ever, in many cases the populations possibly contributing







                                                                                Waples et al.: Genetic Monitoring of Pacific Salmon Hatcheries                              37


                  to a mixture are unknown or cannot be characterized                               Citations
                  genetically. For such a "blind" mixture, gametic dise-
                  quilibrium analysis is a potentially powerful tool for evalu-                     Aebersold, P.B., G.A. Winans, DJ. Teel, G.B. Milner, and F.M. Utter.
                  ating the null hypothesis that the sample could have come                              1987. Manual for starch gel electrophoresis: A method for the detec-
                  from a single gene pool.                                                                 tion of genetic variation. Dep. Commer., NOAA Tech. Rep.
                                                                                                           61:1-19.
                     To evaluate the possibility of genetic admixture in the                        Allendorf, F.W., and N. Ryman.
                  above example, we used a multilocus analysis of gametic                                1987. Genetic management of hatchery stocks. In Population
                  disequilibrium (Waples and Smouse 1990) that considers                                   genetics and fishery management (N. Ryman and F. Utter, eds.),
                  data for all pairs of loci simultaneously. No unusual levels                             p. 141-159. Univ. Washington Press, Seattle.
                  of multilocus gametic disequilibrium were found in the                            Campton, D.E.
                                                                                                         1987. Natural hybridization and introgression in fishes. In Popula-
                  California hatchery or Oregon wild populations (I of 23                                  tion genetics and fishery management (N. Ryman and F. Utter,
                  samples with significant disequilibria at a = 0.05 level;                                eds.), p. 161-192. Univ. Washington Press, Seattle.
                  Waples and Smouse 1990), but the situation was quite                              Crow, J.F., and M. Kimura.
                  different in the samples from the Oregon hatcheries (9 of                              1970. An introduction to population genetics theory. Harper and
                                                                                                           Row, New York, 591 p.
                  18 tests significant; Table 1). These findings are consis-                        Nei, M., and W-H. Li.
                  tent with the hypothesis that, in addition to genetic drift,                           1973. Linkage disequilibriurn in subdivided populations. Genetics
                  a mixture of gene pools contributed to the changes in allele                             75:213-219.
                  frequency observed in some of the Oregon hatchery                                 Northwest Power Planning Council.
                  populations.                                                                           1987. Columbia River Basin Fish and Wildlife Program. Portland,
                     Gametic disequilibrium analysis has considerable poten-                               Oregon, 246 p.
                                                                                                    Stahl, G.
                  tial for assessing the extent of genetic interactions among                            1983. Differences in the amount and distribution of genetic varia-
                  hatcheries and between hatchery and wild populations.                                    tion betw .een natural populations and hatchery stocks of Atlantic
                  It may help in the identification of wild gene pools that                                salmon. Aquaculture 33:23-32.
                  have been relatively unaffected by genes from hatchery                            Utter, F., P. Acbersold, and G. Winans.
                                                                                                         1987. Interpreting genetic variation detected by electrophoresis.
                  populations and therefore merit conservation efforts. In                                 In Population genetics and fishery management (N. Ryman and
                  other cases, where the objective is to supplement and                                    F. Utter, eds.), p. 21-45. Univ. Washington Press, Seattle.
                  enhance wild production, gametic disequilibrium analysis                          Utter, F., G. Milner, G. Stahl, and D. Teel.
                  .provides a means of monitoring the effectiveness of                                   1989. Genetic population structure of chinook salmon in the Pacific
                  transplants from the hatcheries. The possibilities for both                              Northwest. Fish. Bull., U.S. 87:239-263.
                  types of genetic interactions, as well as the necessity for                       Waples, R.S., and P.E. Smouse.
                                                                                                         1990. Genetic disequilibriurn analysis as a means of identifying mix-
                  monitoring them, are likely to increase in the near future.                              tures of salmon populations. American Fish. Soc. Symp.
                  Large scale supplementation of wild populations and                                      7:439-458. (In press.)
                  expansion of hatchery production are planned to achieve                           Waples, R.S., and DJ. Teel.
                  the goal of the current Columbia River Basin Fish and                                  1990. Conservation genetics of Pacific salmon. 1: Temporal changes
                                                                                                           in allele frequency.    Conserv. Biol., Vol. 4:144-156.
                  Wildlife Program (Northwest Power Planning Council                                Winans, G.A.
                  1987)-doubling the run size of anadromous salmonids in                                 1989. Low levels of genetic variability in spring-run chinook salmon
                  the Columbia River basin.                                                                of the Snake River. N. Am. J. Fish. Management 9:47-52.









                                                 Successful Gene Transfer in Fish


                                      S.J. Y00N,1 Z. LIU,3 A.R. KAPUSCINSKI,2 P.B. HACKETT,3
                                                             A. FARAS,4 and K.S. GUISEI
                                                       Departments of Animal Science,' Fisheries and Wildlife,2
                                                            Genetics and Cell Biology,' and Microbiology4
                                                                      University Of Minnesota
                                                                        1988 Fitch Avenue
                                                                       St. Paul, MN 55108




                                                                         ABSTRACT


                                   The neo gene, which confers resistance to the neomycin analog drug G418, has been success-
                                 fully transferred into newly-fertilized dechorionated goldfish eggs by microinjection. Multiple
                                 copies of the gene were incorporated into the high molecular weight fraction of fish DNA (i.e.,
                                 the genomic DNA). RNA dot blots indicate specific neo mRNA synthesis. Gene transfer efforts
                                 using mammalian-derived growth hormone genes, a marker gene for P-galactosidase, and a variety
                                 of promoters are discussed. Current work which also includes the isolation of piscine promoters
                                 and genes for peptide hormones is mentioned.



              Introduction                                                          the United States, four primary groups are known to be
                                                                                    using microinjection of various plasmid constructs to pro-
              Novel genes were first introduced into mice in 1979 by                duce transgenic fish. These laboratories are, in addition
              Gordon et al. (1980). The technology did not receive wide             to our group at the University of Minnesota, those of Drs.
              attention until Palmiter et al. (1982) transferred a rat              Powers and Chen at Johns Hopkins, Dunham at Auburn,
              growth hormone gene linked to a mouse metallothionein                 and Ellinger and Kohler at Southern Illinois. All four U.S.
              promoter, creating a line of mice that grew significantly             groups are pursuing similar goals: production of transgenic
              faster and ultimately larger than control mice. This series           fish by transfer of growth-hormone gene constructs using
              of experiments captured the imagination of a wide variety             microinjection as the primary transfer technique. Our
              of researchers seeking to improve economic traits in domes-           group reports here the successful transfer of a marker gene,
              tic and semidomestic animals. While gene transfer ex-                 neo, into goldfish via microinjection, as a step toward the
              perimentation is currently progressing in most species of             goal of transfer of economically important genes.
              economic importance, including mammalian farm animals
              and poultry, no group of organisms shows more promise
              for the dramatic interaction of transferred genes than fish.          Materials andMethods
              For over three decades it has been known that fish are quite          Egg Preparations
              responsive to injections of crude and purified growth hor-
              mone (Pickford and Thompson 1948; Adelman 1977). Suc-                 Spontaneous ovulation of goldfish was accomplished by the
              cessful transfer and expression of growth-hormone genes               methods of Stacy et al. (1979). Sexually mature fish were
              in fish are thus expected to produce a similar, dramatic              kept under a long photoperiod (16 h light, 8 h dark). On
              response.                                                             day one, breeding fish were transferred from stock aquaria
                 Since 1984, multiple groups worldwide have been pur-               to standing-water breeding aquaria at 13 ï¿½ 1 'C. Aquaria
              suing the goal of producing transgenic fish. Laboratories             were supplied with floating artificial plants, and the water
              in Japan, England, France, and the People's Republic of               temperature was increased to 21 ï¿½ 1'C overnight and kept
              China have published results of these attempts in medaka,             at that temperature through spawning. Spontaneous ovula-
              Oryzias sp. (Ozato et al. 1986), rainbow trout (Oncorhyn-             tion usually occurred during the last half of the dark phase
              chus mykiss) (Maclean and Talwar 1984; Chourrout et al.               on day 3. If no ovulation occurred on day 3, the fish were
              1986), goldfish (Carassius auratus) (Zhu et al. 1985), and            injected intraperitoneally with 3 mg/kg body weight of carp
              loach (Misgurnus anguillicaudatus) (Zhu et al. 1986). Within          pituitary gland extract. About 10 hours later, the fish were

                                                                                                                                                  39








                40        NOAA Technical Report NMFS 92


                artificially spawned and eggs and sperm were collected                Tris - HCI pH 8. 0, 0.1 M EDTA, 0. 5 % SDS buffer with
                separately.                                                           100 yg/mL proteinase K on ice using a Dounce homo-
                  Eggs were fertilized by the milt after they were mixed              genizer. High molecular weight DNA was extracted as per
                with well water in an open petri dish in batches of approx-           Maniatis et at. (1982).
                imately 100 eggs. Ten minutes after fertilization, the eggs             DNA dot blot analysis was used to detect the neo gene.
                were dechorionated by a six minute incubation in 0.217o               Approximately 5 Mg of genomic DNA were denatured in
                trypsin (Zhu et al. 1985). Dechorionation was stopped by              0.4 M NaOH, and following addition of an equal volume
                treatment with 5 % fetal bovine serum in Holtfreter's solu-           of 2 M ammonium acetate, were spotted onto nitrocellu-
                tion (Grand et al. 1941). Dechorionated eggs were washed              lose using a Hybri-dot system. The filter was baked in vacuo
                several times in Holtfreter's solution and transferred to             for 2 hours at 800C. The filter was prehybridized in 5 x
                Holtfreter's solution on a charcoal-agar petri dish, which            SCC (I x SSC = 0. 15 M NaCl, 15 mM Na citrate), 5 x
                provided a dark background for microinjection.                        Denhardt's solution (1 x Denhardt's = 0.02% Ficoll,
                                                                                      0.02% bovine serum albumin, 0.02% polyvinylpyrroll-
                Microinjection                                                        done), 50 mM sodium pyrophosphate pH 6.5, 0.1 % SDS,
                                                                                      0. 1 mg/mL denatured calf thymus DNA, and 0. 1 mg/mL
                Plasmids were prepared by amplification, lysis         in SDS         yeast tRNA at 650C for at least 4 hours. The filter was
                (sodium dodecylsulfate), and CSC12 centrifugation (Mani-              hybridized with I x 106 cpm of 32p labeled probe in the
                atis et al. 1982). Plasmids were linearized with restriction          same solution used for prehybriclization for 20 hours at
                endonuclease Kpnl, extracted with phenol/chloroform,                  65'C. The filter was washed three times in 2 x SSC, 0. 1 %
                ethanol precipitated, and redissolved in 88 mM NaCl, 10               SDS at room temperature for 10 minutes, and three times
                mM Tris - Hcl pH 7.6 to a final concentration of 25 ng/;AL.           in 0. 1 x SSC, 0. 1 17o SDS at 600C for 30 minutes, dried,
                Borosilicate glass needles with an inner tip diameter of ap-          and exposed to Kodak XAR5 x-ray film at - 80'C (Mani-
                proximately 2 Vrn were filled with plasmid solution. Micro-           atus et al. 1982).
                injection was performed with a Brinkman MM33 micro-                     Southern blot analysis was performed by complete diges-
                manipulator with the injection volume controlled by the               tion of 10,ug of genomic DNA with Kpnl, BaniHI, SstI,
                timing of the insertion/withdrawal interval and constant              or Mlu I followed by electrophoresis on a 0. 8 Olo agarose gel.
                fluid flow. It is estimated that 2 nanoliters of soluti'on con-       Transfer to nitrocellulose was made following the method
                taining I X 106 copies of neo gene are delivered. DNA was             of Southern (1975). The Southern blot was probed using
                released into the center of the germinal disc prior to the            the same hybridization conditions as the dot blot analysis.
                first cleavage. Microinjected eggs were allowed to develop
                in Holtfreter's solution until the blastula stage, and re-            RNA Analysis
                turned to well water after the blastula stage. Mock injec-
                tions were performed as controls, using only the buffer               Anterior halves of frozen      90'C) neo DNA positive fish
                component of the injection solution.                                  plus several control and injected fish were homogenized
                                                                                      by Polytron in a solution containing 4 M guanidinium
                Plasmid Construction                                                  isothiocyanate, 0. 1 M P-mercaptoethanol, 0. 5 % sarkosyl,
                                                                                      5 mM sodium citrate, pH 7.0. Total RNA was isolated
                The primary construct used, pRSVneo, contains the neo                 as per Maniatis (1982). RNA dot blot analysis was used
                gene, whose product, aminoglycoside 3'-phosphotrans-                  to test for neo gene expression. Total RNA was dissolved
                ferase, confers resistance to the neomycin analog G418,               in 6 x SSC, and 7.417o (v/v) formaldehyde, and heat de-
                and a Rous sarcoma virus (RSV) promoter. A BamHl-                     natured at 65'C for 15 minutes. Serial amounts of RNA
                HindIll restriction fragment containing the neo gene and              (0.25, 0.5, 1, and 2 Mg) were directly dotted onto nitro-
                mart of SV40 intron was isolated from pSV2neo (Ameri-                 cellulose equilibrated with 6 x SSC. The RNA was fixed
                can Type Culture Collection) and ligated into the poly-               to the filter by baking under vacuum at 80'C for 2 hours.
                linker of pUC 119. A 3 40-bp fragment containing the RSV              The prehybridization was carried out for more than 4 hours
                promoter/enhancer region was ligated to the regenerated               at 60'C in 50 To deionized formamide, 5 x SSC, 5 x Den-
                HindIII site. This construct is termed pRSVneo in this                hardt's solution, 50 mM sodium phosphate, pH 6.5, 0.1 Olo
                paper. Plasmid construction was by standard methods                   SDS, 100 lAg/mL denatured calf thymus DNA, and 100
                (Maniatis et al. 1982).                                               Mg/mL yeast tRNA. The filter was then hybridized with
                                                                                      2 x 106 cpm/mL of 32P-labeled probe in the same buffer
                DNA Analysis                                                          for 20 hours at 601C. After hybridization, the filter was
                                                                                      washed three times with 2 x SSC, 0. 1 % SDS at room
                Genomic DNA was isolated from posterior halves of 1 to                temperature for 10 minutes, three times with 0.2 x SSC,
                2 month-old (approx. 8 g) fish stored at - 900C. Fish were            0. 2 To SDS at 68'C for 30 minutes, and exposed to Kodak
                individually homogenized in three volumes of 0.2 M                    XAR5 x-ray film at - 801C.








                                                                                         Yoon et al.: Successful Gene Transfer in Fish             41


                                            A B C D E F G H I J K L


                                     2
                                     3
                                     4
                                     5
                                     6
                                     7
                                     8


                                                                             Figure 1
                                DNA dot blot analysis of goldfish microinjected with pRSVneo. Total genomic DNA probed with
                                330 bp HindIII-BarnHI restriction fragment of the promoter region of the neo gene. Rows 7 and 8
                                present dilutions of pRSVneo with (row 8) or without (row 7) added genomic DNA. Dilution series
                                is A-B, 0.5 copies; C-D, I copy; E-F, 5 copies; G.H, 25 copies; and K-L, 100 copies per genome.
                                Test fish DNA was spotted twice on filter producing series 1-5, A-F and an identical replicate 1-5,
                                G-L. Dots row 1A-IF (replicate IG-IL) are of six noninjected control goldfish. Dots row 2A-2F
                                and 3A-3E (replicate 2G-2L and 3G-3K) are of pRSVneo-injected goldfish DNA. Dots 3B and 3D
                                indicate presence of approximately five copies of neo gene. Dots 3F, 4A-4F, and 5A-5C (replicate 3L,
                                4G-4L, and 5G-5I) are of pRSVneo-injected fish subjected to G-418 drug selection (4 mg/mL), ar-
                                ranged in increasing survival time from 20 hours (3F) to greater than 8 days (5C). Dots 5D-5F are
                                test dots for detection of possible neo-hybridizable sequences in pink salmon DNA.


               Probe Labeling                                                        tained above 60% saturation by periodic aeration. The
               Plasmid restriction fragments were separated by I 01o aga-            temperature was maintained at 20 ï¿½ I'C. The pRSVneo-
               rose gel electrophoresis in TAE buffer (0. 04 M Tris-acetate I        injected or non-injected control goldfish at a population
               2mM EDTA pH 8.0). Excised bands were phenol:chloro-                   density of 12 g/L were exposed to 4 mg/mL solutions of
               form extracted, and the DNA was precipitated by ethanol               G-418. Mortality of fish was monitored every four hours
               and sodium acetate and redissolved in TE (10 mM                       for the first 24 hours and every 12 hours afterward. Dead
               Tris - HCI pH 7.6, 1 mM EDTA). Fragments were labeled                 individuals were removed upon observation and stored in
               by the procedure of Feinberg and Volgelstein (1983) to                liquid nitrogen. Drug selection was continued until 507o
               0.5-2 x 109 cpm/Mg. DNA probes were used without puri-                of the control fish in 4 mg/mL G-418 had died. Surviving
               fication from unincorporated nucleotides.                             fish were transferred into fresh well water upon termina-
                  The 2.3-kb fragment including the neo gene was also sub-           tion of the test.
               cloned into pTZ 18R under the control of the T7 promoter
               to produce a transcript complementary to neo mRNA. One                Results
               Mg of linear DNA was transcribed using the method of
               Schenborn and Mierendorf (1985) to produce probes used                The survival rate for microinjected fish has ranged from
               in RNA dot blot analysis.                                             10% in the early experiments to nearly 507o at the cur-
                                                                                     rent time. The results reported here are from early injec-
               G-418 Selection                                                       tion studies, where, despite the high mortality rates, we
                                                                                     were still able to effect the transfer and apparent expres-
               For the drug selection screenings, solutions of G-418 in              sion of the transferred gene.
               static water aquaria were aerated to achieve initial satura-             Figure 1 shows the results of DNA dot blot analysis of
               tion levels of dissolved oxygen. Dissolved oxygen was main-           mock- and pRSVneo-injected goldfish. None of the mock-








              42        NOAA Technical Report NMFS 92

                                  P                   Kpo                        Bam                   Sst                 Big
                             P P9                  1 2 3                      1 2 3                1 2 3 1 2                        3
















                                                                           Figure 2
                   Southern analysis of genomic DNA from three goldfish microinjected with pRSVneo. Fish #1 was negative by DNA dot
                   blot analysis, while fish #2 and #3 represent fish from dots 3B and 3D of Figure 1. Lanes p and pg represent linearized pRSVneo
                   and pRSVneo plus genomic DNA, respectively.


              injected fish showed positive hybridizaiton to the neo probe,        (Fig. 1), was confirmed in the RNA dot blot of Figure 3.
              while two of the pRSVneo-injected fish showed approx-                Here, one out of six fish tested showed strong expression
              imately five copies of the gene per genome. Figure 1 also            of neo RNA when probed with a complementary neo RNA
              shows a series of DNA dot blots for pRSVneo-injected fish            probe. In order to rule out the possibility of DNA con-
              that were subjected to drug selection with G-418. The fish           tamination of the RNA used in *he RNA dot blot, we
              were grouped by order of the time of death during drug               treated the RNA from the positive fish of Figure 3 with
              selection. One fish, which survived the full course of selec-        DNase and RNase in a separate experiment (Fig. 4).
              tion, but died on day 8 of fungal infection, showed an ap-           RNase destroyed the hybridization to the neo RNA probe,
              parent hybridization signal of about I copy per genome.              while DNase had little effect. This indicates little if any
              Pink salmon DNA was included as control DNA. Note that               DNA contamination of the RNA.
              only the pink salmon DNA dot blots of Figure 1 (Dots
              5D-5F), show high background hybridization to the probe
              in both control and injected fish. Unlike goldfish, pink             Conclusion and Discussion
              salmon apparently contain genomic sequences that are
              closely related to neo.                                              The results reported here showed successful transfer of a
                 Figure 2 shows results of a Southern blot analysis of             marker gene neo that encodes resistance to the drug G-418.
              genomic DNA cleaved with four different restriction en-              Stable integration of the transferred gene was suggested
              zymes and probed with the 330-bp neo probe. The neo probe            but could not be conclusively proven at this time. Expres-
              detected both linearized plasmid and a higher molecular              sion of the marker gene was confirmed through detection
              weight band in the BamHI and Sstl digests. This may in-              of neo mRNA in the transgenic fish. Functional resistance
              dicate a concatenated integration of the estimated five              conferred by pRSVneo was suggested by the survival
              copies of the gene, but conclusive evidence of integration           through drug selection of a putative transgenic fish con-
              will require breeding studies.                                       taining an extimated single copy of the neo gene.
                 Expression of the transferred neo gene which was sug-               These results indicate that microinjection is a viable
              gested in the resistance of a fish containing neo sequences          method for transfer of selectable genes into fish. Micro-









                                                                                        Yoon et al.: Successful Gene Transfer in Fish             43



                                .25 .5 1, 2
                              A                                                                   ONase                     RNase

                                                                                                         +                         +


                              C
                              D                                                                               5 RNA

                              E


                              F



                                                                                                               Figure 4
                                          Figure 3                                         Total RNA from fish E of Figure 3 probed with
                       RNA dot blot analysis of total RNA from six                         RNA complementary to neo RNA transcript. RNA
                       pRSVneo- injected fish, spotted at 0.25, 0.5, 1                     was spotted before ( - ) and after ( + ) treatment with
                       and 2 ;Ag RNA per dot for each fish.                                RNase-free DNase or DNase-free RNase.



               injection is, however, currently a labor- and patience-              bovine growth hormone constructs into fish eggs, as well
               intensive technique, and promises to remain so for the near          as injecting a second marker gene construct using P-galac-
               future. Microinjection works well upon dechorionated eggs            tosidase. While bovine growth hormone gene constructs
               where needle placement can be monitored, but is less                 may produce growth enhancement in transgenic fish, our
               effective in blind injections. Therefore, we are keenly in-          intent is to effect growth enhancement by transfer of
               terested in development of a selectable marker for use in            species-specific growth hormone genes. To this end, we are
               either blind injection or in mass transfer techniques. Our           isolating piscine growth hormone genes and fish gene pro-
               group has been exploring the use of mass transfer tech-              moters so that the ultimate transgenic, growth-enhanced
               niques to overcome the tedium of injection of the substan-           fish will contain no DNA sequence from outside its own
               tial numbers of eggs necessary to generate the optimal               species. We feel that this will simplify regulatory approval
               transgenic fish. While mass transfer efficiencies may be             of the transgenic fish for human consumption, and enable
               orders of magnitude less than those achieved by microin-             the transferred genes to interact with the natural regula-
               jection, the ability to manipulate tens of thousands of eggs         tory mechanisms in the fish in a more normal fashion.
               at a time should compensate for the loss in transfer effi-
               ciency. The key element necessary to make mass transfer
               a reality is the development of a selection system to differ-        Acknowledgments
               entiate transgenic from nontransgenic fish. The selectable
               marker neo meets this criterion, and should prove effective          The work outlined here was supported in part by Minne-
               in the selectionof transgenic fish when used in co-transfer          sota Sea Grant R/3 to K.S. Guise, by a grant from the
               schemes with other genes.                                            Legislative Commission on Minnesota Resources of the
                 A variety of mass transfer techniques are under inves-             State of Minnesota to A. Faras, K.S. Guise, A.R. Kapu-
               tigation by our group as well as by other laboratories in            scinski, and P.B. Hackett, and by the Minnesota Experi-
               the United States. These range from techniques based on              ment Station (K.S. Guise).
               the CaP04 procedures used in gene transfer in tissue
               culture, to binding of plasmid DNA to sperm and effect-
               ing transfer at the time of fertilization, to electroporation.       Citations
               Studies proceeding along these lines should soon yield
               general mass transfer protocols that will be successful in           Adelman, 1. R.
               a broad spectrum of fish species.                                        1977. Effect of bovine growth hormone on growth of carp (Cyprinus
                 The establishment of the neo gene as a selectable marker                  carpio) and the influences of temperature and photoperiod. J. Fish
                                                                                           Res. Board Can. 34:509-515.
               is a step in the generation of transgenic fish with enhanced         Chourrout, D., R. Guyomard, and L.M. Houdebine.
               growth hormone expression. We are currently injecting                    1986. High efficiency gene transfer in rainbow trout (Salmo gaird-








                 44          NOAA Technical Report NMFS 92


                         neri Rich.) by microinjection into egg cytoplasm. Aquaculture               Palmiter, R.D., R.L. Brinster, R.E. Hammer, M.E. Trumbauer,
                         51:1443-1450.                                                                 M.G. Rosenfeld, N.C. Birnberg, and R.M. Evans.
                 Feinberg, A.P., and B. Vogelstein.                                                       1982. Dramatic growth of mice that develop from eggs microin-
                      1983. A technique for radiolabelling DNA restriction enclonuclease                    jected with metallothionein-growth hormone fusion genes. Nature
                         fragments to high specific activity. Anal. Biochem. 132:                           300:611-615.
                         6-13.                                                                       Pickford, G.E., and E.F. Thompson.
                 Gordon, J.W., G.A. Scangos, D.J. Plotkin, J.A. Barbosa, and                              1948. The effects of purified mammalian growth hormone on the
                    F. H. Ruddle.                                                                           killifish Fundulus heteroclitis (Linn). J. Exp. Zool. 109:367.
                      1980. Genetic transformation of mouse embryos by microin-                      Schenborn, E.T., and R.C. Mierendorf Jr.
                         jection of purified DNA. Proc. Nad. Acad. Sci. U.S.A. 77:                        1985. A novel transcription property of Sp6 and T7 RNA poly-
                         7380-7384.                                                                         merases: Dependence on template structure. Nucleic Acids Res.
                 Grand, C.G., M. Gordon, and G. Cameron.                                                    13:6223-6236.
                      1941. Neoplasma studies: Cell types in tissue culture offish melan-            Southern, E.M.
                         otic tumors compared with mammalian melanomas. Cancer Res.                       1975. Detection of specific sequences among DNA fragments
                         1:660-666.                                                                         separated by gel electrophoresis. J. Mol. Biol. 98:503-517.
                 Maclean, N., and S. Talwar.                                                         Stacy, N.E., A.F. Cook, and R.E. Peter.
                      1984. Injection of cloned genes into rainbow trout eggs. J. Em-                     1979. Spontaneous and gonadotrophin- induced ovulation in the
                         bryol. Exp. Morphol. 82:187.                                                       goldfish, Carassius auratus L. Effects of external factors. J. Fish.
                 Maniatis, T., E.F. Fritsch, and J. Sambrook.                                               Biol. 15:349-361.
                      1982. Molecular cloning: A laboratory manual. Cold Spring Har-                 Zhu, Z., G. Liu, L. He, and S. Chen.
                         bor Laboratory, Cold Spring Harbor, New York.                                    1985. Novel gene transfer into the fertilized eggs of goldfish
                 Ozato, k., H. Kandoh, H. Inohara, T. Iwamatsu, Y. Wakamatsu, and                           (Carassius auratus L. 1758). Z. Angew. Ichthyol. 1:31-34.
                    T. S. Koada.                                                                     Zhu, A., K. Xu, G. Li, Y. Xie, and L. He.
                      1986. Production of transgenic fish: Introduction and expression                    1986. Biological effects of human growth hormone gene micro-
                         of chicken crystallin gene in medaka embryos. Cell Differ. 19:                     injected into the fertilized eggs of loach Misgurnus anguillicaudatus
                         237-244.                                                                           (Cantor). Kexue Tangbao 31:988-990.








                                    Clonal Ginbuna Crucian Carp as a Model for the
                                              Study of Fish Immunology and Genetics


                                                 TERUYUKI NAKANISHI and HIROSHI ONOZATO

                                                                   National Research Institute of Aquaculture
                                                                                   Tomaki
                                                                             Mie 519-04, Japan




                                                                               ABSTRACT


                                       The lack of suitable inbred lines of fish for immunological study in which lymphocytes and
                                    blood samples could be collected have led to the use of clonal ginbuna crucian carp, Carassius
                                    gibelio langsdorfii. Distributed widely in Japan they include both bisexual diploid and tetraploid
                                    types and a unisexual (all female) triploid type which reproduces gynogenetically by omitting
                                    Mciosis I. The kinetics of immunity transfer by immune leukocytes was first examined using
                                    isogeneic crucian carp. Splenic cells were most effective in transferring immune reactivity, followed
                                    by pronephric, then mesonephric cells. However, antibody titres were very low or absent when
                                    the recipients received transferred thymic cells. Antibody productivity was most successfully con-
                                    ferred when cells were taken from 7-day postimmunized donors. The level of antibody titre in
                                    recipients reached its peak on day 7 for mesonephric cells and on day 14 for pronephric and splenic
                                    cells. Transferability of immune leukocytes was also compared in isogeneic, allogeneic and
                                    xenogeneic crucian carp to examine their major histocompatibility complex (MHC) regulation
                                    in adoptive immunity. Adoptive transfer by pronephric cells was successful in isogeneic and weak
                                    histocompatibility (H) gene-disparate transfer systems, while antibody productivity was not
                                    transferable in xenogeneic and strong H gene-disparate transfer systems. In allogeneic fish,
                                    however, antibody productivity was transferable by the transfer of cells in some recipients that
                                    rejected the allograft in an acute fashion.


                Introduction                                                              present review, we describe our recent studies on the fish
                                                                                          immune system using clonal crucian carp and discuss the
                Most of the information on the cellular immune mechan-                    excellent potential of this fish as a model for studying fish
                ism in mammals has been obtained by in vivo and in vitro                  immunology and genetics.
                experiments using histocompatible animals. In fish, how-
                ever, suitable inbred lines for immunological study are not
                available. Although inbred strains of small teleosts have                 Clonal State of Triploid Ginbuna
                been established by sibmating (e.g., platyfish and sword-
                tails, Xiphophorus spp., (Kallman 1964); guppy, Lebistes                  Naturally occurring gynogenetic populations are included
                reticulatus, (Schroder and Holzberg 1972); and medaka,                    in two cyprinid species: silver crucian carp, Carassius auratus
                Oryzias latipes, (Taguchi 1980), they are too small to col-               gibeho (Cherfas 1966), and ginbuna, C. gibeho langsdo!fii(for-
                lect lymphocytes and blood samples. Furthermore, gene-                    mally C. auratus langsdorfil, Kobayashi 19 7 1). Ginbuna have
                tically identical animals are not currently available for                 the widest distribution in Japan, and include both bisex-
                practical use, even though chromosomal set manipulation                   ual diploid (2n = 100) and tetraploid (4n = 206) types, and
                techniques have seen great improvement in recent years                    unisexual (all female) triploid (3n = 156) types (Kobayashi
                and homozygous clonal fish have been obtained by the sup-                 et al. 1970, 1977). In unisexual ginbuna, which reproduce
                pression of meitosis I and successive prevention of Mciosis               gynogenetically, the first polar body formation is skipped
                Il (e.g., zebra fish, Brachydanio rerio, (Streisinger et al.              as the result of lacking Meiosis I (Kobayashi 1976). There-
                1981), medaka (Naruse et al. 1985) and rainbow trout, On-                 fore, the progenies of these fish belong to a clone having
                corhynchus mykiss, (Onozato 1990).                                        the same genotype as the mother, as evidenced by scale
                   Fortunately, clonal crucian carp-naturally occurring                   grafting and electrophoreses (Onozato 1981; Murayama
                gynogenetic fish-are distributed widely in Japan. In the                  et al. 1984). Onozato (1981) has shown that the electro-

                                                                                                                                                            45








                   46           NOAA Technical Report NMFS 92

                   phoretic pattern of isozymes LDH, MDH, a-GPD and the                                                                           Table I
                   muscle protein of all progenies coincide with that of their                                      Antibody response to erythrocyte antigens in crucian carp.
                   mother. Of most interest is the fact that ginbuna was found                                      Fish were given three injections at 2-day intervals with 5
                   to be a heterozygous clone from isozyme analyses (Ono-                                           uL/g body weight of 20% erythrocyte intraperitoneally.
                   zato 1983).
                       Natural populations of triploid ginbuna consist of several                                   Antigens          Individual HA@ titres         1092)      Mean ï¿½ SID
                   clones which differ in strong or weak histocompatibility                                         Bovine                5, 5, 4, 1, 0                           3.0 ï¿½ 2.1
                   genes (H-genes). Allografted scales reciprocally exchanged                                       Horse                 12, 10.5, 10, 9.5, 9.5                 10.3 ï¿½ 0.9
                   among strong H gene-disparate members were rejected                                              Sheep                 7, 7, 7, 7, 6.5                         6.9 ï¿½ 0.2
                   within one week in an acute fashion, whereas allografts                                          Guinea-pig            10, 9.5, 8, 7, 5                        7.9 ï¿½ 1.8
                   among weak H gene-disparate members remained intact                                              Chicken               10, 9.5, 9, 9, 8                        9.1 ï¿½ 0.7
                   until 4 to 5 weeks before rejection (Nakanishi and Ono-                                          'Hemagglutination.
                   zato 1988).


                   Ginbuna Immunology and Genetics                                                             and adjusted to appropriate concentration. All animals
                   Variability in Immune Reactivity                                                            were given an intraperitoneal injection (5 pL/g body
                                                                                                               weight) every 3 days for 6 days. Immunized fish were bled
                   It is a well-known fact that immune responses are gene-                                     from the caudal blood vessels with an heparinized syringe,
                   tically controlled. Our greatest concern was to determine                                   and the plasma samples were analyzed for hemagglutina-
                   to what extent the variation exists in response to erythro-                                 tion titres by a microtiter method. Fish weighing 20-30
                   cyte antigens in genetically identical animals. If the vari-                                g were maintained in tanks with running water at 25 ï¿½ I OC
                   ation is not great, these animals could be used in various                                  and fed pellet. Anesthetization was performed with ethyl
                   studies such as the examination of vaccines and drugs. As                                   p-amino benzoate (100 mg/L) prior to manipulation. Horse
                   well as using fewer animals, we could also expect the results                               red blood cells (HRBC) were found to be the best antigen
                   to be more reproducible compared to those obtained using                                    for inducing high titres of antibody in crucian carp (Table
                   outbred animals.                                                                            1). We also examined the effect of concentration on the
                      We examined several erythrocyte antigens for monitor-                                    diversity in immune responses using outbred nigorobuna
                   ing immune reactivity. Erythrocytes in Alsever's solution                                   crucian carp, Carassius carassius grandoculis. At a high con-
                   were purchased from Teikoku Zoki Ltd (Tokyo), washed                                        centration of 2017o, most of fish elicited similar titres. At
                   three times with phosphate-buffered saline (PBS, pH 7.2)                                    lower concentrations, however, greater variation was ob-




                                                                                                      Table 2
                                                Effect of dosage HRBC (horse red blood cells) antigen on the antibody response of out-
                                                                                bred nigorobuna crucian carp at 25'C.

                                                Concentration
                                                  of antigen               Weeks'             Individual HAb titres         log2)           Mean              C.V.1

                                                    20%                         2                     11, 10, 10, 8.5, 7                     9.3              0.15
                                                                                3                     11, 10.5, 10, 8, 8                     9.5              0.13
                                                      557o                      2                     7.5, 7, 6, 6, 0                        5.3              0.51
                                                                                3                     9, 7, 7, 7, 2.5                        6.5              0.33
                                                      10/0                      2                     9, 7, 5, 5, 0                          5.2              0.58
                                                                                3                     9.5, 7,  7, 6.5, 0                     6.0              0.53
                                                   0.10/0                       2                     7, 5.5, 4, 4, 3                        4.7              0.30
                                                                                3                     8, 7.5,  6.5,6.5, 5                    6.7              0.15
                                                  0.01%                         2                     1, 1, 0, 0,0                           0.4              1.22
                                                                                3                     4.5, 3, 2.5, 2, 1                      2.6              0.45
                                                0.0010/0                        2                     0, 0, 0, 0, 0                          0.0              0.00
                                                                                3                     6, 4, 3, 0, 0                          2.6              0.90

                                                'Interval between the first injection and the         bleeding.
                                                'Hernagglutination.
                                                'Coefficient of variance.







                                                                            Nakanishi and Onozato: Immunology and Genetics of Crucian Carp                                     47



                                                                                              Table 3
                                            Comparison of variability in antibody responses to HRBC between allogeneic and isogeneic
                                                                                         crucian carp.

                                                                         No. of
                                                                        animals          Individual HA' titres      1092 ) b        Mean         C.V.1

                                            20%       Allogeneic            15          13(l), 12(l), 11(2), 10.5(l), 10(4)          10.0        0.14
                                                                                        9.5(3), 9(l), 8.5(l), 7(l)
                                                      Isogencic             15          13(3), 12.5(l), 12(3), 11.5(2)               11.5        0.10
                                                                                        11(3), 10.5(l), 10(l), 9(l)
                                              1 %     Allogeneic            15          9(l), 8(l), 7.5(l), 7(5), 6(3)               6.4         0.22
                                                                                        5.5(l), 4.5(2), 3.5(l)
                                                      Isogeneic             15          B(l), 7.5(l), 7(5), 6(6), 5(i), 4(l)         6.4         0.15
                                             0.017o   Allogeneic            10          7(2), 5(2), 4(l), 3.5(2), 3(l), 4(2)         4.2         0.41
                                                      Isogeneic             10          10(1), 8(l), 7(2), 5(l), 4.5(1)              5.7         0.38
                                                                                        4(3), 3(l)

                                            'Hemmaglutination.
                                            'Number of fish is shown in parenthesis.
                                            'Coefficient of variance.





                  served; some fish showed high titres, and others did not                              Fish source and the procedures of immunization, leuko-
                  respond at all (Table 2). There is no apparent difference                           cyte collection, and cell transfer have been described in
                  between allogeneic and isogeneic crucian carp even at lower                         Nakanishi (1987a). In brief, donors were injected intra-
                  concentrations (Table 3). That is, genetically identical gin-                       peritoneally at 2-day intervals with 5 IiL HRBC/g body
                  buna showed similar considerable variation to that of the                           weight. Donors used for day-3 transfer were given a single
                  outbred nigorobuna in immune responsiveness.                                        injection, those for day-5 transfer were given two injec-
                     Similar results have been obtained from the study of                             tions and those for day-7 or after were given three injec-
                  genetic influence on the diversity in growth rate using                             tions. Recipient fish were subsequently infused with lym-
                  clonal crucian carp (Nakanishi and Onozato 1987). In this                           phoid cells at a rate of 0. 1 mL of 5 x 107 cells/mL per
                  study a great variation in size and weight was found, even                          fish. Pronephric and mesonephric cells from a fish were
                  in clonal crucian carp, indicating the occurrence of a                              injected into two or three fish and thymic and splenic cells
                  superior group when the fry were reared in small tanks at                           were transferred into one fish. Recipients were evaluated
                  high,densities with food of different partical size.                                by measuring the serum antibody titre using standard
                     These results suggest that immune reactivity or growth                           methods of hernagglutination in microtiter plates.
                  is not only dependent on genetic state but is greatly influ-                          In a comparison of transferability between lymphoid
                  enced by other factors which may have been formed in                                organs, splenic cells were most effective in transferring
                  correlation with environmental circumstances. That is,                              immune reactivity, followed by pronephric, then meso-
                  individuals have met with different microenvironments                               nephric cells (Fig. 1). Little or no antibody titres were
                  throughout the life span, including egg state in the ovary,                         elicited when the recipients received transferred thymic cells
                  and the differences in physiological conditions must have                           (Figs. 1, 2). The optimal period for collecting the cells from
                  occurred among individuals later on.                                                the donor was determined by examining the antibody titres
                                                                                                      in the plasma of the recipients which received cells taken
                                                                                                      from immunized donors on days 3, 5, 7, 10, 14, and 2 1.
                  Kinetics of Transfer of                                                             Antibody productivity was most successfully conferred
                  Immunity by Immune Leukocytes                                                       when cells were taken from immune donors 7 days after
                                                                                                      immunization (Fig. 1). In recipients, antibodies were de-
                  Adoptive transfer of immunity can be successful only when                           tected within one day after transfer and the titre reached
                  recipients show no alloimmune response. However, the                                peaked levels on day 7 for mesonephric cells and day 14
                  histocompatibility system of fish has been found to be well                         for phronephric and splenic cells (Fig. 2). These results
                  developed (Kallman 1970; Borysenkc, 1976), with rejec-                              agree well with the kinetics of plaque-forming cells (PFCs
                  tion often occurring in an acute fashion even among sib-                            = antibody-producing cells) and circulating antibodies
                  lings. Therefore, adoptive transfer is only possible when                           described by Nakanishi (1987a). That is, PFCs were
                  inbred or clonal fish are used.                                                     detected on day 3 and peak response was observed 5 to








               48          NOAA Technical Report NMFS 92



                       8



                       7
                       6            /0\  %
                    0  5
                    T
                                               0
                       4        0
                    <
                                                           o
                       3
                    ID


                       2

                                                                                  0                                   Figure I
                                                                                           Kinetics of leukocyte transfer. Pronephros         mesonephros
                                                                                           (0), spleen (A) and thymus (A) cells taken from immunized
                           3          7,       10          14                    21        donors on day 3, 5, 7, 10, 14 and 21 are injected into recip-
                                              Day of Cell Transfer                         ients. Each point represents mean hernagglutination titre in
                                                                                           the plasma for 5 to 10 recipients.






                       9



                       8



                       7



                       6 -
                    0
                    'L                      0-
                       5 -
                                0                                0--
                    <
                    :E 4-
                    C
                                      0

                       3-                                                               0
                                                                                                                         Figure 2
                       2-                                                                         Antibody production kinetics. Recipients receiving
                                                                                                  7-day postimmunized leukocytes from pronephros ( 0
                    <2
                                                                                                  mesonephros (0), spleen (A), and thymus (A) are tested
                           1    3     5     7                    14                    21         for antibody levels in the plasma. Each point represents
                                               Days After Cell Transfer                           mean hernagglutination titre in the plasma for 5 to 10
                                                                                                  recipients.




               7 days after the first injection. On the other hand, cir-                between the PFC response and transferability for individual
               culating antibodies were detected 5 days after the first                 fish was observed in other lymphoid tissues (Nakanishi
               injection and reached their peak on day 14. In the pres-                 1987a). This lack of correlation between the PFC response
               ent study transfer of thymic cells was not successful,                   and transferability might be attributed to the existence of
               even though the number of PFCs of the thymus was sim-                    different developmental stages or of heterogeneous popula-
               ilar to that of the pronephros and the spleen. This findin               tions of antibody-producing cells and the necessity of cell
               g leads to the idea either that thymic PFCs are not the cells            collaboration. In any event this basic infornation. obtained
               involved in the transfer of immunity or that thymic cells                by using isogeneic animals can be useful for further in-
               need collaboration with other sensitized lymphoid cells to               vestigations of cellular immune mechanisms on fish im-
               produce antibodies. In addition, no direct relationship                  mune systems.







                                                                             Nakanishi and Onozato: Immunology and Genetics of Crucian Carp                                       49


                  Transferability of Immune Pronephric                                                  B I to K1 clones, which differed in strong H gene as evi-
                  Cells in Isogeneic, Allogeneic and                                                    denced by the acute rejection of allografts. Little or no
                  Xenogeneic Transfer Systems                                                           antibody titres were detected in the recipients 7 days after
                                                                                                        transfer (Table 4). On the other hand, interclonal transfer
                  The major histocompatibility complex (MHC) of mam-                                    between weak H gene-disparate clones was successful.
                  mals and birds consists of numerous genes involved in acute                           Antibody titre of recipients 7 days after transfer was similar
                  allograft rejection, cell collaboration, and cytotoxic lym-                           to that of intraclonal (isogeneic) transfer (Table 4), although
                  phocyte effective functions (Klein 1977). In lower verte-                             recipients rejected allografted scales from donors 1-2
                  brates, a single genetic region homologous to the MHC                                 months after grafting. Transferability of immune pro-
                  has been described in the anuran amphibian (Du Pasquier                               nephric cells in xenogeneic transfer systems was also ex-
                  et al. 1975; Kaufman et al. 1985; Nakamura et al. 1986).                              amined. Pronephric cell transfer was carried out from
                  The teleost is the lowest vertebrate in which a MHC is                                immunized kinbuna to unimmunized ginbuna. No anti-
                  suspected to exist, owing to their vigorous rejection of                              body was found and all xenografted scales from kinbuna
                  foreign tissue grafts, though knowledge of a MHC in fish                              were rej   .ected within 5 days in ginbuna (Table 4). These
                  is sparse. The present study was undertaken to examine                                results suggest that transferability of immune cells is strictly
                  the transferability of antibody reactivity by immune pro-                             controlled by "MHC."
                  nephric cells among isogeneic, and xenogeneic crucian carp                              The most interesting results were obtained when allo-
                  in order to analyze the correlation between transplanta-                              geneic transfer using pronephric cells was perforned among
                  tion antigens and determinants involved in cell collabora-                            siblings of kinbuna that had rejected allografts of each other
                  tion or cell-mediated lympholysis.                                                    in an acute manner. Two experiments were performed,
                     In these experiments we used two clones of ginbuna (B 1,                           each with different times of cell collection from the immu-
                  D3) from Okushiri Island in the vicinity of Hokkaido Island                           nized donors: 7 days (Fig. 3A) and 14 days (Fig. 3B) after
                  and one clone (Kl) from Lake Kasumigaura, lbaragi                                     immunization. In each study, one of five recipients showed
                  prefecture. In addition, siblings of bisexual diploid kin-                            elevated antibody titre which peaked 2 and 3 weeks after
                  buna, C. carasslus buergeri (formerly C. auratus susp.), were                         transfer from the 7-day (Fig. 3A) and 14-day (Fig. 3B)
                  also used in hopes that some of them might be genetically                             postimmunized donors, respectively. Furthermore, one of
                  related. Fish source and histocompatibility relationship                              the recipients with cells transferred from the 7 days post-
                  among clones have been previously described (Nakanishi                                immunization donor showed a fairly high level of antibody
                  1987b, Nakanishi and Onozato 1988). Cell transfer was                                 titre 5 weeks after transfer, while no antibody was detected
                  carried out according to the methods described above. Scale                           one week and three weeks after transfer. These results sug-
                  transplantation techniques followed Nakanishi (1987c).                                gest that the locus of "class IP antigens involved in cell
                     Interclonal transfer between strong H gene-disparate                               collaboration or cytotoxic lymphocyte effecter function is
                  clones was carried out to determine transferability of im-                            limited in polymorphism in comparison to the transplanta-
                  mune pronephric cells. Transfer of antibody reactivity was                            tion antigens (class I antigens), because antibody reactiv-
                  not successful when pronephric cells were transferred from                            ity was successfully conferred in some donor-host combina-






                                                                                                Table 4
                                              Transferability of immune pronephric cells in isogencic and                  xenogeneic crucian carp.

                                                                    Donor-host             Weeks after                     HA' titre of recipients
                                                                    relationship                transfer                             log')

                                              Isogeneic            BI - BI                        1                7.5     6       8       7     5       5
                                              transfer                                            2                6       6       8       9     6       -

                                              Xenogeneic           kinbuna - Bl                   1                0       0       0       0     1       0
                                              transfer                                            2                0       0       0       0     0       2

                                              Inter-clonal         BI    Kl                       1                0       1       0       2     1       0
                                              transfer V                                          2                0       0       0       0     0       0

                                              Inter-clonal         Bi    D3                       1                9       6       7.5     4     4       J,
                                              transfer 11'                                        2                8.5     5.5     7.3     4     3       +


                                              'Hemagglutination.
                                              'Cells were transferred from 131 clone to a KI      clone that differs in strong H   gene.
                                              'Cells were transferred from Bl clone to a D3       clone that differs in weak H gene.








                  50           NOAA Technical Report NMFS 92


                                                                                                         mice and rats. Indeed, clonal ginbuna are a useful tool from
                             6                                                                           many aspects of fish immunology as shown in this review.
                                                                                                         There are of course some limitations for using ginbuna;
                                                                                                         one is the difficulty of crossing because of their gynogene-
                                                                                                         tic reproduction and the other is their heterozygous state.
                                                                                                         Even so, ginbuna will continue to be one of the best models
                             4                                                                           for the study of fish immunology until the establishment
                                                                                                         of inbred or clonal fish by means of chromosomal manipu-
                             3                                                                           lation technique.

                             2
                                                                                                         Citations
                        .2  -12
                        I                                                                                Borysenko, M.
                        W
                                                                                                               1976. Phylogeny of immunity: an overview. Immunogenetics 3:
                                                                                                                  305-326.
                             7                                                                           Cerfas, N.B.
                                                                                                               1966.   Natural triploidy in the female of the unisexual variety of
                        Z    6                                                                                    the silver crucian carp (C. auratus gibeho Bloch).       Genetika. 5:
                        <                                                                                         16-24.
                        W
                        X    5                                                                           Du Pasquier, L., X. Chardonnens, and V.C. Miggiano.
                                                                                                               1975. A major hostocompatibility complex in the toad Xenopus laevis
                                                                                                                  (Daudin). Immunogenetics 1:482-494.
                             4                                                                           Kallman, K.D.
                                                                                                               1964. An estimate of the number of histocompatibility loci in the
                             3                                                                                    teleost Xiphophorus maculatus. Genetics 50:583-595.
                                                                                                               1970. Genetics of tissue transplantation in Teleostei. Transplant.
                             2                                                                                    Proc. 2:263-271.
                                                                                                         Kaufman, J.F., M.F. Flajnik, L. Du Pasquier, and P. Riegert.
                            <2                                                                                 1985. Xenopus MHC class Il molecules. I: Identification and struc-
                                                                                                                  tural characterization. J. Immunol. 13+:3248-3257.
                                                                                                         Klein, J.
                                  0       1       2       3       4       5       6                            1977. Evolution and function of the major histocompatibility sys-
                                                       WEEKS                                                      tem: facts and speculations. In The major histocompatibility
                                                                                                                  system in man and animals (D. Gotze, ed.), p. 339-378.
                                                                                                                  Springer-Verlag, Berlin, New York.
                                                    Figure 3                                             Kobayashi, H.
                  (A) Transferability of immune pronephric cells from donors 7-days                            1971. A cytological study on gynogenesis of the triploid ginbuna
                  postimmunization in allogencic kinbuna crucian carp. Cells                                      (Carassius auratus langsdo@kz). Zool. Mag. 80:316-322.
                  (5 x 10') per fish were intravascularly transferred and then anti-                           1976. A cytological study on the maturation division in the oogenic
                  body titres in the plasma of recipients were determined every week                              process of the triploid ginbuna (Carassius auratus langsdorfu). Jpn.
                  after transfer. Each line represents the antibody titre of individual                           J. Ichthyol. 22:234-240.
                  crucian carp. (B) Transferability of immune pronephric cells from                      Kobayashi, H., Y. Kawashima, and N. Takeuchi.
                  donors 14 days post-immunization in allogeneic kinbuna crucian                               1970. Comparative chromosome studies in the genus Carassius,
                                                                                                                  especially with a finder of polyploicly in the ginbuna (C. auralus
                  carp.                                                                                           langsdorfit). Jpn. J. Ichthyol. 17:153-160.
                                                                                                         Kobayashi, H., N. Nakano, and M. Nakamura.
                                                                                                               1977. On the hybrids, 4n ginbuna (Carassius auratus langsdorfii) x
                                                                                                                  kinbuna (C. auratus-subsp.), and their chromosomes. Bull. Jpn.
                                                                                                                  Soc. Sci. Fish. 43:31-37.
                  tions even when the recipients rejected an allografted                                 Murayama, Y., M. Hijikata, T. Nomura, and T. Kajishima.
                  donor's scales in an acute manner.                                                           1984. Analyses of histocompatibility and isozyme variations in a
                                                                                                                  triploidfish, Carauius auratus langsdo!fii. J. Fac. Sci., Shinshu Univ.
                                                                                                                  19:9-25.
                  Summarizing Comment                                                                    Nakamura, T., A. Sekizawa, T. Fujii, and C. Katagiri.
                                                                                                               1986. Cosegregation of the polymorphic C4 with the MHC in the
                                                                                                                  frog, Xenopus laevis. Immunogenetics 23:181-186.
                                   / / rI-









                  Many developments have contributed to the understand-                                  Nakanishi, T.
                  ing of the immune system in fish over the last decade.                                       1987a. Kinetics of transfer of immunity by immune leukocytes and
                  However, more progress cannot be expected without using                                         PFC response to HRBC in isogeneic ginbuna crucian carp,
                  isogeneic or genetically defined models. Genetically iden-                                      Carassius auratus langsdorfii. J. Fish Biol. 30:723-729.
                                                                                                               1987b. Transferability of immune plasma and pronephric cells in
                  tical animals offer many additional opportunities for ana-                                      isogeneic, allogeneic and xenogeneic transfer systems in crucian
                  lyzing the immune system of fish, as demonstrated with                                          carp. Dev. Comp. Immunol. 11:521-528.







                                                                              Nakanishi and Onozato: Immunology and Genetics of Crucian Carp                                         51


                        1987c. Histocompatibility analyses in tetraploids induced from                        1983. A story of clonal crucian carp. Tansuigyo 9:33-41. (in
                          clonal triploid crucian carp and in gynogenetic diploid goldfish.                     Japanese.)
                          J. Fish Biol. 31:35-40.                                                             1990. Production of clones of homozygous rainbow trout, Oncorhyn-
                  Nakanishi, T., and H. Onozato.                                                                chus mykiss by gynogenesis. (Abstr.) Annu. Meeting jpn. Soc.
                        1987. Variability in the growth of isogeneic crucian carp, Carassius                    Sci. Fish. (In Japanese.)
                          gibdio langsdorfii. Nippon Suisan Gakkaishi 53:2099-2104.                     Schroder, J.H., and S. Holzberg.
                        1988.   Histocompatibility analyses in a gynogenetic ginbuna cru-                     1972. Population genetics of Lebistes reticulatus Peters (poeciliidae;
                          cian carp, Carassiusgibeho langsdorfii collected from Okushiri Island.                Pisces). 1: Effects of radiation induced mutation on the segrega-
                          (Abstr.) Annu. MeetingJap. Soc. Ichthyol. (injapanese.)                               tion ratio in postirradiation F2. Genetics 70:621-630.
                  Naruse, K_ K. Ijiri, A. Shima, and N. Egami.                                          Streisinger, G., C. Walker, N. Dower, D. Knauber, and F. Singer.
                        1985. The production of cloned fish in the medaka (Oryzias latipes).                  1981. Production of clones of homozygous diploid zebra fish
                          J. Exp. Zool. 236-335-341.                                                            Brachydanio relio. Nature 291:293-296.
                  Onozato, H.                                                                           Taguchi, Y.
                        1981. Gynogenesis in fishes. Fish Genetics and Breeding Science                       1980.   Establishment of inbred strains of the teleost, Oryzias latipes.
                          6:11-18. (in Japanese.)                                                               Zool. Mag. 89:283-301.








                                          Aquaculture of Striped Bass, Morone saxatilis,
                                                    and Its Hybrids in North America"'


                                                                       THEODORE I.J. SMITH

                                                             South Carolina Wildlife and Marine Resources Department
                                                                                  P.O. Box 12559
                                                                               Charleston, SC 29412





                                                                                  ABSTRACT


                                        Increased interest has focused on the culture of striped bass, Morone saxatilis, and its hybrids
                                      (especially M. chrysops) as food fish with the recent decline in fishery landings of striped bass.
                                      Hatchery and pond culture techniques are sufficiently developed to allow initiation of farming
                                      operations throughout many areas of the southeastern U.S. Although aquaculture of these fish
                                      appears economically attractive, there are certain impediments to large-scale culture. Such im-
                                      pediments include issues of seed stock availability and cost, and restrictive laws and regulations.
                                      Such issues are being addressed and progress has been achieved in alleviating some of the im-
                                      pacts of these factors. It is expected that within the near future a sizable industry will be developed
                                      and that cultured striped bass and its hybrids will become readily available in many seafood markets
                                      and restaurants.



                 Introduction                                                                 have been highly encouraging and now there is commer-
                                                                                              cial interest in many states        'to rear these fish for market
                 Native stocks of striped bass, Morone saxattlis, have sup-                   (Smith and Jenkins 1985a).
                 ported major fisheries in the United States along the Atlan-                    This manuscript reviews and discusses the various con-
                 tic coast. However, since 1973 landings have declined                        siderations associated with aquaculture of striped bass and
                 dramatically (Fig. 1) and in an effort to protect the remain-                its hybrids in North America. In particular, information
                 ing stocks, commercial fishing bans have been implemented                    on broodstock acquisition and culture, spawning, hatching,
                 in many states. The striped bass is also an important                        larval rearing, and production data are presented. Also,
                 recreational species and has been stocked in lakes, reser-                   comparative performance data on some of the various
                 voirs, and rivers throughout the United States to support                    hybrid striped bass crosses are include&
                 sport fisheries (Stevens 1984).
                    The striped bass is well known. in the marketplace where
                 it commands a high price (Sport Fishing Institute 1984;                      Status of Aquaculture
                 Swartz 1984). Because of the scarcity of this species and                    Supply of "Seed Stock"
                 its high market value, interest has increased for culturing
                 striped bass or a suitable market substitute. During recent                  A basic impediment to the            large-scale development of
                 years, research and development activities have included                     striped bass and hybrid bass         farms has been the lack of
                 attempts to rear striped bass and its hybrids in ponds, net-                 a dependable supply of seed stock (joint Subcommittee on
                 pens, raceways, and tanks (Powell 1973; Valenti et al.                       Aquaculture 1983; Smith andjenkins 1985b; Smith 1987).
                 1976; Wawronowicz and Lewis 1979; Williams et al. 1981;                      Presently, state and federal hatcheries rely on the capture
                 Kerby et al. 1983a, b; Woods et al. 1983, 1985; Collins                      of wild adults from spawning grounds to support hatchery
                 et al. 1984; Kerby et al. 1987; Smith et al. 1987). Results                  operations (Harrell 1984). However, the private sector is
                                                                                              typically prohibited. from collecting broodstock using the
                                                                                              same techniques (especially electrofishing) and from ob-
                 'Contribution Number 233 from the South Carolina Marine Resource             taining stock from areas used by these public hatcheries.
                  Center.                                                                     Consequently, -acquisition of ripe broodstock by the private
                 'Preparation of this report was supported in part by Dep. of Commerce,
                  NOAA, Office of Sea Grant, under Contract Number NA85AA-D-                  sector is unpredictable, inefficient, and highly regulated.
                  SG-121 and the State of South Carolina.                                     In recent years, there has also been an overall decrease in

                                                                                                                                                                   53








                 54          NOAA Technical Report NMFS 92



                            7



                            6-



                            5-
                      C/)

                      0
                      1---  4--

                      0
                      CC    3
                      F-
                      LU


                            2



                            1 __


                            0                             . . . . . . . . . .                                                                 Figure 1
                                64 66 68 70 72 74 76 78 80 82 84 86                                                     Commercial landings of striped bass on the
                                                                YEARS                                                   Atlantic coast. (Fisheries of the United States,
                                                                                                                        formerly Fisheries Statistics of the U.S.)




                 broodstock numbers such that even state and federal
                 hatchery managers are currently experiencing difficulty in                                                         Table I
                 meeting their production goals.                                                       Age, size, and percent maturity for cultured striped bass
                                                                                                         broodstock (updated from Smith and Jenkins 1986).,

                                                                                                                               Males                       Females
                 Broodstock Development Research
                                                                                                          Age          Mature6        Weight        Mature'        Weight
                 In order to alleviate this "seed stock" problem, South                                (Months)                          (kg)                        (kg)
                 Carolina researchers began in 1982 to examine the feasi-
                 bility of developing domesticated broodstock to support                                   21               22           -                0          -
                 hatchery operations (Smith and Jenkins 1984). Progeny                                     33              100           2.3              25         3.4
                                                                                                           46              100           3.9              75         4.9
                 of wild striped bass were reared in tanks and subjected to                                60              100           5.2            100          6.4
                 controlled temperature and photoperiod regimes (Smith
                 and Jenkins 1986). During a five-year period the growth,                              'Based on number of males and females at 60 months of age. 19 %
                 maturity, and spawning success of these fish were mom-                                 of the fish did not mature by age 60 months.
                                                                                                       6Fish which expressed milt.
                 tored. At 33 months of age males had attained a size of                               'Fish with eggs greater than 700 jA in diameter.
                 2.3 kg and essentially all were mature (Table 1). However,
                 at 3 3 months of age only 25 17o of the females were mature
                 and difficulty was experienced in spawning these fish
                 (Smith and Jenkins 1986). During the following two years                           space required is reduced but effort is intensive and often
                 additional females matured while no additional mature                              broodstock die as a result of handling and stripping pro-
                 males were observed (Table 1).                                                     cedures. In South Carolina, both techniques have been
                    Spawning of wild striped bass broodstock is accom-                              used with 3- to 5-year old cultured broodstock. It is recom-
                 plished by injecting the newly captured fish with human                            mended that the "tank spawning" method be employed
                 chorionic gonadotropin (HCG) at a rate of 330 I.U./kg.                             with striped bass in order to reduce fish injury. Although
                 Then, the fish are either "naturally" tank spawned by pair-                        substantial progress has been achieved, the culture tech-
                 ing up males and females in a tank (Bishop 1975), or the                           niques developed for striped bass females need additional
                 eggs are stripped from the female at time of ovulation and                         research to improve the predictability of spawning success.
                 artificially fertilized with stripped milt (Bayless 1972; Bonn                     In contrast, development of domesticated male broodstock
                 et al. 1976). In the first case, larger facilities are needed                      has been highly successful. Males can be produced in 2
                 to house spawning tanks but effort is less intensive and                           to 3 years and used over several years (Smith and Jenkins
                 damage to broodstock is reduced. In the latter method, the                         1986).







                                                                                                                        Smith: Aquaculture of Striped Bass                       55

                     The techniques described above are used for the pro-                                                               Table 2
                  duction of striped bass fry. However, the striped bass is                               Summary of reciprocal cross fry production using captive
                  not the preferred fish for an aquaculture operation because                             wild white bass females and cultured striped bass males (up-
                  of its slower growth during the first two years and its                                 dated from Smith and Jenkins 1986).
                  reduced environmental tolerances. Concurrent research
                  conducted with the striped bass and white bass (M. @hrysops)                                                    Fish Weight                Larvae Hatched
                  crosses has indicated that these hybrids are the preferred                              Trial  No.                    (kg)                         (no.)
                  fishes for aquaculture development (discussed later in text)                 '                 1                      0.63                       33992
                  In order to make "original cross" hybrids, female striped                                      2                      0.46                       37247
                  bass and male white bass must be stripped because they                                         3                      0.64                       84185
                  can not be induced to tank spawn together. Consequently,                                       4                      0.61                       17870
                  the problems of acquisition, predictability, and reuse of                                      5                      0.64                       87950
                                                                                                                 6                      0.68                       48 100'
                  female striped bass occur. However, the "reciprocal cross"                                     7                      0.73                       48 100'
                  (female white bass x male striped bass) can also be per-                                       8                      0.65                       48 100a
                  formed which results in a hybrid which is quite satisfac-                                      9                      0.66                       481001
                  tory for aquaculture development (see later section). White                             'All   hatches were pooled,   192000 fry produced.
                  bass are smaller than striped bass at maturity (minimum
                  size for females -300 g vs. -3.4 kg for striped bass) and
                  this species is abundant throughout many areas of the U.S.
                  Although this species is of recreational importance, there                          can be left in the spawning tank to hatch or the eggs can
                  is much less public sentiment associated with their removal                         be collected and placed in McDonald jars. Striped bass eggs
                  for private hatchery use compared to the collection of                              are sernibuoyant and are easily kept rolling in the jars. In
                  striped bass (especially females).                                                  contrast, white bass eggs are highly adhesive and will
                     Broodstock development activities with white bass have                           readily form large clumped masses of eggs. Such clump-
                  been based on the use of wild-caught fish, although cultured                        ing is undesirable as dead eggs are not easily removed and
                  white bass may also prove to be suitable. Researchers in                            fungal infections can readily occur. In order to eliminate
                  South Carolina have demonstrated that adult wild fish can                           the adhesiveness, the fertilized eggs are placed in the
                  be captured in the fall, matured in outdoor tank culture                            McDonald jars and aerated in a tannic acid solution (150
                  systems using ambient conditions, and spawned in the                                mg/L) for about 10 minutes before clean fresh water is
                  spring (Smith andjenkins 1986). Unlike striped bass, adult                          flowed through the jars. This technique reduces clumping
                  white bass are either ripe males or ripe females in the spring                      and results in a higher hatch rate of reciprocal cross hybrids
                  with few or no fish of unknown sex. Work-to-date indicates                          (Charles C. Starling, Florida Game and Fresh Water Fish
                  that white bass females and striped bass males are easy to                          Commission, Webster, FL 33597, pers. commun., March
                  spawn and will naturally mature together in outdoor tanks                           1986).
                  (Smith and Jenkins 1985b, 1986, 1987). HCG is also used
                  with white bass but it is administered at a higher level                            Fingerling Production
                  (7 50-1500 1. U. /kg). Average production of reciprocal cross
                  hybrid bass fry is in the range of 35 000 to 90 000 per female                      The fry are reared in either freshwater or brackish water
                  fish 460 to 640 g in weight (Smith and Jenkins 1986; Table                          nursery ponds (Phase I) to a small juvenile size over a
                  2). Recent demonstration of these spawning techniques                               30-45 day period (Bonn et al. 1976; Geiger 1983a, b;
                  using captive, wild broodstock should enhance the develop-                          Parker and Geiger 1984). During the last 2-3 weeks of
                  ment of hybrid striped bass aquaculture farms (Smith and                            nursery rearing, dry feed is added to the ponds to serve
                  Jenkins 1987).                                                                      as a replacement for the diminishing supply of zooplank-
                                                                                                      ton of a suitable size. Typical stocking density for the
                  Incubation and Hatching                                                             Phase I nursery ponds is about 250000 fry/ha. Survival
                                                                                                      ranges from 0 to about 80%, but 25 Olo is common (Parker
                  Eggs of striped bass and its hybrids are typically incubated                        and Geiger 1984). Temperature in the ponds ranges from
                  in McDonald hatchingjars (Bonn et al. 1976). Depending                              about 18 to 25'C. During 1985 and 1986, nursery trials
                  on egg type, approximately 100000-250000 eggs are                                   were conducted at the S.C. Wildlife and Marine Resources
                  placed in each jar. Water injected through a center tube                            Department's Waddell Mariculture Center (WMC). Pro-
                  causes continuous upwelling and oxygenation, and eggs                               duction from these trials conducted at dissimilar densities
                  hatch in about 48 hours at temperatures of 18' to 20'C,                             was 281000/ha for striped bass and 110300/ha on the
                  (Bayless 1972). At time of hatching, fry swim up with the                           average for hybrids (Table 3).
                  outflowing water and are concentrated in outside aquaria.                              The small juveniles are harvested by draining the pond
                  In situations where striped bass are tank-spawned, eggs                             and collecting the fish in catch basins adjacent to the drain








                  56           NOAA Technical Report NMFS 92



                                                                                                Table 3
                                              Results of Phase I nursery trails with striped bass and its hybrids in brackish water (salinity
                                                                                          4-8 ppt) ponds.

                                                                             Stocking data                                      Harvest data

                                                                         Density                      Duration        Density     Survival      Mean wt.
                                              Type of fish               (no./ha)        Date            (d)          (no./ha)      M               (g)

                                              Striped bass               600000         4/28/87          30           281630        46.9            0.36
                                              F, original hybrid         128000         5/08/85          41           96000         75.0            0.45
                                              F, reciprocal hybrid       300000         3/10/86          57           74500         24.8            2.20
                                              F, reciprocal hybrid       300000         3/10/86          56           161030        53.7            0.67




                  structure. Fish produced by private hatcheries are then                                eggs (Stevens 1966, 1967). The focus of the hybridiza-
                  graded and restocked in Phase 11 rearing ponds at a lower                              tion work was to develop a fish that had the growth and
                  density or sold to other farmers. Indoor intensive nursery                             feeding characteristics of the striped bass and the envi-
                  techniques using tanks have also been developed for                                    ronmental adaptability and less stringent spawning re-
                  Phase I rearing of striped bass and its hybrids (Lewis and                             quirements of white bass (Bonn et al. 1976). The first
                  Heidinger 1981; Kerby et al. 1983a; Carlberg et al. 1984;                              hybrid produced was the "original cross." Later other
                  Smith and Jenkins 1984). However, production ofjuveniles                               crosses were made using white perch (M. americana) and
                  from such tank nursery systems is often highly variable and                            yellow bass (M. mississippiensis) males as well as reciprocal
                  the pond-rearing method is currently the preferred tech-                               and backcrosses (Bayless 1972). Early evaluation of these
                  nique for Phase I production.                                                          various hybrids was somewhat opportunistic and not well
                                                                                                         controlled. Nevertheless, from these studies it was con-
                  Performance Characteristics                                                            cluded that the original cross hybrid was a good fish and
                  of Striped Bass and Hybrids                                                            stocking programs were initiated (Bishop 1968; Logan
                                                                                                         1968; Williams 1971; Ware 1975). Field observations in-
                  Hybridization studies with striped               bass were initiated in                dicated that the hybrids outgrew striped bass during the
                  1965 after it was demonstrated that HCG could be used                                  first two years and were both easier to produce and har-
                  to induce final maturation and ovulation of striped bass                               dier than striped bass.




                                                                                                Table 4
                      Summary of indoor tank studies examining performance characteristics of striped bass and its hybrids (updated from Smith
                                                                                        and Jenkins 1985b).

                                              Fish stocked                                                                                 Harvest data


                                                           Size          Density            Duration             Size            Survival           Biomass                Feed
                      Type                                 (g)           (no. /m')              (d)              (g)               M                (kg/m')            conversion

                                                                                           Study number 1
                      Striped bass                         4.4             31.6                 287              289                96                   8.8               2.3
                      F, original hybrid                   6.4             31.6                 287              507                100                16.0                2.2
                      F2 hybrid bass                       12.8            31.6                 287              347                84                   9.2               2.7
                                                                                           Study number 2
                      F, original hybrid                   23.0            31.6                 140              263                99                   8.3               1.9
                      F, white perch hybrid                21.0            31.6                 140              176                99                   5.5               2.5
                                                                                          Study number 3'
                      Striped bass                         6.0             29.2                 56                34                89                   1.4               1.7
                      F, original hybrid                   13.2            29.2                 56                77                98                   3.6               1.6
                      F, reciprocal hybrid                 10.9            29.2                 56                57                93                   2.5               1.9
                      Backcross hybrid                     21.1            29.2                 56                71                92                   3.1               2.4

                      'Study is currently in progress.







                                                                                                      Smith: Aquaculture of Striped Bass               57



                        200



                        1-80


                        160                                                          Reciprocal Hybrids


                        140--



                        120


                        100                                                                 original Hybrids
                   CD
                   LU
                          so--



                          60-



                          40-


                          20-                                                                                                         Figure 2
                                                                                                                              Comparison of F, origi-
                             0                                                                         t                      nal and reciprocal cross
                                   0        28        56       84       1 12       126     140       168       184            hybrids reared in ponds at
                                                                TIME       (days)                                             a density of 10000 fish/
                                                                                                                              ha.




                 With the increasing interest in aquaculture, more                     (T. Smith, unpublished).
               detailed performance information was desired for the                      In summary, the hybrids of striped bass and white bass
               various hybrids. Controlled studies were undertaken                     appear well suited for aquaculture. They can be raised in
               primarily in South Carolina and North Carolina. To date,                fresh or brackish water (Smith et al. 1986) and exhibit high
               not all striped bass hybrids have been tested but sufficient            growth and survival rates. Further, they can be reared in
               information is available to identify suitable candidates for            a broad range of water temperatures and are not killed by
               aquaculture use.                                                        low (7'C) or high (33'C) water temperatures (Smith et al.
                 A number of comparative studies have been undertaken                  1987).
               by the Charleston Laboratory, S.C. Wildlife and Marine
               Resources Department, using indoor tanks which can recir-               Grow-Out      Production
               culate either fresh or brackish water. In the first study
               striped bass were compared to reciprocal cross hybrids and              Research data from production trials to produce market-
               to F2 hybrids (F1 original cross x F, original cross)@                  size fish (>,568 g) have been limited although there is a
               Results of this 287-day study indicated that the reciprocal             commercial operation producing hybrids in tanks using
               cross hybrid grew fastest and had a high survival rate                  geothermal water in California and another company is
               (Table 4, Smith et al. 1985). In the second study the                   using net pens to produce striped bass in New York. Un-
               original cross hybrid was compared to a striped bass x                  fortunately, production data from these two operations are
               white perch hybrid. Again, results indicated that the white             proprietary. In 1984, North Carolina researchers harvested
               bass hybrid grew rapidly and had a high survival rate                   grow-out trials in 0. 1 ha earthen ponds using original cross
               (Table 4). In a current study, the original and reciprocal              hybrids. Survival averaged 83.9% and production ranged
               cross hybrids are outperforming the striped bass. However,              from 5 247 to 5 765 kg/ha (mean 5504 kg/ha) (Kerby et al.
               a backcross hybrid (F1 original x striped bass male) is                 1987). Recently, the S.C. Wildlife and Marine Resources
               also performing well at the present time and may be an                  completed its first pond grow-out trial using original cross
               additional aquaculture candidate (Table 4) (T. Smith, un-               hybrids. This study was conducted in a 0.5 ha pond at
               published). Results of these replicated tank studies are also           WMC. One-year-old juveniles (mean size 220 g) were
               being corroborated in pond culture trials (Figs. 2, 3)                  stocked in March 1986 at a density of 12000/ha for final








                 58         NOAA Technical Report NMFS 92


                                                                                                                                                        4

                            90--



                            80--



                            70--                                                Reciprocal       Hybrids


                            60-



                            50-
                      F_

                      0
                      Lu



                            30-



                            20-
                                                                                                 Striped Bass



                                                                                                                                                     Figure 3
                              0                                                           1 Hill 1 HHHH!   ....         ...                Comparison of striped
                                                                                                                                           bass and reciprocal cross
                                             12          24         36           48          60         72          84          96         hybrids reared in ponds at
                                                                      TIME (days)                                                          a density of 37000 fish/
                                                                                                                                           ha.


                 grow-out to market size. During the growing season                              Market Testing
                 (April-November), fish were fed a commercial trout pellet
                 twice daily and satisfactory water quality conditions were                      The opportunities and constraints associated. with the
                 maintained with paddlewheel aerators and water exchange.                        marketing of hybrid striped bass were recently examined
                 From December, 1986 to January, 1987 the fish were har-                         by Carlberg and Van Olst (1987). Prices received for the
                 vested and marketed. At harvest, mean fish size was 755 g                       cultured hybrids have been in the range of $5.50-1 1.00/kg
                 and survival was 93 To (Table 5). Total production was                          depending on product type (e.g., iced, gutted, live) and
                 8 323 kg/ha with 93 7o of the fish > 568 g (1 Y4 lb). These                     specific market (e.g., wholesaler, retailer, restaurant).
                 results are highly encouraging and represent the highest                        Organoleptic testing of pond-reared cultured hybrids has
                 pond production level yet achieved. Currently, a pond                           been conducted by the Southeast Fisheries Center
                 grow-out trial is underway with reciprocal cross hybrids                        [National Marine Fisheries Service (NMFS)], Charleston,
                 and growth to the present appears similar. From an aqua-                        S.C. Results indicate that the hybrid striped bass is a mild
                 culture perspective, routine production levels of 3900 to                       flavored fish which should have good market appeal
                 7 800 kg/ha should be attainable using techniques similar                       (Michael jahncke, NMFS, Charleston, SC 29412, pers.
                 to those employed for producing channel catfish (Malurus                        commun., June 1987). Restaurant testing has also been
                 nebulosus).                                                                     conducted using our pond-reared hybrid bass. Again,




                                                                                         Table 5
                                        Stocking and harvest data for pond grow-out trial using original cross hybrid striped bass.

                                           Stocking data                                                                       Harvest data

                     Density       Mean wt.         Age of fish       Pond size          Duration      Mean wt.         Survival        Production           Feed
                     (no./ha)          (9)              (d)              (ha)             (d)              (g)            (%)             (kg/ha)         conversion

                      2000           220.2              327               0.6             240              755             93              8323               1.6







                                                                                                                   Smith: Aquaculture of Striped Bass                    59



                                                                                          Table 6
                      Consumer evaluation (N = 216) of hybrid striped bass served in two Flordia restaurants (adapted from Liao et al. 1987).
                                                 Rating scale: 1 = poor, 2 = fair, 3 = good, 4 = very good, 4 = excellent.

                                                                                          Evaluation catergories                           Consumer repurchase (01o)

                      Restaurant        Entree       Price           Appearance         Texture       Smell        Taste       Price       No        Maybe          Yes

                          A             Broiled         10.95            4.7              4.6           4.8         4.7         4.4         2           4           94
                                        Fried           10.95            4.2              4.7           4.2         4.2         4.1        22           0           78
                                        Grilled         10.95            41. 4            4.4           4.4         4.3         4.3         8           0           92

                          B             Broiled          4.95            4.3              4.2           4.4         C 5         4.2         2           5           93
                                        Fried            4.95            4.1              4.2           4.1         4.4         4.2         2           5           93





                  results were favorable indicating that these fish are highly                    Conclusions
                  acceptable to consumers (Table 6, Liao et al. 1987). Thus,                      Commercial farming of striped bass hybrids is an emerg-
                  cultured hybrid striped bass appear to be an excellent                          ing industry in the United States which will need additional
                  market substitute for wild striped bass.                                        research and extension activities to reach its full potential.
                                                                                                  Pond culture technology is currently available and is now
                  Aquaculture Constraints                                                         in the process of being transferred to the private sector.
                  At the present time, there are two major constraints to                         Indeed, during the past year cooperative demonstration
                  the development of striped bass farms: 1) seed stock                            projects have been initiated in several states including
                  availability and cost, and 2) laws and regulations. Recent                      Maryland and North Carolina. Within the next 3-5 years,
                  research in South Carolina on the domestication and                             pond culture operations are expected to develop throughout
                  culture of broodstock has demonstrated commercially                             the southeast and middle Atlantic states as well as in
                  practical techniques for controlled production of "seed                         California.
                  stock. " Thus, in the near future, private hatcheries                              Although the research data base appears adequate for
                  should be able to develop their own captive broodstock                          development of the industry, additional study is needed
                  (especially striped bass males and white bass females)                          in the areas of 1) broodstock development and genetics,
                  and thereby increase the predictability and availability of                     2) identification of other suitable hybrids, 3) nutrition,
                  fry and small juveniles. This use of captive broodstock may                     4) market development, 5) disease treatment and preven-
                  also result in lower production costs and lower seed stock                      tion, and 6) laws and regulations. All these areas can
                  costs.                                                                          substantially impact the economics of bass aquaculture. For
                     The legal issues have been a serious constraint in                           example, profitability could be improved by development
                                                                                                  of hybrids exhibiting faster growth through genetic breed-
                  many states where fishery bans were instituted to protect                       ing or manipulation (induced polyploidy) and by the iden-
                  native fishery stocks including game species such as striped                    tification of more cost-effective rations.
                  bass. In most cases, there was no exclusion for farm-reared                       The future appears highly promising for the develop-
                  fish. During the past year there has been substantial                           ment of hybrid striped bass farms throughout many areas
                  interest expressed by the agricultural community, busi-                         of the United States. For the most part, these fish will be
                  nessmen, and landowners, to commercially farm striped                           reared as a high quality seafood product. However, it is
                  bass and its hybrids. Consequently, pressure has been                           also expected that these fish will be used to support recrea-
                  exerted on legislators to legalize the culture and sale of these                tional fee-imposed fishing operations as well. Such opera-
                  fish with the result that many states are currently re-                         tions will provide income to the operator while at the same
                  examining their laws and making provisions for bass                             time affording recreational fishing opportunity to the public
                  aquaculture (e.g., Florida, Virginia, North Carolina,                           sector. In summary, the striped bass x white bass hybrids
                  Georgia, Mississippi). In addition to the laws concerning                       are not only important recreational fish but they also
                  possession and sale of fish, there are many additional                          appear to be excellent candidates for commercial
                  regulations which affect the aquaculturist. These deal with                     development.
                  site permits, discharge constraints, use of medications, and
                  broodstock collection techniques, among others Uenkins
                  1987). Some of these laws and regulations are also being                        Citations
                  re-examined because of their impact on aquaculture                              Bayless, J.D.
                  development.                                                                        1972. Artificial propagation and hybridization of striped bass,








                 60          NOAA Technical Report NMFS 92


                         Morone saxalilis (Walbaum). South Carolina Wildl. Mar. Re-                          bass. Presented at the Symposium on Markets for Seafood and
                         sources Dep., 135 p.                                                                Aquacultural Products, Charleston, S.C., August 1987. (Mimeo
                 Bishop, R.D.                                                                                report.) S.C. Wildl. and Mar. Resources Dep., Charleston,
                      1968. Evaluation of the striped bass (Roccus saxatilis) and white bass                 SC.
                         (Roccus chrysops) hybrids after two years. Proc. Annu. Conf.                Logan, H.J.
                         Southeast. Assoc. Game Fish Comm. 21:245-254.                                    1968. Comparison of growth and survival rates of striped bass x
                      1975. The use of circular tanks for spawning striped bass (Morone                      white bass hybrids under controlled environments. Proc. Annu.
                         saxatilis). Proc. Annu. Conf. Southeast. Assoc. Game Fish                           Conf. Southeast. Assoc. Game Fish Comm. 21:260-263.
                         Comm. 28:35-44.                                                             Parker, N.C., andJ.C. Geiger.
                 Bonn, E.W., W.M. Bailey, J.D. Bayless, K.E. Erickson, and                                1984. Production methods for striped bass. In Third report to the
                   R.E. Stevens.                                                                             fish farmers (H. K. Dupree and J. V. Huner, eds.), p. 106-118.
                      1976. Guidelines for striped bass culture. Striped Bass Commit-                        U.S. Fish Wild]. Serv., Washington, D.C.
                         tee of the Southern Division. Am. Fisheries Soc., Bethesda, MD,             Powell, M.R.
                         103 p.                                                                           1973. Cage and raceway culture of striped bass in brackish water
                 Carlberg, J.M., and J.C. Van Olst.                                                          in Alabama. Proc. Annu. Conf. Southeast. Assoc. Game Fish
                      1987. Processing and marketing. In Hybrid striped bass culture:                        Comm. 26:345-356.
                         status and perspective (Hodson et al., eds.), p. 73-82. Univ.               Smith,  T.I.J.
                         North Carolina Sea Grant Publ. No. 87-03.                                        1987.   Hatchery. In Hybrid striped bass culture: status and
                 Carlberg, J.M., J.C. Van Olst, MJ. Massingill, and T.A. Hovanec.                            perspective (Hodson et al., eds.), p. 17-22. Univ. North Carolina
                      1984. Intensive culture of striped bass: a review of recent tech-                      Sea Grant Publ. No. 87-03.
                         nological developments. In The aquaculture of striped bass - a              Smith,  T.I.J., and W.E. Jenkins.
                         proceedings (J.P. McCranen, ed.), p. 89-127. Univ. Maryland                      1984. Controlled spawning of F, hybrid striped bass (Morone sax-
                         Sea Grant Publ. No. UM-SG-MAP-84-01.                                                alilis x M. chrysops) and rearing of F2 progeny. J. World Mari-
                 Collins, C.M., G.L. Burton, and R.L. Schweinfor-th.                                         culture Soc. 15:147-161.
                      1984r.    High density culture of white bass x striped bass fingerlings             1985a.   Status of aquaculture of striped bass (Morone saxatilis) and
                         in raceways using power plant heated effluents. In The aqua-                        its white bass (M. chrysops) hybrids and research in South Carolina.
                         culture of striped bass - a proceedings (J.P. McCraren, ed.), p.                    In Proceedings of the 2nd int. conf. warm water aquaculture - fin-
                         129-142. Univ. Maryland Sea Grant Publication No. UM-SG-                            fish. Brigham Young Univ. p. 553-582.
                         MAP-84-01, 262 p.                                                                1985b. Aquaculture research with striped bass (Morone saxatilis) and
                 Geiger, J.G.                                                                                its hybrids in South Carolina. Proc. Annu. Conf. Southeast.
                      1983a. Zooplankton production and manipulation in striped bass                         Assoc. Fish Wild]. Agencies 39:219-227.
                         rearing ponds. Aquaculture 35:331-351.                                           1986. Culture and controlled spawning of striped bass, Moronesax-
                      1983b.    A review of pond zooplankton production and fertilization                    atilis, to produce striped bass, and striped bass x white bass, M.
                         for the culture of larval and fingerling striped bass. Aquaculture                  chrysops hybrids. Proc. Annu. Conf. Southeast. Assoc. Fish WiIdl.
                         35:353-369.                                                                         Agencies 40:152-162.
                 Harrell, R.M.                                                                            1987. Controlled spawning of cultured striped bass, Moronesaxatilis.
                      1984. Review of striped bass broodstock acquisition, spawning                          (Abstract.) 117th Annual Meeting Am. Fisheries Soc.
                         methods and fry production. In The Aquaculture of striped bass              Smith, T. I.J., W. E. Jenkins, and J. F. Snevel.
                         - a proceedings (J.P. McCraren, ed.), p. 45-57. Univ. Maryland                   1985. Production characteristics of striped bass, and F, and F2
                         Sea Grant Publ. No. UM-SG-MAP-84-01.                                                striped bass/white bass hybrids reared in intensive tank systems.
                 Jenkins, W.E.                                                                               J. World Mariculture Soc. 16:57-70.
                      1987. Laws and regulations. In Hybrid striped bass culture: status             Smith, T.I.J., W.E. Jenkins, and R. Haggerty.
                         and perspective (Hodson et al., eds.), p. 93-100. Univ. North                    1986. Growth and survival ofjuvenile striped bass (Moronesaxatilis)
                         Carolina Sea Grant Publ. No. 87-03.                                                 x white bass (M. chrysops) hybrids reared at different salinities.
                 joint Subcommittee on Aquaculture.                                                          Proc. Annu. Conf. Southeast. Assoc. Fish Wild[. Agencies 40:
                      1983.     Striped bass species plan. In National aquaculture develop-                  1+3-151.
                         ment plan, Vol. 2, p. 136-11,5. The joint Subcommittee on                   Smith,  T.I.J., W.E. Jenkins, A.D. Stokes, and R.A. Smiley.
                         Aquaculture of the Federal Coordinating Council on Science,                      1987. Pond production trials with striped bass (Morone saxatilis) and
                         Engineering, and Technology, Washington, DC.                                        white bass (M. chrysops) hybrids. (Abstract.) J. World Aquaculture
                 Kerby, J.H., L.C. Woods 111, and M.T. Huish.                                                Soc. 18(l):8A.
                      1983a. Culture of striped bass and its hybrids: a review of methods,           Sport Fishing Institute.
                         advances and problems. In Proceedings of warm water fish                         1984. An uncertain future. Sport Fishing Inst. Bull. 35(l):1-5.
                         culture workshop (R.R. Stickney and F.P. Myers, eds.), p. 23-54.            Stevens, R.E.
                         World Maricult. Soc., Spec. Publ. No. 3.                                         1966. Hormone-induced spawning of striped bass for reservoir
                      1983b. Pond culture of striped bass x white bass hybrids. J.                           stocking.  Prog. Fish-Cult. 28:19-28.
                         World Maricult. Soc. 14:613-623.                                                 1967. A final report on the use of hormones to ovulate striped bass
                 Kerby, J.H., J.M. Hinshaw, and M.T. Huish.                                                  Roccus saxatilis (Walbaum). Proc. Annu. Conf. Southeast. Assoc.
                      1987. Increased growth and production of striped bass x white                          Game Fish Comm. 18:525-538.
                         bass hybrids in carther ponds. J. World Aquacult. Soc.                           1984. Historical overview of striped bass culture and manage-
                         18(l):35-43.                                                                        ment. In The Aquaculture of striped bass: a proceedings (J.P.
                 Lewis, W.M., and R.C. Heidinger.                                                            McCraren, ed.), p. 1-5. Univ. Maryland Sea Grant Publ. No.
                      1981. Tank culture of striped bass: production manual. Illinois                        UM-SG-MAP-84-01.
                         Striped Bass Project IDC F-2, 6-R, Univ. Carbondale, Illinois,              Swartz, D.
                         115 p.                                                                           1984. Marketing striped bass. In The Aquaculture of striped bass:
                 Liao, D.S., T.I.J. Smith, and W.E. Jenkins.                                                 a proceedings (J.P. McCraren, ed.), p. 233-254. Univ.Mary-
                      1987. Preliminary market analysis for cultured hybrid striped                          land Sea Grant Publ. No. UM-SG-MAP-84-01.







                                                                                                                       Smith: Aquaculture of Striped Bass                       61


                  U.S. Department of Commerce.                                                                Fish-Cult. 41:138-140.
                       1978-87. Fisheries of the United States. (Formerly Fishery Statis-             Williams, H.M.
                         ticsoftheU.S.) U.S. Dep. ofCommer., NOAA,NMFS,var.                                197 1. Preliminary studies of certain aspects of the life history of
                         pagination.                                                                          the hybrid (striped bass x white bass) in two South Carolina reser-
                  U.S. Department of Interior.                                                                voirs. Proc. Annu. Conf. Southeast. Assoc. Game Fish Comm.
                       1965-77.    Fishery Statistics of the U.S.   U.S. Dep. Interior, Fish                  24:424-431.
                         Wild]. Serv., var pagination.                                                Williams, J.E., P.A. Sandifer, and J.M. Lindberg.
                  Valenti, R J., J. Aldred, and J. Liebell.                                                198 1. Net-pen culture of striped x white bass hybrids in estuarine
                       1976. Experimental marine cage culture of striped bass in northern                     waters of South Carolina: a pilot study. J. World Maricult. Soc.
                         waters. Proc. World Maricult. Soc. 7:99-108.                                         12(2):98-110.
                  Ware, Fj.                                                                           Woods, L.C. III, J.H. Kerby, and M.T. Huish.
                       1975. Progress with Morone hybrids in fresh water. Proc. Annu.                      1983. Estuarine cage culture of hybrid striped bass. J. World
                         Conf. Southeast. Assoc. Game Fish Comm. 28:48-54.                                    Maricult. Soc. 14:595-612.
                  Wawronowicz, L.J., and W.M. Lewis                                                        1985. Culture of hybrid striped bass to marketable size in circular
                       1979.   Evaluation of the striped bass as a pond food fish.       Prog.                tanks. Prog.   Fish-Cult. 47(3):147-153.







                      Computerized Image Analysis for Selective Breeding of Shrimp:
                                                                    A Progress Report


                                     L. JAMES LESTER, KIM S. LAWSON, and MARK J. PIOTROWSKI

                                                                       Aquaculture Genetics Laboratory
                                                                     University of Houston - Clear Lake
                                                                            2700 Bay Area Blvd.
                                                                            Houston, TX 77058


                                                                  TENG-CHEONG B. WONG

                                                           Research Institutefor Computing and Information Systems
                                                                     University of Houston - Clear Lake
                                                                            2700 Bay Area Blvd.
                                                                            Houston, TX 77058





                                                                              ABSTRACT


                                       Genetic improvement of aquaculture organisms will be an important component of future
                                     progress by the aquaculture industry. The benefits of selective breeding could be obtained more
                                     rapidly if high selection intensity could be applied. This will be practical with an approach based
                                     on computerized image analysis which can provide an accurate estimate of organism size.
                                     Preliminary studies were conducted with a computer assisted morphometric analysis which ob-
                                     tains more information on size and shape from an image of a shrimp body than more conven-
                                     tional direct measurement. Using computer digitization, a truss network of dimensional variables
                                     was collected from photographs of three species of adult penacid shrimp and used in canonical
                                     discriminant analysis.This approach was found to provide more information for discrimination
                                     than a typically labor intensive conventional morphometric method. The results demonstrate the
                                     usefulness of two-dimensional images for selection or classification. A truss network data set was
                                     also collected from full-sib families of juvenile shrimp using the new method. This study shows
                                     the application of the technique to images from living animals in a format resembling selection.
                                     Phenotypic variation in size is analyzed by analysis of variance, and heritabilities of size based
                                     on principal component scores and single variables are calculated. The multivariate estimator
                                     of size had more of the variance in size attributed to family differences than most single vari-
                                     ables (h' = 0.60 for PC scores vs. a mean of 0.49 for single variables). Ways in which com-
                                     puterized image analysis might be employed in selective breeding of shrimp are discussed.




                Introduction                                                             intensive characteristics of such a program will benefit
                                                                                         greatly from the use of computer image analysis, a valuable
                There has been much discussion about the possible use of                 alternative to human observers. Third, obtaining accuracy
                high selection intensities for aquaculture animals. High                 in the selection process will require the use of multivariate
                selection intensities are possible because the very high                 procedures in the assessment of organism size.
                fecundities of aquacultured organisms (e.g., penaeid                        If we compare the fecundity of shrimp with even the most
                shrimp) permit a very small fraction of the individuals to               fertile domestic animal, they are different by orders of
                be used for replacement of broodstock. This paper will sup-              magnitude. In commercial shrimp culture facilities, female
                port several premises related to the use of high selection               Penaeus vannamei have been observed to average 60000
                intensities in a broodstock selection program. First, the im-            nauplii per spawn and 8-10 spawns during their produc-
                plementation of such selection intensities will be possible              tive life in a maturation facility (B. Ribelin, Laguna Madre
                and should not result in genetic drift. Second, the labor                Shrimp Farm, P.O. Box 4043, Los Fresnos, TX 78566,

                                                                                                                                                          63








               64       NOAA Technical Report NMFS 92


               pers. commun., June 1986). Let us use the figure of 600 000          tical (i.e., high selection intensities would require major
               larvae from an average female and follow the offspring               investments of personnel and time).
               through a production cycle, assuming 50% survival in each               In a previous publication (Lester 1983), it was recom-
               stage. This percentage is low, but not outside the range             mended that a single abdominal measurement, sixth seg-
               of observed values. The numbers would change as follows:             ment depth (SSD), be used as a criterion in selection for
               300 000 postlarvae, 150 000 juveniles, and 75 000 harvested          size of penaeid shrimp (Fig. 1). Several preliminary selec-
               subadults. Assuming a survival of 50% from selection to              tions have been performed using this character. A single
               reproduction, one would only need to select four individual          human observer is capable of applying this criterion to
               subadults to replace the original two breeders.                      300-500 shrimp per hour, but not for many hours con-
                 Selection intensity (0 is related to the response of a             tinuously (pers. observ.). Nevertheless, at a selection in-
               population to selection (R) by the simple equation R =               tensity of 3.0, one would be required to measure approx-
               I.op h2 in which h2 is the heritability of the trait under           imately 300 shrimp, about 40-60 minutes of work, to
               selection and op is the phenotypic variance of the trait.            select a single potential broodstock. In order to obtain a
               The relationship between 1'and the proportion of animals             broodstock of 3000, which is not large by commercial stan-
               selected (p) for truncation selection of a normally distrib-         dards, the observers would have to work for over 2000
               uted trait is i = z1p, where z is the height of the normal           hours. A microcomputer imaging system could perform
               curve at the truncation point (Falconer 1981). Thus the              the measurements and make a decision on whether to cull
               maximum selection intensity for the above example (based             or select in several seconds. Computer analysis would be
               on a proportion of 4 selected from 75000) would be ap-               limited by how fast the shrimp could be moved through
               proximately 4.3, if all the survivors could be measured.             the imaging system. A single unit could measure con-
                 Lasley (1978) gives the percentage of the progeny re-              tinuously and accurately for any amount of time necessary.
               quired for broodstock replacement in domestic animals as                No matter what criterion is employed for the selection
               4-5 % of males and 40-50 % of females in beef cattle; 1-2 %          process, there will be an associated measurement error. At
               of males and 10-15% of females in swine and chickens.                a high selection intensity, the variance due to measurement
               Such broodstock selection programs for domestic animals              error could exceed the true phenotypic variance of the
               obtain selection intensities of 2.0 to 2.7 for males and 0.6         potential broodstock in the distribution. This would repre-
               to 0.8 for females (Falconer 1981). With the very small pro-         sent a serious problem for the classification of animals into
               portion of the population that is required for replacement           potential broodstock and culls. This factor in misclassifi-
               broodstock in aquaculture species, it is possible to achieve         cation can be reduced by moving from univariate to
               selection intensities of 3.0-4.0. Current selection programs         multivariate classification. Thus we are recommending that
               at commercial shrimp farms select the top 1/2 to 1/3 of the          high intensity selection not be based on a single measure-
               size distribution. If we assume that the trait under selec-          ment such as SSD, but rather on a set of measurements.
               tion is heritable, the predicted response under high selec-             The characteristic of commercial importance, rate of
               tion intensity is 3 to 4 times higher than under the current         gain in size, is a latent variable which can not be measured
               system. This could be accomplished using within-family               directly in these selection programs. It can be estimated
               selection to remove the danger of bottlenecks caused by              by analysis of multiple variables which are correlated to
               unrecognized restriction of the gene pool and inbreeding.            it. (See Bookstein et al. 1985, for a thorough discussion
               The limitations on selection intensity are primarily logis-          of multivariate estimation of the latent variable size.) In




                                            AAC
                                                               PCL                                                  Figure I
                             FLF        SAD        FSL         CW                             Diagram of shrimp with measurements used in the first
                          L                                                                   study. Description of measurements: PCL = posterior
                                                                        >                     margin of the orbit to posterior edge of carapace;
                                                                                              CW = carapace width at the level of the last dorsal
                                                                                              rostral tooth; FSL = first abdominal segment length;
                                                                                              AAC = circumference at the intersection of the second
                                                                                              and third abdominal segments; SAD = depth at the in-
                                                                                              tersection of the second and third abdominal segments;
                                       PAC                                                    FLF = fifth segment length with the abdomen maximal-
                              S                                                               ly flexed; PAC = circumference at the intersection of
                                                                                              the fifth and sixth segments; SSL = sixth segment
                                                                                              length; SSD = depth at the midpoint of the sixth
                                                                                              segment.







                                                                                         Lester et al.: Image Analysis for Breeding Shrimp                   65



                                          DM4 DM3 DM2 DMI
                                                                                                                             Figure 2
                                                                                                      Diagram of shrimp with landmarks used in the adult
                         DM5                                                                          truss network study. Descriptions of points: DN11 =
                                                                                                      Posterior rostral tooth; DM2 = dorsal, posterior point
                    DM6                                                                               on carapace; DM3 = dorsal, posterior point on segment
                                                                                                      one; DM4 = dorsal, posterior point on segment three;
                                                                                                      DN15 = dorsal, anterior point on segment six; DN16
                                                                                                      = dorsal, posterior point on segment six; VM1 =
                                                                                                      center of antennal basal segment at insertion; VM2
                                                                                       VM1            - center of basal segment of fifth periopod at insertion;
                                                                                                      VM3 = center of basal segment of first pleopod at in-
                           VM6                                         VM2                            sertion; VM4 = center of basal segment of third pleopod
                                             V 4                                                      at insertion; VM5 = center of basal segment of fifth
                                   VM5                  VM3                                           pleopod at insertion; VM6 = ventral, posterior point
                                                                                                      on segment six.



                our approach to estimation of growth rate, the latent                      variables which would permit the computer to decide
                variable size, is represented by the first principal compo-                whether the animal should be selected or culled. At the
                nent (PC I) score obtained from a set of size-correlated                   present time, we are still at an early stage in the develop-
                dimensions taken from each shrimp. As variables are                        ment, using human observers to digitize still images.
                added, the variance-covariance matrix gets larger, but the                    This report deals with initial analyses of two data sets.
                variance of the multivariate mean (i.e., mean size)                        These analyses are based on still images, not video, and
                decreases according to the factor 1 - R2 (R2 = the surn                    only one uses living animals for the images. The first data
                of the squared partial correlations between the variables).                set was obtained from adults carefully prepared for photo-
                In other words, the multivariate data set becomes a better                 graphing to test the truss network approach on discrimina-
                predictor of the underlying variable size when multiple                    tion of Penaeus species. The second data set was collected
                variables correlated with size are used in the estimation                  from an experiment on growth differences among families
                process.                                                                   of juvenile shrimp. It will be used to examine the way in
                   There is a school of morphometric studies centered                      which the partitioning of the variance in size is affected
                around the work of F.L. Bookstein (Strauss and Bookstein                   by changing from a single to a multivariate approach.
                1982; Bookstein et al. 1985) which has developed an ap-
                proach to the multivariate description of size and shape
                for fish. The approach is based on obtaining a set of                      Materials and Methods
                variables that contains much information about specific                    Testing the Truss Network Approach
                regions of the body and is homologous across conspecifics
                of different sizes and across closely related species. This                Samples of adults were obtained from the following sources
                is accomplished by selecting landmark points on the mor-                   for both data sets: Penaeus seliferus from the wild near Gal-
                phology and arranging them into a set of boxes connected                   veston, TX; Penaeus vannamei collected from harvests of
                by all possible nearest neighbor connections into a truss                  ponds at the King Ranch near Corpus Christi, TX, at the
                network as shown in Figure 2 (Strauss and Bookstein 1982;                  Laguna Madre Shrimp Farm near Harlingen, TX, and
                Bookstein et al. 1985). The resultant network consists of                  at Agromarina de Panama (only in the truss network data
                a set of dimensional variables that covary with the latent                 set) near Aguadulce, Panama; Penaeus stylirostris collected
                variable size and contain information about shape-related                  from harvests of ponds at the King Ranch near Corpus
                deformational changes among a set of individuals. At the                   Christi, TX, and tanks at Marine Culture Enterprises near
                present time, we are interested in using this approach to                  Laie, HI. The standard morphometric study used 104
                precisely estimate genetic differences in size. Later we hope              P. setiferus, 92 P. stylirostris, and 134 P. vannamel'. The truss
                to apply it to the inheritance of shape.                                   network study used 49 P. setiferus, 42 P. stylirostris and 83
                   One objective of the research program that we have                      P. vannamel'. All specimens were preserved by freezing and
                started was the development of a system that would                         thawed for photography.
                automate broodstock selection. This program would use                         The standard morphometric data set contained nine
                a video camera to obtain an image that would be converted                  dimensional variables and two weights, head weight and
                into digital format. This would be measured and analyzed                   tail weight. Dimensional values were obtained with dial
                by a microcomputer to obtain a summary of multiple                         calipers except in the case of circumferences which were








              66        NOAA Technical Report NMFS 92


              obtained by passing a string around the shrimp and                      The postlarvae were weaned from Artemia to a prepared
              measuring the length of string with a ruler. The dimen-               diet from the Texas A&M Shrimp Mariculture Project,
              sional variables are shown in Figure 1. Data sets of this             and reared under the same temperature and salinity con-
              type have been obtained from six species of penaeid shrimp.           ditions for 40 days on a 14L: I OD light cycle. The juveniles
              Only the species P. setiferus, P. stylirostris, and P. vannamei       in each cage were fed once per day at ad libitum levels:
              will be used here for purposes of comparison to truss net-            50 mg/cage in weeks one and two, 100 mg for five days,
              work data sets.                                                       200 mg for five days, 600 mg for five days, and, following
                 The truss network variables are shown in Figure 2. This            sampling for survival and weight at 28 days, 400 mg to
              data set contains 26 dimensional variables, no weights, and           8 cages with survival < 50 To and 600 mg to 14 cages with
              was obtained from pictures of the adult shrimp. The adults            survival > 50 To for the remaining twelve days. Two cages
              were positioned on a light table in the laboratory or on light        had no survivors at 28 days.
              plastic in the field for pictures taken at Agromarina de                After 40 days of growth, the shrimp were harvested and
              Panama. They were photographed with a scale in the frame              survival was determined. Six randomly sampled individ-
              using Kodak Tri-X black and white film. The negatives                 uals from each cage were placed into a viewing cell filled
              were mounted as slides and projected onto a digitizing                with water and photographed using color slide film. Black
              tablet attached to a Tektronix graphics terminal. The                 and white film can be used and the negative mounted as
              operator used the cross-hairs of a mouse to locate 12 land-           a slide. Lighting is critical to the location of landmarks on
              mark points shown in Figure 2. Points were digitized in               small shrimp. Dual fiber optic illuminators positioned to
              order from DM6 to DMI to VMl to VM6. The coor-                        minimize reflection from the exoskeleton were found to give
              dinates were stored in a data file, then converted to mea-            better results than flash lighting. Slides were projected onto
              surements by a Pascal program written by one of the                   a digitizing tablet. The landmarks used were slightly dif-
              authors (Wong, T.-C.).                                                ferent from those employed on the adults. The landmarks
                 All measurements were log transformed before use to                were changed from posterior rostral tooth (DMI in Fig.
              remove the exponential effects of growth. Both the stan-              2) to ventral intersection of rostrum and carapace; from
              dard and the truss network data files were analyzed by                antenna] insertion (VM1 in Fig. 2) to anterior ventral point
              multivariate statistical programs, PRINCOMP for prin-                 on edge of carapace; pleopods marked at anterior edge
              cipal component analysis and CANDISC for canonical                    rather than center point (points VM2 through VM5). The
              discriminant function analysis (SAS Institute 1985). The              coordinates were converted to measurement variables by
              principal component analysis was performed to obtain a                the same software and the log-transformed measurement
              summary estimate of the variation in the latent variable              variables were used in the SAS PRINCOMP program.
              size. Discriminant function analysis was performed with               Principal component scores were stored and ANOVA was
              the two data sets to illustrate the increase in information           performed using the SAS GLM program. Then the vari-
              relevant to species differences contained in the truss net-           ance components were estimated with VARCOMP.
              work data set.                                                        Heritability was estimated from the variance components
                                                                                    according to Becker (1984).
              Multivariate Estimation of Heritability
              Nauplii from ten separate spawns were shipped to the                  Results
              University of Houston - Clear Lake Aquaculture Genetics               Testing the Truss Network Approach
              Laboratory from the Laguna Madre Shrimp Farm. Lar-
              vae were acclimated to Marine Mix sea water at labora-                The correlation among the variables in the standard data
              tory temperature and stocked at 100 larvae/cone in three              set is high. Measurement PAC (Fig. 1) has the lowest
              one-liter Imhoff cones per family. They were treated under            average correlation of 0.68 and SSD the highest, 0.88. This
              standardized conditions: 28'C, 30 ppt, constant light, daily          indicates that these variables are related in their estima-
              changes of water containing Chaetocerosgracilis at 100000             tion of size. The first principal component (PC 1) obtained
              cells/mL and Tetraselmis chuil' at 30000 cells/mL. After the          for the standard data set represents the latent variable
              protozoea-3 stage, Artenzia salina nauplii were fed at 3/mL           "size" as indicated by the equal loadings (0.28 to 0.35)
              (Lester 1988). At the postlarva-2 stage, populations from             of all variables (Bookstein et al. 1985). It explains 8417o of
              those families with more than 120 survivors were trans-               the variance in the standard data set. The second principal
              ferred to 10 liter round cages. Three cages were stocked              component contains very little information, 0.05 of the
              for each family with 40 postlarvae per cage. These 10 liter           variance which primarily represents the posterior ab-
              round cages had screened openings near their bottom and               dominal circumference. The results from discriminant
              continuous water flow from the top. They were distributed             analysis can most easily be seen in the plots of canonical
              among three large tanks (244 cm x 61 cm x 61 cm) which                variate I (CAN1) against canonical variate 2 (CAN2).
              provided common water sources.                                        Figure 3 shows the plot resulting from analysis of the stan-







                                                                                             Lester et al.: Image Analysis for Breeding Shrimp                       67



                                                                                                                              Table I
                              6
                                                                                                   Length of the sixth abdominal segment as an indicator of
                              5                                                                          size differences among species and data sets.

                        v-    4
                        W                                                                                                                  Data Set
                              2                                                                      Species                Standard (cm)               Truss (cm)

                                                                                                   P. setiferus                  1.64                      1.38
                        >     0                                                                    P. stylirostris               1.48                      1.73
                        _j                                                                         P. vannamei                   1.87                      1.56

                        2-2
                        0.3
                        Z
                              -4                        S
                                                                                                   At this point, there appears to be little on which to choose
                                                                                                the truss network over the standard morphometric vari-
                              -6 1                                                              ables. However, results of discriminant analysis on the truss
                              -6 .5 -4 -3 -2 -1 0 1            2 3 4 5 6                        network data set are quite different as shown in Figure 4.
                                     CANONICAL VARIATE 2                                        This analysis results in larger Mahalanobis distances
                                                                                                between the species' centroids, P. setiferus to P. vannarnei
                                               Figure 3                                         = 5.94, P. seliferus to P. slyhrostris = 6.35 and P. vannamel'
                    Plot of canonical variates I and 2 from discriminant analysis               to P. styllrostris = 5.73. The discrimination of species along
                    of the standard data set; The species are represented by                    CANI is primarily due to values of the following variables
                    ellipses based on the 9517o confidence intervals of the                     (between group loadings given in parentheses): DM4-DM5
                    species' centroids. The ellipse for Penaeus setiferus is labeled            (O.M), DM4-VM5 (0.92), DM2-DM3 (0.95), DM5-VM5
                    S, Penaeus stylirostris Y, and Penaeus vannamei V.                          (0.93), DM3-VM2 (0.97). There are 16 variables with
                                                                                                between-group loadings above 0.75 on CAN2. Five are
                                                                                                measurements from the carapace. Four'are from the sixth
                dard data set. The three species show some distinctness,                        abdominal segment; the remaining seven variables are
                but are not well separated. The Mahalanobis distances                           measurements from the first, second, and third abdomenal
                between the centroids of the three species are P. setiferus                     segments. Thus CAN1 represents the first, fourth and fifth
                to P. vannamet = 3.69, P. setiferus to P. stylirostris = 2.51,                  abdomenal segments and CAN2 the head and the second,
                and P. vannarnei to P. styllrostris = 2.32. CANI in Figure                      third, and sixth abdomenal segments. The canonical cor-
                3 shows relatively high loadings of variables AAC (0.86),                       relations of these discriminant variables to the dummy
                FLF (0. 75), SSD (0. 80), CW (0. 73), and PAC (0. 79) which                     classification variables (species identifiers) are higher than
                could be interpreted as a body thickness variable. CAN2                         those obtained with the previous data set, for CAN 1, 0. 93
                shows high loadings of PCL (0. 88), FSL (0. 80), SSL (0. 78),                   and for CAN2, 0.91. This indicates that the discriminant
                and SAD (0.85). It represents the variation in length of                        variables derived from the truss network approach are
                body segments among species. The goodness of fit of each                        better for classifying individuals to species than those
                discriminant variable to the classification variables (iden-                    derived from the labor. intensive approach.
                tifiers for species) can be evaluated by the canonical cor-                        It is unfortunate that these data sets are not the result
                relation which in the case of CANI is 0.84 and in the case                      of measurements on the same individuals. However, the
                of CAN2 = 0.56.                                                                 sizes of animals in both data sets are comparable as in-
                    For the truss network data set, the correlation matrix                      dicated in Table I in which a variable contained in both
                indicated that the correlations were generally lower, and                       data sets is compared (SSL in the standard data set is equal
                problems existed with one of the variables, VM2-VM3.                            to DM5-DM6 in the truss network data set). There are
                This variable showed an average correlation with all other                      also significant differences among the data sets. P. setiferus
                variables of 0.09. The remaining variables had average cor-                     and P. vannamel samples are larger in the standard data
                relations ranging from 0.37 (DM4-DM5) to 0.69 (DM3-                             set, but P. slyhrostris are larger in the truss network data
                VM4). This data set gave results from principal compo-                          set. Size differences among the species could impact the
                nent analysis that were different from the standard data                        discriminant analysis. In this case, the size differences
                set. PC 1 explained 60 To of the variation, PC 2 explains 9                     among species are greater in the standard data set than
                All loadings on PC 1 were between 0. 15 and 0. 23, except                       in the truss network data set and should contribute to
                VM2-VM3 (0.02). Again this indicates that PC I is a size                        greater separation in the discriminant analysis of the stan-
                component. The additional variables do not provide much                         dard data set. The discrimination is better however with
                allometric variation to be explained by PC2.                                    the truss network data set, which only serves to reinforce








                  68          NOAA Technical Report NMFS 92



                                                                                             Table 2
                                            Comparison of variance components and heritabilities obtained from nested ANOVA using
                                            simple variables and the first principal component scores. The eight simple variables were
                                            selected to represent lengths, depths, and diagonals, as well as the range of heritability
                                            estimates. Degrees      of freedom in all cases: Family           7, Cage = 16, and Error = 106.

                                                                     Variance                 Variance              Variance
                                              Variable                (family)                 (cage)                 (error)               Heritability

                                            PCI                     4.42  x 10'                  0                 1.04   x10'                 0.60
                                            DM5-VM5                 1. 40 x 10-'                 0                 4.44 x  10-1                0.48
                                            DM3-VM3                 1.58 x  10-'           0.05  x  10-'           4.36 x  10'                 0.53
                                            DM2-VM2                 1.92 x  10'            0.16 x   10-'           4.66 x  10'                 0.57
                                            DM5-DM6                 1.86 x  10-'                 0                 4.85 x  10-1                0.55
                                            DM5-DM4                 2.50 x  10-'           0.36 x   10-'           5.74 x  10-'                0.58
                                            DM3-DM2                 1.72 x  10-'           0,35 x   10-'           5.56 x  10-1                0.45
                                            DM4-VM3                 2.08 x  10-3                 0                 4.56 x  10'                 0.63
                                            DM3-VM4                 1.98 X  10-3           0.15  x  10-'           4.79 x  10'                 0.57
                                            Mean of 26 variables                                                                               0.49
                                            SE                                                                                                 0.14





                  the conclusion that the truss network approach provides                             of 0. Clearly there are problems with this measurement.
                  additional information about species differences.                                   DM2-VMl and VM2-VM3 have h2 values of only 0.28.
                                                                                                      An average of all the h2 estimates is somewhat lower than
                  Multivariate Estimation of Heritability                                             the one resulting from use of PCI scores.
                  Examination of the correlation matrix output                    from the
                  principal component analysis of the data from families                              Discussion
                  grown under experimental conditions showed that two of
                  the variables, VM2-VM3 and DMI-VMI were weakly                                      Measurement problems are inherent in the development
                  correlated with the remainder of the data set. The correla-                         of a truss network design. In this study, they were reduced
                  tions of VM2-VM3 ranged from 0.25 (with DMI-VMI)                                    by making changes in the landmarks used and by improv-
                  to 0.66 (with DM2-VM2). For DMINM1, the correla-                                    ing photographic technique. Obtaining a simple set of land-
                  tions ranged from - 0.08 (with VM1-VM2) to 0.43 (with                               marks that remain homologous across development from
                  DM5-VM4). These ranges are distinctly lower than the                                postlarva to adult proved more difficult than expected. One
                  other variables and are indicative of measurement prob-                             source of error in the adult data set was the choice of points
                  lems. The juvenile shrimp correlations are higher than                              on movable bodyparts, (e.g., appendage insertions on the
                  those in the truss network data set from the adults. PC 1                           adults). This problem was corrected by adopting new land-
                  explains 82 17o of the variance in the data set and has nearly                      marks on the juveniles that represented stable intersections
                  equal loadings of all of the variables. The coefficients were                       of body parts. In the case of the posterior rostral tooth,
                  between 0. 16 and 0. 23 which is indicative of their similarity                     juvenile shrimp often expressed different degrees of rostral
                  as estimators of size.                                                              tooth development from conspecifics, both adults and other
                     Results from multivariate and univariate scoring were                            juveniles. Thus slightly different landmarks had to be used
                  compared based on the heritabilities and variance com-                              to collect dimensional data from juvenile shrimp of 0. 2 to
                  ponents obtained from the same ANOVA design. Table                                  1.0 gm than were used for the adults. When working with
                  2 shows these results from comparison of eight univariate                           the juvenile shrimp, it was discovered that some points are
                  scores and the first principal component score. The results                         more difficult to see on a live shrimp than on a dead one,
                  show that a univariate approach can yield inaccurate com-                           (e.g., VM2). Better lighting and different photographic
                  parisons among genetic groups. Some variables have                                  techniques were used to minimize this problem. The ex-
                  heritabilities that slightly exceed the estimation based on                         amination of different landmarks is continuing, especially
                  PC 1. However, most variables yield lower estimates of the                          for the juveniles.
                  family component of the variance in size, some drastically                             Several variables used in the truss network analysis ex-
                  lower. Using PC I scores in the ANOVA gives an F value                              hibited their unsuitability as size estimators by a reduced
                  for the model of 4.74 and a heritability (h2) of 0.60. The                          level of correlation with the other variables. The reason
                  most extreme case is VM1-VM2 which has an h2 estimate                               for low correlations from two of the variables, DM I NM I







                                                                                         Lester et al.: Image Analysis for Breeding Shrimp                  69


                and VM2-VM3, was related to difficulty in precisely
                locating the landmark points, DMI, VMI, and VM2, on                                 8
                the pictures of juveniles. Some covariation is expected                             7
                because all of the measurements are dimensions from the                             6
                same image and all will increase with size of the shrimp.                           5
                High correlation can be expected if all variables are reliable                T_    4                S
                estimators of size and can be accurately measured. Vari-                      W     3
                ables in the truss network data sets obtained from juveniles
                                                                                              FC    2
                were all reasonable estimators of size as indicated by the                    <     1
                high correlations and equality of loadings on PCI.                            >                                           Y
                                                                                              _J    0
                   The problems associated with developing a truss network                    4
                approach are outweighed by the advantage obtained frorn                       0     -1
                                                                                              Z
                it. In this study, the three species were used to represent                   0     .2                  V
                                                                                              Z
                any genetically distinct groups about which information                             -3
                is needed on distinguishing characteristics. It was shown                           -4
                that discrimination among Penaeus setiferus, Penaeus vannameil                      -5
                and Penaeus st lirostris is possible using the standard data                        -61
                set as seen in Figure 3. However, discrimination among                              -6 -5 -4 -3 -2 .1 0 1 2 3 4 5 6 7 8
                these groups improved with the truss approach, as one can                                    CANONICAL VARIATE 2
                see by comparing Figures 3 and 4. It appears that the truss
                network approach offers more information for the dis-
                crimination of these genetic groups and the classification                                             Figure 4
                of individuals than the collection of standard measure-                   Plot of canonical variates  I and 2 from discriminant analysis of
                ments. Comparison of a data set of nine variables and one                 the truss network data set. The species are represented by ellipses
                of 26 variables for discriminant analysis is biased against               based on the 95 % confidence intervals of the species' centroids.
                the data set with fewer variables. Although Figure 4 shows                The ellipse for Penaeus setiferus is labeled S, Penaeus slyhrostris Y,
                that classification of individuals by species would be easier             and Penaeus vannamei V.
                from a truss network approach, improved discrimination
                among species could perhaps have been accomplished by
                the addition of random variables to the standard data set.                0.45 (DM2-DM3) to 0.63 (DM4-VM3) is quite large for
                Thus these results should not be taken as sufficient justifica-           heritabilities of measurements taken from the same set of
                tion for the truss network approach. A thorough justifica-                individuals from the same set of families by the same
                tion can be found in Bookstein et al. (1985) and is based                 observers. This variation is indicative of differences in
                on information content and distribution within the data set -             measurement error and information content among the
                   The use of principal component scores derived from the                 variables in the truss network. Principal component anal-
                truss network data set has associated advantages and disad-               ysis is one appropriate way to combine the information in
                vantages. The number of variables obtained is much larger                 many size-related variables and minimize the effect of
                than would normally be collected for a genetic analysis of                measurement error. We believe that the use of multivariate
                size or any other characteristic. If these variables were to              classification analysis will provide a more reliable dis-
                be measured by hand with calipers, the number of variables                crimination based on size. We will continue to pursue this
                would prove prohibitive. The major advantage of using                     approach in our experiments because it provides more ac-
                multivariate classification scores for the selection process              curate estimation of breeding value for size.
                will be realized in the improved accuracy of estimating size                 It is presently impossible to employ the image analysis
                and reduced probability of misclassification. While this ap-              approach for selection of untagged shrimp because the
                proach improves the analysis of size and shape of penaeid                 photography process and the computer analysis process are
                shrimp for experimental purposes, programming a com-                      separated in time and space. Using our current digitizing
                puter to locate homologous landmark points with accuracy                  process, it still takes the technician about two minutes to
                is a major challenge. Some other approach to image anal-                  enter the landmark points from a single image. In addi-
                ysis may be better for commercial application in selective                tion, the time involved in taking good still photographs of
                                                                                                            (D
                                                                                                              @5D





































                breeding.                                                                 living shrimp is considerable. These problems will be solved
                   There is considerable variation among the estimates of                 by the development of an integrated imaging and analysis
                heritability obtained from univariate estimators of size. In              system. The video image can be converted to a still image
                some cases, it is not clear why more of the variance is par-              by a frame grabber which operates in 1/30th of a second.
                titioned into the family component, resulting in a higher                 The ability to locate and record landmark points or to
                heritability. It is clear that the range seen in Table 2 of               employ other image analysis techniques (e.g., definition








                70          NOAA Technical Report NMFS 92


                of the boundary of the shrimp and the area within that                           Benita Waas. Dr. Cecil Hallurn and Dr. Sharon Perkins
                boundary) can be programmed into a fast microcomputer                            critically reviewed the manuscript.
                which could obtain the required measurements. The results
                of a preliminary classification analysis can be stored in the
                computer and used to calculate the selection index. With                         Citations
                a sufficiently powerful computer, the calculation of the in-
                dex and comparison to a truncation value would take a                            Becker, W.A.
                fraction of a second. Thus problems associated with the                               1984. Manual of quantitative genetics, 4th ed. Academic Enter-
                combination of many variables and too complicated an                                     prises, Pullman, WA, 188 p.
                                                                                                 Bookstein, F. L., B. Chernoff, R. L. Elder, J. M. Humphries Jr.,
                analysis could be solved by integration of fairly simple hard-                      G.R. Smith, and R.E. Strauss.
                ware and software.                                                                    1985. Morphometrics in evolutionary biology: The geometry of size
                                                                                                         and shape change, with examples from fish. Acad. Nat. Sciences
                                                                                                         Philadelphia, Spec. Publ. 15, 277 p.
                Acknowledgments                                                                  Falconer, D.S.
                                                                                                      1981. Introduction to quantitative genetics, 2nd ed.       Longman,
                                                                                                         London, 340 p.
                This research was sponsored in part by a grant to L.J.                           Lasley, J.F.
                Lester from the Texas A&M University Sea Grant Col-                                   1978. Genetics of livestock improvement, 3rd ed. Prentice-Hall,
                lege Program, supported by the National Oceanic and                                      Englewood Cliffs, NJ. 492 p.
                Atmospheric Administration, Office of Sea Grant, Depart-                         Lester, L.J.
                ment of Commerce under grant number NA83AA-D-                                         1983. Developing a selective breeding program for penaeid shrimp
                                                                                                         mariculture. Aquaculture 33:41-50.
                00061. Support was also provided by the University of                                 1988. Differences in larval growth among families of Penaeus slyli-
                Houston-Clear Lake. The assistance of the personnel of                                   rostris and Penaeus vannamei. Aquacult. Fish. Manage. 19:243-25 1.
                Laguna Madre Shrimp Farm, Marine Culture Enterprises,                            SAS Institute, Inc.
                Agromarina de Panama, Granada Corp., and the Texas                                    1985. SAS User's Guide: Statistics, Version 5 ed. SAS Institute,
                A&M Shrimp Mariculture Project in obtaining samples                                      Inc., Cary, NC., 956 p.
                                                                                                 Strauss, R.E., and F.L. Bookstein.
                is gratefully acknowledged. Expert technical assistance was                           1982. The truss: body form reconstructions in morphometrics.
                provided by Margaret Dennison, Mary Byam-Smith, and                                      Syst. Zool. 31:113-135.








                                                           Breeding Test on Abalone


                                                                    HARUHIRO MOMMA

                                                             Hokkaido Hakodate Fisheries Experimental Station
                                                                           Yunokawa Hakodate
                                                                          Hokkaido 042, Japan



                Introduction                                                            at maturity. Haliotis gigantea, for instance, is much larger
                                                                                        than H. discus (Inoue et al. 1985) because its growth pat-
                Although production of artificial seed for abalone farming              tern (Takayama 1940) and feeding behavior is different
                in Japan has increased annually over the last 20 years, the             (Momma 1980a). Thus, an increase in H. gigantea growth
                total abalone harvest has continued to decline (Fig. 1)                 rate would need to be proportionally greater in order to
                (Department of Statistics Information (DSI) 1967-86;                    have the same beneficial effect that a smaller increase would
                Japan Sea-Farming Assoc. 1967-1985). In 1985 about 30                   have on H. discus.
                million seeds were produced. The proportion of recaptured                 The faster growing seedlings of a H. d. hannai culture
                artificial seedlings to the total abalones harvested in plant-          were recovered more frequently than the slow ones (Table
                ing areas has usually been reported to be about 30-407c.                3). This higher rate of recovery illustrates that fast-growing
                However, some of these reports have shown values ex-                    seed have higher survival rates than their slower growing
                ceeding 9017o (Table 1). In these cases it is important to              counterparts even though they were the same size at the
                study the genetics of the seed abalone used. Table 2 shows              time of release. It was previously reported by the author
                some examples of recapture ratios of abalone seedlings.                 that juvenile abalone with initially rapid growth rates (until
                Overall, the results suggest that seedling survival is usually          a shell length of 16-17 mm is reached) maintain that trait
                low and a large percentage of the seed suffers mortality.
                   Another reason to study their genetics is the fact that
                differences in recapture ratios occur between species when
                they are planted on the same fishing ground (Inoue et al.
                1985). Are there differences in the quality of the recaptured
                abalones compared to the seedlings which suffered mor-
                tality? In order to increase the recapture ratio, it is impor-
                tant to produce healthy seed abalone, improve seeding
                methods, and control the factors which affect survival on                                                                        25
                the planting ground.

                                                                                               6

                                                                                                                                                 20Z
                                                                                                                                                   C
                Recapture Ratios and Seedling Quality                                                                                              a
                                                                                           X                                                       Cr
                                                                                           W   5.5
                The survival rate within abalone species increases with seed               C
                                                                                           0                                                       0
                size at the time of release. The extent of this increase,                                                                        15
                however, varies between species in relation to their size                                                                          CD
                                                                                                                                                   CL
                                                                                               5


                                                                                                                                                .10
                                               Figure 1                                    12  4.5
                   Total catch (0) and number of artificially produced abalone seed-
                   lings     in Japan. Total catch: after fisheries statistics of Japan                                                          5
                   1967-86. Department of Statistics Information, Ministry of
                   Agriculture, Forestry and Fisheries. Government of Japan.                   4
                   Number of seedlings: after Materials on production, supply and
                                                                                                                                                 0
                   release of fingerlings for enhancement of fisheries resources in              1967   70         '75         so          45
                   Japan, 1967-1985, Japan SeamFarming Association.

                                                                                                                                                       71








                 72          NOAA Technical Report NMFS 92



                                                                                          Table 1
                                                                   Proportion of artificial seed to abalone landed.

                                                                                 Proportion of
                                           Prefecture           Location         seedlings (To)           Species                  References

                                           Hokkaido             Toyoharna              85              H.   d. hannai       Momma (1986)
                                           Iwate                Toni                   39              H.   d. hannai       Takeichi (1988)
                                           Miyagi               Utatsu                 83              H.   d. hannai       Sasaki et al. (1987)
                                           Fkushirna            Nagasaki               59              H.   d. hannai       Sato et al. (1984)
                                           Kanagawa             Nagai                  85              H.   discus          Tauchi (1984)
                                           Fukuoka              Oshima                 54              H.   discus          FutaJima et al. (1985)







                                                                                          Table 2
                                                                         Recapture rate of the planted seedlings.

                                                                                   Recapture
                                           Prefecture           Location           rate (%)             Species                   References

                                           Hokkaido             Shiribeshi               7            H. d. hannai         Miyarnoto et al. (1982)
                                           Iwate                Toni                   18             H. d. hannai         Takeichi (1988)
                                           Yarnagata            Koiwagawa              26             H. d. hannai         Ioka (1983)
                                           Ibaragi              Oarai                  25             H. discus            Kodarna (1985)
                                           Kanagawa             Jogashirna             26             H. gigantea          Inoue (1965)







                                                                                          Table 3
                                           Recapture rate of juvenile H. d. hannai in its nursery ground (from Momma et al. 1980).
                                                                          Term of experiment was 116 days.

                                                          Average growth           Released           Number                              Recapture
                                           Group            ratio (Mm/d)           size (mm)          released        Recovery           rate M

                                             Fast                71                13.8 ï¿½ 1.3            964               170               17.6
                                             Slow                28                13.9 ï¿½ 1.9            185               20                10.8






                 throughout their early development for a period of at least                        method. This method has been repeated several times from
                 116 days (Fig. 2) (Momma 1980b).                                                   1973 to 1989. The 6th generation of H. discus hannai pro-
                    In this paper the effects of artificial selection, h        'ybridiza-          duced by the sib method of inbreeding did not result in
                 tion, and mutation on juvenile abalone growth are dis-                             higher than normal mortality, or growth acceleration (Fig.
                 cussed. The isolated fast-growth qualities were examined                           3) (Momma 1987).
                 through various tests conducted under standardized rear-                             H. discus discus and H. discus hannaiwere introduced for
                 ing conditions with the following results.                                         breeding by the usual method (Kikuchi and Uki 1971).
                                                                                                    These seedlings were reared under the same conditions.
                                                                                                    The growth and mortality for these species was compared.
                 Growth Rates                                                                       Similar shell-growth rates were observed for these two
                                                                                                    species, but mortality of the H. d. discus was higher than
                 The fastest growing individuals were selected from the off-                        H. d. hannal; therefore, the latter produced a greater com-
                 spring of one parent by mass selection when they were                              parative biomass. (Fig. 4) (Momma 1987).
                 150-200 days old: about 15 min in shell length. After these                          Hybridization studies were conducted on H. d. discus,
                 superior individuals were reared about 1 more year, they                           H. d. hannai, and H. kamischatkana using the usual method
                 were introduced for breeding and fertilized by the sib                             on the Ezo-abalone (H. d. hannai). In only one case did







                                                                                                 Momma: Breeding Test on Abalone               73





                       150 -

                                                                    o
                                                                    ooo
                                                                    o

                    MD
                                                                    oo
                       120                                          oo1%o
                                                                    -o o o
                    E                                               o%
                                                                    oooc
                                                                    0-.. %
                                                                    0.
                                                                      0
                                                          0         0
                                                                    0
                                                                    00
                    d) 90                                           00 0
                    E

                    CL
                    X



                       so -
                    M

                    E
                    =L

                    0

                    to 30



                    0
                                                                                                            Figure 2
                                                                                 The relationship between thejuvenile stage (shell length:16-17
                            0       40       60        80           100  120     mm), the growth ratio (pm/d), and the growth ratio during the
                          Juvenile stage (till shell length: 16-17mm)            experimental term (116 d). 0 = fast-growth group at 153-269
                                       growth ratio (pm/day)                     d old and 0     the slow-growth group at 570-686 d old (Mom-
                                                                                 ma 1980b).









                                        30-
                                                    - G2 = 30 June 1986
                                                    - G6 = 7 July 1986
                                     E
                                    E 20m









                                                       50           100          150          200           250          300
                                                                           Age (days)


                                                                           Figure 3
                                 Growth lines of H. discus hannai seedlings; 0 and 0 = the mean shell length of 2nd (G2) and
                                 6th generation (G6), respectively. (G2 fertilized 30 June 1986; G6 fertilized 7 July 1986) (Momma
                                 1987).








                 74          NOAA Technical Report NMFS 92





                                                  4                                                                                             -100






                                                  3                                                                                             75


                                                       - hannai , iomass
                                                       - hannai    :urvival ratio

                                            (A    2    - discus     Biomass                                                                     -50 <
                                            E             discus    Survival ratio
                                            0



                                                                                                                                                25






                                                               204            4"             6H            M             inIll          1260
                                                                                         A4P (days)


                                                                                            Figure 4
                                                   Biomass and survival rate of H. d. hannai and H. d. discus. (Momma 1987).






                                                                                            Table 4
                                            List of the results from analyzed enzymes. (from Fujio 1984; Fujino 1979). D = digestive
                                            diverticula, M = shell-muscle, P             polymorphic, P*          polymorphic less than 517o, M
                                                monomorphic.

                                                           Enzyme                               Locus               Tissue             Polymorphism

                                            Acid phosphatase                                    Acp                   D                       P.
                                            Adenylate Kinase                                    Ak                    M                       M
                                            Aspartrte arninotransferse                          Aat                   M                       P
                                            Esterase                                            Est-I                 D                       P
                                                                                                       -2             D                       M
                                                                                                       -3             D                       M
                                            a-glycerophosphate Dehydrogenase                    aGpd                  M                       M
                                            Glucosephosphate Isornerase                         Gpi                   M                       M
                                            Isocitrate Dehydrogenase                            Idh-I                 M                       M
                                            Lactate Dehydrogenase                               Ldh-I                 M                       p
                                                                                                       -2             M                       M
                                            Leucin Aminopeptidase                               Lap- I                D                       P
                                                                                                       -2             D                       M
                                            Malate Dehydrogenase                                Mdh-I                 M                       M
                                                                                                       -2             M                       P*
                                            Malic Enzyme                                        Me                    M                       M
                                            Mannosephosphate Isomerase                          Mpi                   M                       P*
                                            Octanol Dehydrogenase                               Odh                   D                       P
                                            6-phosphogluconate Dehydrogenase                    6Pgd                  M                       P
                                            Phosphoglucoselsomerase                             Pgi                   D                       P*
                                            Phosphoglucornutase                                 M-1                   M                       P
                                                                                                       -2             M                       P
                                            Superoxide Dismutase                                Sod                   M                       M
                                            Tetrazolium Oxidase                                 To-2                  D                       P*
                                                                                                       -3             D                       P.












                                                     Water temperature (OC)

                                                                                                                                           Ei










                                                                                                                                           bl)




                                                                                                                                                                       co
                                                                                                                                           7S                          0)


                                                                                                                                           o                           M
                                           q*
                                           OD
                                           0)
                                                                                                                                                                       co
                                           >,
                                                                                                                           IM


                                                                                                                                               co
                                                                                                                                               00






                                                                                                                                                                              04         cr)         cf)




                                                                                                                                           cz

                                                           (Luw) LIIBUGI lia4s
                                                                                                                                                                                                 (Luw) LIIBUGI 11GLIS
                                                                                                                                               75








                  76           NOAA Technical Report NMFS 92


                  the hybrid of H. d. hannal and H. kamtschalkana show im-                               Ioka, 1.
                  proved growth rate in cold water. (Fig. 5) (Momma 1988).                                     1983. Yarnagataken ni okeru horyu ezo-awabi no seicho to seizan-
                                                                                                                 ritu no sisanrei. Otsuchi Marine Research Center Report 9:
                     Triploids produced by interference of polar body pro-                                       52-51.
                  jection (both lst and 2nd polar body extrusions) using the                             Japan Sea-Fasming Association.
                  method of Arai et al. (1982) did not differ in growth from                                   1967-1985. SaibaiGyogyoSyubyoSeisan,NyusyuHoryujisseki
                  diploid seed (Fig. 6) (Momma 1988).                                                            Showa 43-52 Nendo. (Materials on production, supply, and release
                                                                                                                 of fingerlings for enhancement of fisheries resources in Japan.)
                                                                                                                 Japan Sea-Farming Assoc., Japan.
                                                                                                         Kikuchi, S., and N. Uki.
                  Abalone Breeding                                                                             1974. Technical study on artificial spawning of abalone, genus
                                                                                                                 Haliods-11. Effect of irradiated sea water with ultraviolet rays
                  Enzyme polymorphism of this species (Table 4) (Fujio                                           on inducing to spawn. Bull. Tohoku Reg. Fish. Res. Lab. 33:
                                                                                                                 79-86.
                  1984; Fujino 1979), and the chromosome handling tech-                                  Kodama, S.
                  niques and growth characteristics of other species have been                                 1985. Awabi no horyu koka ni tuite. Bull. Japanese Soc. Fish.
                  reported. Above all, based on the study of wild abalone                                        Oceanog. 47/48:139-142.
                  populations, as well as the relationship of age to fitness                             Miyamoto, T., K. Saito, M. Ito, and Y. Mizutori.
                  measured at the esterase M locus, the deficient animals                                      1982. On the releasing of the cultured seeds of Ezo-abalone, Haliods
                                                                                                                 discus hannai, in the northern coast of Siribesi, hokkaido, Hokusui-
                  reveal more homozygosity (Fujino 1978). Population                                             shi-geppo. Hokkaido Fish. Exp. Sm. 39(8):169-208.
                  genetics and thremmatological studies may contribute con-                              Momma, H.
                  siderably to the abalone industry.                                                           1980a. On the abalone behavior under the influence of predator.
                                                                                                                 Michurian Seibutsugaku kenkyu 16(l):60-69.
                                                                                                               1980b. Studies on the variation of the abalone-1. On the growth
                                                                                                                 of the different aged young abalone. Aquaculture, 28(3):142-146.
                  Citations                                                                                    1986. Awabijinkoshubyuhouryujigyonokokachosa. Sodateru-
                                                                                                                 gyogyo 159:2-4.
                  Arai, K., H. Tsubaki, and K. Fujino.                                                         1987. Ezo-Awabi. Annu. Rep. Hokkaido Inst. Maricult. 61:
                        1982. Chromosomes of Haliotis discus hannai Ino and H. discus Reeve.                     53-61.
                          Bull. Jap. Soc. Sci. Fish. 48:1689-1692.                                             1988. Kairui shubyo chukan ikusei siken. Ezo-Awabi. Annu.
                  Department of Statistics Information (DSI).                                                    Rep. Hokkaido Inst. Maricult. 62. 57-59.
                        1967-1986. Fishery statistics of Japan 1967-86. DSI,Jpn.Min.                     Momma, H., K. Kobayashi, T. Kato, Y. Sasaki, T. Sakamoto, and
                          Agric., Forest. Fish., Tokyo. (In Japanese.)                                      H. Murata.
                  Fujino, K.                                                                                   1980. On the artificial propagation method of abalone and its ef-
                        1978. Genetic studies on the Pacific Abalone-1. Excessive Homo-                          fects on rocky shore-1. Remaining ratio of the artificial seed
                          zygosity in Deficient Animals. Bull. Jpn. Soc. Sci. Fish., 44(7),                      abalones (Haliotis discus hannai INO) on the lattices artificial reefs.
                          767-770.                                                                               Aquaculture, 27(4):212-216.
                        1979. Apparent physical deficiency associated with inbreeding struc-             Sasaki, R., K. Takahasi, T. Kawamura, and Y. Tasiro.
                          ture suggested by biochemical polymorphism in the Pacific                            1987. Miyagiken hokubu kaiku ni okeru horyu ezo-Awabi no chosa
                          Abalone. In Proc. 16th int. conf. on animal blood group and                            jirei. Tohoku Reg. Fish. Lab., Report 61. 15-29.
                          biochemical polymorphism: Leningrad, USSR (16 August 1978)                     Sato, M., J. Owada, and H. Suzuki.
                          p. 245-256. USSR Organizing Comm. Int. Soc. Animal Blood                             1984. Jinko shubyo awabi, uni horyu koka chosa. Annu. Rep.
                          Group Res.                                                                             Fukushima Pref. Fish. Exp. Stn:181-190.
                  Fujio, Y.                                                                              Takayama, K.
                        1984. Studies on genetic analysis of fishies by isozyme methods.                       1940. On the growth rate of the Abalone at Mie prefecture. Suisan
                          Minist. Agric. Forestry and Fisheries, Government ofJapan, 65 p.                       kenkyushi (precedes Bull. Jpn. Soc. Sci. Fish.) 35(4):99-100.
                  Futajima, K., T. Ito, and G. Kishimoto.                                                Takeichi, M.
                        1985. Awabi no saibai gyogyokanri ni kansuru kenkyu-1. Annu.                           1988. Tairyo horyu sareta ezo-awabi jinko shubyo no kaishuritu
                          Rep. Fukuoka Pref. Fish. Exp. Stn, p. 239-257.                                         to seizanritu Saibai Gyogyo Gijitu Kaihatu Kenkyu (Journal on
                  Inoue, T.                                                                                      technical development for artificial enhancement of fisheries
                        1965. Hyoshiki horyu kara mita Awabi zoku no ishoku ni kansuru                           resources). Saibaigiken 17(l):27-36.
                          1. 2. Suisan-Zoshoku. Ext. 5:23-31.                                            Tauchi, M.
                  Inoue, T., M. Tauchi, and M. Chikayama.                                                      1984. Growth, age composition, survival rate and recapture rate
                        1985. Diagnosis of species participate in effect on artificial seeds                     of Japanise black abalone, Haliods discus Reeve, at Nagai. Sci.
                          release of abalone, Haliods spp. Aquaculture, 32(4), 193-198.                          Rep. Kanagawa Fish. Exp. Sm. 6:17-21.








                                          Two-Stage Hybridization and Introgression
                                     for Improving Production Traits of Red Tilapias


                                 LESLIE L. BEHRENDS, JOHN B. KINGSLEY, and ALBERT H. PRICE III

                                                                        Agriculture Research Department
                                                                           Tennessee Valley Authority
                                                                       Muscle Shoals, AL 35660-1010





                                                                                ABSTRACT


                                       Two-stage hybridization and introgression were evaluated as breeding plans to develop im-
                                     proved hybrid populations of red tilapia. Both plans were successful in overcoming interspecific
                                     breeding barriers, thus allowing development of hybrid populations (both red- and non-nal-colored
                                     phenotypes), with a mix of desirable production traits. The hybrids were evaluated in replicated
                                     polyculture experiments involving blue tilapia, Tilapia aurea, channel catfish, Ictalurus punctatus,
                                     freshwater prawn, Macrobrachium rosenbergii, and Asiatic carps. Yield trials were conducted in earthen
                                     ponds. Two-stage hybridization ( [ T aurea x red tilapia] x [red tilapia x T. nilotica ] ) was used
                                     to produce red- and normal-colored hybrids that grew to average weights of 313 and 292 grams
                                     respectively in 133 days. Under similar conditions (communal stocking at equal density), T aurea
                                     controls averaged 2 52 grams. Introgressive breeding techniques ff T aurea x red tilapia] followed
                                     by two generations of backcrossing red males [F- I and F-2 generations respectively], to female
                                     T. aurea) were used to develop a cold tolerant tilapia hybrid with both red- and normal-colored
                                     phenotypes. After 146 days of culture, both the red- and normal-colored hybrids averaged 296
                                     grams, while T. aurea controls averaged 276 grams. Irrespective of breeding plan, mortality of
                                     the red phenotype was significantly greater     than  either of the normal-colored phenotypes.



                Introduction                                                               stains have not been well documented (Sipe 1979; Fitz-
                                                                                           gerald 1979). However, morphometric and electrophoretic
                Individual (mass) selection for rapid growth in the mater-                 data indicate common ancestry with a red-mutant strain
                nal mouthbrooding genus Tilapia has had limited success,                   of T mossambica (Galman and Avtalion 1983; Halstrom.
                indicating that the fraction of additive genetic variation is              1984). The widely distributed U.S. "Florida strain,"
                small relative to total genetic variation (Chan May Tchien                 derived from the cross female T hornorum x male T mos-
                197 1; Tiechert-Coddington 1983; Hulata et al. 1986). In                   sambica (red mutant), has production traits similar to its
                such instances, hybridization and introgression can be                     parental lines: early sexual maturation, slow growth, and
                utilized to avail for nonadditive sources of genetic varia-                lack of cold tolerance (Halstrom. 1984).
                tion (Brody et al. 1980).                                                     In replicated yield trials, Florida strain young-of-the-year
                   Within the interfertile genus Tilapia, there are species                fingerlings grew only 50 % as fast as blue tilapia, T aurea,
                which exhibit fast growth (T aurea; T nilotica), high fecun-               (Behrends et al. 1982). Thus, it was hypothesized that pro-
                dity (T mossambica), cold tolerance (T aurea), and salinity                duction traits could be improved by hybridizing the Florida
                tolerance (T. mossambica; T aurea). Also, red-colored mu-                  strain with T aurea or T n1lotica, two of the faster-growing
                tant strains of T. mossambica and T nilotica have arisen                   and later-maturing species. Subsequently, Behrends and
                spontaneously from normal-colored populations (Fitzgerald                  Smitherman (1984) reported that cold tolerance and the
                1979; McAndrew et al. 1988). Thus, a diversity of genetic                  red color trait could be incorporated into a single popula-
                resources is available for developing hybrids or synthetic                 tion via hybridization (female T aurea x male Florida
                breeds in which favorable traits are combined.                             strain), followed by recurrent backcrossing of red hybrid
                   Several hybrid strains of red tilapia have been com-                    males to T aurea females.
                mercially produced and widely disseminated throughout                         This paper will present results of studies designed to
                Southeast Asia and the Western Hemisphere during the                       evaluate the use of two-stage hybridization and hybridiza-
                past fifteen years. Breeding plans used in developing these                tion followed by repeated backcrossing (introgression)

                                                                                                                                                              77








               78        NOAA Technical Report NMFS 92
                  -Breeding Plan 1983                                                             Breeding Plan 1984
                                       Hybridization                                                      Introgression
                  ?Tilapia aurea                      ?Red tilapia                            Tilapia aure          x dRed tilapia
                             X                      (Florida strainY                                               W (Florida strainY
                  d Red tilapia                                X                                                   F-1**
                     (Florida strainY               dTilapia nilotica                              (25 50% red; 50% normal)
                           I                                 1                                                     1%
                      F- 1 hybrid                       F- 1 hybrid                                ?T. aurea x (f F-1 (red)
                     (@_-50% red;                       0_--50% red;                                               I
                     50% normal)                       50% normal)                                        F-1 backcross
                                 It                   fr                                          (=-50% red; 50% normal)
                             ?F-1 (red) x dF-1 (red)                                                               1%
                                                                                                     T. aurea x d F- 1 backcross
                                             J1                                                                    I          (red)
                                        FTrihyb                                                          IF-2 back

                   *Florida strain of red tilapia derived from                            *Florida strain of red tilapia derived from
                     9 T. hornorum x o4 T. mossambica (red mutant).                         VT. hornoru x d4T. mosambica (red mutant).
                  "Red phenotypes are heterozygous for red                               "Red broodstock were heterozygous for
                     color. Normal phenotypes are homozygous                                the red color trait, thus approximately
                     for normal color. Pink phenotypes are                                  50% of progeny were red and 50% normal.
                     homozygous for pink color.



                                          Figure I                                                               Figure 2
               Breeding plan for developing cold-tolerant red tilapia hybrids.        Breeding plan for developing fast growing red tilapia hybrids.



               for developing red tilapia hybrids with improved culture               channel catfish (Ictalurus punctatus, 10000/ha), freshwater
               performance.                                                           prawn (Macrobrachium rosenbergii, 20000/ha), grass carp
                                                                                      (Clenopharyngodon idella, 120/ha) and F-1 hybrid Asian carp
                                                                                      (An'stichthys nobilis x Hypopthalmichthys molitrix, 160-500/ha).
               Materials and Methods                                                    Fingerlings developed via two-stage hybridization were
                                                                                      stocked in 1983, while introgressed hybrids were stocked
               Young-of-the-year red- and normal-colored (grey) tilapia               in 1984. Red- and normal-colored hybrids and blue tilapia
               fingerlings were produced at the Tennessee Valley Author-              were costocked into each pond at equal densities (800/ha)
               ity's Research Farm from 1981 to 1984 using breeding                   for a total density of 2 400/ha (Tables 1 and 2). Blue tilapia
               plans illustrated in Figures 1 and 2. In both trials, red              served as within- and between-years controls. Costocking
               broodstock heterozygous for the red color trait were used              of different genetic groups, also referred to as communal
               to ensure production of both red- and normal-colored                   testing, has been shown to be an efficient method for
               phenotypes. By comparing the two hybrid phenotypes, it                 evaluating various tilapia strains and hybrids under con-
               was possible to assess the effect of the red gene complex              ditions of common environment (Wohlfarth et al. 1983).
               on growth, yield, and mortality.                                         Throughout each study, a 32 % crude-protein floating
                  In each of two years (1983 and 1984), three genetically             catfish ration was broadcast onto the pond surfaces daily.
               and phenotypically distinct tilapia populations (Tables I              Feeding rates were adjusted daily based on projected cat-
               and 2), were costocked into four 0.05-ha ponds along with              fish biomass (Behrends et al. 1985). After 130 to 140 days







                                                                                     Behrends et al.: Hybridization and Introgression of Red Tilapias                                    79



                                                                                                   Table I
                                           Stocking and harvest data for three tilapia populations costocked into 0.05-ha earthen ponds.
                                           Polyculture yield trial 1983, Tennessee Valley Authority, Muscle Shoals, Alabama. Each mean
                                           represents the average of four ponds.'

                                                                                     Two-stage hybridization

                                                                                Red hybrid         Normal hybrid             Tilapia aurea

                                           Stocking density (#/ha)              800                  800                     800
                                           Culture duration (days)              133                  133                     133
                                           Initial weight (g/fish)                   2.0                 2.1                    2.3
                                           Final weight (g/fish)                313 a'               292 b                   252 c
                                              Males (mean ï¿½ SD)                 330 ï¿½ 63.0           318 ï¿½   59.0            283 ï¿½  38.0
                                              Females (mean ï¿½ SD)               256  ï¿½ 58.4          254 ï¿½   57.0            225 ï¿½  35.3
                                           Sex ratio (To males)                 77                     59                      48
                                           Daily gain (g/fish/d)
                                              Males (mean)                           2.5                 2.4                    2.1
                                              Females (mean                          1.9                 1.9                    IJ
                                           Survival (To)                        88                     93                    100
                                           Yield (kg/ha)                        219 a                216 a                   200 b               Total 635 kg/ha

                                           'Tilapias were stocked into polyculture ponds containing channel.catfish (10000/ha) freshwater prawn
                                            (20000/ha) and Asiatic carps (300-600/ha) (Behrends et           al. 1985).
                                            Means followed by different letters are significantly different (P<0.05, Student- Newman-Kuels test).
                                            Horizontal comparisons only.






                                                                                                   Table 2
                                           Stocking and harvest data for three tilapia populations costocked into 0.05-ha earthen ponds.
                                           Polyculture yield trial 1984, Tennessee Valley Authority, Muscle Shoals, Alabama. Each mean
                                           represents the average of four ponds             . a

                                                                                                             Introgressed hybrids

                                                                                Red hybrid         Normal    hybrid          T21apia aurea

                                           Stocking density (#/ha)              800                  800                     800
                                           Culture duration (days)              146                  146                     146
                                           Initial weight (g/fish)              . 0.6                    0.9                    0.9
                                           Final weight (g/fish)                296 a'               296 a                   279 b
                                              Males (mean ï¿½ SD)                 299 ï¿½ 65.2           325 ï¿½   65.4            298 ï¿½  63.1
                                              Fema  'les (mean     SD)          268 ï¿½ 44.9           260 ï¿½   62.0            254 ï¿½  46.1
                                           Sex ratio (To males)                 90                     56                      49
                                           Daily gain (g/fish/day)
                                              Males (mean)                           @2. 0               2.2                    2.0
                                              Females (mean)                         1.8                 1.8
                                           Survival                             65                     92                      71
                                           Yield (kg/ha)                        153 b                217 a                   156 b               Total 526 kg/ha

                                           'Tilapias were stocked into polyculture, ponds containing channel catfish (10000/ha) freshwater prawn
                                            (20000/ha) and Asiatic carps (300-600/ha) (Behrends et al. 1985).
                                           'Means followed by different letters are significantly different (P<0.05, Student-Newman-Kuels test). Horizon-
                                            tal comparisons only.




                   of culture (May-October), ponds were drained and har-                                     during 1983 and 1984 included effects due to phenotype,
                   vested. Tilapias within ponds were sorted by phenotype                                    pond nestedwithin phenotype, sex, and all first-order in-
                   and sex within phenotype. Individuals were then weighed                                   teractions. Student-Newman-Kuel's Multiple Comparisons
                   to the nearest gram.                                                                      Test was used to separate means (within years), at a sig-
                      Statistical models used to evaluate growth and yield data                              nificance level of P<0.05 (Barr et al. 1979). Details con-








              80        NOAA Technical Report NMFS 92


              cerning culture and yields of catfish, prawns, and carps            to faster growth and excellent survival. Mortality of
              are presented in a companion paper (Behrends et al. 1985).          normal-colored hybrids averaged only 87o, while mortal-
                                                                                  ity rates for red hybrids and T aurea averaged 35 and 29 7o
                                                                                  respectively (Table 2). Sex ratio of the red hybrid popula-
              Results                                                       -     tion was highly skewed towards males (907o), while the
                                                                                  percentage of males in the normal-colored hybrid and
              Two-Stage Hybridiation                                              T aurea populations was 56 and 497o respectively.
              Two-stage hybridization (Fig.   1), was an effective breeding
              strategy for enhancing growth rate and yield of red- and
              normal-colored tilapia hybrids (Table 1). After 133 days            Discussion
              of culture, mean harvest weights (sexes pooled) of red and
              normal phenotypes were 313 and 292 g respectively. Red              Both two-stage hybridization and introgression were effec-
              hybrids were heavier at harvest (P<0.05) than their                 tive breeding strategies for enhancing growth rates of red-
              normal-colored counterparts, indicating a positive growth           and normal-colored hybrids. However, relative to T aurea
              effect of the red gene complex. In comparison, T aurea con-         controls, two-stage hybridization improved growth rates
              trols averaged 252 g, and weighed less (P<0.05) than either         and yields to a greater extent than did introgression. This
              of the hybrids. Irrespective of phenotype, males were 20            is not surprising because introgression, as practiced, is
              to 307o heavier than females (P<0.05). Relative ranking             analogous to inbreeding; heterozygous hybrid populations
              of mean harvest weights by phenotype were the same in               become progressively more homozygous with repeated
              all ponds, indicating that pond (environment) x phenotype           generations of backcrossing to a small founder population.
              (genotype) interactions were not a significant source of            Introgression is recommended for incorporating a desirable
              variation.                                                          dominant trait from one population into a second, more
                 Net production of the three phenotypes ranged from 200           productive population (Kirpichnikov 1981).
              to 219 kg/ha for a combined yield of 634 kg/ha (Table 1).             During both years, mortality figures for red phenotypes
              Mortality of red hybrids was consistently greater than mor-         were higher than rates for normal-colored phenotypes.
              tality of normal-colored hybrids and T aurea. Sex ratios            Elevated levels of mortality have also been reported in other
              (male:female) of red and normal-colored hybrids averaged            production studies (Behrends et al. 1982, 1988), and in
              7 7 % and 59 01o males respectively, while the T aurea pop-         hatchery research (El-Gamal et al. 1988). This indicates
              ulation averaged 48% males: near the expected 1:1 sex               that the red gene complex, while enhancing growth, may
              ratio.                                                              have had a negative pleiotropic effect on viability. Color
                 Despite their higher rates of mortality, hybrids yielded         mutations in green sunfish, Lepomis cyanellus, and common
              7-10% greater than T aurea (P<0.05). Improved yields                carp, Cyprinus carplo, have resulted in reductions in viabil-
              were due to improved growth of both males and females               ity, growth or both (Dunham and Childers 1990; Kir-
              and male-dominated sex ratios.                                      pichnikov 198 1). Other red mutant strains of tilapia have
                                                                                  been identified (McAndrew et al. 1988; Tave 1989), and
              Introgression                                                       should be evaluated with respect to growth rate, viability,
                                                                                  and use in hybrid breeding programs.
              Introgressive breeding (Fig. 2), similarly improved growth            Breeding plans in this study worked well for combining
              performance of red- and normal-colored hybrids (Table               traits of closely related species. While many species of
              2), although not to the extent of two-stage hybridization.          maternal mouthbrooding tilapia are interfertile, commer-
              After 146 days of culture, mean harvest weights of both             cial production of most F-1 hybrids is difficult owing to
              red- and normal-colored phenotypes (sexes pooled) were              species- specific differences in breeding behavior (Lee 1979;
              296 and 296 g respectively. This differs somewhat from              Hulata et al. 1985). In the present studies, problems related
              results of the 1983 study, where the red hybrid grew                to breeding behavior were overcome by resorting to double
              faster than it's normal-colored counterpart. At harvest,            hybridization or introgression. In both breeding plans, each
              T. aurea controls averaged 279 g and weighed less than              of the respective parents (F-1 and subsequent backcross
              either hybrid (P<0.05). Irrespective of phenotype, males            generations), had a complete or nearly complete set of
              were 15-20% heavier than females (P<0.05). As in the                chromosomes in common. This condition was apparently
              previous years study, relative rankings of mean harvest             sufficient to overcome natural reproductive isolating
              weights (by phenotype) were the same between the four               mechanisms which can impede reproduction between
              replicate ponds.                                                    closely-related species. Similar interspecific breeding plans
                 Net production of the three phenotypes ranged from 153           may be practical for developing unique hybrids or synthetic
              to 217 kg/ha, with a combined yield of 526 kg/ha (Table             strains for special environments, for instance, a fast grow-
              2). Mean yield of the normal-colored hybrid was greater             ing, salt-tolerant red hybrid for net-pen culture in tropical
              (P< 0. 05) than yields of the red hybrid and T aurea owing          estuaries.







                                                                                Behrends et al.: Hybridization and Introgression of Red Tilapias                                81


                  Citations                                                                           Fitzgerald, W.J.
                                                                                                           1979. The red-orange tilapia. Fish Farming Int. 6(l):26-27.
                  Barr Aj., J.H. Goodnight, J.P. Sall, W.H. Blair, D.M. Chilko,                       Galman, 0., and R.R. Avtalion.
                    K.A. Council, and JT. Helwig.                                                          1983. A preliminary investigation of the characteristics of red tdapias
                       1979. SAS User's Guide. SAS Institute Inc., Raleigh, NC,                               from the Philippines and Taiwan. In Int. symposium on tilapia
                          494 p.                                                                              in aquaculture; 8-13 May 1983, Naareth, Israel (L. Fishelson and
                  Behrends, L.L., and R.O. Smitherman.                                                        Z. Yaron, Compilers), p. 291-301. Tel Aviv Univ., Tel Aviv,
                       1984. Development of a cold tolerant population of red tilapia                         Israel,
                          through introgressive hybridization. J. World Maricult. Soc.                Halstrom, M.L.
                          15:172-178.                                                                      1984. Genetic studies of a commercial strain of red tilapia. M.S.
                  Behrends, L.L., R.G. Nelson, R.O. Smitherman, and N.M. Stone.                               Thesis, Auburn Univ., Auburn, AL, 80 p.
                       1982. Breeding and culture of red-gold color phase of tilapia. J.              Hulata, G., S. Rotbbard, J. Itzkovich, G. Woh1farth, and A. Halevy.
                          World Maricult. Soc. 13:210-220.                                                 1985. Differences in hybrid fry production between two strains of
                  Behrends, L.L., J.B. Kingsley, and A.H. Price III.                                          nile tilapia. Prog. Fish Cult. 47(l):42-49.
                       1985. Polyculture of freshwater prawns, tilapia, channel catfish,              Hulata, G., G. Wohlfarth, and A. Halevy.
                          and Chinese carps. J. World Maricult. Soc. 16:437-450.                           1986. Mass selection for growth rate in the Nile tilapia (Oreochromis
                       1988. Bidirectional backcross selection for body weight in red                         nilolicus). Aquaculture 57:177-14.
                          tilapia. In The second international symposium on tilapia in                Kirpichnikov, V.S.
                          aquaculture; 16-20 March 1987, Bangkok, Thailand (R.S.V.                         1981. Genetic Bases of Fish Selection. Springer-Verlag, New
                          Pullin, T. Bhukaswan, K. Tonguthai, and J. L. Maclean, eds.),                       York, NY, 410 p.
                          p. 125-133. Dep. Fisheries, Bangkok, Thailand, and Int. Center              Lee, J.C.
                          for Living Aquatic Resources Manage., Manila, Philippines,                       1979. Reproduction and hybridization of three cichlid fishes, Tilapia
                          ICLARM Conf. Proc. 13.                                                              aurea, T hornorum, and T nilotica in aquaria and plastic pools.
                  Brody, T., N., Storch, D. Kirst, G. Hulata, G. Wohlfarth, and R. Moav.                      Ph.D. Diss., Auburn Univ., Auburn, AL, 84 p.
                       1980. Application of electrophoretic genetic markers to fish breed-            McAndrew, Bj., F.R. Roubal, Rj. Roberts, A.M. Bullock, and
                          ing. III: Diallel analysis of growth rate in carp. Aquaculture                I.M. McEwen.
                          20:371-379.                                                                      1988. The genetics and histology of red, blond and associated color
                  Chan May Tchien.                                                                            variants in Oreochromis niloticus. Genetica 76:127-137.
                       1971. Experience with the determination of realized weight                     Sipe, M.
                          heritability in the tilapia, Tilapia mossambica. Genetica 7(t):                  1979. Golden Perch. Commercial Fish Farmer & Aquaculture
                          53-59.                                                                              News 3(5):56,
                  Dunham, R.A., and W.F. Childers.                                                    Tave, D., M. Rezk, and R.O. Smitherman.
                       1980. Genetics and implications of the golden color morph in green                  1989. Genetics of body color in Tilapia mossambica. J. World
                          sunfish. Prog, Fish Cult. 42(3):160-163.                                            Aquacult. Society 20(4):214-222.
                  El Carnal, A.A., R.O. Smitherman, and L.L. Behrends.                                Teichert-Coddington, D.
                       1988. Viability of red and normal-colored Oreochromis aureus and                    1983.   Bidirectional mass selection for growth rate in Tilapia
                          0. niloticus hybrids. In The second international symposium on                      nilotica. M.S. Thesis, Auburn Univ., Auburn, AL, 48 p.
                          tilapia in aquaculture; 16-20 March 1988, Bangkok, Thailand                 Wohlfarth, G. W., G. Hulata, S. Rothbard, J. Itzkovich, and A. Halevy.
                          (R.S.V. Pullin, T. Bhukaswan, K. Tonguthai, andj.L. Maclean,                     1983. Comparisons between interspecific hybrids for some produc-
                          eds.), p. 153-157. Dep. Fisheries, Bangkok, Thailand, and Int.                      tion traits. In International symposium on tilapia in aquaculture
                          Center for Living Aquatic Resources Manage., Manila, Philip-                        (L. Fishelson and Z. Yaron, Compilers), p. 559-569. Tel Aviv
                          pines, ICLARM Conf. Proc. 15.                                                       Univ., Tel Aviv, Israel.









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