NOAA Technical Report NMFS 92

Genetics in Aquaculture
Proceedings of the Sixteenth
U. S. -Japan Meeting on AquacultureCharleston, South Carolina
October 20 and 21, 1987
Ralph S. Svrjcek (editor)

u.s. Department of Commerce

November 1990

NOAA Technical Report NMFS_

The major responsibilities of the National Marine Fisheries Service (NMFS) are to monitor and assess the abundance and geographic distributionof fishery resources, to understand and predict fluctuations in the quantity and distribution of these resources, and to establish levels for their optimumuse. NMFS is also charged with the development and implementation of policies for managing national fishing grounds, development and enforcementof domestic fisheries regulations, surveillance of foreign fishing off United States coastal waters, and the development and enforcement of international
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69. Environmental quality and aquaculture systems: Proceedings of thethirteenth U.S.-Japan meeting on aquaculture, Mie, Japan, October 24-25,
1984, edited by Carl J. Sindermann. October 1988, 50 p.
70. New and innovative advances in biology/engineering with potential
for use in aquaculture: Proceedings of the fourteenth U.S.-Japan meetingon aquaculture, Woods Hole, Massachusetts, October 16-17,1985, editedby Albert K. Sparks. November 1988, 69 p.
71. Greenland turbot Reinhardtius h.ppoglossoides of the eastern Bering Sea
and Aleutian Islands region, by Miles S. Alton, Richard G. Bakkala, GaryE. Walters, and Peter T. Munro. December 1988, 31 p.
72. Age determination methods for northwest Atlantic species, editedby Judy Penttila and Louise M. Dery. December 1988, 135 p.
73. Marine flora and fauna of the Eastern United States. Mollusca:
Cephalopoda, by Michael Vecchione, Clyde F. E. Roper, and Michael
J. Sweeney. February 1989, 23 p.
74. Proximate composition and fatty acid and cholesterol content of 22
species of northwest Atlantic finfish, by Judith Krzynowek, Jenny Murphy, Richard S. Maney, and Laurie J. Panunzio. May 1989, 35 p.
75. Codend selection of winter flounder Pseudopleuronectes americanus, byDavid G. Simpson. March 1989, JO p.
76. Analysis of fish diversion efficiency and survivorship in the fish returnsystem at San Onofre Nuclear Generating Station, by Milton S. Love,
Meenu Sandhu, Jeffrey Stein, Kevin T. Herbinson, Robert H. Moore,
Michael Mullin, and John S. Stephens Jr. April 1989, 16 p.
77. Illustrated key to the genera of free-living marine nematodes of theorder Enoplida, by Edwin J. Keppner and Armen C. Tarjan. July 1989,
26 p.
78. Survey of fishes and water properties of south San Francisco Bay,
California, 1973-82, by Donald E. Pearson. August 1989, 21 p.
79. Species composition, distribution, and relative abundance of fishes
in the coastal habitat off the southeastern United States, by Charles A.
Wenner and George R. Sedberry. July 1989, 49 p.
80. Laboratory guide to early life history stages of northeast Pacific fishes,by Ann C. Matarese, Arthur W. Kendall Jr., Deborah M. Blood, andBeverly M. Vinter. October 1989, 651 p.
81. Catch-per-unit-effort and biological parameters from the Massachusetts coastal lobster (HDmilrus americanus) resource: Description and Trends,
by Bruce T. Estrella and Daniel J. McKiernan. September 1989, 21 p.
82. Synopsis of biological data on the cobia Rachycentron canadum (Pisces:
Rachycentridae), by Rosalie Vaught Shaffer and Eugene L. Nakamura.
December 1989, 21 p.
83. Celaphopods from the stomachs of sperm whales taken off California, by Clifford H. Fiscus, Dale W. Rice, and Allen A. Wolman. December 1989, 12 p.
84. Results of abundance surveys of juvenile Atlantic and Gulf menhaden, Breuoortia tyrannus and B. patrunus, by Dean W. Ahrenholz, James F.
Guthrie, and Charles W. Krouse. December 1989, 14 p.
85. Marine farming and enhancement: Proceedings of the Fifteenth
U.S.-Japan Meeting on Aquaculture, Kyoto, Japan, October 22-23, 1986,
edited by Albert K. Sparks. March 1990, 127 p.
86. Benthic macrofauna and habitat monitoring on the continental shelfof the northeastern United States. I. Biomass, by Frank Steimle. February 1990, 28 p.
87. Life history aspects of 19 rockfish species (Scorpaenidae: &hastes) fromthe Southern California Bight, by Milton S. Love, Pamela Morris, Merritt McCrae, and Robson Collins. February 1990, 38 p.
88. Early-life-history profiles, seasonal abundance, and distribution offour species of c1upeid larvae from the northern Gulf of Mexico, 1982 and1983, by Richard F. Shaw and David L. Drullinger. April 1990, 60 p.
89. Early-life-history profiles, seasonal abundance, and distribution offour species of carangid larvae off Louisiana, 1982 and 1983, by Richard
F. Shaw and David L. Drullinger. April 1990, 37 p.
90. Elasmobranchs as living resources: Advances in the biology, ecology,
systematics, and the status of the fisheries, edited by Harold L. PrattJr.,
Samuel H. Gruber, and Toru Taniuchi. July 1990, 518 p.
91. Marine flora and fauna of the northeastern United States, Echinodermata: Crinoidea, by Charles G. Messing and John H. Dearborn. August
1990, 30 p.

NOAA Technical Report NMFS 92

Genetics in Aquaculture
Proceedings of the Sixteenth
U. S. -Japan Meeting on AquacultureCharleston) 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 Programin Natural Resources (UJNR)

November 1990

U.S. DEPARTMENT OF COMMERCE
Robert Mosbacher, Secretary

;,
~4

i
-

.5'''-4T£5 Of

"'~

,..+

National Oceanic and Atmospheric Administration
John A. Knauss, Under Secretary for Oceans and Atmosphere

National Marine Fisheries Service
William W. Fox Jr., Assistant Administrator for Fisheries

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 withaquaculture. Charged with exploring and developing bilateral cooperation, the panels havefocused 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 UnitedStates-Japan Committee on Trade and Economic Affairs in January 1964. In addition to aquaculture, current subjects in the program include desalination of seawater, toxic microorganisms,
air pollution, energy, forage crops, national park management, mycoplasmosis, wind andseismic effects, protein resources, forestry, and several joint panels and committees in marine
resources research, development, and utilization.
Accomplishments include: Increased communication and cooperation among technicalspecialists; 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 - JapanThe National Marine Fisheries Service (NMFS) does not approve, recommend or endorse any proprietary product or proprietary material mentioned
in this publication. No reference shall be made to NMFS, or to this publication furnished by NMFS, in any advertising or sales promotion which would
indicate or imply that MFS approves, recommends or endorses any proprietary product or proprietary material memioned herein, or which has
as its purpose an intent to cause direcl1y or indirectly the advertised pro·
duct to be used or purchased because of this NMFS publication.

Text printed on recycled paperii

CONTENTS
Assessment of inbreeding and its implications for salmon broodstock
development

1

Chromosome set manipulation in salmonid fishes

9

R.T. DILLON Jr.
J.J. MANZI

Outcrossed lines of the hard clam Mercenaria mercenaria

11

Y. FU
Y. NATSUKARIK HIRAYAMA

A preliminary study on genetics of two types of the rotifer Brachionus plicatilis

W.K HERSHBERGER
J.M. MYERS
R.N. IWAMOTO
W.C. McAULEY
G.H. THORGAARDK FUKUSHOJ.C. LEONG
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
G.A. WINANS
F.M. UTTER
C. MAHNKEN
S.J. YOON
Z. LID
A.R. KAPUSCINSKI
P.B. HACKETT
A. FARAS
KS. GUISE
T. NAKANISHI
H.ONOZATO
T.LJ. SMITH
L.J. LESTER
KS. LAWSON
M.J. PIOTROWSKI
T.-C. B. WONG
H. MOMMA
L. L. BEHRENDS
J.G. KINGSLEY
A.H. PRICE III

Present status of genetic studies on marine finfish in Japan

21

Recombinant viral vaccines in aquaculture

27

Genetic monitoring of Pacific salmon hatcheries

33

Successful gene transfer in fish

39

Clonal ginbuna crucian carp as a model for the study of fish immunology
and genetics

45

Aquaculture of striped bass, Marone saxatilis, and its hybrids in North America

53

Computerized image analysis for selective breeding of shrimp: a progress
report63

Breeding test on abalone71

Two-stage hybridization and introgression for improving production traits of
red tilapias

77

iii

Assessment of Inbreeding and Its Implications for
Salmon Broodstock Development *WILLIAM K. HERSHBERGER and JAMES M. MYERS
School oj Fisheries WH-10
University oj Washington
Seattle, WA 98195

R.N. IWAMOTO** and w.e. McAULEY
Domsea Farms, Inc.
5500 180th S. W
Rochester, WA 98579

ABSTRACT
Inbreeding is an important part of any selection and breeding program designed to improveaquacultural broodstock. A decrease in freshwater and saltwater growth rate was noted in a strainof 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 thisstrain and to assess different approaches to alleviate problematic levels of inbreeding. Estimationof inbreeding level was conducted via pedigree analysis and change in heterozygosity ofelctrophoretically detected serum 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 tocause the observed depression in growth traits. Two approaches, interstock crosses and crosses
between parallel-selected lines, were assessed for alleviation of inbreeding problems. Both typesof crosses decrease the level of inbreeding, but the performance of the two types of crosses differed 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 fromwhich broodstocks are developed and that subsequent breeding information be collected to produce pedigrees for population main.tenance. Furthermore, the production of parallel" in-house"lines, may provide the best method of minimizing inbreeding without diluting selection gains.

Introduction_

Inbreeding is integral to any selection and breeding program designed for the development of broodstock. Suchprograms generally deal with a "closed" population (i.e.,
migration into the population is eliminated) having a restricted breeding population size. Both of these factors• Contribution No. 760, School of Fisheries WH-IO, University of Washington, Seattle, WA 98195. The Project was supported by U.S. NOAA
Grant NA86AA-D-SG044 A09 to the Washington Sea Grant Program
Project No. R/A-47.
•• Current Address: Ocean Farms of Hawaii, P.O. Box A, Kailua-Kana,
HI 96745

result in increased inbreeding levels (Falconer 1981), where
the magnitude will depend on the genetic characteristicsof the population and the severity of the constraints imposed. Consequently, the factors that influence inbreedingmust be integrated into the design of any program todevelop genetically improved aquacultural stocks.
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 rnykiss(formerly Sa/rna gairdnen), has revealed that increased levels
of inbreeding result in increased egg and fry mortality,
increased numbers of abnormal fry, decreased early
growth, and decreased fishery recovery (Kincaid 1976,
1983; Aulstad and Kittlesen 1971). Research with brook

2

NOAA Technical Report NMFS 92_

trout, Salvelinusfontinalis, has demonstrated a negative impact on weight owing to inbreeding (Cooper 1961). Ryman(1970) reported a decrease in recapture frequency in Atlantic salmon, Salmo salar, with increased levels of inbreeding.
In general, the results of these studies suggest a negative
impact on a variety of biological traits in the populations
studied and, consequently, on production.
No studies have been published on the effects of inbreeding on Pacific salmon, Oncorhynchus spp., nor have any
published reports dealt with the effects of inbreeding in conjunction with a selection and breeding program designed
to develop a genetically improved stock for aquacultural
purposes. To some degree, both of these deficiencies in information are being eliminated as Pacific salmon are used
for captive culture. It is imperative that data be obtainedon inbreeding in these species under defined programs todetermine their response to selection.

Research Rationale

a broodstock with traits that are beneficial to the production of 300-350 g coho salmon for the "plate-size" salmon
market.
The traits that have been emphasized for selective improvement are 1) freshwater growth, 2) smoltification, and3) saltwater growth to harvest size. Genetic analyses ofthese traits in the stock employed by Domsea Farms revealed adequate variability to expect progress from selection (Iwamoto et al. 1982; Hershberger and Iwamoto 1984;
Saxton et al. 1984).
Using estimated genetic values and considering thatthe facilities available to the program would only allow
raising 40 families of 600 individuals or less, a selection
scheme was designed to yield maximum response and to
be useful in a commercial operation (Fig. 1). This scheme
involved several different types of concurrent selection(e.g., family and individual) and used a selection index thatincorporated heritability estimates, relative economic
values, genetic correlations, and mean values on all thetraits of interest. It was recognized early in the development of this scheme that potential inbreeding problems
could arise from the rather severe limitation in breeding
population size (only twenty individuals contribute toeach generation). Consequently, breeding was conductedby a rotational line-crossing procedure (Fig. 2) to minimize
the possibility of crossing within lines. On a theoreticalbasis, these steps should limit the change in inbreeding_

The University of Washington, Domsea Farms, Inc., and
the Washington Sea Grant Program have been conducting a selection and breeding program with coho salmon,
O. kisutch, to develop a broodstock for the marine net-pen
industry in the State of Washington. The major objectiveof this nine year cooperative program has been to develop

3.5 MONTH SALTWATERSAMPLING

FRESHWATER

8MONTH
SALTWAUft

SAMPliNG
SALTWATER
PHASE II,,~MfIlING".,Fish in Excess
of 600 per
Family'................ ",.'25 Families'.' "....... "SALTWATER
PHASE III

INCUBATIONPERFORMANCE
5 Families'

SPAWNING

Figure 1
Diagram of the selection scheme used todevelop coho salmon stocks for marine penculture. The entire cycle represents a twoyear generation interval.

____________________ Hershberger et al.: Assessment of Salmon Broodstock Development

3

IF~ILY I

rFAMiTv
L...!......J

\. I

~
l

FROM

'~\~

TO

':ILY1

6 FLL-

6 FLLSIBFAMILIES

6 FLLSIBFAMILIES

6 FLLSIBFAMILIESSIB
FAMILIES

6 FLLSIBFAMILIESrFAMiiJ1
L...2Ul~. ~ILY6TO

6 FLL-

6 FLLSIBFAMILIES

6 FLLSIBFAMILIES

6 FLLSIBFAMILIESSJB
FAMILIES

to about 1 % per generation (Hershberger and Iwamoto
1984).
In 1983 (for the odd-year line) and 1984 (for the evenyear line) a decrease in the growth of selected fish insaltwater was observed (Fig. 3). One possible explanation

700
600

~
«
c::

C!J

500
400

300
""

200

1977

..

"

1978

1979

1980

1981

1982

1983

1984

1985

1986

6 FLLSIBFAMILIES

Figure 2
Diagram of the rotational line mating
system used in crossing selected individuals. The asterisk indicates thateach family cross is composed of six
single-pair matings to form six double
first-cousin families.

for this growth depression would be the accumulation ofdeleterious alleles through inbreeding. Even with the precautions taken in the design of the selection and breeding
program, there were two potential sources of inbreedingthat could not be quantitated. First, an unknown amountof inbreeding may have been introduced by selection andbreeding that had occurred prior to use of this designed
program. Second, because of some unexpected husbandry
problems with raising fish to maturity there was a strong
probability that a few families contributed disporportionately to the subsequent generations. Prior to the definition of pedigrees for the two lines, the importance of thesefactors was undeterminable.
As a result of these indications, studies were initiatedto 1) determine the actual levels of inbreeding in the twolines and 2) define the best approach to eliminate inbreeding in the selected stocks.

BROODYEAR...' OOD·VEAR LINE

-0- EVEN-YEAR LINE<>- WILD CONTROLS

Determination of Inbreeding Level
Figure 3
Average weight (grams) of selected broodstock and wild controls
after 8 months rearing in marine net-pens. Weights for 1986 aregiven as unadjusted (1) and adjusted (2) for density differencesthat year. N = 1200-2200 for selected broodstock and N
15-35 for wild controls.

_

The level of inbreeding in each of the two selected lines(i.e., odd- and even-year) was determined by two different methods. First, pedigree analyses were employed todetermine the coefficient of inbreeding (F) (Falconer 1981).
Computation of this value is accomplished by tracing the

4

NOAA Technical Report NMFS 92

D0MSEA COHO SALMONSEAWATER BROODSTOCKODD-YEAR LINE PEDIGREE_

BROODYEAR
1977

1979

1981

1983

1985

1987

pedigree back to common ancestors and determining theprobability that a pair of alleles are identical by descent.
Second, the change in genotype frequencies of electrophoretically analyzed protein differences were determined
and the difference in heterozygote frequencies equated toan apparent inbreeding coefficient (Hartl 1980). Electrophoretic analyses were conducted on serum samples from
100-120 adult fish in each offour years (1977, 1978, 1985,
and 1986). The electrophoretic procedures employed were
those reported in Utter et al. (1970) for analysis of serumtransferrins in coho salmon.
Construction of the pedigrees for the two lines of coho
salmon revealed more closely related families than was
originally anticipated (Fig. 4). Calculation of an inbreed-

Figure 4
Pedigree of matings between selected families for the odd-year
broodstock line (1978-1986).
Families enclosed by a stripedbox are double first cousins.

ing coefficient from these pedigrees (Table 1) indicates thatthe current level of inbreeding is not too severe, although
the estimate for the next generation (1987 broodyear) willapproach 8-10 %. These levels of inbreeding would not be
anticipated to cause the level of change found in the response of growth to selection. It has been estimated thatin domesticated animals selection can balance an increase
in inbreeding of approximately 2 % per generation (Pirchner 1969). The estimated levels of inbreeding in coho
salmon lines, to the point where apparent inbreeding
depression was noted (1983 and 1984), are below this value.
However, the coefficients reflect only the inbreeding since
the program was initiated and do not provide a measureof prior inbreeding. Further, it is difficult to determine what

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Hershberger et a1.: Assessment of Salmon Broodstock Development

5

Table 1Inbreeding estimates based on pedigree analysis for both odd- and even-year lines, andbased solely on effective population size (Ne). The estimates are calculated assuming theinitial inbreeding coefficient (F) is equal to O.
!J.F = (1I2N + 4)'

Pedigree estimates
Odd-yearControl

0.00
0.00
0.32
2.34
4.86
8.34

2.50
4.71
8.68
11.00
13.79

1977
1979
1981
1983
1985
1987

Even-year

Control

Odd

Even

0.00
0.00
0.63
4.22
5.90

2.50
5.75
9.11
12.20

0.00
2.27
4.49
8.78
10.86
12.88

0.00
2.27
4.41
6.58
8.52

1978
1980
1982
1984
1986

'Theoretical !J. F excluding sib-matings.

the effects of an incremental change in inbreeding may bein a species that has been recently developed from naturally
reproducing populations (Soule 1980).
The second type of inbreeding assessment employed electrophoretic analysis of the transferrin locus, which has beenshown to have three variant alleles (Utter et al. 1970) andis one of the few genetically variable protein loci found incoho salmon (Utter et al. 1980). Comparison of the genotype and gene frequency values in the original adult population with those from the fourth generation of selected
stock (Table 2) revealed changes that would be anticipatedin an inbred population (Falconer 1981); that is, there wasa decrease in the frequency ofheterozygotes and, with one
exception, there was little change in the gene frequencies.
Calculation of apparent inbreeding coefficients based on
the frequency change in heterozygotes (Fig. 5) yields amuch larger value than was obtained from the pedigree
analyses (Table 1).
It is possible to rationalize the discrepancy in these values
on two bases. First, there is evidence suggesting selective
differences among the various alleles of the transferrin locus(Suzumoto et al. 1977; Pratschner 1977). The results ofPratschner's research indicated that fish with the "A" and"C" alleles were more resistant to challenges by Vibrio
bacteria than those with the" B" allele, and Suzumoto etal (1977) found that the "A" allele imparted higher survival to BKD (bacterial kidney disease) challenge. If suchselective pressures were applied to the selected coho salmonlines, analyses based on the genotype frequencies would
tend to overestimate the inbreeding coefficient. The datafrom the current study support the hypothesis that fish withthe "A" and "C" alleles have a selective advantage, andTable 2
Observed transferrin gene and genotype frequencies in the odd- and even-year lines ofcoho salmon and their changes over four generations of selection (N = 100-120).
Odd-year broodstock line
Genotype
Year
1977
1985

ChangeGene frequency

AA

AB

AC

BB

BC

CC

JA

JB

JC

0.00
0.05

0.08
0.03

0.33
0.08

0.00
0.00

0.13
0.18

0.48
0.68

0.20
0.10

0.10
0.10

0.70
0.80

+ 0.05- 0.05- 0.25+ 0.00+ 0.05+ 0.20- 0.100.00+ 0.10Even-year broodstock line
Genotype
Year
1978
1986

ChangeGene frequency

AA

AB

AC

BB

BC

CC

JA

JB

JC

0.10
0.12

0.05
0.00

0.45
0.42

0.05
0.00

0.25
0.04

0.10
0.42

0.35
0.33

0.20
0.02

0.45
0.65

+ 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

Estimates of the apprent inbreeding coefficients for the odd- andeven-year lines based on the changes in observed and expected
genotype frequencies of the coho transferrin alleles.

ODD-YEARACTUAL HETEROZYGOSITY VS. EXPECTED

1977.525·.46/.46 =+14.1 %1985.275 - .525 1.525 = • 47.6 %Table 3

CHANGE IN OBSERVED HETEROZYGOSITY

The relative growth and survival of interstrain (Domsea
x Univ. ofWA) and intrastrain (Domsea odd- x evenyear) crosses after 8 months rearing in marine net-pens.
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/IOO. N
8-45 for each cross.

CHANGE 1977 to 1985: .275· .525/.525 = - 47.6 %ESTIMATED t.F = 41.9 %EVEN-YEAR

Outcrossing schemes
RelativeweightACTUAL HETEROZYGOSITY VS. EXPECTED
1978.75 - .635/.635 = + 18.1 %

Index

100
25
25
21.4

100
36.8
35.3
11.7

DOMSEA line crosses
Relative
Relativeweightsurvival

Index

DOMSEA (D)D x UW (9 x et)
UW x D (9 x et)University of Wash. (UW)1986 .461 - .465 I .465 = - 0.8 %CHANGE IN OBSERVED HETEROZYGOSITY
CHANGE 1978 to 1986: .461 - .75/.75 = - 38.5 %ESTIMATED t.F =29.3 %

vibriosis is a common problem in the marine net-pen
culture of salmon. The directed selection practiced on thestock 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 coefficient is the absence of selection. Such an assumption isclearly not valid in this situation and may result in the
inflation of the calculated value.
To summarize, it appears that pedigree analysis is thebest approach to determine inbreeding levels in coho
salmon. Thus, it would seem wise to assure that a selection and breeding program incorporates the mechanismsthat define accurate pedigrees of the breeding population.
Further, caution should be exercised in the use of genotype frequency changes to determine absolute values for
inbreeding coefficients. The potential effects of direct andindirect selection must be determined for these values tobe considered as valid measurements of inbreeding.

Elimination of Inbreeding

Relativesurvival_

Although the apparent levels of inbreeding in the selected
stocks of coho salmon were not large, two approaches toelimination of accumulated inbreeding were investigated:
outcrossing between stocks and outcrossing between lines
within stocks. Test crosses were made between the Domsea

DOMSEA (2DOMSEA (2DOMSEA (3DOMSEA (3

x 2)
x 3)
x 2)
x 3)

100
147
141
55.1

100
116.1
101.4·
128.7

lOa
ISO
225

lOa

lOa
174.5
174.1
128.7

coho salmon stock and the hatchery stock of the University of Washington, and between the Domsea odd- andeven-year parallel-selected lines. Progeny from these crosses
were reared in conjunction with the broodstock line.
It is apparent from the data (Table 3) that the progeny
from the crosses derived from the Domsea intrastock crosses
were superior to the interstock cross at the time of harvest.
Although both of the University of Washington x Domsea
hybrids were larger after eight months of saltwater rearing, relative to the Domsea controls, the overall survivalof both the hybrids and the University of Washington fish
was extremely poor under net-pen conditions. The highvalues reported reflect the survival of a few large hybrids
which biased the weight measurements. The University ofWashington x Domsea hybrids may not necessarily be
indicative of all interstock crosses, but the results suggestthat extensive hybrid testing may be necessary to identify
a complementary stock. The Domsea intrastock hybrids,
however, showed both good growth and greatly improved
survival relative to controls. Maintaining "in-house"
parallel selection lines may be a more efficient expenditureof effort relative to testing outcrosses. The "odd x even"_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Hershberger et aI.: Assessment of Salmon Broodstock Development

crosses would appear to be the method-of-choice for alleviating the inbreeding "load" while preserving selection
gams.

Implications for
Broodstock Development_

The coho salmon stocks that have been developed as aresult of this research program have, apparently, not yet
reached a level of inbreeding which would result in a strong
negative impact on their performance. The depression in
growth observed in both lines appears to have been environmentally generated and subsequent generations have
performed well (Fig. 3). However, analyses of inbreedingin these lines have demonstrated several areas requiring
special consideration in the development of aquaculture
broodstocks. Where possible, a selection and breeding program should be initiated with a large enough population
size to completely address the combined needs of a reasonable selection differential and elimination of close familial
relationships. Otherwise, definitive steps must be taken in
the formulation of the selection and breeding program tominimize the accumulation of inbreeding from thesefactors.
Further, a broodstock program should be initiated fromeither an outbred population with an inbreeding coefficient (F) equal, or close to 0, or from a stock with a defined
and well maintained pedigree. This would insure that the
inbreeding level could be unquestionably determined andthe effects of any increases could be well defined. In addition, research is needed to determine the response of aquacultural species recently derived from wild populations toan increase in inbreeding level. While the response ofdomesticated animals to increases in inbreeding has been
quantitated to some degree (Pirchner 1969), there is noa priori method by which to predict the magnitude of
responses in natural populations. As indicated by Gall
(1987), the best information will be obtained from inducing high levels of inbreeding in such stocks and quantifying the effects. However, inbreeding effects observed in
the progeny of sib-matings are indicative of, but not highly
correlated with the performance of individuals with equal
inbreeding levels produced through generations of matings.
Finally, it appears that using parallel selection in at least
two separate lines of broodstock would be a valuable approach to incorporate into a selection and breeding program. This provides an additional data set with which toevaluate a selection program and also incorporates amechanism that has the potential to eliminate inbreeding
effects without the loss of advances made in the traits thatare beneficial to aquaculture production.

Citations

7_

Aulstad, D., and Kittlesen.
1971. Abnormal body curvatures of rainbow trout (Salmo gairdnen)
inbred fry. J. Fish. Res. Board Can. 28:1918-1920.
Cooper, E. L.
1961. Growth of wild and hatchery strains of brook trout. Trans.
Am. Fish. Soc. 23:614-617.
Falconer, D.S.
1981. Introduction to quantitative genetics. Longman, Inc. New
York, NY, 340 p.
Gall, G.A.E.
1987. Inbreeding. In Population genetics & fishery management(N. Ryman and F. Utter, eds.), p. 47-87. Univ. Washington
Press, Seattle, W A.
Hartl, D.L.
1980. Principles of population genetics. Sinauer Assoc., Inc.
Sunderland, MA, 488 p.
Hershberger, W.K., and R.N. Iwamoto.
1984. Systematic genetic selection and breeding in salmonid culture
and enhancement programs. In Proceedings of the 11th U.S.Japan Meeting on Aquaculture, salmon enhancement; 19-20
October 1982, Tokyo, Japan, p. 29-32. U.S. Dep. Commer.,
NOAA Tech. Rep. NMFS 27.
Iwamoto, R.N., A.M. Saxton, and W.K. Hershberger.
1982. Genetic estimates for length and weight of coho salmon(Oncorhynchus kisutch) during freshwater rearing. J. Hered.
73:187-191.
Kincaid, H.L.
1976. Effects of inbreeding on rainbow trout populations. Trans.
Am. Fish. Soc. 105:273-280.
1983. Inbreeding in fish populations used for aquaculture. Aquaculture 33:215-227.
Pirchner, F.
1969. Population Genetics in Animal Breeding. W.H. Freeman
and Co., San Francisco, CA, 274 p.
Pratschner, G.A.
1977. Relative resistance of six transferrin phenotypes of coho
salmon to cytophagosis, furunculosis and vibriosis. M.S. Thesis,
Univ. Washington, Seattle, WA, 71 p.
Ryman, N.
1970. A genetic analysis of recapture frequencies of released youngof salmon (Salmo salar L.). Hereditas 65: 159-160.
Saxton, A.M., W.K. Hershberger, and R.N. Iwamoto.
1984. Smoltification in the net-pen culture of accelerated coho
salmon (Oncorhynchus kisutch); quantitative genetic analysis. Trans.
Am. Fish. Soc. 113:339-347.
Soule, M.E.
1980. Thresholds for survival: maintaining fitness and evolutionary
potential. In Conservation Biology (M.E. Soule and B.A. Wilcox,eds.), p. 151-169. Sinauer Assoc., Inc., Sunderland, MA.
Suzumoto, B.K., C.B. Schreck, and J.D. McIntyre.
1977. Relative resistances of three transferrin genotypes of coho
salmon (Oncorhynchus kistuch) and their hematological responses tobacterial kidney disease. J. Fish. Res. Board Can. 34:1-8.
Utter, F.M., W.E. Ames, and H.O. Hodgins.
1970. Transferrin polymorphism in coho salmon (Oncorhynchus
kisutch). J. Fish. Res. Board Can. 27:2371-2373.
Utter, F.M., D. Campton, S. Grant, G. Milner,]. Seeb, and L. Wishard.
1980. Population structures of indigenous salmonis species of the
Pacific Northwest. In Salmonid ecosystems of the North Pacific(W.J. McNeil and D.C. Himsworth, eds.), p. 285-304. Oregon
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

ABSTRACTTechniques to manipulate chromosome sets and produce polyploid fishes or fishes with all theinheritance 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 wehave explored in salmonids in our laboratory and in collaboration with other laboratories havebeen 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 thechum 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 hematopoietic
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 inactivation of the paternal genome by radiation or chemical treatment of the sperm. However, we havedemonstrated 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 andisozyme 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 potentiallybe 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 homozygous diploids. We have successfully induced androgenesis in rainbow trout and have also produced androgenetic progeny from homozygous androgenetic males. Androgenesis has a numberof 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 mercenarza
ROBERT T. DILLON jr.
Department of Biology
College of Charleston
Charleston, SC 29424

JOHN j. MANZIMarine Resources Research InstituteCharleston, SC 29412

ABSTRACTA large-scale breeding program has been initiated in South Carolina to achieve improved growthand 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 ResearchInstitute, 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 production 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 24 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 averaged 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 aremarking the entire genomes of their parents, and that variation in the growth rates of the offspring from individual clams may be obscuring any relationship with overall heterozygosity.

11

A Preliminary Study on Genetics of Two Types of the Rotifer
Brachionus plicatilis
YONG FU, YUTAKA NATSUKARI, and KAZUTSUGU HIRAYAMA
Faculty oj Fisheries
Nagasaki University
Bunkyomachi, Nagasaki
Nagasaki 852, Japan

ABSTRACTThe domesticated rotifer Brach ion us plicatilis can be divided roughly into two types, called
Land 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 toenvironmental factors make this method unreliable. We have, therefore, tried to clarify differences 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 (quantitative). To accomplish this, morphological features were measured, the ratios of which createdan 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 Land 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 consisted of only S-type strains, the other group was subdivided again into 3 clusters. One of thesethree 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 Land S strains.

Introduction_

Since the introduction of the rotifer Brachionus plicatilis tonourish larval fish, aquaculturists have increased scientificattention on this organism. In Japan a significant achievement in rotifer biology was the discovery that the domesticated rotifers can be divided roughly into two so-called
Sand L types as shown in Figure 1 (Fukusho 1983). The
main morphological differences between the two types arelorica size, lorica shape, and the shape of the anterior spines
on the lorica. They also exhibit differences in growth withrespect to temperature. The morphological and physiological differences in the two types were summarized in aprevious review (Hirayama 1987). The rotifer, especiallythe domesticated rotifer, exhibits cyclomorphosis (seasonalvariation in size) and also polymorphosis (change in size
influenced by variations in diet) (Fukusho and Iwamoto
1980, 1981). So, there is a probability that observed differences could be attributed to cyclo- or poly-morphosis,

not to genetic differences. However, Fukusho and Okauchi(1982, 1983, 1984) have provided evidence that differences
may be genetic and that the two types can be isolated fromeach other. In countries outside Japan, many scientists
recognize the variation of rotifers which is due to polymorphosis. Scientific approaches concerning analysis ofallozyme variation have therefore been investigated (Serraand Miracle 1983, 1985, 1987; Snell and Carrillo 1984;
Snell and Winkler 1984; Suzuki 1983, 1987; King andZhao 1987), while in Japan there have been no studies todetect allozyme variation in the two types by means of electrophoretic procedures.
Using strains collected from many locations, we attempted to distinguish Land S types using morphologicalcomparisons. In order to confirm the genetic differences
between strains, allozyme variations were detected byhorizontal starch gel electrophoresis. Then, the genetic
distances among collected strains were compared for morphological similarities.
13

14

NOAA Technical Report NMFS 92

_

5

L

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

Materials and Methods_

We collected many strains from all over the world. On themap (Fig. 2), the localities of 34 strains used in this study
are shown. Table 1 shows the abbreviated names andorigins of the strains. In the tables and figures, L- andS-type strains are shown by abbreviation wid. capital .md
small letters, respectively.

collected those eggs into test tubes reculturing them again
with marine Chlorella. After the offspring hatching fromthose eggs grew and laid their first eggs, we performed morphological measurements. We removed 20 individuals per
sample and measured seven morphological features (Fig.
3, A through G). The ratios of these measurements wereused to cr~ate indices for a cluster analysis.

Allozyme Analysis
Morphological Analysis
We first observed the anterior spines of each of the 34
strains and qualitatively divided them into the two types,
Land S, according to whether they had obtuse angled orpointed spine~, respectively. We classified 15 strains into
the L type and 19 strains into the S type. After the initial
screening, one individual from each ~train was selected for
culturing parthenogenetically and was regarded as one
genetic strain for further study. Each strain was cultured
with marine Chlorella (NannochloTOPsis oculata). We collected
eggs and recultured each strain in marine Chlorella suspensions in 23°C. The first eggs were laid after 48 hours. We

The same 34 strains were used both for electrophoretic andmorphological analysis. Allozyme analysis for each strain
was conducted with a population grown from one individual and cultured with marine Chlorella and baker's
yeast. The population was harvested with a net, washedwith clean seawater several times, blotted dry using filter
paper and frozen at - 30°C until analyzed. Before harvesting, the group was starved for one day to remove theinfluences of food. Immediatel~' prior to electrophoretic
analysis, we thawed the sample and used a small amountof the drip absorbed by filter paper as a crude extract of
enzyme for allozyme aiayisis. Electrophoresis were carried

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

15

j-amp

J-NSGT
J-NSGT-II

J-TKU

~#,
i=~~g J.
J-NSZ
j -nszlocality 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
by Clayton and Tretiak (1972) with minor modifications(Table 2). Staining procedures were from Shaw and Prasad(1970) and Siciliano and Shaw (1976). The following 18
enzymes were tested: a-Glycerophosphate dehydrogenase(aGPD, EC 1.1.1.8); D-Sorbitol dehydrogenase (SDH, EC
1.1.1.14); Lactate dehydrogenase (LDH, EC 1.1.1. 27;
3-Hydrooxybutyrate dehydrogenase (HBDH, EC
1.1.1.30); Malate dehydrogenase (MDH, EC 1.1.1.37);
Malic enzyme (ME, EC 1.1.1.40); Isocitrate dehydrogenase (IDH, EC 1.1.1. 42); 6-Phosphogluconate
dehydrogenase (6PGD, EC 1.1.1.44); Glucose-6-phosphate
dehydrogenase (G6PD, EC 1.1.1.49); Superoxide
dismutase (SOD, EC 1.15.1.1); Aspartate aminotransferase (AAT, EC 2.6.1.1); Adenylate kinase (AK, EC
2.7.4.3); Phosphoglucomutase (PGM, EC 2.7.5.1);
Esterase (EST, EC 3.1.1.1); Alkaline phosphatase (ALP,EC 3.1.3.1); Acid phosphatase (ACP, EC 3.1((/~

11 J::.3

I~
L-..J

J-'t;

Leucine aminopeptidase (LAP, EC 3.4.11.1); and Glucose
phosphaste isomerase (GPI, EC 5.3.1.9). t.d-....

~ {2f' Q. (I.-(.. (f=Results_2.~~
Morphological Analysis
In Figure 3, are shown the average morphological measurements and standard deviations of the 15 Land 19 S
strains. The results indicate that the strains of the rotifer
could be divided clearly into the two types by quantitating
the shape of the anterior spine (E/D, GIF). The results ofthe cluster analysis (Fig. 4A) are identical to the classification judging by the anterior spine shape (15 L types, 19
S types). Each cluster can be divided again into 2 small
clusters. These results indicate that with statistical treatment of the morphologica charcteristics, the varieties of the

if

16

NOAA Technical Report NMFS 92_

Table 1Abbreviated names and origins of 34 strains of Brachionus plicalilis testrd for morphological and genetic differences. PE: Prefectural 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. Capitaland small letters mean that the strain belongs to Land S type respectively .

_-----------------------.

AbbreviatednameCountry

j-amp
j-kay
j-ehp
j-otk
j-nsz
j-kgkoj-kgko '86
j-kgs
a-sal
a-mk
c-xm
p-ilo
p-Ie

japan
japan
japan
japan
japan
japan
japan
japan
USA
USA
China
Philippines
Philippines

Aomori
KagawaEhime
Oita
NagasakiKagoshima (Kai Lake)
Kagoshima (Kai Lake)
Kagoshima (Shibushi
California (Salton Sea)
Florida (Makay Bay)Fujian Fish. Res. Inst.
Panay Island
Panay Island

p-ot
i-ja

Philippines
Indonesia
Singapore
ThailandThailandIsrael
japan
japan
japan
japan
japan
japan
japan
japan
japan
japan
Japan
FranceFranceFranceFranceOton River (Panay Island)
java
Nat!. Inst. of Aquaculture
SonkiaPuket Marine Inst.
Eilat
Shizuoka
Univ. Tokyo
Oita (kamiura)
Saga
Saga
Nagasaki Univ.
NagasakiNagasakiNagasaki (Goto Island)
Nagasaki (Goto Island)
Kagoshima Univ.
Palavas-Ies- Flots
Palavas-Ies- Flots
Palavas-les- Flots
Palavas-les- Flots

S-Sln

(-son
t-pu
is-eil
j-SOI
j-TKU
j-OTK
j-SAP
j-SAP'86
j-NSU
j-NSZ
j-NSC
j-NSGT
j-NSGT-II
J-KAU
F-PA
F-PA-Il
F-PA-IlI
F-PA-IV

Station or
hatchery

LocalityL-

Wild (w) ordomesticated (d)

'87
'87
'87
'87
'86
'78
'86
'87
'78
'80
'87
'84
'84

d
d
d
d
d
w
w
d
w
w
d
d
d

'84
'86
'86
'87
'87
'87
'78
'78
'87
'84
'86
'69
'86
'86
'87
'87
'86
'87
'87
'87
'87

w
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d

PE
PE
PE
SFC
PE

SFC

AQD SEAFDEC
Leganes Stn.
AQD SEAFDEC

NICA

PE
SFC
PE
PE
PE
CE
SFC
SFC

Table 2
Buffer systems used for electrophoresis of enzymes.
AbbreviatednameYear ofcollection

Electrode buffer------------

Gel buffer

Components

pH

Components

pH

References

C-A

0.04 m Citric acid,adjust pH up to 6.1 with
N-(3-aminopropyl)-morpholine.

6.1

Dilute 50 mL of electrode buffer
to I liter (Citric acid, 0.002 M).

6.1

Clayton and Tretiak (1972)

C-A

0.04 m Citric acid,adjust pH up (0 6.1 withN -(3-aminopropyl)-morpholine,
then to 6.9 with NaOH.

6.9

Dilute 50 mL of electrode buffer
to I liter (Citric acid, 0.002 M).

6.9

Clay and Tretiak (1972)

C-T

0.04 m Citric acid,adjust pH up to 8.0 with
Tris-(hydroxymethyl)-methylamine.

8.0

Dilute 50 mL of electrode buffer
to I liter (Citric acid, 0.002 M).

8.0

Clayton and Tretiak (1972)_

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

10

1M
§

rotifer can be divided into two groups, and that the strains
within the same type display further variation.

s

A

L

206

277

f---<)---------

,

f

•

220

200

100

2fJJ

240
L

•

I

,

,

,

,
0.6

085

L

f------O---l

05

•

0.662

,
01

L

1'1

E~
F

T"

~

0.8

0~9

10

,'2

1:1

L

G

H

0:7

0.8

S
1.095
0

•

5

Among 18 enzymes tested, 10 enzymes showed clear banding patterns (Table 3). However, bandings for 3 enzymes(AK, EST, and IDH) were not genetically interpretable.
The number of alleles of each locus are summarized inTable 4. On MDH, 3 isozyme loci were recognized, although no alleles were detected at 2 loci. In Table 5 areshown the number of L- and S-type strains and the alleles
they posses at each locus. The Land S strains differ considerably in allele profiles. For instance, at Ldh where 8
alleles were observed, 9 of 15 L strains possessed the A
allele whereas none of S strains possessed the A allele. In
contrast, B allele appeared only in the S strains. There wereconsiderable genetic differences between Land S strainsfor 15 alleles at 6 loci. Allele frequencies for each allele at
9 loci affecting 7 enzymes were estimated for each strain
in which individuals were considered to be genetically identical. For MDH, however, three zones of banding patterns
appeared. Although two of those three zones were not interpretable as showing allozyme variation, we regarded
allele frequency as one if the strain had the bandings inS
I 0.821

0

S
0.619

J1

Allozyme Analysis)Cxx/,rrI

rl.ao

0.755

0

2ro

0.792

0.75

C

S
0.568
0

0.348
~

0')

,

,

0·4

05

07

0:6

Figure .3
Averages and standard deviations of 5 varieties of measurements
considered for differentiating L- and S-type strains.

(A)

80

60

morphological

40

20

a

0

I

J-amp

t-s 0 ni s-e i 1
i-j a
p-o t
s-s i nj -k g k 0 ·86

t-pu
c-xm
p-i 10j-kay
J-k g sJ -0 S z
J-e h pj-at kp-Ie
a-mk
a-s a I

j-kgkoJ-TKUF-PA-n
F-PA
J-SAP
J-NSZ
J-NSGT-n
J-NSGT
J-KAU[~=;~~.86
J-OTK
F-PA-mF-PA-IV
J-NSC
J-NSU

80

60

17

40
20
fusion level( B)
genetical
0.2
, 0.4
, 0.6
, 0.8 1.0 1.2 1.4 1.6 1.8
i

i

i

i

i

i

t-s 0 np-o t
j -k g k 0 ·86

a-s a 1
j -k g k 0
i-j a
j-k ayj-e h pa-mk

J=~~ k]~= ~ ~ 0]s-s i nt-p u
j-k g sj-n s 7.
j-amp

i s-e i 1
F-PA-D
F-PA
J-TKU
J-SAP
J-NSZ

~=~;g:::-nl
J-NSC
J-SOI
~
J-SAP· 86
J-OTK
J-KAU

~=~~:~]

J-NSU

I

!

I

I

i

i

i

I

!

I

a 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
genetical distance

Figure 4
Dendrograms of similarities among 34 strains by morphological and genetic snalyses. Abbreviated names by capital and small lettersmean that the strain belongs to Land S type, respectively.

18

NOAA Technical Report NMFS 92

Table .3
The different enzyme systems of Brachionus plicatilis screened
with various buffers. x = no detectable bandings; ...
= unclear bandings; • = find bandings.

Table 5The number of L- and S-type strains for each allele at different enzyme loci. • = Great difference in allele possession between L- and S-type strains.

BufferEnzymeRelative mobility
Locus
C-T
(pH 8.0)

x
x

x
x

x
x

• A
• C
B

A

eD

SDH
LDH
HBDH
MDH
ME
IDH
6PGD
G6PD
SOD
AAT
AK
PGM
EST
ALP
ACP
LAP
GPr

•
•
A

A
x

x

A
x

A
A

•
A

x

•
A
•
A
A
A

x

A

A

0,

A
A
A
A
A
A
A

A

x

E

A
A
x
x

•
••
•
•
A

x

x

•

A

Mdh-ll

O2
A
B
C
e ?

Mdh-lll

e ?

6Pgd

A
e B
BL
C
CL

Mdh-l

x

•

• D
DL
e E
F
Sod

Enzyme

Locus

LDH
MDH

eA

2
9
0
4
1
1
0
1

0
0
5
4
12
3
2
0

100
83
62

0
13
15

3
16
9
17

0
15

6

100
91
75
68
55
( - )39(- )52(- )68( - ) 75

0
10
1
4
2
0
0
0
0

1
0
1
1
0
10
1
13
1

100
95

6
0
17
0

eD

49

0
10
0
5

e B
e C

Table 4
Isozyme loci and number of observed alleles.

100
81
69
64
47
39

X

C-A
(pH 6.9)

aGPD

S-Type
(19)

(%)

C-A
(pH 6.1)

Ldh

L-Type(15)

Allele74

AlleleSubunit structure

Aat-l

A
B

11
15

8
15

Ldh

8

Pgm-l

Mdh-l
Mdh-llMdh-lll3

Tetramer
Dimer

100
75

9
4

A
B
C
D
E

100
93
89
86
79

4
3
1
7
3
0
0
1

6
2
2
6
9
1
2
0
6
1
6
13
0
2

Dimer

6PGD

6Pgd

SOD
AAT
PGM

Sod
Pgm-l

2
8

Dimer
Dimer
Monomer

GPr

Gpi

6

Dimer

Aat-l

Gpi

F

77

G

70

°

e A
B
C

e D
e E
F

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 genefrequencies including estimated values for MDH. The dendrogram expressing similarities among the 34 strains was
also drawn from genetic distances (Fig. 4B). The 34 strains
can therefore be divided into two major groups. One groupconsists only of the strains which had been identified as
S type judging by the anterior spine shape. The othercluster can be divided again into 3 smaller clusters, one

100
78
67
43
28
0

0
0
2
0
13
7

of which consists only of S type strains, and the other two
clusters consist only of L-type strains. Although one of thetwo large clusters includes the two types of rotifers, theclassification by the genetic distances also pointed out that
there are great genetical distances between Land S strains.
Some of the strains which are genetically identical (e. g.,
genetic distance = 0) were collected from neighboring loca-

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Fu et al.: Genetics of the Rotifer BrachiQnus plicatilis

tions or from the same hatchery, for instances between thetwo strains of p-ilo and p-le or between J-NSGT and
J-NSGT-II. However, in one instance (c-xm and j-otk),
the samples were geographically unrelated.

Discussion_

For comparison, the two dendrograms are shown in thesame frame (Fig. 4). The dendrogram patterns for the two
methods are very similar, especially with respect to the
L-type strains.
The results indicate that the rotifer Brachionus plicatilis
can be divided into the two types of genetic constitution.
The results in this report were drawn from 34 strains,
collected mainly from western Japan. In the case of theL-type, the overseas strains obtained came from only one
locality. We are now collecting more strains from all over
the world in order to make a more unequivocal conclusion.

Acknowledgments
The authors wish to express their sincere thanks to
H. Kayano, Nagasaki University, for his kind advice on
the interpretation of allozyme variation, to K. Fukusho who
kindly provided photos of Land S strains, and also to thescientists who kindly sent us live samples of the rotifers.

Citations
Clayton, j.W., and D.N. Tretiak.
1972. Amine-citrate buffers for pH control in starch gel electrophoresis. j. Fish. Res. Board Canada 29: 1169-1172.
Fukusho, K.
1983. Present status and problems in culture of the rotifer Brachionus
plicatilis for fry production of marine fishes injapan. Symp. IntI.
Acuacultura Coquimbo, Chike-Septiembre:361-373.
Fukusho, K., and I. Iwamoto.
1980. Cyclomorphosis in size of the cultured rotifer, Brachionus
plicatilis. Bull. Natl. Res. Inst. Aquacult. 1:29-37.

19

1981. Polymorphosis in size of rotifer, Brachionus plicatilis, cultured
with various feeds. Bull. Natl. Res. Inst. Aquacult. 2:1-10.
Fukusho, K., and M. Okauchi.
1982. Strain and size of the rotifer, Brachionus plicatilis, being
cultured in southeast asian countries. Bull. Natl. Res. Inst.
Aquacult.3:107-109.
1983. Sympatry in natural distribution of two strains of a rotifer,
Brachionus plica/ilis. 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.
Hirayama, K.
1987. An Approach from the physiological aspect to the problems
in present mass culture technique of the rotifer. In Proceedingsof the 15th (1986) U.S. japan meeting on aquaculture (AI Sparks,ed.). U.S. Dep. Commer., NOAA Tech. Rep.
King, C.E., and Y. Zhao.
1987. Coexistence of rotifer (Brachionus plicatilis) clones in Soda Lake,
Nevada. Hydrobiologia 147:57-64.
Nei, M.
1972. Genetic distance between populations. Am. Nat. 106:283-292.
Serra, M., and M.R. Miracle.
1983. Biometric analysis of Brachionus plicatilis ecotypes from Spanish
lagoon. H ydrobiologia 104: 279-29l.
1985. Enzyme polymorphism in Brachionus plicatilis populations fromseveral 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:
83-90.
Shaw, C.R., and R. Prasad.
1970. Starch gel electrophoresis of enzymes - a compilation ofrecipes. Biochem. Genet. 4:297-320.
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
Ltd, London.
Snell, T.W., and K. Carrillo.
1984. Body size variation among strains of the rotifer Brachionus
plica/ilis. Aquaculture 37 :359-367.
Snell, T.W., and B.C. Winkler.
1984. Isozyme analysis of rotifer proteins. Biochem Syst Eco
12: 199-202.
Suzuki, M.
1983. Taxonomical study on rotifers cultured for fry production.
Zool. Mag. 91:657.
1987. Intraspecific variability of Brachionus plica/ilis. H ydrobiologia
147:45-48.

Present Status of Genetic Studies on Marine Finfish in JapanKUNIHIKO FUKUSHONational Research Institute oj Aquaculture
Fisheries Agency
Nakatsuhama, Nansei-cho, Watarai-gun, Mie
516-01 Japan

ABSTRACTThe 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 techniqueof genetic breeding for marine finfish, even though limited scientific data and experimental results
have been reported. Selection was conducted on red sea bream, Pagrus major, and Japanese flounder,
Paralichthys olivaceus, and their selected strains were supplied for industrial culture. Experimentson hybridization leading to heterosis were conducted as well as interspecific, intergeneric, interfamily, 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 manipulation 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 industrial 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 areimported 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 gynogenesis, and triploid production. This technology will
no doubt be adopted in androgenesis and gene bank projects for marine finfish.

Introduction_

Mariculture of finfish is well developed in Japan as reflectedby the total harvest in 1986 of nearly 2 x 10 5 tons. The
number of cultured species is approximately 30 (Table 1)(Fukusho 1981). Yellowtail, Seriola quinqueradiata (14.6 x
104 tons), and red sea bream (3.4 x 10 4 tons), are the mostimportant species. The Japanese flounder and coho
salmon, Oncorhynchus kisutch, have shown great promise ascultured species, with their production levels increasing
rapidly in recent years (0.3 x 10 4 and 1.2 x 10 4 tons, respectively for 1987 data). All the marine species are cultured in net cages except the Japanese flounder which isusually raised in land based tanks.
At present, the total supply is adequate to satisfy demandfor yellowtail and red sea bream, taking into consideration the total comsumptiom plus the carrying capacity of
the culture ground. Therefore, research presently focuseson the improvement of fish quality (e. g., high growth rate,
high resistance to pollution, good taste and flavor, andfavorable color) based on the requirements of culturists andconsumers. Genetic breeding is one of the most effective

tools for improving fish quality. Therefore, various kindsof experiments on genetics, including chromosome manipulation have been intensively conducted in recent years
for marine finfish, despite the short history of mariculture.
The objective of the present paper is to provide a brief
introduction on the status of genetic breeding of marine
finfish, except salmonids in Japan. Introductions leading
to domestication are considered part of the study of genetics for the purpose of this review.

The Introduction of Exotic Species_

Exotic species or strains of marine finfish, such as
yellowtail, red sea bream, rockfish, Sebasticus spp., grouper
Epinephelus spp., knifejaw Oplegnathus spp., have been introduced to Japan and cultured in recent years. Marine
finfish have been introduced for different reasons than havefreshwater fish. Most marine species are imported to supply
seed where the local production is inadequate, not to introduce a new industrial target species of strain. An exception is the case of the red sea bream.
21

22

NOAA Technical Report NMFS 92_

Table 1Marine finfish cultured in Japan (Fukusho 1981).
FamilyCommon nameScientific name

Pref. No.'

Salmonidae
Mugilidae
Oplegnathidae

Coho salmon
Grey mullet
Japanese striped knifejaw
Japanese spotted knifejaw
Sea bass
Sea bass
Groupers
Nibbler
Red sea bream
Porgy
Crimson sea bream
Porgy
Porgy
Grunt
Yellowtail
AmberjackAmberjackHorse mackerel
Striped jackBluefin tuna
Rabbit fish
FilefishPufferRockfishRockfishRockfishFlounderOncorhynchus kisutch
M ugi! cephalus
Oplegnathus fasciatus
O. punctatus
Lateolabrax japonicus
L. latus
Epinephelus spp.
Cirelta punctata
Pagrus major
Sparus sarba
Evynnis japonica
Acanthopagrus schiegeli
A. latus
Parapristipoma trilinea
Seriola quinqueradiata
S. purpurascens
S. aureovittata
Trachurus japonicus
Longiros/rum delicatissimus
Thunnus thynnus
Siganus fuscessens
Stephanolepis cirrhifer
Fugu rubripes
Sebastiscus marmora/us
Sebas/es inermis
S. schlegeli
Paralichthys olivaceus

11Serranidae
Girellidae
SparidaePomadasyidae
Carrangidae

Scombridae
Siganidae
Aluteridae
Tetradontidae
Scorpaenidae
Bothidae

19
7
5
1

3
11

25
2
11

17

2
1

26

8
2

20
9
3
3

2
9
3
1

10

'Number of prefectures where the species was cultured.

A deep reddish color is highly prized in cultured red sea
bream by the Japanese consumers. A Korean strain, which
is identical to the Japanese strain taxonomically, shows amuch deeper reddish color than the latter (Harada et al.
1988, Harada et al. 1985, Kumai et al. 1986). No difference
has been found between the two strains in electrophoresis
analysis of isozymes. The Korean strain is preferred by fish
farmers because of its deeper reddish color, even though
the japanese strain is superior to the Korean strain in termsof growth rate (Kumai et al. 1986). The deep reddish color
is caused by its higher content of carotenoid and astaxanthine in the skin, which results even under the same rearing and feeding conditions as the japanese strain (Kumai
et al. 1986). Hybridization between the japanese andKorean strains has been conducted to provide a hybrid witha deeper reddish color and higher growth rate (Haradaet al. 1988).
Both fertilized eggs and juveniles of japanese red sea
bream have been exported to foreign countries; Thus
marine finfish have been introduced world-wide and mariculture has expanded. In each country, they are being
cultured as an introduced species. Precise investigation andresearch of a new marine species' biological characteristics

and response to new environmental conditions should always be
considered prior to introduction, as with freshwater fish.

Hybridization_

Experiments on hybridization leading to heterosis have contributed to the development of larval rearing techniques formarine finfish (Fujita 1961,1967; Harada 1974,1975,1978).
Interspecific, intergeneric, interfamily, back and reciprocal
crosses have also been attempted (Harada 1978).
Hybridization of marine finfish was initiated on puffers(1961-67) of which several species are a high prized luxury fooddespite the fact that parts of these fish are highly toxic (Fujita1967) (Table 2). The Fisheries Laboratory of Kinki University
has further promoted hybridization to improve fish quality.
Several successful and promising hybrids were produced that aresuperior in growth rate, survival rate, body color, and meat
quality to each parent fish (Table 3) (Harada 1974, 1975, 1978).
The "kindai" (Oplegnathus jasciatus x O. punctatus) , which abbreviates the name of Kinki Vniversity and which means sea
bream and sounds like "golden fish" in japanese, is significantly
superior in growth rate, survival rate, and handling to each

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Fukusho: Genetic Studies on Marine Finfish in Japan

Table 3Hybrids of marine finfish produced at the Kinki U niversity (Harada 1974, 1975, 1978).

Table 2Hybridization of puffers (Fujita 1967).
FemaleTakifugu pardalis x
T. poecilonotus x
T. rubripes x
Lagocephalus lunaris spadiceus x
L. I. spadiceus x

Male

T. poecilonotus
T. pardalis
T. xanthopterus
T. niphob/es
Canthigaster rivulata

Hatching rate
(%)

95
95
93
0
0

parent fish (Kumai 1984; Harada et al. 1986). Because red
sea bream are usually cultured in protected bays with varying salinity, tolerance to low salinity is an important characteristic. Therefore, a useful hybrid of the red sea bream,
and the porgy, Acanthopagrus shulegeli, was developed whichshowed both improved tolerance to lower salinity than thematernal fish plus faster growth and better taste than thepaternal one (Harada 1975). The Nagasaki Prefectural
Institute of Fisheries has also conducted studies on the
hybridization of marine finfish. A hybrid of a sparid, Sparus
sarba, and the porgy, A. shulegeli, was produced in order
to combine the highest growth characteristics of S. sarba
and the low salinity resistance of A. schulegeli (Kitajima andTsukashima 1983). However, the hybrid showed maternal characteristics in both its morphological and physiological characteristics (Kitajima and Tsukashima 1983).
The same phenomenon occurred in the hybrid of P. major
and the crimson sea bream, Evynnisjaponicus (Arakawa andYoshida 1986, Arakawa et al. 1988).
Few of these hybrids have been widely used by industry,
except the hybrid of P. major x E. japonicus. The reason
may be due to 1) conservative consumers to whom appearance is very important (red sea bream must look like thewild red sea bream because the Japanese people always eat
the whole and raw fish for sashimi and sushi; 2) limited
attempts to show clearly the difference in quality between
the hybrid and parent fish; and 3) too short a period ofmarine finfish culture for the industry to evaluate andutilize new strains or hybrids as well as exotic species.

Selection

23_

Selection is the most successful technique of genetic breeding for marine finfish, even though limited scientific dataand experimental results have been reported. Selectedstrains of red sea bream have been supplied by the Fisheries Laboratory of Kinki University. These selections arehighly desired by fish farmers because their growth rates
are approximately 30-40% higher than the wild forms.
Mass selection has been conducted at the Fisheries Laboratory of Kinki University over several generations from

Femalex

MalePagrus major
P. major
Oplegnathus fasciatus
O. fasciatus'
Seriola quinqueradiata'
S. quinqueradiata
S. purpurascens'S. aureovittata
P. major'
O. puntatus'
Auxis thazardx

Acanthopagrus schulegeli
Sparus sarba
A. schlegeli
O. punctatus
S. auroeovittata
S. purpurascens
S. aureovittata
S. purpurascens
Evynnis Japonicus
O. fasciatus
Euthynnus afjinis

x
x
x
x
x
x
x
x
x
x

1964
67
68
69
70
70
71
72
73
73
76

'Promising hybrid.

wild red sea bream caught in the Akashi area in the Seto
Inland Sea. Production of selected strains has also been
intensively conducted on the japanese flounder and theJapanese sriped knifejaw, a.fasciatus, at the Fisheries Laboratory of Kinki University (Harada 1975, 1978).

Chromosome Manipulation_

Since 1984, chromosome manipulation studies such as
triploid production, all female production by gynogenesis,
extraction and synthesis of growth hormone, productionof cloned fish, and cell fusion have been conducted injapan to produce new strains of marine finfish. These
technologies have been called' 'Fisheries Biotechnology."
In 1985, the Ministry of Agriculture, Forestry, andFisheries (MAFF) designed and organized a large-scalescientific project on chromosome manipulation titled"Development of new breeding techniques by inductionof gynogenesis in fish." The National Research Instituteof Aquaculture was the leading institution to promote theproject, along with three Universities (Tokyo Universityof Fisheries, Hokkaido University, and Kansei Gakuin
University), two Regional Fisheries Laboratories (Nansei
Regional Fisheries Laboratory and Hokkaido Regional
Fisheries Laboratory of the Fisheries Agency-MAFF), andthree Prefectural Institutes of Fisheries (Saitama, Hyogo,
and Hokkaido). The target species included the japaneseflounder, red sea bream, plaices, Verasper moseri, Limanda
shrenki, L. punctatissima, Platichthys stellatus, and filefish,
Stephanolepis cirrhifer, Thamnaconus modestus, Aluterus monoceros.
The Fisheries Agency has also organized and initiated ascientific project addressing fisheries biotechnology. Several
Prefectural fisheries laboratories have also joined this"Local Biotechnological Study Project" where marine

24

NOAA Technical Report NMFS 92_

finfish such as the red sea bream and flounder are being
studied.
Triploid and gynogenetic diploid induction techniques
which use cold shock have been used to block the second
polar body extrusion for red sea bream (Arakawa et al.
1987; Arakawa and Miyahara 1988; Fukusho et aI. 1987b),
porgy (Arakawa et al. 1987), and flounder (Tabata, 1988;
Tabata and Corie 1988a, Tabata et al. 1986). The duration of cold shock intervals are as follows: for red sea bream,
15-20 min. duration ofO°C, starting 3 min. after insemination; for porgy, 25 min. duration, starting 1.5 min. after
insemination; and for flounder, 45 min. duration, starting 3-5 min. after insemination. Suppression of the 1stcleavage was achieved by using increased hydrostaticpressure (Tabata and Corie 1988b). UV irradiation has
been effective for genetic inactivation of sperm (e. g.,
1000-2000 erg/mm 2 for red sea bream). Also, sperm ofdifferent species have often been used as an indicator of
the induction of gynogenesis (Fukusho et al. 1987a; Yanoand Sakai 1988). Triploids were produced in red sea bream
and porgy (Arakawa et al. 1987, 1988; Fukusho et al.
1987b). Thus, various conditions for induction of triploidand gynogenesis have been examined for several marine
finfish, and comparisons of growth rate, survival rate, andother biological characteristics have been conducted
through larval rearing (Arakawa and Yoshida 1986; Fukusho et al. 1987a; Tabata et al. 1986; Tabata and Corie
1988a). Comparison of growth rate during rearing to young
stage and commercial size was also conducted (Tabata andCorie 1988a), but there is little information to evaluate
gynogenetic and triploid fish in view of industrial culture
because the scientific activities have only just started.
Reports and papers on the comparison of growth andmaturation rates and sex ratio between chromosome manipulated fish and common diploid fish are expected topromote these techniques in industrial mariculture of finfish. Techniques for examination of ploidy have been
established, but with conflicting result (e.g., appearanceof males among gynogenetic diploids of the flounder in spiteof a theoretical expectation of all female production)(Tabata 1988; Tabata and Corie 1988b). These phenomena could not be explained by the XX and XY sex
chromosome theory. Further investigation on embryology
and sexual differentiation might be required as well as
genetic studies. The mechanism of sexual differentiation
should be clarified to advance the technology of chromosome manipulation.

Cryopreservation of Sperm_

Experiments on androgenesis have been conducted for
freshwater fish in Japan. Cryopreservation of sperm incombination with androgenesis is useful in preserving endangered species, and also in all male production. Cryo-preservation is also used in most hybridization studies,
induction of gynogenesis, and triploid production.
In marine fish, cryopreservation has been conducted ona variety of species, such as: two species of goby, Glossogobius olivaceus, Acanthogobiusflavimanus; porgy; mullet, Mugil
cephalus; mackerel, Scomber japonicus; bluefin tuna, Thunnus
thynnus; and puffer, Takaifugu nipholbles (Doi et al. 1982;
Kurokura 1983). A recent study on the hybridization of
red sea bream and crimson sea bream showed positive
results with high survival rates and increased growth rates
observed when sperm preserved for 6 months was used
(Kurokura et al. 1986).
Thus, gamete preservation is useful for hybridization of
species which spawn in different seasons, genetic breedingby chromosome manipulation, transplantation (introduction), and gene bank projects for marine finfish.

Acknowledgments_

I wish to express my sincere thanks to Dr. F. Brian Davy,
IDRC, Canada and Dr. Ryo Suzuki, National Res. Inst.
Aquaculture, Japan for their critical reading of thismanuscript.

Citations_Arakawa, T., and J. Miyahara.
1988. Induction of gynogenesis with ultra violet rays in red sea
bream, Pagrus major. Bull. Nagasaki Pref. Ins!. Fish. 14:37-42.
(In Japanese; English summ.)
Arakawa, T., and Y. Yoshida.
1986. Growth, survival and morphologic comparison between fry
cross bred Pagrus major with Evynnis Japonica and hatchery reared
Pagrusu major. Bull. Nagasaki Pref. Ins!. Fish. 12:27-35. (InJapanese; English summ.)
Arakawa, T., M. Tanaka, K. Inoue, 1. Takami, and K. Yamashita.
1987. An examination of the conditions for triploid induction bycold shocck in red sea bream and black sea bream. Bull. Nagasaki
pref. Ins!. Fish. 132:25-30. (In Japanese; English summ.)
Arakawa, T., C. Kitajima, K. Yamashita, A. Ikeda, and H. Iimura.
1988. Growth and morphology of crossbred Pagrus major with Evynnis
Japonica. Bull. Nagasaki Pref. Ins!. Fish. 14:31-35. (In Japanese;
English summ.)
Doi, M., T. Hoshino, Y. Taki, and Y. Ogasawara.
1982. Activity of the sperm of the bluefin tuna Thunnus thynnus underfresh and preserved conditions. Bull. Japan. Soc. Sci. Fish. 48:
495-498. (In Japanese; English summ.)
Fujita, S.
1961. Studies on life history and aquaculture of important puffers
in Japan. Special report of the Nagasaki Pref. Ins!. Fisheries,
No.2, 121 p. (In Japanese)
1967. Artificial interspecific and intergeneric hybridization among
the tetradontid puffer (Prelim. rep.). Jpn. J. Michurin BioI.,
3:5-11. (In Japanese; English summ.)
Fukusho, K.
1981. Present status and view of fry production and genetic breeding of marine finfish. Fish Genetics and Breeding 6:1-10. (InJapanese.)___________________________ Fukusho: Genetic Studies on Marine Finfish in Japan
Fukusho, K., M. Okauchi, H. Nanba, M. Hoshi, and H. Tsubaki.
1987a. Comparison in growth and survival rate among gynogenetic
larvae of red sea bream, being induced by sperm of red sea bream,
flounder, and striped knifejaw. Proc. Meetingjapan. Soc. Sci.
Fish. 1987 (Hakodate), p. 149. (In japanese.)
1987b. An attempt of triploid induction of red sea bream, using
fertilized eggs by natural spawning in net cage. Proc. Meetingjapan. Soc. Sci. 1987 (Hakodate), p. 149. (In japanese.)
Harada, T.
1974. Genetic improvement of sea bream. Yoshoku (Midorishobow), 11:50-54. (In japanese.)
1975. Target species of marine finfish for fry production. In
Feeding and development of larvae and juvenile Oapan. Soc. Sci.
Fish., eds.), p. 90-96. Koseisha-Koseikaku, Tokyo, japan. (Injapanese.)
1978. Cross breeding of marine finfish. Yoshoku (Midori-shobow)
15:32-35. (In japanese.)
Harada, T., O. Murata, S. Miyashita, S. Oda, and S. Maeda.
1985. Incubation and larval rearing of Korean red sea bream.
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Harada, T., H. Kumi, and O. Murata.
1986. Artificial hybrids between japanese parrot fish and spottedparrot fish. Bull. japan. Soc. Sci. Fish. 52:613-621. (Injapanese;
English summ.)
Harada, T., O. Murata, and S. Miyashita.
1988. Artificial hybridization between japanese red sea bream andKorean red sea bream, and three years culture of the hybrids.
Proc. Meetingjapan. Soc. Sci. Fish., 1988 (Tokyo), p. 276. (Injapanese.)
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1983. Morphology, growth and low temperature and low salinity
tolerance of sparid hybrids. jpn. J. Ichthyol. 30:275-283. (Injapanese; English summ.)
Kumai, H.
1984. Biological studies on culture of the japanese parrot fish,

25

Oplegnathusfasciatus (Temminck and Schlegel). Bull. Fish. Lab.
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Kumai, H., M. Nakamura, Y. Kubo, and Asada.
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Kurokura, H.
1983. Cryopreservation of fish sperm. Fish Genetics and Breeding
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Kurokura H., S. Kasahara, H. Kumai, and M. Nakamura.
1986. Hybridization of red sea bream and crimson sea bream bycryopreservation of sperm. Proc. Meetingjapan. Soc. Sci. Fish.
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Tabata, K.
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Tabata, K., and S. Gorie.
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1988b. Induction of gynogenetic diploids in Paralichthys olivaceus by
suppression of the 1st cleavage with special reference to their survival and growth. Bull. jpn. Soc. Sci. Fish. 54: 1867-1872. (Injapanese; English summ.)
Tabata, K., S. Gorie, and N. Taniguchi.
1986. Verification by isozyme gene marker for gynogenetic
diploidization and triploidization in Hirame, Paralighthys
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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 oj Microbiology
Oregon State University
Corvallis, Oregon 97331-3804

ABSTRACT
Viral pathogens in aquaculture have largely been controlled by the culling and destructionof carriers and infected animals and eggs. Because most viral pathogens in aquaculture are transmitted via water and because sensitive animals reside in the neighboring waters, the administration 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 aquacultureindustry. 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 development 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_

One of the major factors that will have an impact on the
success of the aquaculture industry is the control of
diseases. As the industry grows and greater productivity
demands are made on facilities, the incidence of disease
outbreaks will increase. Thus, the need for more effective
disease controls has been receiving more attention. The
viral diseases are particularly important because there areno suitable treatments available. In the United States, there
are no approved antiviral drugs or vaccines that can beused in the aquaculture industry today.
At the present time, the control of viral diseases is basedlargely on management. Current recommendations for thecontrol of viral disease outbreak include the destruction of
diseased stocks, drainage of ponds, disinfection of contaminated areas with chlorine, treatment with sunlight or
lime, and the restocking of the facility with disease-freestock. These procedures are very expensive and instituted
with understandable reluctance by the industry. With

'Oregon Agricultural Experiment Station Technical Paper No. 8961.

earthen ponds and stable viruses, like the baculoviruses andthe picornaviruses, these disinfection procedures may not
work.
Another facet of present day controls for viral diseases
in aquaculture is the requirement for certified pathogenfree stocks and eggs and the use of specific pathogen-freewater in the facility. When available, these requirements
have been very effective in preventing disease outbreaks.
However, it has not always been possible to obtain diseasefree stocks for highly-prized strains nor economically practical to use specific pathogen-free water. Thus, the aquaculture industry has a definite need for viral vaccines. Ourgroup reports here the successful development of two prototype viral vaccines by recombinant DNA techniques.
Two viruses were selected for vaccine development
because these viruses affect economically important aquaculture species (salmon and trout) in the United States,
Europe, and Japan. In addition, these viruses, infectious
hematopoetic necrosis virus (IHNV), and infectious pancreatic necrosis virus (IPNV), affect very young fish, andimmunization of large numbers of fish at this size by immersIOn IS fairly easy. IHNV is a rhabdovirus with anenveloped virion and glycoprotein peplomers on the27

28

NOAA Technical Report NMFS 92_

envelope surface; it has a viral genome of single-stranded
RNA of negative sense (McAllister and Wagner 1977). In
contrast, IPNV is a nonenveloped virus with a single capsid and a genome of two segments of double-stranded RNA(Dobos 1975). The techniques that were involved in theconstruction of recombinant plasmids containing the genes
for the major immunogenic proteins of these two viruseshave been described (Kurath and Leong 1985; Huang
et a1. 1986). The expression of these proteins in bacteria
and the use of these expressed proteins as vaccines will be
described here.

Materials and Methods

pared against purified IHNV and IPNV were made as
described (Engelking and Leong 1989).

Construction of Recombinant Plasmids
The construction of a recombinant plasmid containing thetrpE promoter and the trpE gene fused to an immunogenic
region of the gene for IHNV glycoprotein gene or the
IPNV VP2 gene is shown schematically in Figure 2. The
isolation, cloning and sequence analysis of these genes havebeen reported (Koener et al. 1987; D. S. Manning 1988).
The pATH vectors were the generous gift of A. Tzagaloff(Dieckmann and Tzagoloff 1985). The constructions wereverified by DNA sequence analysis by the dideoxy method(Sanger et al. 1977). The plasmid pUC 19, which served
as the negative control for pTAI in Figure 1 was obtained
from Pharmacia, Inc., Piscataway, NJ.

_

Cells, Virus, and Antisera
The following viruses here used in this study: the IHNV
isolate from Round Butte was obtained from W. Groberg(Oregon Department of Fisheries and Wildlife) and the
IPNV isolates, Sp and Buhl, were obtained from R. Hedrick, University of California at Davis. The virus used forchallenge studies was prepared by infecting rainbow trout(Oncorhynchus mykiss) fry and reisolating the virus from fishdying of IHN disease in the case of the IHNV isolates and
IPN disease in the case of the IPNV isolates. Subsequently,
the virus was grown for two passages in chinook salmon
embryo cells (CHSE-214 cells) (Fryer et a1. 1978). The
tissue-culture supernatant fluid containing the virus wasused as the challenge virus. The IHNV and IPNV used
as molecular weight markers in Figure 1 were prepared
as described in Kurath and Leong (1985) for IHNV and
Huang et al. (1986) for IPNV. The rabbit antisera pre-

~

ClI

A.

B.

Bacterial crude lysates were prepared as described (Kleid
et al. 1981). Proteins were analyzed by sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
and Western immunoblotting as previously described(Gilmore et al. 1988). The crude lysates were used to immunize fish by immersion. Rainbow trout fry at 0.4 g were
immunized in sets of 100 fish. Immunization was accomplished by bathing groups of 100 fry in 25 mL of thevaccine preparation (ca. 3 mg/mL total protein concentration) for 1 minute. At that time, the immersion solution volume was increased to 250 mL with water and fish
were incubated in this diluted solution for an additional

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Immunization Trials in Fish

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-VP3
-VP3A

abcdefgh

Anti-IPNV Sera

Figure 1
Analysis of bacterial production of trp E-viral genefusion proteins by antibody reactivity on an electrophorectic transfer blot of a 10% SDS-polyacrylamide gel of bacterial extracts. (A) Developmentof the blot made with anti-IHNV sera. Lane ais the trpE-G fusion protein detected in cells containing the recombinant plasmid, p52G; Lane b
are proteins detected in cells containing the expression vector pATH3, without a viral gene insert; and Lane c is purified IHNV. (B) Development of the blot made with anti-IPNV sera.
Lanes a and i contain the molecular weightmarker proteins: phosphorylase B (110000 Da);
bovine serum albumin (66000 Da); ovalbumin(+5000 Da); and carbonic anhydrase (31 000 Da).
In lanes b, c, and dare celllysates from bacterial
cells containing the plasmid pUC19 with no viral
insert; the samples were loaded at 1, 5 and 25
ilL respectively in lanes b, c, and d. In lanes e,

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Leong et al.: Recombinant Viral Vaccines in Aquaculture

2 minutes. These fish were then placed in aquaria of 5
gallons with a water flow rate of 0.25 gal/min in a constant water temperature of lOoC. The control fish were
exposed to saline in the same procedure or left undisturbed.
Approximately one month after immunization, the experimental and control fish were placed in separate aquaria
in groups of 25. The fish were exposed to serial log virus
dilutions in 1 liter of water. The challenge virus was
prepared as described by Engelking and Leong (1981). In
Figure 3, the data for fish exposed to 7.2 x lO + 5 plaqueforming units per mL (PFU/mL) is presented. The data
represents the mean of duplicate experiments. All dead fish
were assayed for the presence of infectious virus in chinook
salmon embryo cells (CHSE-214) as described by Engelking and Leong (1981).

Results_

Antigen Production in Bacteria
The size and quantity of virus-specific antigen produced
in bacteria hosting the recombinant plasmids was estimated29

from stained gels and Western blots of total bacterial extract. In Figure 1, the product of a trpE-IHNV glycoprotein fusion gene from the plasmid p52G and the major capsid protein ofIPNV from the plasmid pTAI is shown inWestern blots of the appropriate bacteriallysates. A determination of the DNA sequence of p52G indicated that a
264 bp fragment of the IHNV glycoprotein gene had been
inserted in-frame with the trpE protein to produce a fusion
protein of 49 000 daltons (49 kDa = 37 kDa [trp E 1 + 11
kDa [glycoprotein gene fragment]). In addition, a second
fragment of the IHNV glycoprotein gene had been inserted
out-of-frame adjacent to the 264 bp fragment and this additional nucleic acid resulted in 1 kDa (84 bp extra) moreof amino acid residue owing to the fusion protein (Gilmore
et al. 1988).
The IPNV expressing plasmid, pTAl, contained theentire coding region of the A segment of the viralgenome for the isolate Sp. It was constructed so thatthe viral genetic information was fused in-frame to thetrpE protein (Figure 2) and all the proteins encoded by the
A segment were synthesized in the bacteria. Thus, VP2(major capsid protein), and VP3 (minor capsid protein)

EcoRI
B<mHI
Hind IIIVIRAL GENE
Figure 1 (Continued)
f, and g are 1, 5 and 25 j.lL of cell Iysates frombacterial cells containing the plasmid pTA!. Laneh contains purified IPNV. The arrow indicates the
trpE-VP2 fusion protein found in lanes e, f, and g.
The symbol VP 1 indicates virion protein 1; VP2,
virion protein 2; VP3, virion protein 3; and VP3a,
breakdown product of VP3.

~
VIRAL GENE

Restrict with compatible
enzymeRestrict with either
Eco RIBam HI
Hind III

TrpE

Figure 2
Construction of the expression vectors for trp E-viralgene fusions. The cDNA cloned insert of the IHNV
glycoprotein gene or the A segment of the IPNV
genome was restricted with a compatible nucleaseto 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 expression of a trp E-viral gene fusion protein in E. coli.

VIRAL GENE
INSERTIMMUNOLOGICAL-----+ SCREENING FOR
EXPRESSIONNOAA Technical Report NMFS 92

30

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Figure 3
Immunization of rainbow trout with a subunit vaccine against IHNV. Rainbow trout fry (0.4 g) were
immersed in a bacterial lysate (3 mg/mL, containing 10% expressed trpE-G fusion protein) as described in Materials and Methods section. The
results are expressed as mean percent total mortality
on the ordinate and days after the initiation of viralchallenge on the abscissa. There were 25 fish in thecontrol group and 26 fish in the vaccinated group.
In this particular challenge, the fish received 7.2 x
10+ 5 plaque forming units/mL.

50
CONTROL

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20

30

40

50

DAYS AFTER VIRAL CHALLENGE

of IPNV-Sp were expressed by this recombinant plasmidin bacteria.

Immunization Trials with Subunit Vaccine
Viral challenges provided data on the efficacy of thebacterially expressed protein as vaccines. A significant
level of protection (69 %) was conferred on fish immunized with p52G versus unimmunized fish when challenged with the Round Butte isolate of IHNV (Figure 3).
The glycoprotein used in constructing the fusion protein was derived from this strain. In Figure 4, the protection that was achieved by immunization with pTA1
against the Buhl isolate of IPNV, a heterologous virus
strain, is shown. A decrease in virus-induced mortalities
from 45 % to 3 % was found for the immunized group of
fish.

60

Figure 4
Immunization of rainbow trout with a subunit vaccine against IPNV. Rainbow trout fry (0.3 g) were
immersed in a bacterial lysate (3 mg/mL) containing the trp E-VP2 fusion protein as described inMaterials and Methods section. The results are
expressed as percent total mortality on the ordinate
and days after the initiation of viral challenge on theabscissa. There were 25 fish in both control and vaccinated groups. The fish received 10+ 6 plaqueforming units/mL of IPNV-Buhl strain for viralchallenge.

Discussion_

We have presented initial findings on the efficacy of bacterially expressed viral proteins as subunit vaccines for fish.
Both the IHNV and the IPNV vaccines were effective in
immunizing fish against lethal viral challenge in laboratory
trials. Moreover, the vaccinations were carried out on rainbow trout fry that were 0.4 g in size. These fish were able
to respond effectively to the viral vaccine. Previous studiesof immunization in fish have indicated that the minimum
size for successful immunization by immersion was 0.8 g
for chinook salmon (Fryer et al. 1978) and 1-2.5 g for rainbow trout Oohnson et al. 1982).
The use of these vaccines with different species of fish
and against a variety of different viral strains must be
tested. In addition, the duration of effective immunity must
be determined. However, the possibility now exists for

_________________________ Leong et al.: Recombinant Viral Vaccines in Aquaculture

developing an inexpensive and effective vaccme for fishusing recombinant DNA technology.
The development of any vaccine must have safety as wellas efficacy as one of its primary considerations. The safetyof live attenuated vaccines has been questioned for theaquaculture industry because of the nature of the environment where the vaccine would be applied. The vaccine has
to be completely safe for cultured and wild salmonid fishin the watershed. Moreover, the vaccine has to be economical and a subunit vaccine produced in bacteria seems
to be a viable alternative. The initial trials of the subunit
vaccines reported here suggest that bacterially expressedviral proteins, even in crude lysates, can be used as effective and economical viral vaccines.

Acknowledgments_

This publication is the result of research sponsored by
Bonneville Power Administration Contract DE-A17984BP16479, Project 84-43 (G.R. Bouck and R. Morinaka
served as the Contracting Office Technical Representatives
On the project) and, in part, by Oregon Sea Grant withfunds from the National Oceanic and Atmospheric Administration, Office of Sea Grant, Department of Commerce, under grant nO. NA85AA-D-SG095 (project nO.
R/FSD-l1) and from appropriations made by the Oregon
State Legislature. We thank Rebecca Day for typing themanuscript.

Citations_

Dieckmann, C.L., and A. Tzagoloff.
1985. Assembly of the mitochondrial membrane system. J. BioI.
Chern. 260:1513-1520.
Dobos, P.
1975. Size and structure of the genome of infectious pancreatic

31

necrosis virus. Nucl. Acid Res. 3:/903-1919.
Engelking. H.M., andj.C. Leong.
1981. IHNV persistently infects chinook salmon embryo cells.
Virol. 109:47-58.
1989. The glycoprotein of infectious hematopoeitic necrosis virus
elicits neutralizing antibody and protective responses. Virus Res.
13:213-230.
Fryer, j.F., j.S. Nelson, and R.L. Garrison.
1978. Immunization of salmonids for control of vibriosis. Mar.
Fish. Rev. 40:20-23.
Gilmore, R.D. Jr., H.M. Engelking, D.S. Manning, andJ.C. Leong.
1988. Expression in Escherichia coli of an epitope of the glycoprotein of infectious hematopoietic necrosis virus protects against viralchallenge. Bio/Technology 6:295-300.
Huang, M., D.S. Manning, M. Warner, E.B. Stephens, andJ.C. Leong.
1986. A physical map of the viral genome for infectious pancreatic
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 immersion in Vibrio anguillarum and Yersinia ruckeri bacterins. J. Fish.
Dis. 5: 197-205.
Kleid, D.G., D. Yansura, B. Small, D. Dowbenko, D.M. Moore,
M.J. Grubman, P.D. McKercher, D.O. Morgan, B.H. Robertson,and H.L. Bachrach.
1981. Cloned viral protein vaccine for foot-and-mouth disease:
Responses in cattle and swine. Science 214: 1125-1129.
Koener, j.F., C.W. Passavant, G. Kurath, and J.C. Leong.
1987. Nucleotide sequence of a cDNA clone carrying the glycoprotein gene of infectious pancreatic necrosis virus, a fish rhabdoVlfUS.
j. Virol. 61(5):1342-1349.
Kurath, G., and J.C. Leong.
1985. Characterization of infectious hematopoietic necrosis virus
in mRNA species reveals a non-virion rhabdovirus protein. J.
Virol. 53:462-468.
McAllister, P.E., and R.R. Wagner.
1977. Virion RNA polymerases of two salmonid rhabdoviruses. J.
Virol. 22(3):839-843.
Manning, D.S.
1988. Deletion mapping and Expression of the Large Genomic segment of Infectious Pancreatic Necrosis Virus. Doctoral Diss.,
Oregon State Univ., Corvallis, Oregon.
Sanger, F., S. Nicklen, and A.R. Coulson.
1977. DNA sequencing with chain-terminating inhibitors. Proc.
Natl. Acad. Sci. USA 74(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

ABSTRACTIn 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 areoften 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 important concerns for hatchery populations: levels of genetic variability, stability of allele frequencies, and genetic interactions (due to straying or overplanting) between hatchery and wild populations. 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 populations 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 population size, and (in some cases) the infusion of genes from other populations through straying ortransfer of broodstock between hatcheries.

Introduction_

As a consequence of increased fishing pressure, loss of
spawning habitat, and blockage of migratory routes, returns of wild anadromous salmonids in the Pacific Northwest have declined substantially in this century. In partto mitigate these losses, an extensive public hatchery system has been developed during the last several decades.
Throughout most of this period, management practices at
the hatcheries have been dictated primarily by production
demands, and relatively little consideration has been given
to the genetic quality of released fish and their effects onwild fish. The availability of large amounts of data produced by protein electrophoresis over the last decade has
made possible a critical evaluation of the genetic status ofPacific coast hatchery populations of salmonids. Here, wesummarize results from several recent studies which are
pertinent to three important concerns: 1) levels of genetic
variability found in hatchery and wild populations; 2)stability of allele frequencies in hatchery and wild populations; and 3) genetic interactions (due to straying or overplanting) between hatchery and wild populations.

Materials and Methods_

The electrophoretic data discussed here were collected overthe last decade at the National Marine Fisheries Service
laboratory in Seattle. A considerable database exists for
all the North American species of Pacific salmon, Oncorhynchus, but here we consider only data for chinook salmon,
O. tshawytscha; for this species, data are available for populations from California to Alaska. Whole juvenile fish or
tissue samples (muscle, liver, eye, heart) from adult fish
were collected in the field and stored at - 70°C until
analyzed. Starch gel electrophoresis was performed as
described by Aebersold et al. (1987). Each sample wassurveyed for genetic variation at up to 100 presumptive
gene loci, and genotypes inferred from the phenotypic
banding patterns (see Utter et al. 1987 for discussion) wereused to compute allele frequencies and a variety of standard indices of genetic variability and differentiation.

Levels of Genetic Variability_

Recent policy statements (e.g., Northwest Power Planning
33

34

NOAA Technical Report NMFS 92

Council, 1987) regarding anadromous salmonids express
two major concerns: that existing levels of genetic diversity be maintained, and that unique gene pools be preserved. Loss of genetic variability is a real concern for
managed populations because constraints on money, space,and other resources often limit the size of the breeding
population. In a closed population, approximately 112Neof the existing genetic variation is lost each generation, with
N e being the effective number of breeders (Crow andKimura 1970). The effective population size (Ne ) is lessthan the actual number (N) if the sex ratio is uneven or
if the variance in reproductive success among families islarge-both factors that might be influenced by hatcherymanagement procedures. Furthermore, if population sizechanges over time, long-term N e is determined primarily
by the effective number of breeders in the generation(s)with smaller size. Therefore, a population bottleneck (reduced effective breeding size in one or a few generations)can contribute appreciably to the long-term erosion ofgenetic variability.
To determine whether these effects are important in
Pacific salmon, we examined two measures of genetic
variability (average heterozygosity and effective numberof alleles per locus) in a series of hatchery and wild populations of chinook salmon. The occurrence of consistently lower levels of genetic variability in hatchery stocks
would suggest that artificial propagation has caused population bottlenecks. The heterozygosity data, however,provide no evidence of the erosion of genetic variability
in cultured populations of chinook salmon in the PacificNorthwest. In each case where data are available for acomparison (Fig. 1), hatchery and wild populations fromthe same area have very similar levels of heterozygosity.
This result differs from that reported in a number of studies
of Atlantic salmon, Sa/rno safar, and rainbow, Oncorhynchus
rnykiss, cutthroat, O. clarkii, and brown trouts, Sa/rno trulla(review, Allendorf and Ryman 1987); some cultured
populations of these species have been found to have greatly
reduced levels of heterozygosity relative to the ancestralwild stocks.
Some interesting trends are apparent in the heterozygosity data for chinook salmon but these relate to geographic differences rather than to differences between
hatchery and wild populations. In the Columbia Riverbasin, coastal populations have higher heterozygosity than
do lower river populations, which in turn retain moregenetic variability than Snake River populations from farther upstream (Fig. 1). Populations from the Klamath andupper Fraser rivers also show reduced levels of genetic
variability relative to those closer to the coast (Georgia
Strait, Puget Sound). Presumably, these differences reflect
the essentially independent evolutionary histories of the different areas and, perhaps, the smaller population size or
increased frequency of population bottlenecks in the upriver populations (Winans 1989).

_

One drawback to the above analysis is that average
heterozygosity is not very sensitive to the presence orabsence of uncommon alleles. Although they contributelittle to the measurement of heterozygosity, such alleles arepotentially very important to a population because they
allow a greater degree of plasticity in response to changes
in the environment. The presence of numerous alleles (eventhose at low frequency) in a population ensures that eachgeneration, many genotypic combinations are produced
upon which natural selection might act. Because alleles at
low frequency are easily lost if the effective breeding size
is small, the average number of alleles per locus is a moresensitive indicator than average heterozygosity of undesirable changes in the genetic makeup of a population.
According to Utter et al. (1989), the average number ofalleles per locus for seven hatchery and six wild populations from Oregon were similar (1.74 and 1.68, respectively). This lends additional support to our conclusion thatthe wholesale reduction of genetic variability reported insome hatchery populations of Sa/rno (e.g., Stahl 1983) has
apparently not occurred in chinook salmon hatcheries in
the Pacific Northwest.
This result is encouraging, but by no means constitutes
a clean bill of health for hatchery populations. If the genetic
makeup of the source populations is to be perpetuated as
accurately as possible, it is important not only to conserve
overall levels of genetic variability, but also to avoid largechanges in frequency of the alleles present. For example,
consider a locus with two alleles (A and a), sampled in a
population at two times, with the following frequencies
observed-time 1: A = 0.8, a = 0.2; time 2: A = 0.2,
a = 0.8. Hardy-Weinberg expected heterozygosity (2Aa
= 0.32) remains unchanged, but allele frequencies haveshifted drastically. Clearly, it is also important to monitor
allele frequencies over time in artificially propagatedpopulations.

Temporal Stabilityof Allele Frequencies_

To evaluate the temporal stability of allele frequencies, we
examined electrophoretic data for 21 coastal chinook
salmon populations from Oregon and California that were
sampled in each of two years (Waples and TeeI1990). For
each population, allele frequencies in the two samples
were compared at an average of 10 polymorphic loci. For
each locus, a contingency chi-square test was used to test
the hypothesis that the population frequencies were unchanged. Results of these tests are very revealing (Table1). For the three California hatchery and the nine Oregon
wild populations, the number of single locus tests showing a significant change in allele frequency (1/ 16 = 6 %to 7/88 = 8 %) was close to the value (5 %) expected toarise from sampling error, while the figure for the nine

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

15
10

35

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Heterozygosity in hatcheryand wild chinook salmon

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Oregon hatchery populations was much higher (29/81 =36 % of all tests showing significant allele frequencychange). In addition, combined chi-square tests over allloci indicate very significant (P<O.Ol) or highly significant(P<O.OOl) changes in allele frequency between 1981 and1985 samples in eight of the nine Oregon hatcheries (Table
1).
Possible causes of short-term allele frequency change include natural selection, genetic drift, and migration. In thepresent example, selection appears to be an unlikely cause,
given Waples and Teel's (1990) demonstration that it isnecessary to invoke unrealistically large selection coeffi-

Figure 1
Comparison of average heterozygosity values forhatchery and wild stocks of chinook salmon bygeographic area.

cients to explain such large frequency shifts in a single
generation. Waples and Teel also showed that the observed
differences can be accounted for by genetic drift only if theeffective number of breeders in the Oregon hatcheries
averaged about 50 or less. Examination of brood stock data
indicates that effective population size may indeed havebeen quite low in at least some of the hatcheries (Waples
and Teel 1990).
Another possibility is that some of the genetic changes
resulted from the infusion of new genes during the transfer
of fish between hatcheries, or from natural straying into
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, andsignificance levels are given for combined chi-square test over all loci and a test for gametic
JOldisequilibria (n.s. = not significant).
:;c....... :fi: -+- z: 11'

prPopulation'

Between-year cmparisons'

Sample size
1981 1985 6

Single locus(no. sig./total)

Gametic disequilibria"

All loci

1981

0/11

n.s.

1/7
1/12
1/9
1/9

n.S.

n.s.
n.s.
n.s.
n.s.

1985 6

Oregon wild populations
Alsea
ChetcoCoquille
Nehalem
Nestuca
SiletzSixes
Siuslaw
Tillamook
No. sig./total

94
100
115
141

60
92
100
82
88

50
93
50
50
50
50
50
34
50

n.S.

n.s.

n.s.
n.s.
n.s.

n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.

1/9

0/9

0/9

0.001

0.01

0.001

n.s.

n.s.
n.s.
n.s.

n.s.
n.s.
n.s.
n.s.

n.s.
n.s.
n.s.
n.s.

0/11
1/8

0.01

1/11

n.S.

1/10
7/87

n.S.
n.S.

Oregon hatchery populations
Cedar Creek
Cole R. (S)Cole R.
Elk R.
Fall Creek
Rock Creek (S)SalmonTrask (S)Trask
No. sig./total

99
113
50
100
100
100
99
100
100

Iron Gate
Trinity (S)TrinityNo. sig./total

99
50
100

100
50
100
100
100
100
100
100
100

50
100
50

4/9
1/9
5/13
2/9

3/7

0.01
0.001
0.01
0.001
0.001
0.001
0.01

29/81

2/7
4/9
5/8
3/10

0.001
0.05

0.01

0.001
0.001
0.05
0.001

819

4/9

5/9

California hatchery populations
1/8
n.s.

n.s.

n.S.

n.s.

n.s.

0.05

0/5
0/3

n.s.

n.s.

n.S.

1/16

0/3

0/2

1/3

n.S.

'Spring run denoted by (S); all others are fall run stocks.
6Samples taken in 1983 for Oregon wild populations, 1984 for California populations.
'Data from Waples and Tee! (in press).
"Data from Waples and Smouse (1990).

from gametic disequilibrium analysis, a powerful meansof detecting samples which are actually a mixture of
distinct gene pools. Gametic disequilibrium (the nonrandom association of alleles at different gene loci)occurs as the result of a mixture of gene pools that differ in allele frequency at two or more loci (Nei and Li1973).

Genetic Interactions Between" yHatchery and Wild Populations
~7V

,-1

r

dd Z-

q): ~mi~tures.(mixtures offish fro~ more than a single gene
1,/" pool) lllvolvlllg hatchery populatIOns are a concern for two

reasons. First, the transfer of eggs, fry, and brood stock
among hatcheries is a common occurrence that complicatesthe problem of identifying the genetic makeup of hatcherypopulations. Second, strays of hatchery or transplanted fish
may have an adverse effect on wild populations adapted
to local conditions. The genetic consequences of such
admixtures are difficult to evaluate by traditional methods(physical tags, behavioral observations) because the presence of exotic fish in a population does not ensure that they
will interbreed with the residents and produce viable offspring. If the potential source populations can be identified
and adequate genetic markers are available, estimates ofthe mixture fractions are possible (Campton 1987). However, in many cases the populations possibly contributing

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

to a mixture are unknown or cannot be characterized
genetically. For such a "blind" mixture, gametic disequilibrium analysis is a potentially powerful tool for evaluating the null hypothesis that the sample could have comefrom a single gene pool.
To evaluate the possibility of genetic admixture in theabove example, we used a multilocus analysis of gametic
disequilibrium (Waples and Smouse 1990) that considers
data for all pairs of loci simultaneously. No unusual levelsof multilocus gametic disequilibrium were found in the
California hatchery or Oregon wild populations (1 of 23
samples with significant disequilibria at a = 0.05 level;
Waples and Smouse 1990), but the situation was quite
different in the samples from the Oregon hatcheries (9 of
18 tests significant; Table 1). These findings are consistent with the hypothesis that, in addition to genetic drift,
a mixture of gene pools contributed to the changes in allele
frequency observed in some of the Oregon hatcherypopulations.
Gametic disequilibrium analysis has considerable potential for assessing the extent of genetic interactions among
hatcheries and between hatchery and wild populations.
It may help in the identification of wild gene pools thathave been relatively unaffected by genes from hatcherypopulations and therefore merit conservation efforts. In
other cases, where the objective is to supplement andenhance wild production, gametic disequilibrium analysis
provides a means of monitoring the effectiveness oftransplants from the hatcheries. The possibilities for bothtypes of genetic interactions, as well as the necessity formonitoring them, are likely to increase in the near future.
Large scale supplementation of wild populations andexpansion of hatchery production are planned to achieve
the goal of the current Columbia River Basin Fish andWildlife Program (Northwest Power Planning Council
1987)-doubling the run size of anadromous salmonids in
the Columbia River basin.

Citations37_

Aebersold, P.B., G.A. Winans, D.]' Teel, G.B. Milner, and F.M. Utter.
1987. Manual for starch gel electrophoresis: A method for the detection of genetic variation. Dep. Commer., NOAA Tech. Rep.
61 :1-19.
Allendorf, F.W., and N. Ryman.
1987. Genetic management of hatchery stocks. In Population
genetics and fishery management (N. Ryman and F. Utter, eds.),
p. 141-159. Univ. Washington Press, Seattle.
Campton, D.E.
1987. Natural hybridization and introgression in fishes. In Population genetics and fishery management (N. Ryman and F. Utter,eds.), p. 161-192. Univ. Washington Press, Seattle.
Crow, J.F., and M. Kimura.
1970. An introduction to population genetics theory. Harper andRow, New York, 591 p.
Nei, M., and W-H. Li.
1973. Linkage disequilibrium in subdivided populations. Genetics
75:213-219.
Northwest Power Planning Council.
1987. Columbia River Basin Fish and Wildlife Program. Portland,
Oregon, 246 p.
Stahl, G.
1983. Differences in the amount and distribution of genetic variation between natural populations and hatchery stocks of Atlantic
salmon. Aquaculture 33:23-32.
Utter, F., P. Aebersold, and G. Winans.
1987. Interpreting genetic variation detected by electrophoresis.
In Population genetics and fishery management (N. Ryman andF. Utter, eds.), p. 21-45. Univ. Washington Press, Seattle.
Utter, F., G. Milner, G. Stahl, and D. Tee!.
1989. Genetic population structure of chinook salmon in the PacificNorthwest. Fish. Bull., U.S. 87:239-263.
Waples, R.S., and P.E. Smouse.
1990. Genetic disequilibrium analysis as a means of identifying mixtures of salmon populations. American Fish. Soc. Symp.
7:439-458. (In press.)
Waples, R.S., and D.]. Teel.
1990. Conservation genetics of Pacific salmon. 1: Temporal changes
in allele frequency.
Conserv. BioI., Vol. 4:144-156.
Winans, G.A.
1989. Low levels of genetic variability in spring-run chinook salmonof the Snake River. N. Am. J. Fish. Management 9:47-52.

Successful Gene Transfer in Fish
S.J. YOON,I Z. LIU,3 A.R. KAPUSCINSKI,2 P.B. HACKETT,3
A. FARAS,4 and K.S. GUISE!
Departments of Animal Science, 1 Fisheries and Wildlife,2Genetics and Cell Biology, 3 and Microbiology 4
University of Minnesota
1988 Fitch Avenue
St. Paul, MN 55108

ABSTRACTThe neo gene, which confers resistance to the neomycin analog drug G418, has been successfully 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 [3-galactosidase, and a variety
of promoters are discussed. Current work which also includes the isolation of piscine promotersand genes for peptide hormones is mentioned.

Introduction_

Novel genes were first introduced into mice in 1979 byGordon et al. (1980). The technology did not receive wide
attention until Palmiter et al. (1982) transferred a rat
growth hormone gene linked to a mouse metallothionein
promoter, creating a line of mice that grew significantly
faster and ultimately larger than control mice. This seriesof experiments captured the imagination of a wide varietyof researchers seeking to improve economic traits in domestic and semidomestic animals. While gene transfer experimentation is currently progressing in most species of
economic importance, including mammalian farm animals
and poultry, no group of organisms shows more promisefor the dramatic interaction of transferred genes than fish.
For over three decades it has been known that fish are quite
responsive to injections of crude and purified growth hormone (Pickford and Thompson 1948; Adelman 1977). Successful transfer and expression of growth-hormone genes
in fish are thus expected to produce a similar, dramatic
response.
Since 1984, multiple groups worldwide have been pursuing the goal of producing transgenic fish. Laboratories
in Japan, England, France, and the People's Republic ofChina have published results of these attempts in medaka,
Oryzias sp. (Ozato et al. 1986), rainbow trout (Oncorhynchus mykiss) (Maclean and Talwar 1984; Chourrout et al.
1986), goldfish (Carassius auratus) (Zhu et al. 1985), andloach (Misgurnus anguillir.audatus) (Zhu et al. 1986). Within

the United States, four primary groups are known to beusing microinjection of various plasmid constructs to produce transgenic fish. These laboratories are, in addition
to our group at the University of Minnesota, those of Drs.
Powers and Chen at Johns Hopkins, Dunham at Auburn,and Ellinger and Kohler at Southern Illinois. All four U.S.
groups are pursuing similar goals: production of transgenic
fish by transfer of growth-hormone gene constructs using
microinjection as the primary transfer technique. Ourgroup reports here the successful transfer of a marker gene,neo, into goldfish via microinjection, as a step toward thegoal of transfer of economically important genes.

Materials and Methods_

Egg Preparations
Spontaneous ovulation of goldfish was accomplished by themethods of Stacy et al. (1979). Sexually mature fish were
kept under a long photoperiod (16 h light, 8 h dark). On
day one, breeding fish were transferred from stock aquaria
to standing-water breeding aquaria at 13 ± 1°C. Aquaria
were supplied with floating artificial plants, and the watertemperature was increased to 21 ± 1°C overnight and kept
at that temperature through spawning. Spontaneous ovulation usually occurred during the last half of the dark phaseon day 3. If no ovulation occurred on day 3, the fish were
injected intraperitoneally with 3 mg/kg body weight of carp
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
separately.
Eggs were fertilized by the milt after they were mixed
with well water in an open petri dish in batches of approximately 100 eggs. Ten minutes after fertilization, the eggs
were dechorionated by a six minute incubation in 0.2 %trypsin (Zhu et a!. 1985). Dechorionation was stopped bytreatment with 5 % fetal bovine serum in Holtfreter's solution (Grand et al. 1941). Dechorionated eggs were washedseveral times in Holtfreter's solution and transferred to
Holtfreter's solution on a charcoal-agar petri dish, which
provided a dark background for microinjection.

Microinj ection
Plasmids were prepared by amplification, lysis in SDS(sodium dodecylsulfate), and CsCl 2 centrifugation (Maniatis et a!. 1982). Plasmids were linearized with restriction
endonuclease Kpnl, extracted with phenol/chloroform,
ethanol precipitated, and redissolved in 88 mM NaCl, 10
mM Tris' Hcl pH 7.6 to a final concentration of25 ng/J.lL.
Borosilicate glass needles with an inner tip diameter of approximately 2 J.lm were filled with plasmid solution. Microinjection was performed with a Brinkman MM33 micromanipulator with the injection volume controlled by thetiming of the insertion/withdrawal interval and constant
fluid flow. It is estimated that 2 nanoliters of solution containing 1 x 106 copies of neo gene are delivered. DNA wasreleased into the center of the germinal disc prior to thefirst cleavage. Microinjected eggs were allowed to develop
in Holtfreter's solution until the blastula stage, and returned to well water after the blastula stage. Mock injections were performed as controls, using only the buffer
component of the injection solution.

Plasmid Construction
The primary construct used, pRSVneo, contains the neo
gene, whose product, aminoglycoside 3'-phosphotransferase, confers resistance to the neomycin analog G418,
and a Rous sarcoma virus (RSV) promoter. A Bam HIHindIII restriction fragment containing the neo gene andmart of SV40 intron was isolated from pSV2neo (American Type Culture Collection) and ligated into the polylinker of pUC 119. A 340-bp fragment containing the RSV
promoter/enhancer region was ligated to the regenerated
HindlII site. This construct is termed pRSVneo in thispaper. Plasmid construction was by standard methods(Maniatis et a!. 1982).

DNA Analysis
Genomic DNA was isolated from posterior halves of 1 to
2 month-old (approx. 8 g) fish stored at - 90°C. Fish wereindividually homogenized in three volumes of 0.2 M_

Tris'HCl pH 8.0, 0.1 M EDTA, 0.5% SDS buffer with
100 J.lg/mL proteinase K on ice using a Dounce homogenizer. High molecular weight DNA was extracted as per
Maniatis et a!. (1982).
DNA dot blot analysis was used to detect the neo gene.
Approximately 5 J.lg of genomic DNA were denatured in
0.4 M NaOH, and following addition of an equal volumeof 2 M ammonium acetate, were spotted onto nitrocellulose using a Hybri-dot system. The filter was baked in vacuo
for 2 hours at 80°C. The filter was prehybridized in 5 x
SCC (1 x SSC = 0.15 M NaCl, 15 mM Na citrate), 5 x
Denhardt's solution (1 x Denhardt's = 0.02% Ficoll,
0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone), 50 mM sodium pyrophosphate pH 6.5, 0.1 % SDS,
0.1 mg/mL denatured calf thymus DNA, and 0.1 mg/mL
yeast tRNA at 65°C for at least 4 hours. The filter was
hybridized with 1 x 10 6 cpm of 32p labeled probe in thesame solution used for prehybridization for 20 hours at
65°C. The filter was washed three times in 2 x SSC, 0.1 %SDS at room temperature for 10 minutes, and three times
in 0.1 x SSC, 0.1 % SDS at 60°C for 30 minutes, dried,and exposed to Kodak XAR5 x-ray film at - 80°C (Maniatus et a!. 1982).
Southern blot analysis was performed by complete digestion of 10 J.lg of genomic DNA with Kpnl, Bam HI, Sstl,
or Mlul followed by electrophoresis on a 0.8% agarose gel.
Transfer to nitrocellulose was made following the methodof Southern (1975). The Southern blot was probed using
the same hybridization conditions as the dot blot analysis.

RNA Analysis
Anterior halves of frozen ( - 90°C) neo DNA positive fishplus several control and injected fish were homogenizedby Polytron in a solution containing 4 M guanidinium
isothiocyanate, 0.1 M (3-mercaptoethanol, 0.5 % sarkosyl,
5 mM sodium citrate, pH 7.0. Total RNA was isolated
as per Maniatis (1982). RNA dot blot analysis was used
to test for neo gene expression. Total RNA was dissolvedin 6 x SSC, and 7.4% (v/v) formaldehyde, and heat denatured at 65°C for 15 minutes. Serial amounts of RNA(0.25,0.5, 1, and 2 J.lg) were directly dotted onto nitrocellulose equilibrated with 6 x SSC. The RNA was fixed
to the filter by baking under vacuum at 80°C for 2 hours.
The prehybridization was carried out for more than 4 hours
at 60°C in 50% deionized formamide, 5 x SSC, 5 x Denhardt's solution, 50 mM sodium phosphate, pH 6.5, 0.1 %SDS, 100 I-Ig/mL denatured calf thymus DNA, and 100
J.lg/mL yeast tRNA. The filter was then hybridized with
2 x lOll cpm/mL of 32P-labeled probe in the same buffer
for 20 hours at 60°C. After hybridization, the filter waswashed three times with 2 x SSC, 0.1 % SDS at room
temperature for 10 minutes, three times with 0.2 x SSC,
0.2% SDS at 68°C for 30 minutes, and exposed to Kodak
XAR5 x-ray film at - 80°C.

41

Yoon et al.: Successful Gene Transfer in FishA B C 0 E F G H I J K l

1
2
3
4

5
6
7
8

•

Figure 1
DNA dot blot analysis of goldfish microinjected with pRSVneo. Total genomic DNA probed with330 bp HindlII-BamHI 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, 1 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 lA-1F (replicate lG-1L) 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), arranged 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
Plasmid restriction fragments were separated by 1 % agarose gel electrophoresis in TAE buffer (0.04 M Tris-acetate,
2mM EDTA pH 8.0). Excised bands were phenol:chloroform extracted, and the DNA was precipitated by ethanoland sodium acetate and redissolved in TE (10 mM
Tris' HCl pH 7.6, 1 mM EDTA). Fragments were labeledby the procedure of Feinberg and Volgelstein (1983) to
0.5-2 x 109 cpm/Jig. DNA probes were used without purification from unincorporated nucleotides.
The 2.3-kb fragment including the neo gene was also subcloned into pTZ18R under the control of the T7 promoter
to produce a transcript complementary to neo mRNA. One
Jig of linear DNA was transcribed using the method of
Schenborn and Mierendorf (1985) to produce probes usedin RNA dot blot analysis.

G-418 Selection
For the drug selection screenings, solutions of G-418 instatic water aquaria were aerated to achieve initial saturation levels of dissolved oxygen. Dissolved oxygen was main-tained above 60 % saturation by periodic aeration. The
temperature was maintained at 20 ± 1
The pRSVneoinjected or non-injected control goldfish at a population
density of 12 giL were exposed to 4 mglmL solutions of
G-418. Mortality of fish was monitored every four hours
for the first 24 hours and every 12 hours afterward. Dead
individuals were removed upon observation and stored inliquid nitrogen. Drug selection was continued until 50%of the control fish in 4 mg/mL G-418 had died. Surviving
fish were transferred into fresh well water upon termination of the test.

ac.

Results_

The survival rate for microinjected fish has ranged from
10% in the early experiments to nearly 50% at the current time. The results reported here are from early injection studies, where, despite the high mortality rates, wewere still able to effect the transfer and apparent expression of the transferred gene.
Figure 1 shows the results of DNA dot blot analysis ofmock- and pRSVneo-injected goldfish. None of the mock-

42

NOAA Technical Report NMFS 92 _~

p

I,.

P PI

I 2 3

_

Sst
II.
I Z 3 I 2 3

Figure 2Southern 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 pRSVneoand pRSVneo plus genomic DNA, respectively.
injected fish showed positive hybridizaiton to the neo probe,
while two of the pRSVneo-injected fish showed approximately five copies of the gene per genome. Figure 1 also
shows a series of DNA dot blots for pRSVneo-injected fishthat were subjected to drug selection with G-418. The fish
were grouped by order of the time of death during drug
selection. One fish, which survived the full course of selection, but died on day 8 offungal infection, showed an apparent hybridization signal of about 1 copy per genome.
Pink salmon DNA was included as control DNA. Note that
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, pinksalmon apparently contain genomic sequences that areclosely related to neo.
Figure 2 shows results of a Southern blot analysis of
genomic DNA cleaved with four different restriction enzymes and probed with the 330-bp neo probe. The neo probe
detected both linearized plasmid and a higher molecularweight band in the BamHI and Sstl digests. This may indicate a concatenated integration of the estimated fivecopies of the gene, but conclusive evidence of integration
will require breeding studies.
Expression of the transferred neo gene which was suggested in the resistance of a fish containing neo sequences(Fig. 1), was confirmed in the RNA dot blot of Figure 3.
Here, one out of six fish tested showed strong expressionof neo RNA when probed with a complementary neo RNAprobe. In order to rule out the possibility of DNA contamination of the RNA used in ~he RNA dot blot, we
treated the RNA from the positive fish of Figure 3 with
DNase and RNase in a separate experiment (Fig. 4).
RNase destroyed the hybridization to the neo RNA probe,
while DNase had little effect. This indicates little if any
DNA contamination of the RNA.

Conclusion and Discussion_

The results reported here showed successful transfer of amarker gene neo that encodes resistance to the drug G-418.
Stable integration of the transferred gene was suggested
but could not be conclusively proven at this time. Expression of the marker gene was confirmed through detectionof neo mRNA in the transgenic fish. Functional resistanceconferred by pRSVneo was suggested by the survival
through drug selection of a putative transgenic fish containing an extimated single copy of the neo gene.
These results indicate that microinjection is a viable
method for transfer of selectable genes into fish. Micro-

_________________________=='-__ Yoon et al.: Successful Gene Transfer in Fish

.25

.5

1

43

2

RNase
- +

DNase
+

A

•
C

5 RN I

D

E
F
Figure 4
Figure 3

RNA dot blot analysis of total RNA from six
pRSVneo-injected fish, spotted at 0.25, 0.5, 1
and 2 lAg RNA per dot for each fish.

lllJection is, however, currently a labor- and patienceintensive technique, and promises to remain so for the near
future. Microinjection works well upon dechorionated eggs
where needle placement can be monitored, but is lesseffective in blind injections. Therefore, we are keenly interested in development of a selectable marker for use ineither blind injection or in mass transfer techniques. Ourgroup has been exploring the use of mass transfer techniques to overcome the tedium of injection of the substantial numbers of eggs necessary to generate the optimaltransgenic fish. While mass transfer efficiencies may beorders of magnitude less than those achieved by microinjection, the ability to manipulate tens of thousands of eggs
at a time should compensate for the loss in transfer efficiency. The key element necessary to make mass transfera reality is the development of a selection system to differentiate transgenic from nontransgenic fish. The selectable
marker neo meets this criterion, and should prove effectivein the selectionof transgenic fish when used in co-transfer
schemes with other genes.
A variety of mass transfer techniques are under investigation by our group as well as by other laboratories in
the United States. These range from techniques based on
the CaP0 4 procedures used in gene transfer in tissue
culture, to binding of plasmid DNA to sperm and effecting transfer at the time of fertilization, to electroporation.
Studies proceeding along these lines should soon yield
general mass transfer protocols that will be successful ina broad spectrum of fish species.
The establishment of the neo gene as a selectable marker
is a step in the generation of transgenic fish with enhanced
growth hormone expression. We are currently injecting

Total RNA from fish E of Figure 3 probed withRNA complementary to neo RNA transcript. RNA
was spotted before ( - ) and after ( + ) treatment with
RNase-free DNase or DNase-free RNase.

bovine growth hormone constructs into fish eggs, as wellas injecting a second marker gene construct using f3-galactosidase. While bovine growth hormone gene constructs
may produce growth enhancement in transgenic fish, ourintent is to effect growth enhancement by transfer of
species-specific growth hormone genes. To this end, we are
isolating piscine growth hormone genes and fish gene promoters so that the ultimate transgenic, growth-enhanced
fish will contain no DNA sequence from outside its own
species. We feel that this will simplify regulatory approvalof the transgenic fish for human consumption, and enable
the transferred genes to interact with the natural regulatory mechanisms in the fish in a more normal fashion.

Acknowledgments_

The work outlined here was supported in part by Minnesota Sea Grant R/3 to K.S. Guise, by a grant from theLegislative Commission on Minnesota Resources of theState of Minnesota to A. Faras, K.S. Guise, A.R. Kapuscinski, and P.B. Hackett, and by the Minnesota Experiment Station (K.S. Guise).

Citations_

Adelman, I.R.
1977. Effect of bovine growth hormone on growth of carp (Cyprinus
carpio) and the influences of temperature and photoperiod. J. FishRes. Board Can. 34:509-515.
Chourrout, D., R. Guyomard, and L.M. Houdebine.
1986. High efficiency gene transfer in rainbow trout (Sa/ma gaird-

44

NOAA Technical Report NMFS 92

neri Rich.) by microinjection into egg cytoplasm. Aquaculture
51: 1443-1450.
Feinberg, A.P., and B. Vogelstein.
1983. A technique for radiolabelling DNA restriction endonucleasefragments to high specific activity. Anal. Biochem. 132:
6-13.
Gordon, j.W., G.A. Scangos, D.J. Plotkin, J.A. Barbosa, andF.H. Ruddle.
1980. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc. Natl. Acad. Sci. U.S.A. 77:
7380-7384.
Grand, C.G., M. Gordon, and G. Cameron.
1941. Neoplasma studies: Cell types in tissue culture of fish melanotic tumors compared with mammalian melanomas. Cancer Res.
1:660-666.
Maclean, N., and S. Talwar.
1984. Injection of cloned genes into rainbow trout eggs. J. Embryol. Exp. Morphol. 82:187.
Maniatis, T., E.F. Fritsch, and J. Sambrook.
1982. Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Ozato, K., H. Kandoh, H. Inohara, T. Iwamatsu, Y. Wakamatsu, andT.S. Koada.
1986. Production of transgenic fish: Introduction and expressionof chicken crystallin gene in medaka embryos. Cell Differ. 19:
237-244.

_
Palmiter, R.D., R.L. Brinster, R.E. Hammer, M.E. Trumbauer,
M.G. Rosenfeld, N.C. Birnberg, and R.M. Evans.
1982. Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature
300:611-615.
Pickford, G.E., and E.F. Thompson.
1948. The effects of purified mammalian growth hormone on thekillifish Fundulus hetaoelitis (Linn). J. Exp. Zool. 109:367.
Schenborn, E.T., and R.C. Mierendorf Jr.
1985. A novel transcription property of Sp6 and T7 RNA polymerases: Dependence on template structure. Nucleic Acids Res.
13 :6223-6236.
Southern, E.M.
1975. Detection of specific sequences among DNA fragments
separated by gel electrophoresis. J. Mol. BioI. 98:503-517.
Stacy, N.E., A.F. Cook, and R.E. Peter.
1979. Spontaneous and gonadotrophin-induced ovulation in the
goldfish, Carassius auratus L. Effects of external factors. J. Fish.
BioI. 15:349-361.
Zhu, Z., G. Liu, L. He, and S. Chen.
1985. Novel gene transfer into the fertilized eggs of goldfish(Carassius auratus L. 1758). Z. Angew. Ichthyol. 1:31-34.
Zhu, A., K. Xu, G. Li, Y. Xie, and L. He.
1986. Biological effects of human growth hormone gene microinjected into the fertilized eggs of loach Misgurnus anguillicaudatus(Cantor). Kexue Tangbao 31 :988-990.

Clonal Ginbuna Crucian Carp as a Model for the
Study of Fish Immunology and GeneticsTERUYUKI NAKANISHI and HIROSHI ONOZATONational Research Institute oj Aquaculture
TamakiMie 519-04, Japan

ABSTRACTThe lack of suitable inbred lines of fish for immunological study in which lymphocytes andblood samples could be collected have led to the use of clonal ginbuna crucian carp, Carassius
gibelio langsdorJii. Distributed widely in Japan they include both bisexual diploid and tetraploidtypes and a unisexual (all female) triploid type which reproduces gynogenetically by omitting
Meiosis 1. The kinetics of immunity transfer by immune leukocytes was first examined using
isogeneic crucian carp. Splenic cells were most effective in transferring immune reactivity, followedby pronephric, then mesonephric cells. However, antibody titres were very low or absent when
the recipients received transferred thymic cells. Antibody productivity was most successfully conferred when cells were taken from 7-day postimmunized donors. The level of antibody titre inrecipients 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 andxenogeneic crucian carp to examine their major histocompatibility complex (MHC) regulationin adoptive immunity. Adoptive transfer by pronephric cells was successful in isogeneic and weakhistocompatibility (H) gene-disparate transfer systems, while antibody productivity was nottransferable 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_

Most of the information on the cellular immune mechanism in mammals has been obtained by in vivo and in vitro
experiments using histocompatible animals. In fish, however, suitable inbred lines for immunological study are notavailable. Although inbred strains of small teleosts havebeen established by sibmating (e. g., platyfish and swordtails, Xiphophorus spp., (Kallman 1964); guppy, .Lebistes
reticulatus, (Schroder and Holzberg 1972); and medaka,
Oryzias latipes, (Taguchi 1980), they are too small to collect lymphocytes and blood samples. Furthermore, genetically identical animals are not currently available forpractical use, even though chromosomal set manipulation
techniques have seen great improvement in recent years
and homozygous clonal fish have been obtained by the suppression of meitosis I and successive prevention of Meiosis
II (e.g., zebra fish, Brachydanio rerio, (Streisinger et al.
1981), medaka (Naruse et al. 1985) and rainbow trout, Oncorhynchus mykiss, (Onozato 1990).
Fortunately, clonal crucian carp-naturally occurring
gynogenetic fish-are distributed widely in Japan. In thepresent review, we describe our recent studies on the fish
immune system using clonal crucian carp and discuss theexcellent potential of this fish as a model for studying fish
immunology and genetics.

Clonal State of Triploid Ginbuna_

Naturally occurring gynogenetic populations are included
in two cyprinid species: silver crucian carp, Garasslus auratus
gibelio (Cherfas 1966), and ginbuna, C. gibelio iangsdoifii (formally C. auratus langsdorfii, Kobayashi 1971). Ginbuna nave
the widest distribution in Japan, and inciude both bisexual diploid (2n = 100) and tetraploid (4n = 206) types, andunisexual (all female) triploid (3n = 156) types (Kobayashi
et al. 1970, 1977). In unisexual ginbuna, which reproduce
gynogenetically, the first polar body formation is skipped
as the result oflacking Meiosis I (Kobayashi 1976). Therefore, the progenies of these fish belong to a clone having
the same genotype as the mother, as evidenced by scale
grafting and electrophoreses (Onozato 1981; Murayama
et al. 1984). Onozato (1981) has shown that the electro45

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Nakanishi and Onozato: Immunology and Genetics of Crucian Carp47

Table 3Comparison of variability in antibody responses to HRBC between allogeneic and isogeneic
crucian carp.
No. ofanimals

Individual HA' titres ( - log2) b

Mean

C.Y.'

Allogeneic

15

13(1), 12(1), 11(2), 10.5(1), 10(4)9.5(3), 9(1), 8.5(1), 7(1)

10.0

0.14

Isogeneic

15

13(3),12.5(1), 12(3), 11.5(2)11(3),10.5(1),10(1),9(1)

11.5

0.10

Allogeneic

15

9(1),8(1),7.5(1),7(5),6(3)5.5(1),4.5(2),3.5(1)

6.4

0.22

Isogeneic

158(1),7.5(1),7(5),6(6),5(1),4(1)

6.4

0.15

0.01% Allogeneic
Isogeneic

107(2),5(2),4(1),3.5(2),3(1),4(2)

4.2

0.41

10

10(1),8(1),7(2),5(1),4.5(1)
4(3), 3(1)

5.7

0.38

20%

1%

Hemmaglutination.
bNumber of fish is shown in parenthesis.
'Coefficient of variance.
aserved; some fish showed high titres, and others did not
respond at all (Table 2). There is no apparent difference
between allogeneic and isogeneic crucian carp even at lower
concentrations (Table 3). That is, genetically identical ginbuna showed similar considerable variation to that of theoutbred nigorobuna in immune responsiveness.
Similar results have been obtained from the study ofgenetic influence on the diversity in growth rate using
clonal crucian carp (Nakanishi and Onozato 1987). In thisstudy a great variation in size and weight was found, evenin clonal crucian carp, indicating the occurrence of asuperior group when the fry were reared in small tanks athigh densities with food of different partical size.
These results suggest that immune reactivity or growth
is not only dependent on genetic state but is greatly influenced by other factors which may have been formed incorrelation with environmental circumstances. That is,
individuals have met with different microenvironments
throughout the life span, including egg state in the ovary,and the differences in physiological conditions must haveoccurred among individuals later on.

Kinetics of Transfer of
Immunity by Immune Leukocytes
Adoptive transfer of immunity can be successful only whenrecipients show no alloimmune response. However, thehistocompatibility system of fish has been found to be welldeveloped (Kallman 1970; Borysenko 1976), with rejection often occurring in an acute fashion even among siblings. Therefore, adoptive transfer is only possible when
inbred or clonal fish are used.

Fish source and the procedures of immunization, leukocyte collection, and cell transfer have been described in
Nakanishi (1987a). In brief, donors were injected intraperitoneally at 2-day intervals with 5 ilL HRBC/g bodyweight. Donors used for day-3 transfer were given a single
injection, those for day-5 transfer were given two injections and those for day-7 or after were given three injections. Recipient fish were subsequently infused with lymphoid cells at a rate of 0.1 mL of 5 x 10 7 cells/mL per
fish. Pronephric and mesonephric cells from a fish were
injected into two or three fish and thymic and splenic cells
were transferred into one fish. Recipients were evaluatedby measuring the serum antibody titre using standard
methods of hemagglutination in microtiter plates.
In a comparison of transferability between lymphoidorgans, splenic cells were most effective in transferring
immune reactivity, followed by pronephric, then mesonephric cells (Fig. 1). Little or no antibody titres wereelicited when the recipients received transferred thymic cells(Figs. 1, 2). The optimal period for collecting the cells fromthe donor was determined by examining the antibody tit res
in the plasma of the recipients which received cells takenfrom immunized donors on days 3,5,7, 10, 14, and 21.
Antibody productivity was most successfully conferred
when cells were taken from immune donors 7 days after
immunization (Fig. 1). In recipients, antibodies were detected within one day after transfer and the titre reachedpeaked levels on day 7 for mesonephric cells and day 14
for phronephric and splenic cells (Fig. 2). These results
agree well with the kinetics of plaque-forming cells (PFCs=
antibody-producing cells) and circulating antibodies
described by Nakanishi (1987a). That is, PFCs weredetected on day 3 and peak response was observed 5 to

48

NOAA Technical Report NMFS 92

_

8
7

6
N

0>
0

I

.~

5
4

~

I

c
0

Cl>

3

::;;

2

3

5

7

10

14

Figure 1
Kinetics of leukocyte transfer. Pronephros (.), mesonephros(0), spleen (A) and thymus (~) cells taken from immunizeddonors on day 3,5,7, 10, 14 and 21 are injected into recipients. Each point represents mean hemagglutination titre in
the plasma for 5 to 10 recipients.

21

Day of Cell Transfer

9•/'-.....

8

/e.~
Je
~ i

7
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./

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.2

-.!..
~ 5
:;:
~

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::;;

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~.
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2
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6 - -- - - -- - - 6

3

5

7

14
Days After Cell Tronsfer

7 days after the first injection. On the other hand, Circulating antibodies were detected 5 days after the first
injection and reached their peak on day 14. In the present study transfer of thymic cells was not successful,even though the number of PFCs of the thymus was similar to that of the pronephros and the spleen. This finding leads to the idea either that thymic PFCs are not the cells
involved in the transfer of immunity or that thymic cells
need collaboration with other sensitized lymphoid cells to
produce antibodies. In addition, no direct relationship

21

Figure 2
Antibody production kinetics. Recipients recelvmg
7-day postimmunized leukocytes from pronephros (.),
mesonephros (0), spleen (A), and thymus (~) are tested
for antibody levels in the plasma. Each point representsmean hemagglutination titre in the plasma for 5 to 10
recipients.

between the PFC response and transferability for individual
fish was observed in other lymphoid tissues (Nakanishi
1987a). This lack of correlation between the PFC response
and transferability might be attributed to the existence ofdifferent developmental stages or of heterogeneous populations of antibody-producing cells and the necessity of cellcollaboration. In any event this basic infomation obtainedby using isogeneic animals can be useful for further investigations of cellular immune mechanisms on fish immune systems.

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Nakanishi and Onozato: Immunology and Genetics of Crueian Carp

B1 to K1 clones, which differed in strong H gene as evidenced by the acute rejection of allografts. Little or no
antibody titres were detected in the recipients 7 days after
transfer (Table 4). On the other hand, interclonal transfer
between weak H gene-disparate clones was successful.
Antibody titre of recipients 7 days after transfer was similar
to that of intraclonal (isogeneic) transfer (Table 4), although
recipients rejected allografted scales from donors 1-2
months after grafting. Transferability of immune pronephric cells in xenogeneic transfer systems was also examined. Pronephric cell transfer was carried out from
immunized kinbuna to unimmunized ginbuna. No antibody was found and all xenografted scales from kinbuna
were rejected within 5 days in ginbuna (Table 4). These
results suggest that transferability of immune cells is strictly
controlled by "MHC."
The most interesting results were obtained when allogeneic transfer using pronephric cells was perfomed among
siblings of kinbuna that had rejected allografts of each otherin an acute manner. Two experiments were performed,
each with different times of cell collection from the immunized donors: 7 days (Fig. 3A) and 14 days (Fig. 3B) after
immunization. In each study, one of five recipients showedelevated antibody titre which peaked 2 and 3 weeks after
transfer from the 7-day (Fig. 3A) and 14-day (Fig. 3B)
postimmunized donors, respectively. Furthermore, one of
the recipients with cells transferred from the 7 days postimmunization donor showed a fairly high level of antibody
titre 5 weeks after transfer, while no antibody was detected
one week and three weeks after transfer. These results suggest that the locus of "class II" antigens involved in cellcollaboration or cytotoxic lymphocyte effecter function islimited in polymorphism in comparison to the transplantation antigens (class I antigens), because antibody reactivity was successfully conferred in some donor-host combina-

Transferability of Immune PronephricCells in Isogeneic, Allogeneic and
Xenogeneic Transfer Systems
The major histocompatibility complex (MHC) of mammals and birds consists of numerous genes involved in acuteallograft rejection, cell collaboration, and cytotoxic lymphocyte effective functions (Klein 1977). In lower vertebrates, a single genetic region homologous to the MHC
has been described in the anuran amphibian (Du Pasquieret al. 1975; Kaufman et al. 1985; Nakamura et al. 1986).
The teleost is the lowest vertebrate in which a MHC issuspected to exist, owing to their vigorous rejection offoreign tissue grafts, though knowledge of a MHC in fishis sparse. The present study was undertaken to examine
the transferability of antibody reactivity by immune pronephric cells among isogeneic, and xenogeneic crucian carpin order to analyze the correlation between transplantation antigens and determinants involved in cell collaboration or cell-mediated lympholysis.
In these experiments we used two clones of ginbuna (B 1,
D3) from Okushiri Island in the vicinity of Hokkaido Islandand one clone (K1) from Lake Kasumigaura, Ibaragi
prefecture. In addition, siblings of bisexual diploid kinbuna, C. carassius buergeri (formerly C. aura/us susp.), werealso used in hopes that some of them might be geneticallyrelated. Fish source and histocompatibility relationshipamong clones have been previously described (Nakanishi
1987b, Nakanishi and Onozato 1988). Cell transfer was
carried out according to the methods described above. Scaletransplantation techniques followed Nakanishi (1987c).
Interclonal transfer between strong H gene-disparate
clones was carried out to determine transferability of immune pronephric cells. Transfer of antibody reactivity was
not successful when pronephric cells were transferred from

Table 4
Transferability of immune pronephric cells iq isogeneic and xenogeneic crucian carp.
Donor-hostrelationship
Isogeneictransfer

Bl - B1

Xenogeneictransfer

kinbuna - Bl

Inter-clonaltransfer I'

Bl - KI

Inter-clonaltransfer II'

BI - D3

49

Weeks after
transfer
1

2
1

2
1

2
1

2

HAa titre of recipients( -10g2)
7.5
6

6
6

8

7
9

5
6

5

8

0
0

0
0

0
0

0
0

I

0

0

2

0
0

1

2
0

1

0

0
0

0

0
0

9
8.5

6
5.5

7.5
7.5

4
4

3

a Hemagglutination.
'Cells were transferred from B I clone to a K1 clone that differs in strong H gene.
'Cells were transferred from B1 clone to a 03 clone that differs in weak H gene.

4

4
4

NOAA Technical Report NMFS 92

50

_

6

A

5

4

/

3

I

:0 . v·- ~--_

2

.....

N

'" <2
0

~

w

lr

.....
.....

«

:r

z

A.

/ '··v·'

B

6

«

w

~

........

/

5

/

4

/

3
2

/""-.

~// ..... ,,;/"~~~~~

<2

o

2

3

4

5

6

WEE K 5Figure 3(A) Transferability of immune pronephric cells from donors 7-days
postimmunization in allogeneic kinbuna crucian carp. Cells(5 x 106) per fish were intravascularly transferred and then antibody titres in the plasma of recipients were determined every week
after transfer. Each line represents the antibody titre of individual
crucian carp. (B) Transferability of immune pronephric cells fromdonors 14 days post-immunization in allogeneic kinbuna crucian
carp.

tions even when the reCIpients rejected an allografted
donor's scales in an acute manner.

Summarizing Comment
Many developments have contributed to the understanding of the immune system in fish over the last decade.
However, more progress cannot be expected without using
isogeneic or genetically defined models. Genetically identical animals offer many additional opportunities for analyzing the immune system of fish, as demonstrated withmice and rats. Indeed, clonal ginbuna are a useful tool frommany 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 gynogenetic reproduction and the other is their heterozygous state.
Even so, ginbuna will continue to be one of the best models
for the study of fish immunology until the establishmentof inbred or clonal fish by means of chromosomal manipulation technique.

Citations
Borysenko, M.
1976. Phylogeny of immunity: an overview. Immunogenetics 3:
305-326 .
Cerfas, N.B.
1966. Natural triploidy in the female of the unisexual variety ofthe silver crucian carp (C. aura/us gibelio Bloch). Genetika. 5:
16-24.
Du Pasquier, L., X. Chardonnens, and V.C. Miggiano.
1975. A major hostocompatibility complex in the toad Xenopus laevis(Daudin). Immunogenetics 1:482-494.
Kallman, K.D.
1964. An estimate of the number of histocompatibility loci in the
teleost Xiphophorus maculatus. Genetics 50:583-595.
1970. Genetics of tissue transplantation in Teleostei. Transplant.
Proc. 2:263-271.
Kaufman, ].F., M.F. Flajnik, L. Du Pasquier, and P. Riegert.
1985. Xenopus MHC class II molecules. I: Identification and structural characterization. ]. Immunol. 134:3248-3257.
Klein, ].
1977. Evolution and function of the major histocompatibility system: facts and speculations. In The major histocompatibility
system in man and animals (D. Gotze, ed.), p. 339-378.
Springer-Verlag, Berlin, New York.
Kobayashi, H.
1971. A cytological study on gynogenesis of the triploid ginbuna(Carassius auratus langsdorfii). Zool. Mag. 80:316-322.
1976. A cytological study on the maturation division in the oogenic
process of the triploid ginbuna (Carassius aura/us langsdorfi.). ]pn.
]. Ichthyol. 22 :234-240.
Kobayashi, H., Y. Kawashima, and N. Takeuchi.
1970. Comparative chromosome studies in the genus Carassius,
especially with a finder of polyploidy in the ginbuna (C. aura/us
langsdorjii). ]pn.]. 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. ]pn.
Soc. Sci. Fish. 43:31-37.
Murayama, Y., M. Hijikata, T Nomura, and T Kajishima.
1984. Analyses of histocompatibility and isozyme variations in a
triploidfish, Carassius aura/us langsdorfii. ]. Fac. Sci., Shinshu Univ.
19:9-25.
Nakamura, T, A. Sekizawa, T. Fujii, and C. Katagiri.
1986. Cosegregation of the polymorphic C4 with the MHC in thefrog, Xenopus laevis. Immunogenetics 23: 181-186.
Nakanishi, T
1987a. Kinetics of transfer of immunity by immune leukocytes and
PFC response to HRBC in isogeneic ginbuna crucian carp,
Carassius aura/us langsdorfii. ]. Fish BioI. 30:723-729.
1987b. Transferability of immune plasma and pronephric cells in
isogeneic, allogeneic and xenogeneic transfer systems in cruciancarp. Dev. Compo Immunol. 11:521-528.

____________________ Nakanishi and Onozato: Immunology and Genetics of Crucian Carp
1987c. Histocompatibility analyses in tetraploids induced fromclonal triploid crucian carp and in gynogenetic diploid goldfish.
J. Fish BioI. 31: 35-40.
Nakanishi, T., and H. Onozato.
1987. Variability in the growth of isogeneic crucian carp, Carassius
gibelio langsdorfi·i.
ippon Suisan Gakkaishi 53:2099-2104.
1988. Histocompatibility analyses in a gynogenetic ginbuna cru·
cian carp, Carassius gibelio langsdorfii collected from Okushiri Island.
(Abstr.) Annu. Meeting Jap. Soc. Ichthyol. (in Japanese.)
Naruse, K., K. Ijiri, A. Shima, and N. Egami.
1985. The production of cloned fish in the medaka (Oryzias latipes).
J. Exp. ZooI. 236-335-341.
Onozato, H.
1981. Gynogenesis in fishes. Fish Genetics and Breeding Science
6:11-18. (in Japanese.)

51

1983. A story of clonal crucian carp. Tansuigyo 9:33-41. (inJapanese.)
1990. Production of clones of homozygous rainbow trout, Oncorhynchus mykiss by gynogenesis. (Abstr.) Annu. Meeting Jpn. Soc.
Sci. Fish. (In Japanese.)
Schroder, J.H., and S. Holzberg.
1972. Population genetics of Lebistes reticulatus Peters (poeciliidae;
Pisces). 1: Effects of radiation induced mutation on the segregation ratio in postirradiation F2 . Genetics 70:621-630.
Streisinger, G., C. Walker, N. Dower, D. Knauber, and F. Singer.
1981. Production of clones of homozygous diploid zebra fish
Brachydanio relio. Nature 291 :293-296.
Taguchi, Y.
1980. Establishment of inbred strains of the teleost, Oryzias latipes.
Zool. Mag. 89:283-301.

Aquaculture of Striped Bass, Morone saxatilis,and Its Hybrids in North America 1,2
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 impediments 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 impacts 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_

Native stocks of striped bass, Morone saxatilis, have supported major fisheries in the United States along the Atlantic coast. However, since 1973 landings have declined
dramatically (Fig. 1) and in an effort to protect the remaining stocks, commercial fishing bans have been implemented
in many states. The striped bass is also an important
recreational species and has been stocked in lakes, reservoirs, and rivers throughout the United States to support
sport fisheries (Stevens 1984).
The striped bass is well known in the marketplace where
it commands a high price (Sport Fishing Institute 1984;
Swartz 1984). Because of the scarcity of this species andits high market value, interest has increased for culturing
striped bass or a suitable market substitute. During recent
years, research and development activities have included
attempts to rear striped bass and its hybrids in ponds, netpens, raceways, and tanks (Powell 1973; Valenti et al.
1976; Wawronowicz and Lewis 1979; Williams et al. 1981;
Kerby et al. 1983a, b; Woods et al. 1983, 1985; Collins
et al. 1984; Kerby et al. 1987; Smith et al. 1987). Results

Contribution Number 233 from the South Carolina Marine Resource
Center.
2 Preparation of this report was supported in part by Dep. of Commerce,
NOAA, Office of Sea Grant, under Contract Number NA85AA-DSG-121 and the State of South Carolina.
1have been highly encouraging and now there is commercial interest in many states to rear these fish for market(Smith and Jenkins 1985a).
This manuscript reviews and discusses the various considerations associated with aquaculture of striped bass andits hybrids in North America. In particular, informationon broodstock acquisition and culture, spawning, hatching,larval rearing, and production data are presented. Also,
comparative performance data on some of the various
hybrid striped bass crosses are included.

Status of Aquaculture_

Supply of "Seed Stock"
A basic impediment to the large-scale development ofstriped bass and hybrid bass farms has been the lack ofa dependable supply of seed stock Ooint Subcommittee on
Aquaculture 1983; Smith and Jenkins 1985b; Smith 1987).
Presently, state and federal hatcheries rely on the captureof wild adults from spawning grounds to support hatcheryoperations (Harrell 1984). However, the private sector istypically prohibited from collecting broodstock using thesame techniques (especially electrofishing) and from obtaining stock from areas used by these public hatcheries.
Consequently, acquisition of ripe broodstock by the private
sector is unpredictable, inefficient, and highly regulated.
In recent years, there has also been an overall decrease in
53

54

NOAA Technical Report NMFS 92

_

7

6

5
CI)

Z

0

I-

4

()

a::

I-

3

UJ

~

2

1

o
64 66 68

70

72

74 76 78 80 82 84 86
YEARS

broodstock numbers such that even state and federal
hatchery managers are currently experiencing difficulty inmeeting their production goals.

Figure 1
Commercial landings of striped bass on the
Atlantic coast. (Fisheries of the United States,
formerly Fisheries Statistics of the U.S.)

Table 1Age, size, and percent maturity for cultured striped bass
broodstock (updated from Smith and Jenkins 1986).a
Males

Broodstock Development Research
In order to alleviate this "seed stock" problem, South
Carolina researchers began in 1982 to examine the feasibility of developing domesticated broodstock to support
hatchery operations (Smith and Jenkins 1984). Progenyof wild striped bass were reared in tanks and subjected tocontrolled temperature and photoperiod regimes (Smithand Jenkins 1986). During a five-year period the growth,maturity, and spawning success of these fish were monitored. At 33 months of age males had attained a size of
2.3 kg and essentially all were mature (Table 1). However,at 33 months of age only 25 % of the females were matureand difficulty was experienced in spawning these fish
(Smith and Jenkins 1986). During the following two years
additional females matured while no additional mature
males were observed (Table 1).
Spawning of wild striped bass broodstock is accomplished by injecting the newly captured fish with humanchorionic gonadotropin (HCG) at a rate of 330 I.U.lkg.
Then, the fish are either "naturally" tank spawned by pairing up males and females in a tank (Bishop 1975), or theeggs are stripped from the female at time of ovulation andartificially fertilized with stripped milt (Bayless 1972; Bonnet al. 1976). In the first case, larger facilities are needed
to house spawning tanks but effort is less intensive anddamage to broodstock is reduced. In the latter method, theAge(Months)

Mature'
(%)

21
33
46
60

lOa
lOa
lOa

Females
Weight(kg)

Mature'
(%)

2.3
3.9
5.2

25
75
100

Weight
(kg)

a

22

3.4
4.9
6.4

'Based on number of males and females at 60 months of age. 19%of the fish did not mature by age 60 months.
'Fish which expressed milt.
'Fish with eggs greater than 700 !J in diameter.

space required is reduced but effort is intensive and often
broodstock die as a result of handling and stripping procedures. In South Carolina, both techniques have beenused with 3- to 5-year old cultured broodstock. It is recommended that the "tank spawning" method be employedwith striped bass in order to reduce fish injury. Althoughsubstantial progress has been achieved, the culture techniques developed for striped bass females need additional
research to improve the predictability of spawning success.
In contrast, development of domesticated male broodstock
has been highly successful. Males can be produced in 2
to 3 years and used over several years (Smith and Jenkins
1986).

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Smith: Aquaculture of Striped Bass

The techniques described above are used for the production of striped bass fry. However, the striped bass is
not the preferred fish for an aquaculture operation becauseof its slower growth during the first two years and its
reduced environmental tolerances. Concurrent research
conducted with the striped bass and white bass (M. chrysops)
crosses has indicated that these hybrids are the preferredfishes for aquaculture development (discussed later in text).
In order to make "original cross" hybrids, female stripedbass and male white bass must be stripped because they
can not be induced to tank spawn together. Consequently,
the problems of acquisition, predictability, and reuse of
female striped bass occur. However, the" reciprocal cross"(female white bass x male striped bass) can also be performed which results in a hybrid which is quite satisfactory for aquaculture development (see later section). White
bass are smaller than striped bass at maturity (minimum
size for females ""300 g vs. ""3.4 kg for striped bass) andthis species is abundant throughout many areas of the U.S.
Although this species is of recreational importance, there
is much less public sentiment associated with their removal
for private hatchery use compared to the collection of
striped bass (especially females).
Broodstock development activities with white bass havebeen based on the use of wild-caught fish, although cultured
white bass may also prove to be suitable. Researchers inSouth Carolina have demonstrated that adult wild fish canbe captured in the fall, matured in outdoor tank culture
systems using ambient conditions, and spawned in thespring (Smith and Jenkins 1986). Unlike striped bass, adultwhite bass are either ripe males or ripe females in the springwith few or no fish of unknown sex. Work-to-date indicatesthat white bass females and striped bass males ace easy tospawn and will naturally mature together in outdoor tanks(Smith and Jenkins 1985b, 1986, 1987). HCG is also usedwith white bass but it is administered at a higher level(750-1500 1. U ./kg). Average production of reciprocal crosshybrid bass fry is in the range of35000 to 90000 per female
fish 460 to 640 g in weight (Smith and Jenkins 1986; Table2). Recent demonstration of these spawning techniques
using captive, wild broodstock should enhance the development of hybrid striped bass aquaculture farms (Smith andJenkins 1987).

Incubation and Hatching
Eggs of striped bass and its hybrids are typically incubatedin McDonald hatching jars (Bonn et al. 1976). Dependingon egg type, approximately 100000-250000 eggs areplaced in each jar. Water injected through a center tube
causes continuous upwelling and oxygenation, and eggs
hatch in about 48 hours at temperatures of 18° to 20°C,(Bayless 1972). At time of hatching, fry swim up with theoutflowing water and are concentrated in outside aquaria.
In situations where striped bass are tank-spawned, eggs

55

Table 2
Summary of reciprocal cross fry production using captive
wild white bass females and cultured striped bass males (updated from Smith and Jenkins 1986).

Trial No.

Fish Weight(kg)

Larvae Hatched
(no.)

1
2
3
4
5
6
7
8
9

0.63
0.46
0.64
0.61
0.64
0.68
0.73
0.65
0.66

33992
37247
84185
17870
87950
48100'
48100'
48100'
48100''All hatches were pooled, 192000 fry produced.

can be left in the spawning tank to hatch or the eggs canbe collected and placed in McDonald jars. Striped bass eggs
are semibuoyant and are easily kept rolling in the jars. In
contrast, white bass eggs are highly adhesive and willreadily form large clumped masses of eggs. Such clumping is undesirable as dead eggs are not easily removed andfungal infections can readily occur. In order to eliminate
the adhesiveness, the fertilized eggs are placed in the
McDonald jars and aerated in a tannic acid solution (150mg/L) for about 10 minutes before clean fresh water isflowed through the jars. This technique reduces clumping
and results in a higher hatch rate of reciprocal cross hybrids(Charles C. Starling, Florida Game and Fresh Water FishCommission, Webster, FL 33597, pers. commun., March1986).

Fingerling Production
The fry are reared in either freshwater or brackish water
nursery ponds (Phase I) to a small juvenile size over a
30-45 day period (Bonn et al. 1976; Geiger 1983a, b;Parker and Geiger 1984). During the last 2-3 weeks ofnursery rearing, dry feed is added to the ponds to serve
as a replacement for the diminishing supply of zooplankton of a suitable size. Typical stocking density for thePhase I nursery ponds is about 250000 fry/ha. Survival
ranges from 0 to about 80%, but 25% is common (Parker
and Geiger 1984). Temperature in the ponds ranges fromabout 18 to 25°C. During 1985 and 1986, nursery trials
were conducted at the S.C. Wildlife and Marine Resources
Department's Waddell Mariculture Center (WMC). Production from these trials conducted at dissimilar densities
was 281000/ha for striped bass and 110300/ha on the
average for hybrids (Table 3).
The small juveniles are harvested by draining the pond
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

Type of fish

Density(no.lha)Date

Duration(d)Density(no.lha)Survival(%)

Mean wt.
(g)

Striped bass
F, original hybrid
F, reciprocal hybrid
F, reciprocal hybrid

6000001280003000003000004/28/87
5/08/85
3/10/86
3/10/86

30
41
57
56

28163096000
74500
161030

46.9
75.0
24.8
53.7

0.36
0.45
2.20
0.67

structure. Fish produced by private hatcheries are thengraded and restocked in Phase II rearing ponds at a lower
density or sold to other farmers. Indoor intensive nursery
techniques using tanks have also been developed forPhase I rearing of striped bass and its hybrids (Lewis andHeidinger 1981; Kerby et al. 1983a; Carlberg et al. 1984;
Smith and Jenkins 1984). However, production ofjuveniles
from such tank nursery systems is often highly variable andthe pond-rearing method is currently the preferred technique for Phase I production.

Performance Characteristicsof Striped Bass and Hybrids
Hybridization studies with striped bass were initiated in1965 after it was demonstrated that HCG could be used
to induce final maturation and ovulation of striped bass

eggs (Stevens 1966, 1967). The focus of the hybridization work was to develop a fish that had the growth andfeeding characteristics of the striped bass and the environmental adaptability and less stringent spawning requirements of white bass (Bonn et al. 1976). The firsthybrid produced was the "original cross." Later othercrosses were made using white perch (M. americana) andyellow bass (M. mississippiensis) males as well as reciprocaland backcrosses (Bayless 1972). Early evaluation of thesevarious hybrids was somewhat opportunistic and not wellcontrolled. Nevertheless, from these studies it was concluded that the original cross hybrid was a good fish andstocking programs were initiated (Bishop 1968; Logan
1968; Williams 1971; Ware 1975). Field observations indicated that the hybrids outgrew striped bass during thefirst two years and were both easier to produce and hardier than striped bass.

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

Fish stocked

Type

Size
(g)

Density(no.lm 3)

Striped bass
F, original hybrid
F2 hybrid bass

4.46.412.8

F, original hybrid
F, white perch hybrid
Striped bass
F, original hybrid
F, reciprocal hybrid
Backcross hybrid'Study is currently in progress.

Duration
(d)

Size
(g)

Survival(%)

Biomass(kg/m 3)FeedconverSion

31.6
31.6
31.6

Study number 1
287
287
287

289
507
34-7

96
100
84-

8.8
16.0
9.2

2.3
2.2
2.7

23.0
21.0

31.6
31.6

Study number 2
140
140

263
176

99
99

8.3
5.5

1.9
2.5

6.0
13.2
10.9
21.1

29.2
29.2
29.2
29.2

Study number 3'
56
56
56
56

34-

89
98
93
92

1.43.6
2.5
3.1

1.7
1.6
1.9
2.4

77

57
71

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Smith: Aquaculture of Striped Bass

57

200
1BO
/

/

Reciprocal

160

Hybrids

/
/

/

/

/

/

/

140
0>

120

:I:

100

---t(!)

w

:;:

80
60
40
20
0

o

28

56

84
1 12
126
TIME (days)

With the increasing interest in aquaculture, moredetailed performance information was desired for thevarious hybrids. Controlled studies were undertaken
primarily in South Carolina and North Carolina. To date,
not all striped bass hybrids have been tested but sufficient
information is available to identify suitable candidates foraquaculture use.
A number of comparative studies have been undertakenby the Charleston Laboratory, S.C. Wildlife and Marine
Resources Department, using indoor tanks which can recirculate either fresh or brackish water. In the first study
striped bass were compared to reciprocal cross hybrids andto F 2 hybrids (F 1 original cross x F 1 original cross).
Results of this 287 -day study indicated that the reciprocal
cross hybrid grew fastest and had a high survival rate
(Table 4, Smith et al. 1985). In the second study theoriginal cross hybrid was compared to a striped bass x
white perch hybrid. Again, results indicated that the whitebass hybrid grew rapidly and had a high survival rate
(Table 4). In a current study, the original and reciprocal
cross hybrids are outperforming the striped bass. However,a backcross hybrid (F J original x striped bass male) is
also performing well at the present time and may be an
additional aquaculture candidate (Table 4) (T. Smith, unpublished). Results of these replicated tank studies are also
being corroborated in pond culture trials (Figs. 2, 3)

140

168

184

Figure 2
Comparison of F I onglnal and reciprocal crosshybrids reared in ponds at
a density of 10 000 fish/ha.

(T. Smith, unpublished).
In summary, the hybrids of striped bass and white bass
appear well suited for aquaculture. They can be raised in
fresh or brackish water (Smith et al. 1986) and exhibit highgrowth and survival rates. Further, they can be reared ina broad range of water temperatures and are not killed bylow (7°C) or high (33°C) water temperatures (Smith et al.
1987).

Grow-Out Production
Research data from production trials to produce marketsize fish (~568 g) have been limited although there is acommercial operation producing hybrids in tanks using
geothermal water in California and another company isusing net pens to produce striped bass in New York. Unfortunately, production data from these two operations areproprietary. In 1984, North Carolina researchers harvested
grow-out trials in 0.1 ha earthen ponds using original crosshybrids. Survival averaged 83.9% and production ranged
from 5247 to 5765 kg/ha (mean 5504 kg/ha) (Kerby et al.
1987). Recently, the S.C. Wildlife and Marine Resources
completed its first pond grow-out trial using original crosshybrids. This study was conducted in a 0.5 ha pond at
WMC. One-year-old juveniles (mean size 220 g) were
stocked in March 1986 at a density of 12 OOO/ha for final

58

NOAA Technical Report NMFS 92

_

90
80
1

70

Reciprocal Hybrids

1

1

1

1

1

1

1

1

/
1

1

--

1/

60

0>

I::I:

C'

w

;;

1

50
1

/

1

1

1

1

1

1

1

1

1

1

1

1

1

/
/

40
/

30
//

/

/

/

/

/

/

//

20

/

//
/

10

/

Striped Bass/-------------------0
12

24

36
48
60
TIME (days)

grow-out to market SIze. During the growing season(April-November), fish were fed a commercial trout pellet
twice daily and satisfactory water quality conditions weremaintained with paddlewheel aerators and water exchange.
From December, 1986 to January, 1987 the fish were harvested and marketed. At harvest, mean fish size was 755 g
and survival was 93 % (Table 5). Total production was
8323 kg/ha with 93% of the fish ~568 g (1 Vi. Ib). These
results are highly encouraging and represent the highest
pond production level yet achieved. Currently, a pond
grow-out trial is underway with reciprocal cross hybrids
and growth to the present appears similar. From an aquaculture perspective, routine production levels of 3900 to
7800 kg/ha should be attainable using techniques similar
to those employed for producing channel catfish (lctalurus

72

84

96

Figure 3
Comparison of stripedbass and reciprocal crosshybrids reared in ponds at
a density of 37000 fish/ha.

Market Testing
The opportumties and constraints associated with themarketing of hybrid striped bass were recently examinedby Carlberg and Van Olst (1987). Prices received for thecultured hybrids have been in the range of $5.50-11.00/kg
depending on product type (e.g., iced, gutted, live) andspecific market (e. g., wholesaler, retailer, restaurant).
Organoleptic testing of pond-reared cultured hybrids has
been conducted by the Southeast Fisheries Center[National Marine Fisheries Service (NMFS)], Charleston,
S.C. Results indicate that the hybrid striped bass is a mild
flavored fish which should have good market appeal(Michael Jahncke, NMFS, Charleston, SC 29412, pers.
commun., June 1987). Restaurant testing has also been
conducted using our pond-reared hybrid bass. Again,

nebulosus) .

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

Harvest data

Density(no.lha)Mean wI.
(g)

Age of fish(d)Pond size(ha)

Duration(d)Mean wI.
(g)

Survival(%)

Production(kg/ha)Feedconversion

2000

220.2

327

0.6

240

755

93

8323

1.6

_________________________________ Smith: Aquaculture of Striped Bass

59

Table 6
(N -- 216) of hybrid striped bass served in two Flordia restaurants
from Liao et al. 1987).
.
Consumer evaI uatlOn
4 (adaptedII
Rating scale: 1poor, 2 = fair, 3 = good, 4 = very good,= exce ent.
Consumer repurchase (%)

Evaluation catergories
Restaurant

EntreePrice ($)

Appearance

Texture

Smell

Taste

Price

No

Maybe

Yes

A

BroiledFried
Grilled

10.95
10.95
10.95

4.7
4.2
4.4-

4.6
4.7
4.4

4.8
4.2
4.4

4.7
4.2
4.3

4.44.1
4.3

2
22
8

4
0
0

94
78
92

B

Broiled
Fried

4.95
4.95

4.3
4.1

4.2
4.2

4.44.1

4.5
4.4-

4.2
4.2

2
2

5
5

93
93

results were favorable indicating that these fish are highly
acceptable to consumers (Table 6, Liao et al. 1987). Thus,
cultured hybrid striped bass appear to be an excellent
market substitute for wild striped bass.

Aquaculture Constraints
At the present time, there are two major constraints tothe development of striped bass farms: 1) seed stock
availability and cost, and 2) laws and regulations. Recent
research in South Carolina on the domestication andculture of broodstock has demonstrated commerciallypractical techniques for controlled production of "se.ed
stock." Thus, in the near future, private hatchenes
should be able to develop their own captive broodstock(especially striped bass males and white bas~ fe~ales)
and thereby increase the predictability and availabilIty offry and small juveniles. This use of captive broodstock may
also result in lower production costs and lower seed stock
costs.
The legal issues have been a serious constraint In
many states where fishery bans were instituted to protect
native fishery stocks including game species such as striped
bass. In most cases, there was no exclusion for farm-reared
fish. During the past year there has been substantial
interest expressed by the agricultural community, businessmen, and landowners, to commercially farm stripedbass and its hybrids. Consequently, pressure has beenexerted on legislators to legalize the culture and sale of thesefish with the result that many states are currently reexamining their laws and making provisions for bass
aquaculture (e.g., Florida, Virginia, North Caroli?a,Georgia, Mississippi). In addition to the laws concerning
possession and sale of fish, there are many additio~alregulations which affect the aquaculturist. These deal Withsite permits, discharge constraints, use of medications, andbroodstock collection techniques, among others Oenkins
1987). Some of these laws and regulations are also being
re-examined because of their impact on aquaculture
development.

Conclusions_

Commercial farming of striped bass hybrids is an emerging industry in the United States which wil! need additio.nal
research and extension activities to reach Its full potential.
Pond culture technology is currently available and is now
in the process of being transferred to the private sect.or.
Indeed, during the past year cooperative demonstratIOn
projects have been initiated in several states including
Maryland and North Carolina. Within the next 3-5 years,
pond culture operations are expected to develop througho~t
the southeast and middle Atlantic states as well as In
California.
Although the research data base appears adequate fordevelopment of the industry, additional study is nee~edin the areas of 1) broodstock development and genetics,
2) identification of other suitable hybrids, 3) nutrition,4) market development, 5) disease treatment and prevention, and 6) laws and regulations. All these areas cansubstantially impact the economics of bass aquaculture. For
example, profitability could be improved by developmentof hybrids exhibiting faster growth through genetic breeding or manipulation (induced polyploidy) and by the identification of more cost-effective rations.
The future appears highly promising for the development of hybrid striped bass farms throughout many areasof the United States. For the most part, these fish will bereared as a high quality seafood product. However, it isalso expected that these fish will be used to support recreational fee-imposed fishing operations as well. Such operations will provide income to the operator while at the sametime affording recreational fishing opportunity to the public
sector. In summary, the striped bass x white bass hybrids
are not only important recreational fish but they also
appear to be excellent candidates for commercial
development.

Citations_

Bayless, J.D.
1972. Artificial propagation and hybridization of striped bass,

60

NOAA Technical Report NMFS 92

Morone saxatilis (Walbaum). South Carolina Wild!. Mar. Resources Dep., 135 p.
Bishop, R.D.
1968. Evaluation of the striped bass (Roccus saxatilis) and white bass
(Roccus chrysops) hybrids after two years. Proc. Annu. Conf.
Southeast. Assoc. Game Fish Comm. 21:245-254.
1975. The use of circular tanks for spawning striped bass (Morone
saxatilis). Proc. Annu. Conf. Southeast. Assoc. Game Fish
Comm. 28:35-44.
Bonn, E.W., W.M. Bailey, J.D. Bayless, K.E. Erickson, andR.E. Stevens.
1976. Guidelines for striped bass culture. Striped Bass Committee of the Southern Division. Am. Fisheries Soc., Bethesda, MD,
103 p.
Carlberg, J.M., and J.C. Van Olst.
1987. Processing and marketing. In Hybrid striped bass culture:status and perspective (Hodson et a!., eds.), p. 73-82. Univ.
North Carolina Sea Grant Pub!. No. 87-03.
Carlberg, J.M., J.C. Van Olst, M.]. Massingill, and TA. Hovanec.
1984. Intensive culture of striped bass: a review of recent technological developments. In The aquaculture of striped bass - aproceedings (J.P. McCranen, ed.), p. 89-127. Univ. Maryland
Sea Grant Pub!. No. UM-SG-MAP-84-01.
Collins, C.M., G.L. Burton, and R.L. Schweinforth.
1984. High density culture of white bass x striped bass fingerlings
in raceways using power plant heated effluents. In The aquaculture of striped bass - a proceedings (J.P. McCraren, ed.), p.
129-142. Univ. Maryland Sea Grant Publication No. UM-SGMAP-84-01, 262 p.
Geiger, J.G.
1983a. Zooplankton production and manipulation in striped bass
rearing ponds. Aquaculture 35:331-351.
1983b. A review of pond zooplankton production and fertilization
for the culture of larval and fingerling striped bass. Aquaculture
35: 353-369.
Harrell, R.M.
1984. Review of striped bass broodstock acquisition, spawning
methods and fry production. In The Aquaculture of striped bass
- a proceedings (J.P. McCraren, ed.), p. 45-57. Univ. Maryland
Sea Grant Pub!. No. UM-SG-MAP-84-01.
Jenkins, W.E.
1987. Laws and regulations. In Hybrid striped bass culture: status
and perspective (Hodson et a!., eds.), p. 93-100. Univ. North
Carolina Sea Grant Pub!. No. 87-03.
Joint Subcommittee on Aquaculture.
1983. Striped bass species plan. In National aquaculture development plan, Vo!. 2, p. 136-145. The Joint Subcommittee on
Aquaculture of the Federal Coordinating Council on Science,
Engineering, and Technology, Washington, DC.
Kerby, J.H., L.C. Woods III, and M.T Huish.
1983a. Culture of striped bass and its hybrids: a review of methods,
advances and problems. In Proceedings of warm water fishculture workshop (R.R. Stickney and F.P. Myers, eds.), p. 23-54.
World Maricult. Soc., Spec. Pub!. No.3.
1983b. Pond culture of striped bass x white bass hybrids J.
World Maricult. Soc. 14:613-623.
Kerby, J.H., J.M. Hinshaw, and M.T Huish.
1987. Increased growth and production of striped bass x whitebass hybrids in earther ponds. J. World Aquacult. Soc.
18(1):35-43.
Lewis, W.M., and R.C. Heidinger.
1981. Tank culture of striped bass: production manua!. Illinois
Striped Bass Project IDC F-2, 6-R, Univ. Carbondale, Illinois,
115 p.
Liao, D.S., TI.]. Smith, and W.E. Jenkins.
1987. Preliminary market analysis for cultured hybrid striped_
bass. Presented at the Symposium on Markets for Seafood andAquacultural Products, Charleston, S.C., August 1987. (Mimeo
report.) S.C. Wild!. and Mar. Resources Dep., Charleston,SC.
Logan, H.].
1968. Comparison of growth and survival rates of striped bass x
white bass hybrids under controlled environments. Proc. Annu.
Conf. Southeast. Assoc. Game Fish Comm. 21 :260-263.
Parker, N.C., and J.C. Geiger.
1984. Production methods for striped bass. In Third report to thefish farmers (H.K. Dupree andJ.V. Huner, eds.), p. 106-118.
U.S. Fish Wild!. Serv., Washington, D.C.
Powell, M.R.
1973. Cage and raceway culture of striped bass in brackish waterin Alabama. Proc. Annu. Conf. Southeast. Assoc. Game Fish
Comm. 26:345-356.
Smith, TI.].
1987. Hatchery. In Hybrid striped bass culture: status andperspective (Hodson et aI., eds.), p. 17-22. Univ. North Carolina
Sea Grant Pub!. No. 87-03.
Smith, T!.J., and W.E. Jenkins.
1984. Controlled spawning of F, hybrid striped bass (Morone saxatilis x M. chrysops) and rearing of F2 progeny. J. World Mariculture Soc. 15:147-161.
1985a. Status of aquaculture of striped bass (Morone saxatilis) and
its white bass (M. chrysops) hybrids and research in South Carolina.
In Proceedings of the 2nd int. conf. warm water aquaculture - finfish. Brigham Young Univ. p. 553-582.
1985b. Aquaculture research with striped bass (Morone saxatilis) and
its hybrids in South Carolina. Proc. Annu. Conf. Southeast.
Assoc. Fish Wild!. Agencies 39:219-227.
1986. Culture and controlled spawning of striped bass, Morone saxatilis, to produce striped bass, and striped bass x white bass, M.
chrysops hybrids. Proc. Annu. Conf. Southeast. Assoc. Fish WildJ.
Agencies 40: 152-162.
1987. Controlled spawning of cultured striped bass, Morone saxatilis.
(Abstract.) 117th Annual Meeting Am. Fisheries Soc.
Smith, TI.]., W.E. Jenkins, and J.F. Sneve!.
1985. Production characteristics of striped bass, and F, and F 2
striped bass/white bass hybrids reared in intensive tank systems.
J. World Mariculture Soc. 16:57-70.
Smith, TI.]., W.E. Jenkins, and R. Haggerty.
1986. Growth and survival ofjuvenile striped bass (Moronesaxatilis)
x white bass (M. chrysops) hybrids reared at different salinities.
Proc. Annu. Conf. Southeast. Assoc. Fish Wild!. Agencies 40:
143-151.
Smith, T.I.]., W.E. Jenkins, A.D. Stokes, and R.A. Smiley.
1987. Pond production trials with striped bass (Morone saxatilis) andwhite bass (M. chrysops) hybrids. (Abstract.) J. World Aquaculture
Soc. 18(1):8A.
Sport Fishing Institute.
1984. An uncertain future. Sport Fishing Inst. Bull. 35(1):1-5.
Stevens, R.E.
1966. Hormone-induced spawning of striped bass for reservoir
stocking. Prog. Fish-Cult. 28: 19-28.
1967. A final report on the use of hormones to ovulate striped bass
Roccus saxatilis (Walbaum). Proc. Annu. Conf. Southeast. Assoc.
Game Fish Comm. 18:525-538.
1984. Historical overview of striped bass culture and management. In The Aquaculture of striped bass: a proceedings (J.P.
McCraren, ed.), p. 1-5. Univ. Maryland Sea Grant Pub!. No.
UM -SG-MAP-84-0 1.
Swartz, D.
1984. Marketing striped bass. In The Aquaculture of striped bass:
a proceedings (J.P. McCraren, ed.), p. 233-254. Univ. Maryland Sea Grant Pub!. No. UM-SG-MAP-84-01.

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Smith: Aquaculture of Striped Bassu.s. Department of Commerce.
1978-87. Fisheries of the United States. (Formerly Fishery Statistics of the U.S.) U.S. Dep. of Com mer., NOAA, NMFS, var.
pagination.
u. S. Department of Interior.
1965-77. Fishery Statistics of the U.S. U.S. Dep. Interior, Fish
Wild!. Serv., var pagination.
Valenti, R.J.,]. Aldred, and J. Liebell.
1976. Experimental marine cage culture of striped bass in northernwaters. Proc. World Maricult. Soc. 7:99-108.
Ware, F.J.
1975. Progress with Morone hybrids in fresh water. Proc. Annu.
Conf. Southeast. Assoc. Game Fish Comm. 28:48-54.
Wawronowicz, L.J., and W.M. Lewis
1979. Evaluation of the striped bass as a pond food fish. Prog.

61

Fish-Cult. 41: 138-140.
Williams, H.M.
1971. Preliminary studies of certain aspects of the life history of
the hybrid (striped bass x white bass) in two South Carolina reservoirs. Proc. Annu. Conf. Southeast. Assoc. Game .Fish Comm.
24:424-431.
Williams, J.E., P.A. Sandifer, and J.M. Lindberg.
1981. Net-pen culture of striped x white bass hybrids in estuarine
waters of South Carolina: a pilot study. J. World Maricult. Soc.
12(2):98-110.
Woods, L.C. III, J.H. Kerby, and M.T. Huish.
1983. Estuarine cage culture of hybrid striped bass. J. World
Maricult. Soc. 14:595-612.
1985. Culture of hybrid striped bass to marketable size in circular
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 Institute for 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 basedon computerized image analysis which can provide an accurate estimate of organism size.
Preliminary studies were conducted with a computer assisted morphometric analysis which obtains more information on size and shape from an image of a shrimp body than more conventional direct measurement. Using computer digitization, a truss network of dimensional variables
was collected from photographs of three species of adult penaeid shrimp and used in canonicaldiscriminant analysis. This approach was found to provide more information for discrimination
than a typically labor intensive conventional morphometric method. The results demonstrate theusefulness of two-dimensional images for selection or classification. A truss network data set wasalso 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 basedon principal component scores and single variables are calculated. The multivariate estimatorof size had more of the variance in size attributed to family differences than most single variables (h 2 = 0.60 for PC scores vs. a mean of 0.49 for single variables). Ways in which computerized image analysis might be employed in selective breeding of shrimp are discussed.

Introduction_

There has been much discussion about the possible use ofhigh selection intensities for aquaculture animals. High
selection intensities are possible because the very highfecundities of aquacultured organisms (e.g., penaeid
shrimp) permit a very small fraction of the individuals to
be used for replacement of broodstock. This paper will support several premises related to the use of high selection
intensities in a broodstock selection program. First, the implementation of such selection intensities will be possible
and should not result in genetic drift. Second, the labor

intensive characteristics of such a program will benefit
greatly from the use of computer image analysis, a valuable
alternative to human observers. Third, obtaining accuracy
in the selection process will require the use of multivariate
procedures in the assessment of organism size.
If we compare the fecundity of shrimp with even the mostfertile domestic animal, they are different by orders ofmagnitude. In commercial shrimp culture facilities, female
Penaeus vannamei have been observed to average 60000
nauplii per spawn and 8-10 spawns during their productive life in a maturation facility (B. Ribelin, Laguna Madre
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
larvae from an average female and follow the offspringthrough a production cycle, assuming 50% survival in eachstage. This percentage is low, but not outside the rangeof observed values. The numbers would change as follows:
300000 postlarvae, 150000 juveniles, and 75000 harvested
subadults. Assuming a survival of 50% from selection to
reproduction, one would only need to select four individual
subadults to replace the original two breeders.
Selection intensity (i) is related to the response of a
population to selection (R) by the simple equation R =i Op h 2 in which h 2 is the heritability of the trait underselection and Op is the phenotypic variance of the trait.
The relationship between i and the proportion of animals
selected (P) for truncation selection of a normally distributed trait is i = zip, where z is the height of the normal
curve at the truncation point (Falconer 1981). Thus the
maximum selection intensity for the above example (basedon a proportion of 4 selected from 75000) would be approximately 4.3, if all the survivors could be measured.
Lasley (1978) gives the percentage of the progeny required for broodstock replacement in domestic animals as
4-5% of males and 40-50% offemales in beef cattle; 1-2 %of males and 10-15 % of females in swine and chickens.
Such broodstock selection programs for domestic animals
obtain selection intensities of2.0 to 2.7 for males and 0.6
to 0.8 for females (Falconer 1981). With the very small proportion of the population that is required for replacement
broodstock in aquaculture species, it is possible to achieve
selection intensities of3.0-4.0. Current selection programs
at commercial shrimp farms select the top 1/2 to 1/3 of thesize distribution. If we assume that the trait under selection is heritable, the predicted response under high selection intensity is 3 to 4 times higher than under the current
system. This could be accomplished using within-family
selection to remove the danger of bottlenecks caused byunrecognized restriction of the gene pool and inbreeding.
The limitations on selection intensity are primarily logis-

tical (i.e., high selection intensities would require major
investments of personnel and time).
In a previous publication (Lester 1983), it was recommended that a single abdominal measurement, sixth segment depth (SSD), be used as a criterion in selection forsize of penaeid shrimp (Fig. 1). Several preliminary selections have been performed using this i:haracter. A single
human observer is capable of applying this criterion to300-500 shrimp per hour, but not for many hours continuously (pers. observ.). Nevertheless, at a selection intensity of 3.0, one would be required to measure approximately 300 shrimp, about 40-60 minutes of work, toselect a single potential broodstock. In order to obtain abroodstock of 3000, which is not large by commercial standards, the observers would have to work for over 2000
hours. A microcomputer imaging system could performthe measurements and make a decision on whether to cullor select in several seconds. Computer analysis would be
limited by how fast the shrimp could be moved throughthe imaging system. A single unit could measure continuously and accurately for any amount of time necessary.
No matter what criterion is employed for the selection
process, there will be an associated measurement error. At
a high selection intensity, the variance due to measurement
error could exceed the true phenotypic variance of thepotential broodstock in the distribution. This would represent a serious problem for the classification of animals into
potential broodstock and culls. This factor in misclassification can be reduced by moving from univariate tomultivariate classification. Thus we are recommending thathigh intensity selection not be based on a single measurement such as SSD, but rather on a set of measurements.
The characteristic of commercial importance, rate ofgain in size, is a latent variable which can not be measured
directly in these selection programs. It can be estimatedby analysis of multiple variables which are correlated toit. (See Bookstein et al. 1985, for a thorough discussionof multivariate estimation of the latent variable size.) In_ _ _ _ _ _ _ _ _ _ _.

Diagram of shrimp with measurements used in the firststudy. 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 intersection of the second and third abdominal segments;
FLF = fifth segment length with the abdomen maximally 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.

Figure 1---'

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Lester et al.: Image Analysis for Breeding Shrimp

DM4

VM6
VMS

VM4

DM3 DM2

DM1

VM2
VM3

our approach to estimation of growth rate, the latentvariable size, is represented by the first principal component (PC 1) score obtained from a set of size-correlated
dimensions taken from each shrimp. As variables areadded, the variance-covariance matrix gets larger, but thevariance of the multivariate mean (i.e., mean size)
decreases according to the factor 1 - R 2 (R 2 = the sumof the squared partial correlations between the variables).
In other words, the multivariate data set becomes a better
predictor of the underlying variable size when multiple
variables correlated with size are used in the estimation
process.
There is a school of morphometric studies centeredaround the work of F. L. Bookstein (Strauss and Bookstein
1982; Bookstein et al. 1985) which has developed an approach to the multivariate description of size and shapefor fish. The approach is based on obtaining a set ofvariables that contains much information about specificregions of the body and is homologous across conspecifics
of different sizes and across closely related species. Thisis accomplished by selecting landmark points on the morphology and arranging them into a set of boxes connectedby all possible nearest neighbor connections into a truss
network as shown in Figure 2 (Strauss and Bookstein 1982;
Bookstein et al. 1985). The resultant network consists ofa set of dimensional variables that covary with the latentvariable size and contain information about shape-related
deformational changes among a set of individuals. At thepresent time, we are interested in using this approach toprecisely estimate genetic differences in size. Later we hope
to apply it to the inheritance of shape.
One objective of the research program that we havestarted was the development of a system that would
automate broodstock selection. This program would use
a video camera to obtain an image that would be converted
into digital format. This would be measured and analyzed
by a microcomputer to obtain a summary of multiple

65

Figure 2

Diagram of shrimp with landmarks used in the adulttruss network study. Descriptions of points: DMl =Posterior rostral tooth; DM2 = dorsal, posterior pointon carapace; DM3 = dorsal, posterior point on segment
one; DM4 = dorsal, posterior point on segment three;
DMS = dorsal, anterior point on segment six; DM6= dorsal, posterior point on segment six; VMl =center of antennal basal segment at insertion; VM2= center of basal segment of fifth periopod at insertion;
VM3 = center of basal segment of first pleopod at insertion; VM4 = center of basal segment of third pleopod
at insertion; VMS = center of basal segment of fifthpleopod at insertion; VM6 = ventral, posterior point
on segment six.

variables which would permit the computer to decide
whether the animal should be selected or culled. At thepresent time, we are still at an early stage in the development, using human observers to digitize still images.
This report deals with initial analyses of two data sets.
These analyses are based on still images, not video, andonly one uses living animals for the images. The first data
set was obtained from adults carefully prepared for photographing to test the truss network approach on discrimination of Penaeus species. The second data set was collectedfrom an experiment on growth differences among families
of juvenile shrimp. It will be used to examine the way inwhich the partitioning of the variance in size is affectedby changing from a single to a multivariate approach.

Materials and Methods_

Testing the Truss Network Approach
Samples of adults were obtained from the following sources
for both data sets: Penaeus setiferus from the wild near Galveston, TX; Penaeus vannamei collected from harvests ofponds at the King Ranch near Corpus Christi, TX, at theLaguna Madre Shrimp Farm near Harlingen, TX, andat Agromarina de Panama (only in the truss network dataset) near Aguadulce, Panama; Penaeus stylirostris collectedfrom harvests of ponds at the King Ranch near Corpus
Christi, TX, and tanks at Marine Culture Enterprises nearLaie, HI. The standard morphometric study used 104
P. setiferus, 92 P. stylirostris, and 134 P. vannamei. The truss
network study used 49 P. setiferus, 42 P. stylirostris and 83
P. vannamei. All specimens were preserved by freezing andthawed for photography.
The standard morphometric data set contained ninedimensional variables and two weights, head weight and
tail weight. Dimensional values were obtained with dialcalipers except in the case of circumferences which were

66

NOAA Technical Report NMFS 92

obtained by passing a string around the shrimp andmeasuring the length of string with a ruler. The dimensional variables are shown in Figure 1. Data sets of thistype have been obtained from six species ofpenaeid shrimp.
Only the species P. setiferus, P. stylirostris, and P. vannamei
will be used here for purposes of comparison to truss network data sets.
The truss network variables are shown in Figure 2. Thisdata set contains 26 dimensional variables, no weights, andwas obtained from pictures of the adult shrimp. The adults
were positioned on a light table in the laboratory or on lightplastic in the field for pictures taken at Agromarina dePanama. They were photographed with a scale in the frame
using Kodak Tri-X black and white film. The negatives
were mounted as slides and projected onto a digitizing
tablet attached to a Tektronix graphics terminal. The
operator used the cross-hairs of a mouse to locate 12 landmark points shown in Figure 2. Points were digitized inorder from DM6 to DM1 to VM1 to VM6. The coordinates were stored in a data file, then converted to measurements by a Pascal program written by one of theauthors (Wong, T.-C.).
All measurements were log transformed before use toremove the exponential effects of growth. Both the standard and the truss network data files were analyzed bymultivariate statistical programs, PRINCOMP for principal component analysis and CANDISC for canonicaldiscriminant function analysis (SAS Institute 1985). The
principal component analysis was performed to obtain asummary estimate of the variation in the latent variable
size. Discriminant function analysis was performed withthe two data sets to illustrate the increase in information
relevant to species differences contained in the truss network data set.

_

The postlarvae were weaned from Artemia to a prepareddiet from the Texas A&M Shrimp Mariculture Project,
and reared under the same temperature and salinity conditions for 40 days on a 14L: 10D light cycle. The juveniles
in each cage were fed once per day at ad libitum levels:
50 mg/cage in weeks one and two, 100 mg for five days,
200 mg for five days, 600 mg for five days, and, following
sampling for survival and weight at 28 days, 400 mg to
8 cages with survival <50 % and 600 mg to 14 cages withsurvival >50% for the remaining twelve days. Two cages
had no survivors at 28 days.
After 40 days of growth, the shrimp were harvested andsurvival was determined. Six randomly sampled individuals from each cage were placed into a viewing cell filledwith water and photographed using color slide film. Blackand white film can be used and the negative mounted asa slide. Lighting is critical to the location of landmarks on
small shrimp. Dual fiber optic illuminators positioned tominimize reflection from the exoskeleton were found to give
better results than flash lighting. Slides were projected onto
a digitizing tablet. The landmarks used were slightly different from those employed on the adults. The landmarks
were changed from posterior rostral tooth (DM1 in Fig.
2) to ventral intersection of rostrum and carapace; fromantennal insertion (VM1 in Fig. 2) to anterior ventral pointon edge of carapace; pleopods marked at anterior edge
rather than center point (points VM2 through VM5). The
coordinates were converted to measurement variables by
the same software and the log-transformed measurement
variables were used in the SAS PRINCOMP program.
Principal component scores were stored and ANOVA was
performed using the SAS GLM program. Then the variance components were estimated with VARCOMP.
Heritability was estimated from the variance components
according to Becker (1984).

Multivariate Estimation of Heritability
Nauplii from ten separate spawns were shipped to the
University of Houston - Clear Lake Aquaculture Genetics
Laboratory from the Laguna Madre Shrimp Farm. Larvae were acclimated to Marine Mix sea water at laboratory temperature and stocked at 100 larvae/cone in three
one-liter Imhoff cones per family. They were treated understandardized conditions: 28°C, 30 ppt, constant light, dailychanges of water containing Chaetoceros gracilis at 100000
celis/mL and Tetraselmis chuii at 30000 celis/mL. After the
protozoea-3 stage, Artemia salina nauplii were fed at 3/mL
(Lester 1988). At the postlarva-2 stage, populations fromthose families with more than 120 survivors were transferred to 10 liter round cages. Three cages were stocked
for each family with 40 postlarvae per cage. These 10 literround cages had screened openings near their bottom andcontinuous water flow from the top. They were distributed
among three large tanks (244 cm x 61 cm x 61 cm) which
provided common water sources.

Results_

Testing the Truss Network Approach
The correlation among the variables in the standard data
set is high. Measurement PAC (Fig. 1) has the lowest
average correlation of 0.68 and SSD the highest, 0.88. This
indicates that these variables are related in their estimation of size. The first principal component (PC 1) obtained
for the standard data set represents the latent variable"size" as indicated by the equal loadings (0.28 to 0.35)
of all variables (Bookstein et al. 1985). It explains 84% of
the variance in the standard data set. The second principal
component contains very little information, 0.05 of thevariance which primarily represents the posterior abdominal circumference. The results from discriminant
analysis can most easily be seen in the plots of canonicalvariate 1 (CAN 1) against canonical variate 2 (CAN2).
Figure 3 shows the plot resulting from analysis of the stan-

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Lester et al.: Image Analysis for Breeding Shrimp

67

Table 1
Length of the sixth abdominal segment as an indicator ofsize differences among species and data sets.

65.... 4

Data Set~3~ 2

SpeciesStandard (em)Truss (em)~1p setiferus
P stylirostrisP vannamel

1.64

1.38
1.73
1.56

II:

>
..J 0
~ -1

1.48
1.87

()

Z-2

0-3
Z

~-4

()

-5

-61-6

_
-5

-4

-3

·2

-1

0

2

3

4

5

6

CANONICAL VARIATE 2
Figure 3
Plot of canonical variates 1 and 2 from discriminant analysisof the standard data set. The species are represented byellipses based on the 95 % confidence intervals of thespecies' centroids. The ellipse for Penaeus setiferus is labeled
S, Penaeus stylirostris Y, and Penaeus vannamei V.

dard data set. The three species show some distinctness,but are not well separated. The Mahalanobis distances
between the centroids of the three species are P. setiferus
to P. vannamei = 3.69, P. setiferus to P. stylirostris = 2.51,
and P. vannamei to P. stylirostris = 2.32. CAN1 in Figure
3 shows relatively high loadings of variables AAC (0.86),
FLF (0.75), SSD (0.80), CW (0.73), and PAC (0.79) which
could be interpreted as a body thickness variable. CAN2
shows high loadings of PCL (0.88), FSL (0.80), SSL (0.78),
and SAD (0.85). It represents the variation in length ofbody segments among species. The goodness of fit of eachdiscriminant variable to the classification variables (identifiers for species) can be evaluated by the canonical correlation which in the case of CAN 1 is 0.84 and in the caseof CAN2 = 0.56.
For the truss network data set, the correlation matrix
indicated that the correlations were generally lower, andproblems existed with one of the variables, VM2-VM3.
This variable showed an average correlation with all othervariables of 0.09. The remaining variables had average correlations ranging from 0.37 (DM4-DM5) to 0.69 (DM3VM4). This data set gave results from principal component analysis that were different from the standard data
set. PC1 explained 60% of the variation, PC2 explains 9%.
All loadings on PC1 were between 0.15 and 0.23, except
VM2-VM3 (0.02). Again this indicates that PC1 is a size
component. The additional variables do not provide muchallometric variation to be explained by PC2.

At this point, there appears to be little on which to choose
the truss network over the standard morphometric variables. However, results of discriminant analysis on the truss
network data set are quite different as shown in Figure 4.
This analysis results in larger Mahalanobis distances
between the species' centroids, P. setiferus to P. vannamei= 5.94, P. setiferus to P. stylirostris = 6.35 and P. vannamei
to P. stylirostris = 5.73. The discrimination of species along
CAN 1 is primarily due to values of the following variables(between group loadings given in parentheses): DM4-DM5(0.84), DM4-VM5 (0.92), DM2-DM3 (0.95), DM5-VM5(0.93), DM3-VM2 (0.97). There are 16 variables with
between-group loadings above 0.75 on CAN2. Five aremeasurements from the carapace. Four are from the sixth
abdominal segment; the remaining seven variables aremeasurements from the first, second, and third abdomenal
segments. Thus CAN1 represents the first, fourth and fifth
abdomenal segments and CAN2 the head and the second,
third, and sixth abdomenal segments. The canonical correlations of these discriminant variables to the dummyclassification variables (species identifiers) are higher thanthose obtained with the previous data set, for CANl, 0.93
and for CAN2, 0.91. This indicates that the discriminantvariables derived from the truss network approach are
better for classifying individuals to species than thosederived from the labor intensive approach.
It is unfortunate that these data sets are not the resultof measurements on the same individuals. However, thesizes of animals in both data sets are comparable as indicated in Table 1 in which a variable contained in bothdata sets is compared (SSL in the standard data set is equal
to DM5-DM6 in the truss network data set). There arealso significant differences among the data sets. P. setiferus
and P. vannamei samples are larger in the standard dataset, but P. stylirostris are larger in the truss network data
set. Size differences among the species could impact thediscriminant analysis. In this case, the size differencesamong species are greater in the standard data set thanin the truss network data set and should contribute to
greater separation in the discriminant analysis of the standard data set. The discrimination is better however withthe truss network data set, which only serves to reinforce

68

NOAA Technical Report NMFS 92_

Table 2Comparison of variance components and heritabilities obtained from nested ANOVA using
simple variables and the first principal component scores. The eight simple variables wereselected 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.
Variable

Variance(family)

4.42 x 10- 3
PCI
1.40 x 10- 3
DM5-VM5
1.58 x 10- 3
DM3-VM3
1.92 x 10- 3
DM2-VM2
1.86 x 10- 3
DM5-DM6
2.50 x 10- 3
DM5-DM4
1.72 x 10- 3
DM3-DM2
2.08 x 10- 3
DM4-VM3
1.98 x 10- 3
DM3-VM4
Mean of 26 variables

Variance(cage)

Variance(error)00

1.04 X 10- 1
4.44 X 10- 3
4.36 X 10- 3
4.66 X 10- 3
4.85 X 10- 3
5.74 X 10- 3
5.56 X 10- 3
4.56 X 10- 3
4.79 X 10- 3

0.05 X 10- 3
0.16 X 10- 3
00.36 X 10- 3
0.35 X 10- 3
00.15 X 10- 3

SEthe conclusion that the truss network approach provides
additional information about species differences.

Multivariate Estimation of Heritability
Examination of the correlation matrix output from theprincipal component analysis of the data from families
grown under experimental conditions showed that two of
the variables, VM2-VM3 and DMI-VMl were weakly
correlated with the remainder of the data set. The correlations ofVM2-VM3 ranged from 0.25 (with DMI-VM1)
to 0.66 (with DM2-VM2). For DMI-VM1, the correlations ranged from - 0.08 (with VMI-VM2) to 0.43 (withDM5-VM4). These ranges are distinctly lower than theother variables and are indicative of measurement problems. The juvenile shrimp correlations are higher thanthose in the truss network data set from the adults. PC 1
explains 82 % of the variance in the data set and has nearly
equal loadings of all of the variables. The coefficients were
between 0.16 and 0.23 which is indicative of their similarity
as estimators of size.
Results from multivariate and univariate scoring werecompared based on the heritabilities and variance components obtained from the same ANOVA design. Table
2 shows these results from comparison of eight univariate
scores and the first principal component score. The results
show that a univariate approach can yield inaccurate comparisons among genetic groups. Some variables haveheritabilities that slightly exceed the estimation based on
PC 1. However, most variables yield lower estimates of thefamily component of the variance in size, some drasticallylower. Using PC 1 scores in the ANOVA gives an F value
for the model of 4.74 and a heritability (h 2) of 0.60. The
most extreme case is VM 1-VM2 which has an h2 estimate

Heritability
0.60
0.48
0.53
0.57
0.55
0.58
0.45
0.63
0.57
0.49
0.14

of O. Clearly there are problems with this measurement.
DM2-VMl and VM2-VM3 have h 2 values of only 0.28.
An average of all the h 2 estimates is somewhat lower thanthe one resulting from use of PC 1 scores.

Discussion_

Measurement problems are inherent in the developmentof a truss network design. In this study, they were reducedby making changes in the landmarks used and by improving photographic technique. Obtaining a simple set oflandmarks that remain homologous across development frompostlarva to adult proved more difficult than expected. One
source of error in the adult data set was the choice of points
on movable bodyparts, (e.g., appendage insertions on theadults). This problem was corrected by adopting new landmarks on the juveniles that represented stable intersectionsof body parts. In the case of the posterior rostral tooth,juvenile shrimp often expressed different degrees of rostral
tooth development from conspecifics, both adults and otherjuveniles. Thus slightly different landmarks had to be used
to collect dimensional data from juvenile shrimp of 0.2 to
1.0 gm than were used for the adults. When working withthe juvenile shrimp, it was discovered that some points are
more difficult to see on a live shrimp than on a dead one,(e. g., VM2). Better lighting and different photographic
techniques were used to minimize this problem. The ex·amination of different landmarks is continuing, especiallyfor the juveniles.
Several variables used in the truss network analysis exhibited their unsuitability as size estimators by a reduced
level of correlation with the other variables. The reason
for low correlations from two of the variables, DMI-VMl

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Lester et al.: Image Analysis for Breeding Shrimp

and VM2-VM3, was related to difficulty in precisely
locating the landmark points, DM 1, VM 1, and VM2, on
the pictures of juveniles. Some covariation is expected
because all of the measurements are dimensions from thesame image and all will increase with size of the shrimp.
High correlation can be expected if all variables are reliable
estimators of size and can be accurately measured. Variables in the truss network data sets obtained from juveniles
were all reasonable estimators of size as indicated by thehigh correlations and equality of loadings on PC 1.
The problems associated with developing a truss network
approach are outweighed by the advantage obtained fromit. In this study, the three species were used to represent
any genetically distinct groups about which informationis needed on distinguishing characteristics. It was shownthat discrimination among Penaeus setiferus, Penaeus vannamei,
and Penaeus stylirostris is possible using the standard data
set as seen in Figure 3. However, discrimination among
these groups improved with the truss approach, as one cansee by comparing Figures 3 and 4. It appears that the truss
network approach offers more information for the discrimination of these genetic groups and the classificationof individuals than the collection of standard measurements. Comparison of a data set of nine variables and oneof 26 variables for discriminant analysis is biased against
the data set with fewer variables. Although Figure 4 showsthat classification of individuals by species would be easierfrom a truss network approach, improved discrimination
among species could perhaps have been accomplished by
the addition of random variables to the standard data set.
Thus these results should not be taken as sufficient justification for the truss network approach. A thorough justification can be found in Bookstein et al. (1985) and is basedon information content and distribution within the data set.
The use of principal component scores derived from thetruss network data set has associated advantages and disadvantages. The number of variables obtained is much larger
than would normally be collected for a genetic analysis ofsize or any other characteristic. If these variables were to
be measured by hand with calipers, the number of variables
would prove prohibitive. The major advantage of using
multivariate classification scores for the selection process
will be realized in the improved accuracy of estimating size
and reduced probability ofmisclassification. While this approach improves the analysis of size and shape of penaeid
shrimp for experimental purposes, programming a computer to locate homologous landmark points with accuracy
is a major challenge. Some other approach to image analysis may be better for commercial application in selective
breeding.
There is considerable variation among the estimates ofheritability obtained from univariate estimators of size. In
some cases, it is not clear why more of the variance is partitioned into the family component, resulting in a higher
heritability. It is clear that the range seen in Table 2 of

69

8
7
6

5

....

4

W

3

~

2

~

ct

>

...J

ct

0

o ·1
oZ ·2
Z

ct

o

·3
-4

·5

_----------------~ .....
~

·5

-4

·3

·2

·1

0

2345678CANONICAL VARIATE 2

Figure 4
Plot of canonical variates 1 and 2 from discriminant analysis of
the truss network data set. The species are represented by ellipses
based on the 95 % confidence intervals of the species' centroids.
The ellipse for Penaeus setijerus is labeled S, Penaeus stylirostris Y,
and Penaeus vannamei V.

0.45 (DM2-DM3) to 0.63 (DM4-VM3) is quite large forheritabilities of measurements taken from the same set ofindividuals from the same set of families by the sameobservers. This variation is indicative of differences inmeasurement error and information content among thevariables in the truss network. Principal component analysis is one appropriate way to combine the information in
many size-related variables and minimize the effect ofmeasurement error. We believe that the use of multivariateclassification analysis will provide a more reliable discrimination based on size. We will continue to pursue thisapproach in our experiments because it provides more accurate estimation of breeding value for size.
It is presently impossible to employ the image analysis
approach for selection of untagged shrimp because thephotography process and the computer analysis process are
separated in time and space. Using our current digitizing
process, it still takes the technician about two minutes toenter the landmark points from a single image. In addition, the time involved in taking good still photographs ofliving shrimp is considerable. These problems will be solvedby the development of an integrated imaging and analysis
system. The video image can be converted to a still image
by a frame grabber which operates in 1I30th of a second.
The ability to locate and record landmark points or toemploy other image analysis techniques (e.g., definition

70

NOAA Technical Report NMFS 92

of the boundary of the shrimp and the area within thatboundary) can be programmed into a fast microcomputer
which could obtain the required measurements. The resultsof a preliminary classification analysis can be stored in thecomputer and used to calculate the selection index. Witha sufficiently powerful computer, the calculation of the index and comparison to a truncation value would take afraction of a second. Thus problems associated with the
combination of many variables and too complicated ananalysis could be solved by integration of fairly simple hardware and software.

Acknowledgments
This research was sponsored III part by a grant to L.J.
Lester from the Texas A&M University Sea Grant College Program, supported by the National Oceanic andAtmospheric Administration, Office of Sea Grant, Department of Commerce under grant number NA83AA-D00061. Support was also provided by the University of
Houston-Clear Lake. The assistance of the personnel of
Laguna Madre Shrimp Farm, Marine Culture Enterprises,
Agromarina de Panama, Granada Corp., and the Texas
A&M Shrimp Mariculture Project in obtaining samples
is gratefully acknowledged. Expert technical assistance wasprovided by Margaret Dennison, Mary Byam-Smith, and_Benita Waas. Dr. Cecil Hallum and Dr. Sharon Perkins
critically reviewed the manuscript.

Citations_

Becker, W.A.
1984. Manual of quantitative genetics, 4th ed. Academic Enterprises, Pullman, WA, 188 p.
Bookstein, F.L., B. Chernoff, R.L. Elder, J.M. Humphries Jr.,
C.R. Smith, and R.E. Strauss.
1985. Morphometries in evolutionary biology: The geometry of sizeand shape change, with examples from fish. Acad. Nat. Sciences
Philadelphia, Spec. Pub!. 15, 277 p.
Falconer, D.S.
1981. Introduction to quantitative genetics, 2nd ed. Longman,
London, 340 p.
Lasley, J .F.
1978. Genetics of livestock improvement, 3rd ed. Prentice-Hall,Englewood Cliffs, NJ. 492 p.
Lester, L.J.
1983. Developing a selective breeding program for penaeid shrimp
mariculture. Aquaculture 33:41-50.
1988. Differences in larval growth among families of Penaeus stylirostn's and Penaeus vannam.ei. Aquacult. Fish. Manage. 19:243-251.
SAS Institute, Inc.
1985. SAS User's Guide: Statistics, Version 5 ed. SAS Institute,Inc., Cary, NC., 956 p.
Strauss, R.E., and F.L. Bookstein.
1982. The truss: body form reconstructions in morphometries.
Syst. Zoo!' 31:113-135.

Breeding Test on Abalone
HARUHIRO MOMMA
Hokkaido Hakodate Fisheries Experimental Station
Yunokawa Hakodate
Hokkaido 042, Japan

Introduction_

Although production of artificial seed for abalone farming
inJapan has increased annually over the last 20 years, thetotal abalone harvest has continued to decline (Fig. 1)(Department of Statistics Information (DSI) 1967-86;
Japan Sea-Farming Assoc. 1967-1985). In 1985 about 30
million seeds were produced. The proportion of recaptured
artificial seedlings to the total abalones harvested in planting areas has usually been reported to be about 30-40 %.
However, some of these reports have shown values exceeding 90 % (Table 1). In these cases it is important tostudy the genetics of the seed abalone used. Table 2 showssome examples of recapture ratios of abalone seedlings.
Overall, the results suggest that seedling survival is usually
low and a large percentage of the seed suffers mortality.
Another reason to study their genetics is the fact thatdifferences 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 mortality? In order to increase the recapture ratio, it is important to produce healthy seed abalone, improve seeding
methods, and control the factors which affect survival on
the planting ground.

Recapture Ratios and Seedling Quality _ _The survival rate within abalone species increases with seedsize at the time of release. The extent of this increase,
however, varies between species in relation to their size

at maturity. Haliotis gigantea, for instance, is much larger
than H. discus (Inoue et al. 1985) because its growth pattern (Takayama 1940) and feeding behavior is different(Momma 1980a). Thus, an increase in H. gigantea growthrate would need to be proportionally greater in order tohave the same beneficial effect that a smaller increase would
have on H. discus.
The faster growing seedlings of a H. d. hannai culture
were recovered more frequently than the slow ones (Table3). This higher rate of recovery illustrates that fast-growing
seed have higher survival rates than their slower growing
counterparts even though they were the same size at thetime of release. It was previously reported by the authorthat juvenile abalone with initially rapid growth rates (untila shell length of 16-17 mm is reached) maintain that trait

I

I'

<-~

r

/
/

/0

......

/0--

'"0
><
1/1 5.5c:--0/\I /\ I

,f

0

:e.

-

.r:.
(,l

III

u 5

/\/

0

~

/ .

\"

r-~/
'70

20

Z

c:

3

0'

...

CD

0

15

1/1
CD
CD
Co

><......
100....:f

0- 0

~

4.5

1967

I

.to

j

iii
Figure 1
Total catch (0) and number of artificially produced abalone seedlings (.) in Japan. Total catch: after fisheries statistics ofJapan
1967-86. Department of Statistics Information, Ministry ofAgriculture, Forestry and Fisheries. Government of Japan.
Number of seedlings: after Materials on production, supply andrelease of fingerlings for enhancement of fisheries resources inJapan, 1967-1985, Japan Sea-Farming Association.

I
.

o

/

25

'75

'80

'85

71

72

NOAA Technical Report NMFS 92_

Table 1
Proportion of artificial seed to abalone landed.

Prefecture

Location

Proportion of
seedlings (%)

Hokkaido
Iwate
MiyagiFkushima
Kanagawa
Fukuoka

Toyohama
Toni
UtatsuNagasaki
Nagai
Oshima

85
39
83
59
85
54

Species

References

H. d. hannai
H. d. hannai
H. d. hannai

Momma (1986)
Takeichi (1988)
Sasaki et al. (1987)
Sato et al. (1984)
Tauchi (1984)
Futajima et aI. (1985)

H d. hannai
H discus
H. discus

Table 2
Recapture rate of the planted seedlings.

Prefecture

Location

Recapture
rate (%)

Hokkaido
Iwate
Yamagata
Ibaragi
Kanagawa

Shiribeshi
Toni
Koiwagawa
Oarai
Jogashima

7
18
26
25
26

Species

References

H d. hannai

Miyamoto et aI. (1982)
Takeichi (1988)
loka (1983)
Kodama (1985)
Inoue (1965)

H. d. hannai
H. d. hannai
H. discus
H. gigan/ea

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

Group

Average growthratio (I'm/d)Releasedsize (mm)Numberreleased

Recovery

Recapture
rate (%)

Fast
Slow

71
28

13.8 ± 1.3
13.9 ± 1.9

964
185

170
20

17.6
10.8

throughout their early development for a period of at least
116 days (Fig. 2) (Momma 1980b).
In this paper the effects of artificial selection, hybridization, and mutation on juvenile abalone growth are discussed. The isolated fast-growth qualities were examinedthrough various tests conducted under standardized rearing conditions with the following results.

Growth Rates
The fastest growing individuals were selected from the offspring of one parent by mass selection when they were150-200 days old: about 15 mm in shell length. After thesesuperior individuals were reared about 1 more year, they
were introduced for breeding and fertilized by the sib

method. This method has been repeated several times from1973 to 1989. The 6th generation of H. discus hannai produced by the sib method of inbreeding did not result inhigher than normal mortality, or growth acceleration (Fig.
3) (Momma 1987).
H. discus discus and H. discus hannai were introduced forbreeding by the usual method (Kikuchi and Uki 1974).
These seedlings were reared under the same conditions.
The growth and mortality for these species was compared.
Similar shell-growth rates were observed for these two
species, but mortality of the H. d. discus was higher thanH. d. hannai; therefore, the latter produced a greater comparative biomass. (Fig. 4) (Momma 1987).
Hybridization studies were conducted on H. d. discus,
H. d. hannai, and H. kamtschatkana using the usual methodon the Ezo-abalone (H. d. hannal)' In only one case did

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Momma: Breeding Test on Abalone

73

150

Cil
>I'Cl

..

'0
to

....

~

o
a

120

-

0

o
o

c9 0

0

co

..

...E

~

"iii
c:
Q)
E

o

o

00% 0
0 0

a

0

00 CO

o
00

0

o

o
o

o

90

o

';:

Q)

Co
)(

Q)

,:
60
>:
I'Cl

.

'0

E::l..

-

...
....

30

.
-ti..... .
... I

0

.

40

60

,

.

80

100

,
120

Juvenile stage (till shell length: 16-17mm)
growth ratio (/-1m/day)Figure 2
The relationship between the juvenile stage (shell length: 16-17mm), the growth ratio (!Jm/d), and the growth ratio during theexperimental term (116 d). 0 = fast-growth group at 153-269
d old and. = the slow-growth group at 570-686 d old (Momma 1980b).

30

-

E
.§.. 20

G2 = 30 June 1986
• G6 = 7 July 1986

o

~~//

/.~

-

.c

tn
C

.!!

Qi 10

.c

en

/./"
/~'/

~~/

"==='-=--'·---5~0----1~0-0------c1..c.50:------=-20O-:0------,2=-=5,.,.0-----::c300

Age (days)
Figure 3
Growth lines of H. discus hannai seedlings; 0 and • = the mean shell length of 2nd (G2) and6th generation (G6), respectively. (G2 fertilized 30June 1986; G6 fertilized 7 July 1986) (Momma
1987).

74

NOAA Technical Report NMFS 92

_

0

100

\:\:-_.~.-------------: :::::: :~;:;::;:;~

Cl
~
l fj
lfj

CI:I

E

2

o discusr. discus/\

BIomass
Survival ratio

o
iii

~.

"~
50

~

6

_______

25/

O,---------~~...--0-0:::::::::::::~_o-o

200

400

<'

!.

600

1000

800

1200

Age (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 5 %, M
monomorphic.
EnzymeAcid phosphatase
Adenylate Kinase
Aspartrte aminotransferse
Esterasea-glycerophosphate Dehydrogenase
Glucosephosphate Isomerase
Isocitrate Dehydrogenase
Lactate Dehydrogenase
Leucin Aminopeptidase
Malate Dehydrogenase
Malic Enzyme
Mannosephosphate Isomerase
Octanol Dehydrogenase
6-phosphogluconate Dehydrogenase
Phosphoglucoselsomerase
Phosphoglucomutase
Superoxide Dismutase
Tetrazolium Oxidase

Locus

TissuePolymorphism

Acp
Ak
Aat
Est-l
-2
-3
aGpd
Gpi
Idh-l
Ldh-l
-2
Lap-I
-2
Mdh-l
-2
Me
Mpi
Odh
6Pgd
Pgi
m-I
-2
Sod
To-2
-3

D
M
M
D
D
D
M
M
M
M
M
D
D
M
M
M
M
D
M
D
M
M
M
D
D

P'
M
P
P
M
M
M
M
M
P
M
P
M
M
P'
M
P'
P
P
P'
P
P
M
P'
P'

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Momma: Breeding Test on Abalone85

20

o : hannai X hannai°

11 July 1984--//\0 --• : hannai X kamtschatkana

70

15

...

7 Aug. 1984

E
E

CD
CD

.c

Ol
C

::E

Q)

3

10

55

~

"C

...
c...

CD

Q)

G>
.c:
en

-CD

I

0

0----.------0
350

650

550

450

0

850

750

Age (days)
Figure 5
Growth rate in hybrid (.) and H. d. hannai (0) in the fishing ground. Curved line indicates
the water temperature (Momma 1988).

18 May 1984

02N
70

-e
E

--

.3N-2p b
60

.c:

Ol
C

~

G>

.c
en

50

1"

,

!

550

600

650

700

750

800

850

900

950

1000

1050

1100

1150

Age (days)
Figure 6
Growth rate in triploid and normal diploid seedlings; 0 = diploid, /::" = triploid produced byinterference of 1st polar body, • = triploid produced by interference of 2nd polar body, fertilized 18 May 1984, respectively (Momma 1987).

75

76

NOAA Technical Report NMFS 92

the hybrid of H. d. hannai and H. kamtschatkana show improved growth rate in cold water. (Fig. 5) (Momma 1988).
Triploids produced by interference of polar body projection (both 1st and 2nd polar body extrusions) using themethod of Arai et al. (1982) did not differ in growth from
diploid seed (Fig. 6) (Momma 1988).

Abalone Breeding
Enzyme polymorphism of this species (Table 4) (Fujio
1984; Fujino 1979), and the chromosome handling techniques and growth characteristics of other species have beenreported. Above all, based on the study of wild abalone
populations, as well as the relationship of age to fitness
measured at the esterase M locus, the deficient animals
reveal more homozygosity (Fujino 1978). Population
genetics and thremmatological studies may contribute considerably to the abalone industry.

Citations
Arai, K., H. Tsubaki, and K. Fujino.
1982. Chromosomes of Haliotis discus hannai Ino and H. discus Reeve.
Bull. Jap. Soc. Sci. Fish. 48:1689-1692.
Department of Statistics Information (DSI).
1967-1986. Fishery statistics ofJapan 1967-86. DSI,Jpn. Min.
Agric., Forest. Fish., Tokyo. (In Japanese.)
Fujino, K.
1978. Genetic studies on the Pacific Abalone-I. Excessive Homozygosity in Deficient Animals. Bull. Jpn. Soc. Sci. Fish., 44(7),767-770.
1979. Apparent physical deficiency associated with inbreeding structure suggested by biochemical polymorphism in the PacificAbalone. In Proc. 16th int. conf. on animal blood group andbiochemical polymorphism: Leningrad, USSR (16 August 1978)
p. 245-256. USSR Organizing Comm. Int. Soc. Animal BloodGroup Res.
Fujio, Y.
1984. Studies on genetic analysis of fishies by isozyme methods.
Minist. Agric. Forestry and Fisheries, Government ofJapan, 65 p.
Futajima, K., T Ito, and G. Kishimoto.
1985. Awabi no saibai gyogyokanri ni kansuru kenkyu-1. Annu.
Rep. Fukuoka Pref. Fish. Exp. Stn, p. 239-257.
Inoue, T.
1965. Hyoshiki horyu kara mita Awabi zoku no ishoku ni kansuru
1. 2. Suisan-Zoshoku. Ext. 5:23-31.
Inoue, T, M. Tauchi, and M. Chikayama.
1985. Diagnosis of species participate in effect on artificial seeds
release of abalone, Haliotis spp. Aquaculture, 32(4), 193-198.

_
loka, I.
1983. Yamagataken ni okeru horyu ezo-awabi no seicho to seizanritu no sisanrei. Otsuchi Marine Research Center Report 9:
52-54.
Japan Sea-Farming Association.
1967-1985. Saibai Gyogyo Syubyo Seisan, Nyusyu HoryuJissekiShowa 43-52 Nendo. (Materials on production, supply, and releaseof fingerlings for enhancement of fisheries resources in Japan.)Japan Sea-Farming Assoc., Japan.
Kikuchi, S., and N. Uki.
1974. Technical study on artificial spawning of abalone, genus
Haliotis-II. Effect of irradiated sea water with ultraviolet rays
on inducing to spawn. Bull. Tohoku Reg. Fish. Res. Lab. 33:
79-86.
Kodama, S.
1985. Awabi no horyu koka ni tuite. Bull. Japanese Soc. Fish.
Oceanog.47/48:139-142.
Miyamoto, T, K. Saito, M. Ito, and Y. Mizutori.
1982. On the releasing of the cultured seeds of Ezo-abalone, Haliotis
discus hannai, in the northern coast of Siribesi, hokkaido, Hokusuishi-geppo. Hokkaido Fish. Exp. Stn. 39(8): 169-208.
Momma, H.
1980a. On the abalone behavior under the influence of predator.
Michurian Seibutsugaku kenkyu 16(1):60-69.
1980b. Studies on the variation of the abalone-I. On the growthof the different aged young abalone. Aquaculture, 28(3): 142-146.
1986. Awabi jinko shubyu houryu jigyo no koka chosa. Sodaterugyogyo 159:2-4.
1987. Ezo-Awabi. Annu. Rep. Hokkaido Inst. Maricult. 61:
53-61.
1988. Kairui shubyo chukan ikusei siken. Ezo-Awabi. Annu.
Rep. Hokkaido Inst. Maricult. 62. 57-59.
Momma, H., K. Kobayashi, T Kato, Y. Sasaki, T. Sakamoto, andH. Murata.
1980. On the artificial propagation method of abalone and its effects on rocky shore-I. Remaining ratio of the artificial seedabalones (Haliotis discus hannai INa) on the lattices artificial reefs.
Aquaculture, 27(4):212-216.
Sasaki, R., K. Takahasi, T. Kawamura, and Y. Tasiro.
1987. Miyagiken hokubu kaiku ni okeru horyu ezo-Awabi no chosajirei. Tohoku Reg. Fish. Lab., Report 61. 15-29.
Sato, M., J. Owada, and H. Suzuki.
1984. Jinko shubyo awabi, uni horyu koka chosa. Annu. Rep.
Fukushima Pref. Fish. Exp. Stn:181-190.
Takayama, K.
1940. On the growth rate of the Abalone at Mie prefecture. Suisan
kenkyushi (precedes Bull. Jpn. Soc. Sci. Fish.) 35(4):99-100.
Takeichi, M.
1988. Tairyo horyu sareta ezo-awabi jinko shubyo no kaishuritu
to seizanritu Saibai Gyogyo Gijitu Kaihatu Kenkyu Oournal on
technical development for artificial enhancement of fisheries
resources). Saibaigiken 17(1):27-36.
Tauchi, M.
1984. Growth, age composition, survival rate and recapture rateof Japanise black abalone, Haliotis discus Reeve, at Nagai. Sci.
Rep. Kanagawa Fish. Exp. Stn. 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

ABSTRACTTwo-stage hybridization and introgression were evaluated as breeding plans to develop improved hybrid populations of red tilapia. Both plans were successful in overcoming interspecificbreeding barriers, thus allowing development of hybrid populations (both red- and normal-coloredphenotypes), with a mix of desirable production traits. The hybrids were evaluated in replicatedpolyculture 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 252 grams. Introgressive breeding techniques ([ T. aurea x red til apia] followedby two generations of backerossing red males [F-l and F-2 generations respectively], to female
T. aurea ) were used to develop a cold tolerant tilapia hybrid with both red- and normal-coloredphenotypes. 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_

Individual (mass) selection for rapid growth in the maternal mouthbrooding genus Tilapia has had limited success,indicating that the fraction of additive genetic variation issmall relative to total genetic variation (Chan May Tchien
1971; Tiechert-Coddington 1983; Hulata et al. 1986). In
such instances, hybridization and introgression can beutilized to avail for nonadditive sources of genetic variation (Brody et al. 1980).
Within the interfertile genus Tilapia, there are species
which exhibit fast growth (T. aurea; T. nilotica), high fecundity (T. mossambica), cold tolerance (T. aurea), and salinity
tolerance (T. mossambica; T. aurea). Also, red-colored mutant strains of T. mossambica and T. nilotica have arisen
spontaneously from normal-colored populations (Fitzgerald
1979; McAndrew et al. 1988). Thus, a diversity of genetic
resources is available for developing hybrids or synthetic
breeds in which favorable traits are combined.
Several hybrid strains of red tilapia have been commercially produced and widely disseminated throughout
Southeast Asia and the Western Hemisphere during thepast fifteen years. Breeding plans used in developing thesestains have not been well documented (Sipe 1979; Fitzgerald 1979). However, morphometric and electrophoretic
data indicate common ancestry with a red-mutant strain
of T. mossambica (Galman and Avtalion 1983; Halstrom
1984). The widely distributed U.S. "Florida strain,"derived from the cross female T. homOTUm x male T. mossambica (red mutant), has production traits similar to its
parental lines: early sexual maturation, slow growth, andlack of cold tolerance (Halstrom 1984).
In replicated yield trials, Florida strain young-of-the-year
fingerlings grew only 50 % as fast as blue tilapia, T. aurea,(Behrends et al. 1982). Thus, it was hypothesized that production traits could be improved by hybridizing the Florida
strain with T. aurea or T. nilotica, two of the faster-growing
and later-maturing species. Subsequently, Behrends andSmi therman (1984) reported that cold tolerance and thered color trait could be incorporated into a single population via hybridization (female T. aurea x male Florida
strain), followed by recurrent backcrossing of red hybrid
males to T. aurca females.
This paper will present results of studies designed toevaluate the use of two-stage hybridization and hybridization followed by repeated backcrossing (introgression)
77

78

NOAA Technical Report NMFS 92~_~Breeding

Breeding Plan 1983
Hybridization? Tila~ia aureaxPlan 1984
Introgression ~~~~~.

cf Red tilapia(Florida strain) *? Red tilapia(Florida strain)*x<1 Red tilapia(Florida strain)*<1 Tila~ia nilotica

F-1 hybrid

F-1 hybrid(=50% red;
50% normal)(=50% red;
50% normal)

F-1 **(== 50% red; 50% normal)

• •
, ,
•
?F-1 (red)

9 T. aurea"•,x€I F-1 (red)F-1 backcross(== 50% red; 50% normal)

x cfF-1 (red)

9 T. aurea

x•

ITrihybrid 1**

cf F-1 backcross(red)

IF-2 backcross I* Florida strain of red tilapia derived from~ T. hornorum x ~ T. mossambica (red mutant),**Red phenotypes are heterozygous for redcolor. Normal phenotypes are homozygous
for normal color. Pink phenotypes arehomozygous for pink color.

·Florida strain of red tilapia derived from~T. hornorum x ~L mosambica (red mutant).
**Red broodstock were heterozygous forthe red color trait, thus approximately
50% of progeny were red and 50% normal.

Figure 1
Breeding plan for developing cold-tolerant red tilapia hyb~ids.

Figure 2
Breeding plan for developing fast growing red tilapia hybrids.

for developing red tilapia hybrids with improved culture
performance.

channel catfish (Ictalurus punctatus, 10000/ha), freshwater
prawn (Macro brachium rosen bergii, 20000/ha), grass carp
(Ctenopharyngodon idella, 120/ha) and F-1 hybrid Asian carp
(Aristichthys nobilis x Hypopthalmichthys molitrix, 160-500/ha).
Fingerlings developed via two-stage hybridization were
stocked in 1983, while introgressed hybrids were stocked
in 1984. Red- and normal-colored hybrids and blue tilapia
were costocked into each pond at equal densities (800/ha)
for a total density of 2 400/ha (Tables 1 and 2). Blue tilapia
served as within- and between-years controls. Costockingof different genetic groups, also referred to as communal
testing, has been shown to be an efficient method forevaluating various tilapia strains and hybrids under conditions of common environment (Wohlfarth et al. 1983).
Throughout each study, a 32 % crude-protein floating
catfish ration was broadcast onto the pond surfaces daily.
Feeding rates were adjusted daily based on projected catfish biomass (Behrends et al. 1985). After 130 to 140 days

Materials and Methods_

Young-of-the-year red- and normal-colored (grey) tilapia
fingerlings were produced at the Tennessee Valley Authority's Research Farm from 1981 to 1984 using breedingplans illustrated in Figures 1 and 2. In both trials, redbroodstock heterozygous for the red color trait were used
to ensure production of both red- and normal-coloredphenotypes. By comparing the two hybrid phenotypes, it
was possible to assess the effect of the red gene complex
on growth, yield, and mortality.
In each of two years (1983 and 1984), three geneticallyand phenotypically distinct tilapia populations (Tables 1
and 2), were costocked into four 0.05-ha ponds along with

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

79

Table 1
Stocking and harvest data for three tilapia populations costocked into O.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

Stocking density (#/ha)Culture duration (days)
Initial weight (g/fish)
Final weight (g/fish)
Males (mean ± SD)
Females (mean ± SD)
Sex ratio (% males)
Daily gain (g/fish/d)Males (mean)
Females (mean)
Survival (%)
Yield (kg/ha)

Red hybrid

Normal hybrid

Tilapia aurea

800
133
2.0
313 a'
330 ± 63.0
256 ± 58.4

800
133
2.1
292 b
318 ± 59.0
254 ± 57.0
59

800
133
2.3
252 c
283 ± 38.0
225 ± 35.3
48

2.4
1.9
93
216 a

2.1
1.7
100
200 b

77

2.5
1.9
. 88
219 a

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 O.05-ha earthen ponds.
Polyculture yield trial 1984, Tennessee Valley Authority, Muscle Shoals, Alabama. Each mean
represents the average of four ponds.'
Introgressed hybrids

Stocking density (#/ha)Culture duration (days)
Initial weight (g/fish)
Final weight (g/fish)
Males (mean ± SD)
Females (mean ± SD)
Sex ratio (% males)
Daily gain (g/fish/day)Males (mean)
Females (mean)
Survival (%)
Yield (kg/ha)

Red hybrid

Normal hybrid

Tilapia aurea

800
146
0.6
296 a'
299 ± 65.2
268 ± 44.9
90

800
146
0.9
296 a
325 ± 65.4
260 ± 62.0
56

800
146
0.9
279 b
298 ± 63.4254 ± 46.1
49

2.0
1.8
65
153 b

2.2
1.8
92
217 a

2.0
1.7
71
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). Horizontal comparisons only.

of culture (May-October), ponds were drained and harvested. Tilapias within ponds were sorted by phenotype
and sex within phenotype. Individuals were then weighedto the nearest gram.
Statistical models used to evaluate growth and yield data

during 1983 and 1984 included effects due to phenotype,pond nested within phenotype, sex, and all first-order interactions. Student-Newman-Kuel's Multiple Comparisons
Test was used to separate means (within years), at a significance level of P<O.05 (Barr et al. 1979). Details con-

80

NOAA Technical Report NMFS 92_

cerning culture and yields of catfish, prawns, and carps
are presented in a companion paper (Behrends et al. 1985).

Results_

Two-Stage H ybridiation
Two-stage hybridization (Fig. 1), was an effective breeding
strategy for enhancing growth rate and yield of red- andnormal-colored tilapia hybrids (Table 1). After 133 daysof culture, mean harvest weights (sexes pooled) of red andnormal phenotypes were 313 and 292 g respectively. Red
hybrids were heavier at harvest (P<0.05) than theirnormal-colored counterparts, indicating a positive growtheffect of the red gene complex. In comparison, T. aurea controls averaged 252 g, and weighed less (P<0.05) than eitherof the hybrids. Irrespective of phenotype, males were 20
to 30% heavier than females (P<0.05). Relative rankingof mean harvest weights by phenotype were the same inall ponds, indicating that pond (environment) x phenotype(genotype) interactions were not a significant source ofvariation.
Net production of the three phenotypes ranged from 200
to 219 kg/ha for a combined yield of 634 kg/ha (Table 1).
Mortality of red hybrids was consistently greater than mortality of normal-colored hybrids and T. aurea. Sex ratios(male:female) of red and normal-colored hybrids averaged
77 % and 59 % males respectively, while the T. aurea population averaged 48 % males: near the expected 1: 1 sex
ratio.
Despite their higher rates of mortality, hybrids yielded
7-10% greater than T. aurea (P<0.05). Improved yields
were due to improved growth of both males and females
and male-dominated sex ratios.

Introgression
Introgressive breeding (Fig. 2), similarly improved growth
performance of red- and normal-colored hybrids (Table2), although not to the extent of two-stage hybridization.
After 146 days of culture, mean harvest weights of bothred- and normal-colored phenotypes (sexes pooled) were
296 and 296 g respectively. This differs somewhat fromresults of the 1983 study, where the red hybrid grewfaster than it's normal-colored counterpart. At harvest,
T. aurea controls averaged 279 g and weighed less thaneither hybrid (P<0.05). Irrespective of phenotype, males
were 15-20% heavier than females (P<0.05). As in theprevious years study, relative rankings of mean harvest
weights (by phenotype) were the same between the four
replicate ponds.
Net production of the three phenotypes ranged from 153
to 217 kg/ha, with a combined yield of 526 kg/ha (Table2). Mean yield of the normal-colored hybrid was greater(P<0.05) than yields of the red hybrid and T. aurea owing

to faster growth and excellent survival. Mortality ofnormal-colored hybrids averaged only 8 %, while mortality rates for red hybrids and T. aurea averaged 35 and 29%
respectively (Table 2). Sex ratio of the red hybrid population was highly skewed towards males (90%), while thepercentage of males in the normal-colored hybrid and
T. aurea populations was 56 and 49 % respectively.

Discussion_

Both two-stage hybridization and introgression were effective breeding strategies for enhancing growth rates of redand normal-colored hybrids. However, relative to T. aurea
controls, two-stage hybridization improved growth rates
and yields to a greater extent than did introgression. This
is not surprising because introgression, as practiced, isanalogous to inbreeding; heterozygous hybrid populations
become progressively more homozygous with repeatedgenerations of backcrossing to a small founder population.
Introgression is recommended for incorporating a desirable
dominant trait from one population into a second, moreproductive population (Kirpichnikov 1981).
During both years, mortality figures for red phenotypes
were higher than rates for normal-colored phenotypes.
Elevated levels of mortality have also been reported in otherproduction studies (Behrends et al. 1982, 1988), and inhatchery research (EI-Gamal et al. 1988). This indicatesthat the red gene complex, while enhancing growth, may
have had a negative pleiotropic effect on viability. Colormutations in green sunfish, Lepomis cyanellus, and common
carp, Cyprinus carpio, have resulted in reductions in viability, growth or both (Dunham and Childers 1980; Kirpichnikov 1981). Other red mutant strains oftilapia havebeen identified (McAndrew et al. 1988; Tave 1989), andshould be evaluated with respect to growth rate, viability,
and use in hybrid breeding programs.
Breeding plans in this study worked well for combining
traits of closely related species. While many species ofmaternal mouthbrooding tilapia are interfertile, commercial production of most F-I hybrids is difficult owing to
species-specific differences in breeding behavior (Lee 1979;
Hulata et al. 1985). In the present studies, problems related
to breeding behavior were overcome by resorting to double
hybridization or introgression. In both breeding plans, eachof the respective parents (F-I and subsequent backcrossgenerations), had a complete or nearly complete set ofchromosomes in common. This condition was apparently
sufficient to overcome natural reproductive isolating
mechanisms which can impede reproduction between
closely-related species. Similar interspecific breeding plans
may be practical for developing unique hybrids or synthetic
strains for special environments, for instance, a fast growing, salt-tolerant red hybrid for net-pen culture in tropicalestuaries.

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

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