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Genetic heterozygosity and growth rate in Louisiana oysters (Crassostrea virginica)
57 (1986) 261-269 Elsevier Science Publishers B.V., Amsterdam -
261
Aquaculture,
Printed in The Netherlands
Genetic Heterozygosity and Growth Rate in Louisiana Oysters (Crassostrea uirginica) DAVID W. FOLTZ and MARK CHATRY’ Department
of Zoology
and Physiology,
Louisiana
(U.S.A.) ‘Louisiana Department of Wildlife and Fisheries, Office Box 37, Grand Isle, LA 70358 (U.S.A.)
State University,
Baton Rouge, LA 70803
Lyle S. St. Amant Marine
Laboratory,
Post
ABSTRACT Foltz, D.W. and Chatry, M., 1986. Genetic heterozygosity and growth rate in Louisiana oysters (Crassostrea
virginica).
Aquaculture,
57: 261-269.
Heterozygosity at nine eleetrophoretically variable allozyme loci was studied in relation to weight and length in approximately 1600 sixteen-week-old oysters (Crassostrea virginica) produced from five pair crosses. There was little evidence that heterozygosity itself was important in determining growth rates within crosses, either in single-locus or in multi-locus comparisons. There was evidence that some allelic substitutions at the aminopeptidase-1 locus may have had an effect on growth. This result indicates either that some allelic substitutions within an allozyme locus affect growth and others do not, or that the effect is due to a growth-affecting gene that is linked to the allozyme locus.
INTRODUCTION There is now abundant evidence that enzyme heterozygosity is positively correlated with fitness-related traits such as growth, viability and fertility in many different organisms [summarized by Mitton and Grant (1984) and Zouros and Foltz (1986)]. In the oyster Crussostrea uirginica (Koehn and Shumway, 1982) and the clam Mulinicz lateralis (Garton et al., 1984) there is a negative correlation between oxygen consumption and enzyme heterozygosity, suggesting that heterozygous individuals grow faster than more homozygous ones because heterozygotes have a higher metabolic efficiency and a lower cost of routine metabolism. These observations raise several questions. What is the biochemical basis of this apparent heterozygote superiority? Does allozyme heterozygosity directly affect growth rate or is it simply a marker for heterozygosity at linked loci affecting growth? And how (if at all) can these results be used for the genetic improvement of bivalve stocks? The last question has been discussed by Newkirk (1983) and, by Gaffney and Scott (1984).
0044-8486/86/$0X50
0 1986 Elsevier Science Publishers B.V.
262
The present study addresses the possible effect on growth rate of genes linked to allozyme loci by measuring the correlation between growth and heterozygosity in five pair crosses of Louisiana oysters (C. virgin&). If allozyme heterozygosity directly affects growth rate, then significant associations between heterozygosity and growth should appear both in samples from natural populations and in samples of progeny produced by pair crosses. METHODS
Hatchery procedures
Broodstock oysters were collected from Barataria Bay, Louisiana in May 1984 and brought to the oyster hatchery at the Lyle S. St. Amant Marine Laboratory on Grand Terre Island. Each oyster was cleaned of -fouling organisms, placed overnight in a 4-l glass jar containing 2 1of 5 pm-filtered, UV-irradiated seawater and allowed to purge itself. Spawning was induced by raising the temperature 2-4 ’ C above ambient. Eggs from one female were collected and divided into 12 batches. All but one batch of eggs were fertilized separately by sperm from 11 different males. The last batch was left unfertilized and served as a control. Development of the unfertilized eggs would indicate either parthenogenesis or the presence of foreign sperm. Each of the 11 cultures was divided into two (A and B) or three (A, B and C) replicates 5 days after fertilization. However, crosses 1,3,4,7,10 and 11 plus one replicate of cross 5 had very low survivorship and produced few or no spat. Also, for both cross 2 and cross 8, both replicates were pooled in all analyses because small sample sizes precluded meaningful between-replicate comparisons. The larvae were reared using the brown water culture technique of Ogle (1982). Each replicate was cultured in a 20-l polyethylene bucket containing 14 1 of aerated 5 ,um-filtered, UV-irradiated seawater. The water was changed every 3 days. The salinity of the water varied over time between 11 and 22%. Each culture was provided with individually labelled clam (Rarzgia) shells as cultch material. After metamorphosis, each oyster was transferred to a common tank supplied with seawater filtered to remove foreign oyster larvae. At 16 weeks of age, the oysters were taken to Baton Rouge and processed for electrophoretic analysis. Laboratory procedures Each oyster (minus the left valve) was weighed on an electronic balance and its length was measured using dial calipers. In most instances, more than one oyster settled on a cultch shell. To adjust for any effect of crowding on growth rate, the number of oysters per cultch shell (which we will refer to as the ‘
263
and weight) data. Each oyster was processed and analyzed by starch-gel electrophoresis using standard methods (e.g., Schaal and Anderson, 1974; Buroker et al., 1975). Nine allozyme loci were segregating in the five crosses: aminopeptidase-1 (Ap-l), glutamate oxaloacetate transaminase-2 (Got-2, also known as Aat-2), leucine aminopeptidase-1 (Lap-l), Lap-2, mannosephosphate isomerase (Mpi), 6-phosphogluconate dehydrogenase (6-Pgd), phosphoglucose isomerase (Pgi), phosphoglucomutase-2 (Pgm-2), and xanthine dehydrogenase (Xdh). Within each locus, alleles were designated by numbers indicating mobility relative to the allele found to be most common in a preliminary survey of electrophoretic variation in oysters from Barataria Bay (Foltz, 1986), which was designated as “100”. Negative numbers referred to cathodally migrating bands and “00” was used to designate the null (non-reactive) allele at the Lap-2 locus (Foltz, 1986).
Statistical procedure Analyses were performed on log-transformed weight and length measurements using the Statistical Analysis System (SAS 82.4) as implemented on the System Network Computer Center’s IBM 370/3081 at Louisiana State University. The partial correlation coefficient between heterozygosity and logtransformed size measurements, with the crowding index held constant, was calculated using Sokal and Rohlf’s (1981, p. 656) formula. In addition to looking at the effect of multi-locus heterozygosity on growth, we were also interested in determining if there were size differences among single-locus genotypes within crosses. The number of genotypic classes occurring among the offspring of a pair cross will vary from one to four, depending on parental genotypes. The seven possible mating types are listed in Table 1, where A, B, C and D have been used to designate alleles at a hypothetical locus. The types of contrasts that are possible will vary among these mating types, and only some contrasts will be biologically meaningful. Table 1 lists the contrasts that can be interpreted in terms of additive or non-additive models of gene action. However, additive and non-additive effects of allele substitutions cannot always be separated (e.g., mating type III). Also, in some mating types (IV, VI, and VII), onlyadditive effects can be testedContrasts of these sorts are easily performed using, for example, the General Linear Models (GLM) procedure in SAS. RESULTS
In all, 1387 oysters were examined for all nine variable allozyme loci. The mean number of heterozygous loci per individual was 4.47 (range: 1 - 7). The corresponding values for each cross (including replicates within crosses) are reported in Table 2, which- also gives the partial correlation between log-trans-
264 TABLE 1 Single-locus mating types, progeny genotypes and contrasts among means for analysis of size data (all contrasts have 1 degree of freedom, except for the second contrast in V, which has 2) Mating type male X female (and reciprocal)
Possible effects leading to rejection of null hypothesis
Progeny Genotype(s) (contrast)
I
AA x AA
AA
None; no contrast possible
II
AA x BB (BB x AA)
AB
None; no contrast possible
III
AA x AB (AB x AA)
AA AB (+1 -1)
AA x BC (BC x AA)
AB AC (+1 -1)
Additive effect of allele substitution
AA AB BB +2 -1) (-1 0 +1)
Non-additive effect of allele substitution (a) Additive effect of allele substitution (b)
IV
V
AB x AB
Additive effect of allele substitution Non-additive effect of allele substitution
(-1
VI
AB x AC (AC x AB)
AA AB AC BC (+1 -1 +1 -1) (+1
VII
AB x CD (CD x AB)
+1
-1
-1)
AC AD BC BD (+1 $1 -1 -1) (+l
-1
+1
-1)
Additive effect of allele substitution in male gamete (a) Additive effect of allele substitution in female gamete (b)
Additive effect of allele substitution in male gamete (a) Additive effect of allele substitution in female gamete (b)
formed weight and heterozygosity. In two of eight instances, there was a sigcorrelation between log-transformed weight and nificant positive heterozygosity. However, the results were not consistent among replicates within crosses. For cross 6, there was a significant result in replicate A, but not in B. Similarly, there was a significant positive correlation in 9B, but 9A and 9C both had (non-significant) negative correlations between log-transformed weight and heterozygosity. The weighted mean correlation between log-transformed weight and heterozygosity was 0.052. Analysis of the log-transformed lengths gave similar results but are not presented here. Table 3 shows the outcome of single-locus tests for differences in size among
265 TABLE 2 Sample sizes (NJ, number of heterozygous loci per individual and correlation between log-transformed weight and heterozygosity (r) in five pair crosses of Louisiana oysters (Crassostrea virginica) (A, B, and C refer to replicates within crosses) Cross
N
Number of heterozygous loci Mean
Range
2 5 6A 6B
47 426 162 41
3.34 4.65 2.95 2.98
2-6 2-7 14
0.213 0.061 0.203**
14
0.001
8
41 222
5.00 4.82
3-7 3-7
0.039 -0.008
104
5.11 4.80
3-7 3-7
0.200* - 0.052
9A 9B 9c
344
*p < 0 .05., **p < 0.01. progeny genotypes. The only clear effect was at the Ap-1 locus, in which there ivas an additive effect of substituting the “127” allele for the “100” allele in the male gamete in cross 9. This result was consistent among all three replicates of that cross, with the 127/108 heterozygote having a lower mean weight than the lOSjlO0 heterozygote. Because both progeny genotypes were heterozygous, there was no effect of heterozygosity per se. The other nominally significant results (at Got-Z, Lap-2 and Mpi) involved cross 5 or 6. Because the female was heterozygous at Got-S, Lap-2, and Mpi, the iesults should have been consistent among crosses, because all crosses had the same female parent. The fact that inconsistent results were found at the Got-2, Lap-2 and Mpi loci suggests either that the effect was spurious or else due to an interaction between the female gamete and the male gamete. Further research is necessary to distinguish between these two alternatives. When each cross or replicate was stratified according to time of larval settlement, there was a significant difference (P < 0.05) in heterozygosity between early settling larvae and late settling ones in one (6A) dut of seven comparisons, with early settlers being more heterozygous than late settlers. However, a similar trend was not observed in the other replicate (6B) of this cross. A locus-by-locus test for differences in genotype frequencies between early and late settling larvae gave mostly negative results except for cross 5, in which three loci (Got-2, Pgi, and Xdh) exhibited significant differences. DISCUSSION
The finding that in natural populations there is a positive association between level of allozyme heterozygosity and growth rate has been confirmed in several
266 TABLE
3
Contrasts
of mean weights among progeny genotypes.
Roman
numerals refer to mating types as
listed in Table 1 (Arabic numerals refer to the identity of alleles being contrasted, further explanation; - indicates that no contrast was possible) Cross
2
5
Locus Ap-1
Got-2
III
Via
108/100
78/-
100
VIb 78/-
219
100/00
-
Via 78/-
Lap-l
Lap-2
Mpi
6-Pgd
Pgi
Pgm-2
Xdh
-
VIIa
III
III
IV
-
-
115/108 VIIb
100/91
100155
IV
III
III
VIIa
-
III
100/00*
100/91*
100/72
112/100 VIIb
100
VIb
6B
-
-
-
-
lOO**
VIb 78/-
100/91 Vb
279
100/91 -
-
78/- 100 VIb 781-279 8
Va
Via 78/-
Via
IV
IV
III
116/100
781-279
100/88
-
Va
-
9B
9c
IV
IV
127/100*
781-279
IV
IV
127/100*
18/-279
IV 127/100**
IV 78/-279
115/100
-
-
-
Via
-
-
-
-
-
100/78 VIb 78158 -
Via
100/91 Vb
100178 VIb
100/91
78158
Via
III
VIIa
III
lll/lOO
100/72
106/100
100/82
VIb 100/91 9A
78158
78158
781-279 6A
see text for
VIIb 78/58
IV
III
III
100/00
100/91
100172
IV
IV
III
III
100/00
100/91
100172
78158
IV 100/00
III 100/91
III 100/72
IV 78/58
78158 IV
III
-
100182 III
-
100/82* III 100/82
-
*P < 0.05; **p < 0.01.
different species, although counter-examples are known [see Zouros and Foltz (1986) for a review of this literature]. In bivalves, evidence that heterozygotes have an advantage in growth rate comes from studies of samples from natural populations (Singh and Zouros, 1978; Zouros et al., 1980; Singh, 1982; Green
261
et al., 1983; Koehn and Gaffney, 1984), from studies of ploidy manipulation (Stanley et al., 1984)) and from studies of the physiological energetics of growth (Koehn and Shumway, 1982; Garton et al., 1984). The associations between allozyme heterozygosity and growth rate observed in natural populations of bivalves are not necessarily present in hatchery populations produced from a limited number of parents. In offspring of pair crosses in Myths edulis (Beaumont et al., 1983) and Mulinia lateralis (Gaffney and Scott, 1984), there was no association between size and heterozygosity, although positive associations were observed in natural populations of both species. Similarly, there was little or no association between size and heterozygosity in offspring of mass spawnings in C. virginica, Spisula solidissima, M. lateralis (Gaffney and Scott, 1984) and Mercenaria mercenaria (Adamkewicz et al., 1984). The present study confirms these findings. When comparing the present study with earlier studies of heterozygosity and growth in oysters, it is important to realize that the sample sizes used here (both number of individuals and number of polymorphic loci) are as large as or larger than those employed in the earlier studies. Thus, the largely non-significant results are not due to inadequate sample sizes. As Gaffney and Scott (1984) have emphasized, these results indicate that associations between enzyme heterozygosity and growth depend strongly on the number of parental genomes contributing to the progeny (see also Koehn and Gaffney, 1984). The failure to observe heterozygosity-growth associations in hatchery populations produced from limited numbers of p*arents suggests that the associations observed in natural populations are not directly due to allozyme heterozygosity itself. Although it might be argued that progeny from pair crosses have lower heterozygosity than individuals from a natural population and that this phenomenon accounts for the lack of heterozygosity-growth associations in the former, the average heterozygosity is not reduced in pair crosses or mass spawnings, although the variance in heterozygosity among loci may be increased. This result follows from the fact that the loss of heterozygosity is 1 -f, where f is the inbreeding coefficient (Crow and Kimura, 1970). If the parents have been chosen at random from the population, which is presumably the case in most hatchery matings involving wild-collected broodstock, then f = 0. Only if the offspring from a pair mating are themselves crossed to produce a second generation in the hatchery would there be a reduction in average heterozygosity. Even though average heterozygosity is not reduced when a limited number of parents contribute to a cross, particular alleles may easily be lost. There can be no more than four different alleles at a locus present in offspring of a pair cross, and if both parents share one or both alleles there will be even less allelic diversity among the offspring. Thus, a possible explanation for the lack of association between heterozygosity and growth in pair crosses is that some allelic substitutions at allozyme loci affect growth and others do not, and that the growth-affecting alleles were absent
268
from the crosses which gave non-significant results. However, this model shifts attention away from heterozygosity per se and toward the effect of particular alleles. Although the results of the single-locus tests reported here (particularly for Ap-1) and some of the results of Beaumont et al. (1983), Adamkewicz et al. (1984), and Gaffney and Scott (1984) are consistent with the suggestion that only some allelic substitutions at an allozyme locus have an effect on growth rate, the possibility that the effect is due to a gene linked to the allozyme locus cannot be ruled out. Either explanation could account for the finding that correlations between allozyme heterozygosity and growth appear only sporadically in offspring of pair crosses. ACKNOWLEDGEMENTS
We thank B. Glidewell and P. Larsen for help in the laboratory and Dr. A. Bull for assistance in the hatchery. D.W. Foltz would like to thank the director and staff of the St. Amant Marine Laboratory for their support and hospitality. This research was supported by NSF Grant no. BSR-8407450 to D.W. Foltz. Additional support was provided by Louisiana State University and by the Louisiana Department of Wildlife and Fisheries.
REFERENCES Adamkewicz, L., Taub, S.R. and Wall, J.R., 1984. Genetics of the clam Mercenaria mercenaria. II. Size and genotype. Malacologia, 25: 525533. Beaumont, A.R., Beveridge, C.M. and Budd, M.D., 1983. Selection and heterozygosity within single families of the mussel Mytilus edulis (L.). Mar. Biol. I.&t., 4: 151-161. Buroker, N.E., Hershberger, W.E. and Chew, K.K., 1975. Genetic variation in the Pacific oyster, Crassostreagigas. J. Fish. Res. Board Can., 32: 2471-2477. Crow, J.F. and Kimura, M., 1970. An Introduction to Population Genetics Theory. Harper & Row, Publishers, Inc., New York, NY, 591 pp. Foltz, D.W., 1986. Null alleles as a possible cause of heterozygote deficiencies in the oyster Crussostrea virginica and other bivalves. Evolution, in press. Gaffney, P.M. and Scott, T.M., 1984. Genetic heterozygosity and production traits in natural and hatchery populations of bivalves. Aquaculture, 42: 289-302. Garton, D.W., Koehn, R.K. and Scott, T.M., 1984. Multiple-locus heterozygosity and the physiological energetics of growth in the coot clam, Mulinia lateralis, from a natural population. Genetics, 108: 445-455. Green, R.H., Singh, S.M., Hicks, B. and McCuaig, J., 1983. An arctic intertidal population of Mucoma balthica (Mollusca, Pelecypoda): genotypic and phenotypic components of population structure. Can. J. Fish. Aquat. Sci., 40: 1360-1371. Koehn, R.K. and Gaffney, P.M., 1984. Genetic heterozygosity and growth rate in Mytilus edulis. Mar. Biol., 82: l-7. Koehn, R.K. and Shumway, S.E., 1982. A genetic/physiological explanation for differential growth rate among individuals of the American oyster, Crassostrea virginica (Gmelin). Mar. Biol. Lett., 3: 35-42.
269
Mitton, J.B. and Grant, M.C., 1984. Associations among protein heterozygosity, growth rate, and developmental homeostasis. Annu. Rev. Ecol. Syst., 15: 479-499. Newkirk, G.F., 1983. Applied breeding of commercially important molluscs: a summary of discussion. Aquaculture, 33: 415-422. Ogle, J.T., 1982. Operation of an oyster hatchery utilizing a brown water culture technique. J. Shellfish Res., 2(2): 153-156. Schaal, B.A. and Anderson, W.W., 1974. An outline of techniques for starch gel electrophoresis of enzymes from the American oyster Crassostrea uirginica Gmelin. Univ. Georgia Mar. Sci. Cen. Tech. Rep. Ser. No. 74-3,17 pp., unpubl. Singh, S.M., 1982. Enzyme heterozygosity associated with growth at different developmental stages in oysters. Can. J. Genet. Cytol., 24: 451-458. Singh, S.M. and Zouros, E., 1978. Genetic variation associated with growth rate in the American oyster (Crmsostrea virginica). Evolution, 32: 342-353. Sokal, R.R. and Rohlf, F.J., 1981. Biometry, 2nd edition. Freeman, San Francisco, CA, 859 pp. Stanley, J.G., Hidu, H. and Allen, S.K., Jr., 1984. Growth of American oysters increased by polyploidy induced by blocking meiosis I but not meiosis II. Aquaculture, 3’7(2): 147-155. Zouros, E. and Foltz, D.W., 1986. The use of allelic isozyme variation for the study of heterosis. In: M.C. Rattazzi, J.G. Scandalios and G.S. Whitt (Editors), Isozymes: Current Topics in Biological and Medical Research, 13. Alan R. Liss, New York, NY. Zouros, E., Singh, S.M. and Miles, H.E., 1980. Growth rate in oysters: an overdominant phenotype and its possible explanations. Evolution, 34: 856-867.
261
Aquaculture,
Printed in The Netherlands
Genetic Heterozygosity and Growth Rate in Louisiana Oysters (Crassostrea uirginica) DAVID W. FOLTZ and MARK CHATRY’ Department
of Zoology
and Physiology,
Louisiana
(U.S.A.) ‘Louisiana Department of Wildlife and Fisheries, Office Box 37, Grand Isle, LA 70358 (U.S.A.)
State University,
Baton Rouge, LA 70803
Lyle S. St. Amant Marine
Laboratory,
Post
ABSTRACT Foltz, D.W. and Chatry, M., 1986. Genetic heterozygosity and growth rate in Louisiana oysters (Crassostrea
virginica).
Aquaculture,
57: 261-269.
Heterozygosity at nine eleetrophoretically variable allozyme loci was studied in relation to weight and length in approximately 1600 sixteen-week-old oysters (Crassostrea virginica) produced from five pair crosses. There was little evidence that heterozygosity itself was important in determining growth rates within crosses, either in single-locus or in multi-locus comparisons. There was evidence that some allelic substitutions at the aminopeptidase-1 locus may have had an effect on growth. This result indicates either that some allelic substitutions within an allozyme locus affect growth and others do not, or that the effect is due to a growth-affecting gene that is linked to the allozyme locus.
INTRODUCTION There is now abundant evidence that enzyme heterozygosity is positively correlated with fitness-related traits such as growth, viability and fertility in many different organisms [summarized by Mitton and Grant (1984) and Zouros and Foltz (1986)]. In the oyster Crussostrea uirginica (Koehn and Shumway, 1982) and the clam Mulinicz lateralis (Garton et al., 1984) there is a negative correlation between oxygen consumption and enzyme heterozygosity, suggesting that heterozygous individuals grow faster than more homozygous ones because heterozygotes have a higher metabolic efficiency and a lower cost of routine metabolism. These observations raise several questions. What is the biochemical basis of this apparent heterozygote superiority? Does allozyme heterozygosity directly affect growth rate or is it simply a marker for heterozygosity at linked loci affecting growth? And how (if at all) can these results be used for the genetic improvement of bivalve stocks? The last question has been discussed by Newkirk (1983) and, by Gaffney and Scott (1984).
0044-8486/86/$0X50
0 1986 Elsevier Science Publishers B.V.
262
The present study addresses the possible effect on growth rate of genes linked to allozyme loci by measuring the correlation between growth and heterozygosity in five pair crosses of Louisiana oysters (C. virgin&). If allozyme heterozygosity directly affects growth rate, then significant associations between heterozygosity and growth should appear both in samples from natural populations and in samples of progeny produced by pair crosses. METHODS
Hatchery procedures
Broodstock oysters were collected from Barataria Bay, Louisiana in May 1984 and brought to the oyster hatchery at the Lyle S. St. Amant Marine Laboratory on Grand Terre Island. Each oyster was cleaned of -fouling organisms, placed overnight in a 4-l glass jar containing 2 1of 5 pm-filtered, UV-irradiated seawater and allowed to purge itself. Spawning was induced by raising the temperature 2-4 ’ C above ambient. Eggs from one female were collected and divided into 12 batches. All but one batch of eggs were fertilized separately by sperm from 11 different males. The last batch was left unfertilized and served as a control. Development of the unfertilized eggs would indicate either parthenogenesis or the presence of foreign sperm. Each of the 11 cultures was divided into two (A and B) or three (A, B and C) replicates 5 days after fertilization. However, crosses 1,3,4,7,10 and 11 plus one replicate of cross 5 had very low survivorship and produced few or no spat. Also, for both cross 2 and cross 8, both replicates were pooled in all analyses because small sample sizes precluded meaningful between-replicate comparisons. The larvae were reared using the brown water culture technique of Ogle (1982). Each replicate was cultured in a 20-l polyethylene bucket containing 14 1 of aerated 5 ,um-filtered, UV-irradiated seawater. The water was changed every 3 days. The salinity of the water varied over time between 11 and 22%. Each culture was provided with individually labelled clam (Rarzgia) shells as cultch material. After metamorphosis, each oyster was transferred to a common tank supplied with seawater filtered to remove foreign oyster larvae. At 16 weeks of age, the oysters were taken to Baton Rouge and processed for electrophoretic analysis. Laboratory procedures Each oyster (minus the left valve) was weighed on an electronic balance and its length was measured using dial calipers. In most instances, more than one oyster settled on a cultch shell. To adjust for any effect of crowding on growth rate, the number of oysters per cultch shell (which we will refer to as the ‘
263
and weight) data. Each oyster was processed and analyzed by starch-gel electrophoresis using standard methods (e.g., Schaal and Anderson, 1974; Buroker et al., 1975). Nine allozyme loci were segregating in the five crosses: aminopeptidase-1 (Ap-l), glutamate oxaloacetate transaminase-2 (Got-2, also known as Aat-2), leucine aminopeptidase-1 (Lap-l), Lap-2, mannosephosphate isomerase (Mpi), 6-phosphogluconate dehydrogenase (6-Pgd), phosphoglucose isomerase (Pgi), phosphoglucomutase-2 (Pgm-2), and xanthine dehydrogenase (Xdh). Within each locus, alleles were designated by numbers indicating mobility relative to the allele found to be most common in a preliminary survey of electrophoretic variation in oysters from Barataria Bay (Foltz, 1986), which was designated as “100”. Negative numbers referred to cathodally migrating bands and “00” was used to designate the null (non-reactive) allele at the Lap-2 locus (Foltz, 1986).
Statistical procedure Analyses were performed on log-transformed weight and length measurements using the Statistical Analysis System (SAS 82.4) as implemented on the System Network Computer Center’s IBM 370/3081 at Louisiana State University. The partial correlation coefficient between heterozygosity and logtransformed size measurements, with the crowding index held constant, was calculated using Sokal and Rohlf’s (1981, p. 656) formula. In addition to looking at the effect of multi-locus heterozygosity on growth, we were also interested in determining if there were size differences among single-locus genotypes within crosses. The number of genotypic classes occurring among the offspring of a pair cross will vary from one to four, depending on parental genotypes. The seven possible mating types are listed in Table 1, where A, B, C and D have been used to designate alleles at a hypothetical locus. The types of contrasts that are possible will vary among these mating types, and only some contrasts will be biologically meaningful. Table 1 lists the contrasts that can be interpreted in terms of additive or non-additive models of gene action. However, additive and non-additive effects of allele substitutions cannot always be separated (e.g., mating type III). Also, in some mating types (IV, VI, and VII), onlyadditive effects can be testedContrasts of these sorts are easily performed using, for example, the General Linear Models (GLM) procedure in SAS. RESULTS
In all, 1387 oysters were examined for all nine variable allozyme loci. The mean number of heterozygous loci per individual was 4.47 (range: 1 - 7). The corresponding values for each cross (including replicates within crosses) are reported in Table 2, which- also gives the partial correlation between log-trans-
264 TABLE 1 Single-locus mating types, progeny genotypes and contrasts among means for analysis of size data (all contrasts have 1 degree of freedom, except for the second contrast in V, which has 2) Mating type male X female (and reciprocal)
Possible effects leading to rejection of null hypothesis
Progeny Genotype(s) (contrast)
I
AA x AA
AA
None; no contrast possible
II
AA x BB (BB x AA)
AB
None; no contrast possible
III
AA x AB (AB x AA)
AA AB (+1 -1)
AA x BC (BC x AA)
AB AC (+1 -1)
Additive effect of allele substitution
AA AB BB +2 -1) (-1 0 +1)
Non-additive effect of allele substitution (a) Additive effect of allele substitution (b)
IV
V
AB x AB
Additive effect of allele substitution Non-additive effect of allele substitution
(-1
VI
AB x AC (AC x AB)
AA AB AC BC (+1 -1 +1 -1) (+1
VII
AB x CD (CD x AB)
+1
-1
-1)
AC AD BC BD (+1 $1 -1 -1) (+l
-1
+1
-1)
Additive effect of allele substitution in male gamete (a) Additive effect of allele substitution in female gamete (b)
Additive effect of allele substitution in male gamete (a) Additive effect of allele substitution in female gamete (b)
formed weight and heterozygosity. In two of eight instances, there was a sigcorrelation between log-transformed weight and nificant positive heterozygosity. However, the results were not consistent among replicates within crosses. For cross 6, there was a significant result in replicate A, but not in B. Similarly, there was a significant positive correlation in 9B, but 9A and 9C both had (non-significant) negative correlations between log-transformed weight and heterozygosity. The weighted mean correlation between log-transformed weight and heterozygosity was 0.052. Analysis of the log-transformed lengths gave similar results but are not presented here. Table 3 shows the outcome of single-locus tests for differences in size among
265 TABLE 2 Sample sizes (NJ, number of heterozygous loci per individual and correlation between log-transformed weight and heterozygosity (r) in five pair crosses of Louisiana oysters (Crassostrea virginica) (A, B, and C refer to replicates within crosses) Cross
N
Number of heterozygous loci Mean
Range
2 5 6A 6B
47 426 162 41
3.34 4.65 2.95 2.98
2-6 2-7 14
0.213 0.061 0.203**
14
0.001
8
41 222
5.00 4.82
3-7 3-7
0.039 -0.008
104
5.11 4.80
3-7 3-7
0.200* - 0.052
9A 9B 9c
344
*p < 0 .05., **p < 0.01. progeny genotypes. The only clear effect was at the Ap-1 locus, in which there ivas an additive effect of substituting the “127” allele for the “100” allele in the male gamete in cross 9. This result was consistent among all three replicates of that cross, with the 127/108 heterozygote having a lower mean weight than the lOSjlO0 heterozygote. Because both progeny genotypes were heterozygous, there was no effect of heterozygosity per se. The other nominally significant results (at Got-Z, Lap-2 and Mpi) involved cross 5 or 6. Because the female was heterozygous at Got-S, Lap-2, and Mpi, the iesults should have been consistent among crosses, because all crosses had the same female parent. The fact that inconsistent results were found at the Got-2, Lap-2 and Mpi loci suggests either that the effect was spurious or else due to an interaction between the female gamete and the male gamete. Further research is necessary to distinguish between these two alternatives. When each cross or replicate was stratified according to time of larval settlement, there was a significant difference (P < 0.05) in heterozygosity between early settling larvae and late settling ones in one (6A) dut of seven comparisons, with early settlers being more heterozygous than late settlers. However, a similar trend was not observed in the other replicate (6B) of this cross. A locus-by-locus test for differences in genotype frequencies between early and late settling larvae gave mostly negative results except for cross 5, in which three loci (Got-2, Pgi, and Xdh) exhibited significant differences. DISCUSSION
The finding that in natural populations there is a positive association between level of allozyme heterozygosity and growth rate has been confirmed in several
266 TABLE
3
Contrasts
of mean weights among progeny genotypes.
Roman
numerals refer to mating types as
listed in Table 1 (Arabic numerals refer to the identity of alleles being contrasted, further explanation; - indicates that no contrast was possible) Cross
2
5
Locus Ap-1
Got-2
III
Via
108/100
78/-
100
VIb 78/-
219
100/00
-
Via 78/-
Lap-l
Lap-2
Mpi
6-Pgd
Pgi
Pgm-2
Xdh
-
VIIa
III
III
IV
-
-
115/108 VIIb
100/91
100155
IV
III
III
VIIa
-
III
100/00*
100/91*
100/72
112/100 VIIb
100
VIb
6B
-
-
-
-
lOO**
VIb 78/-
100/91 Vb
279
100/91 -
-
78/- 100 VIb 781-279 8
Va
Via 78/-
Via
IV
IV
III
116/100
781-279
100/88
-
Va
-
9B
9c
IV
IV
127/100*
781-279
IV
IV
127/100*
18/-279
IV 127/100**
IV 78/-279
115/100
-
-
-
Via
-
-
-
-
-
100/78 VIb 78158 -
Via
100/91 Vb
100178 VIb
100/91
78158
Via
III
VIIa
III
lll/lOO
100/72
106/100
100/82
VIb 100/91 9A
78158
78158
781-279 6A
see text for
VIIb 78/58
IV
III
III
100/00
100/91
100172
IV
IV
III
III
100/00
100/91
100172
78158
IV 100/00
III 100/91
III 100/72
IV 78/58
78158 IV
III
-
100182 III
-
100/82* III 100/82
-
*P < 0.05; **p < 0.01.
different species, although counter-examples are known [see Zouros and Foltz (1986) for a review of this literature]. In bivalves, evidence that heterozygotes have an advantage in growth rate comes from studies of samples from natural populations (Singh and Zouros, 1978; Zouros et al., 1980; Singh, 1982; Green
261
et al., 1983; Koehn and Gaffney, 1984), from studies of ploidy manipulation (Stanley et al., 1984)) and from studies of the physiological energetics of growth (Koehn and Shumway, 1982; Garton et al., 1984). The associations between allozyme heterozygosity and growth rate observed in natural populations of bivalves are not necessarily present in hatchery populations produced from a limited number of parents. In offspring of pair crosses in Myths edulis (Beaumont et al., 1983) and Mulinia lateralis (Gaffney and Scott, 1984), there was no association between size and heterozygosity, although positive associations were observed in natural populations of both species. Similarly, there was little or no association between size and heterozygosity in offspring of mass spawnings in C. virginica, Spisula solidissima, M. lateralis (Gaffney and Scott, 1984) and Mercenaria mercenaria (Adamkewicz et al., 1984). The present study confirms these findings. When comparing the present study with earlier studies of heterozygosity and growth in oysters, it is important to realize that the sample sizes used here (both number of individuals and number of polymorphic loci) are as large as or larger than those employed in the earlier studies. Thus, the largely non-significant results are not due to inadequate sample sizes. As Gaffney and Scott (1984) have emphasized, these results indicate that associations between enzyme heterozygosity and growth depend strongly on the number of parental genomes contributing to the progeny (see also Koehn and Gaffney, 1984). The failure to observe heterozygosity-growth associations in hatchery populations produced from limited numbers of p*arents suggests that the associations observed in natural populations are not directly due to allozyme heterozygosity itself. Although it might be argued that progeny from pair crosses have lower heterozygosity than individuals from a natural population and that this phenomenon accounts for the lack of heterozygosity-growth associations in the former, the average heterozygosity is not reduced in pair crosses or mass spawnings, although the variance in heterozygosity among loci may be increased. This result follows from the fact that the loss of heterozygosity is 1 -f, where f is the inbreeding coefficient (Crow and Kimura, 1970). If the parents have been chosen at random from the population, which is presumably the case in most hatchery matings involving wild-collected broodstock, then f = 0. Only if the offspring from a pair mating are themselves crossed to produce a second generation in the hatchery would there be a reduction in average heterozygosity. Even though average heterozygosity is not reduced when a limited number of parents contribute to a cross, particular alleles may easily be lost. There can be no more than four different alleles at a locus present in offspring of a pair cross, and if both parents share one or both alleles there will be even less allelic diversity among the offspring. Thus, a possible explanation for the lack of association between heterozygosity and growth in pair crosses is that some allelic substitutions at allozyme loci affect growth and others do not, and that the growth-affecting alleles were absent
268
from the crosses which gave non-significant results. However, this model shifts attention away from heterozygosity per se and toward the effect of particular alleles. Although the results of the single-locus tests reported here (particularly for Ap-1) and some of the results of Beaumont et al. (1983), Adamkewicz et al. (1984), and Gaffney and Scott (1984) are consistent with the suggestion that only some allelic substitutions at an allozyme locus have an effect on growth rate, the possibility that the effect is due to a gene linked to the allozyme locus cannot be ruled out. Either explanation could account for the finding that correlations between allozyme heterozygosity and growth appear only sporadically in offspring of pair crosses. ACKNOWLEDGEMENTS
We thank B. Glidewell and P. Larsen for help in the laboratory and Dr. A. Bull for assistance in the hatchery. D.W. Foltz would like to thank the director and staff of the St. Amant Marine Laboratory for their support and hospitality. This research was supported by NSF Grant no. BSR-8407450 to D.W. Foltz. Additional support was provided by Louisiana State University and by the Louisiana Department of Wildlife and Fisheries.
REFERENCES Adamkewicz, L., Taub, S.R. and Wall, J.R., 1984. Genetics of the clam Mercenaria mercenaria. II. Size and genotype. Malacologia, 25: 525533. Beaumont, A.R., Beveridge, C.M. and Budd, M.D., 1983. Selection and heterozygosity within single families of the mussel Mytilus edulis (L.). Mar. Biol. I.&t., 4: 151-161. Buroker, N.E., Hershberger, W.E. and Chew, K.K., 1975. Genetic variation in the Pacific oyster, Crassostreagigas. J. Fish. Res. Board Can., 32: 2471-2477. Crow, J.F. and Kimura, M., 1970. An Introduction to Population Genetics Theory. Harper & Row, Publishers, Inc., New York, NY, 591 pp. Foltz, D.W., 1986. Null alleles as a possible cause of heterozygote deficiencies in the oyster Crussostrea virginica and other bivalves. Evolution, in press. Gaffney, P.M. and Scott, T.M., 1984. Genetic heterozygosity and production traits in natural and hatchery populations of bivalves. Aquaculture, 42: 289-302. Garton, D.W., Koehn, R.K. and Scott, T.M., 1984. Multiple-locus heterozygosity and the physiological energetics of growth in the coot clam, Mulinia lateralis, from a natural population. Genetics, 108: 445-455. Green, R.H., Singh, S.M., Hicks, B. and McCuaig, J., 1983. An arctic intertidal population of Mucoma balthica (Mollusca, Pelecypoda): genotypic and phenotypic components of population structure. Can. J. Fish. Aquat. Sci., 40: 1360-1371. Koehn, R.K. and Gaffney, P.M., 1984. Genetic heterozygosity and growth rate in Mytilus edulis. Mar. Biol., 82: l-7. Koehn, R.K. and Shumway, S.E., 1982. A genetic/physiological explanation for differential growth rate among individuals of the American oyster, Crassostrea virginica (Gmelin). Mar. Biol. Lett., 3: 35-42.
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Mitton, J.B. and Grant, M.C., 1984. Associations among protein heterozygosity, growth rate, and developmental homeostasis. Annu. Rev. Ecol. Syst., 15: 479-499. Newkirk, G.F., 1983. Applied breeding of commercially important molluscs: a summary of discussion. Aquaculture, 33: 415-422. Ogle, J.T., 1982. Operation of an oyster hatchery utilizing a brown water culture technique. J. Shellfish Res., 2(2): 153-156. Schaal, B.A. and Anderson, W.W., 1974. An outline of techniques for starch gel electrophoresis of enzymes from the American oyster Crassostrea uirginica Gmelin. Univ. Georgia Mar. Sci. Cen. Tech. Rep. Ser. No. 74-3,17 pp., unpubl. Singh, S.M., 1982. Enzyme heterozygosity associated with growth at different developmental stages in oysters. Can. J. Genet. Cytol., 24: 451-458. Singh, S.M. and Zouros, E., 1978. Genetic variation associated with growth rate in the American oyster (Crmsostrea virginica). Evolution, 32: 342-353. Sokal, R.R. and Rohlf, F.J., 1981. Biometry, 2nd edition. Freeman, San Francisco, CA, 859 pp. Stanley, J.G., Hidu, H. and Allen, S.K., Jr., 1984. Growth of American oysters increased by polyploidy induced by blocking meiosis I but not meiosis II. Aquaculture, 3’7(2): 147-155. Zouros, E. and Foltz, D.W., 1986. The use of allelic isozyme variation for the study of heterosis. In: M.C. Rattazzi, J.G. Scandalios and G.S. Whitt (Editors), Isozymes: Current Topics in Biological and Medical Research, 13. Alan R. Liss, New York, NY. Zouros, E., Singh, S.M. and Miles, H.E., 1980. Growth rate in oysters: an overdominant phenotype and its possible explanations. Evolution, 34: 856-867.