Dennis Hedgecock

Genetic drift and effective population sizes of hatchery-propagated stocks of the Pacific oyster, Crassostrea gigas

Abstract Using starch-gel electrophoresis, we scored genetic differences for 14 polymorphic enzymes among individuals from three Pacific oyster populations: natural set from Dabob Bay, Washington, and two cultivated stocks. The stocks were each derived from the Dabob Bay wild population and reproductively isolated from this population and from each other for three generations via hatchery propagation and separate rearing on commercial growout beds in Willapa Bay, Washington, and Humboldt Bay, California. Compared with the wild population, one of the two cultivated stocks had significantly fewer alleles per locus, but proportions of polymorphic loci and average heterozygosities were not statistically different among the three population samples. Hatchery-propagated stocks differed markedly in allelic frequencies from each other and from the wild population at most loci. Allelic frequencies in the Dabob Bay sample are assumed to represent those in the progenitors of the hatchery stocks. Average per locus allele-frequency variances between the progenitor and derived hatchery populations are normally distributed after appropriate transformation, indicating that divergence of hatchery stocks from Dabob Bay population owes to random genetic drift. Based on the inverse relationship between the magnitude of random genetic drift and effective population size (Ne), the per-generation effective sizes of the two commercial oyster stocks are calculated to be only 40.6±13.9 (s.d.) and 8.9±2.2 (s.d.) for the Willapa Bay and Humboldt Bay stocks, respectively. These estimates account well for the loss of alleles in these hatchery-propagated stocks. Analysis of allele-frequency drift is recommended over simple comparisons of genetic diversity for revealing the extent and nature of genetic change in reproductively isolated aquaculture stocks.

Effective population numbers of shellfish broodstocks estimated from temporal variance in allelic frequencies

Abstract Effective population number (Ne) can be estimated from temporal changes in the frequencies of selectively neutral alleles in isolated populations. The utility and limitations of this temporal method for aquaculture broodstocks are examined using published allozyme data for 16 shellfish stocks and their wild progenitors. Estimates of Ne for these broodstocks are all less than 100, and 13 are less than 50. For eight stocks, estimated Ne agrees with records of breeding numbers (Nb), but in the remaining eight cases, Nb lies outside of the 95% confidence interval for estimated Ne. Assumptions of the temporal method are evaluated. First, two independent tests, one based on the distribution of temporal variances at individual loci, the other on the expected loss of alleles from finite populations suggest that allozymes behave as selectively neutral genetic markers suitable for estimating genetic drift and Ne. Second, migration into a stock can have diverse effects on estimates of Ne, depending on the genetic similarity of immigrants and the captive broodstock population. Exchange among diverged hatchery stocks of Kuruma shrimp appears to have inflated temporal change, reducing estimated Ne; on the other hand, additions of wild hard clams to a hatchery stock appear to have retarded temporal change, increasing estimated Ne. Four cases in which Nb is smaller than the lower confidence limit of estimated Ne are explained by contamination among stocks propagated simultaneously in the same hatchery. Finally, comparison of captive broodstocks with contemporary samples from natural sources confounds genetic drift that has occurred in both lineages; genetic drift is not negligible in some natural populations, leading to uncertainty in some Ne estimates. For four cases in which Nb is much larger than estimated Ne, variance in reproductive success is proposed as the most likely explanation of loss of genetic diversity over time. The extremely high fecundities and variable fertilities of aquatic organisms must be taken into account in broodstock management, particularly if hatchery products are used to enhance natural populations. The temporal method, when correctly applied and interpreted, provides new insight into the genetics of aquaculture broodstock populations.

HYBRID VIGOR IN PACIFIC OYSTERS: AN EXPERIMENTAL APPROACH USING CROSSES AMONG INBRED LINES

Two competing genetic hypotheses for heterosis, dominance and overdominance, have been championed to explain positive correlations between allozyme heterozygosity and fitness-related traits for bivalve molluscs. To begin to test these hypotheses, we made controlled crosses among inbred lines of the Pacific oyster Crassostrea gigas. In such mating experiments, heterosis (hp) can be defined and quantified through ANOVA as QL > 1.0 or < − 1.0, where L is the trait-difference between the two parental inbred lines and Q is twice the deviation of the hybrid from the mid-parent value (Griffing, 1990). Inbred lines of the Pacific oyster were made by selfing hermaphrodites; brood stock pedigrees and a grand mean fixation index of F = 0.5 were confirmed by allozymes. In two separate experiments pairs of inbred lines were crossed in 2 X 2 fashion to produce two inbred and two hybrid progeny genotypes. Each genotype was replicated by pairwise matings and replicates were grown in multiple containers (8 1 plastic bags), from which data on larval mortality (proportional decrement in population number per day) and size (shell-height) were obtained. In the first experiment, the two inbred genotypes differed significantly from each other in daily larval mortality (−0.115 vs. −0.881) and both hybrids had lower mortalities than inbred genotypes (−0.034 and −0.078); mean hp, 1.15, was significantly greater than 1.0. By Day 14, larvae of one hybrid group were significantly smaller than those of the inbred genotypes (241.0 μm vs. 253.5 and 259.2 μm, respectively); only a very small part of this difference could be attributed to negative correlation between larval density and growth. Negative heterosis for larval size was highly significant (hp = −5.36, P < 0.001) and persisted to the juvenile stage (mean hp = −1.34, ns, for shell-height at Day 154 and hp = −7.77, P < 0.001, for live-weight at Day 163). In the second experiment, inbred genotypes again differed significantly in daily larval mortality (−0.341 vs. −0.654). One hybrid group had significantly less daily mortality than either parent (−0.110), but the other was similar to the best parent (−0.347), so that for one hybrid, hp = 2.47, P < 0.01, and for the other, hp = 0.96, ns). Variance among genotypes for larval shell-length was highly significant on Days 2, 7, and 14, with heterosis evident on Days 2 and 7 (hp = 1.04 and hp = 3.84, respectively). At 11 months of age, hybrid shell-height averaged 150% of the shell-height for the best inbred parent and hp ≈ 7.7. These measurements of heterosis, both positive and negative, implicate a third explanation of heterosis, epistasis. Intercrosses of F1 hybrids can be used to discriminate among dominance, overdominance and epistasis hypotheses for quantitative trait loci (QTL) causing hybrid vigor.

Quantitative and molecular genetic analyses of heterosis in bivalve molluscs

Abstract Associations of allozyme-heterozygosity with growth and its physiological underpinnings have been well documented for bivalve molluscs. The associations are correlational, however, derived almost entirely from studies of wild-caught juveniles or adults. Such studies cannot resolve alternative genetic explanations of heterosis. Four experimental approaches have recently been made to this problem; (1) a correlational study contrasting allozyme and presumably selectively neutral nuclear DNA polymorphisms; (2) detailed studies of allozyme inheritance in families; (3) a study contrasting the performance of meiosis-I and meiosis-II triploids with diploids and (4) a classical quantitative genetic study of the performance of hybrids produced by crosses among inbred lines. The last approach has uncovered remarkable heterosis in growth and its physiological components, both for the larval and juvenile or adult stages, and has implicated epistasis as a significant cause of this heterosis. More importantly, this approach now permits dissection of heterosis into quantitative trait loci (QTL) mapped by the co-segregation of allozyme and nuclear DNA markers with growth phenotypes in the F2 hybrid and backcross generations.

Feeding behaviour and metabolic efficiency contribute to growth heterosis in Pacific oysters [Crassostrea gigas (Thunberg)]

Abstract Physiological measurements were made on Pacific oysters from controlled crosses between inbred lines. Hybrid individuals were expected to perform better than inbred oysters, for a variety of traits related to feeding behaviour. The oysters were offered a diet simulating natural suspended particulate matter. By quantifying the organic and inorganic fractions of food, faeces and pseudofaeces, various aspects of feeding were elucidated. The results agreed with expectation; on average, hybrid oysters had higher rates and efficiencies of feeding and growth than inbreds. In one experiment there were significant differences between hybrids and inbreds for seventeen out of twenty cases; in another experiment hybrids performed better than inbreds for eight out of sixteen cases. In both experiments, we find significant differences between the reciprocal hybrids, though heterosis for growth is evident for all hybrids. Our experiments therefore confirmed heterosis for growth and hybrid superiority for physiological traits, independent of ration level; emphasised the complexity of these relationships amongst genotypes; and demonstrated the segregation of physiological traits in the F 2 generation.

Genetic effects of artificial propagation: signals from wild and hatchery populations of red abalone in California

Abstract Numerous attempts have been made to restore the declining California abalone fisheries by outplanting of hatchery-produced seed. Poor survival of planted seed has been generally attributed to predation and emigration, lessening enthusiasm for reseeding efforts. We present genetic evidence that one large-scale outplanting of red abalone (Haliotis rufescens) in southern California was effective, and document changes in the genetic composition of red abalone populations accompanying hatchery propagation. Allozymes appeared to behave as neutral and therefore effective markers for tracking population bottlenecks in hatchery production and reseeded populations. Careful monitoring of hatchery breeding practices is essential for both commercial production and artificial reseeding programs.

Photoperiod-induced changes in hemolymph vitellogenins in female lobsters (Homarus americanus)

Abstract 1. 1. The course of changes of vitellogenin (VG) concentrations in the hemolymph of female lobsters, Homarus americanus , was studied following a photoperiodic stimulation of secondary vitellogenesis. 2. 2. Two immunochemically related VGs were found, each corresponding to an ovarian lipovitellin (LV). 3. 3. Indirect quantification of VGs was possible by virtue of their relationship of partial immunological identity in quantitative immunoelectrophoresis. 4. 4. Changes in VG hemolymph concentration could be related to photoperiodic schedule, and to the occurence of molting, egg extrusion, ovarian resorption, and to VG clearance from the hemolymph. 5. 5. Both VG influx from an extraovarian source and uptake by the ovary appeared to be episodic in nature and not closely correlated in time. 6. 6. The two VGs appear to be produced in the same amounts (stoichiometrically), perhaps as subunits of a larger precursor which differ from one another mainly in astaxanthin content or state and in tertiary or quaternary configuration. 7. 7. During the weeks of vitellogenesis hemolymph VG 1 appears to change slowly into VG 2 l in the oocytes this change appears to be arrested. 8. 8. Clearance and ovarian uptake and resorption all seem to be unbiased with respect to VG species.

Mass Extinctions and Genetic Polymorphism in the “Killer Clam,” Tridacna

Mass extinctions of marine invertebrates have been attributed to genetic depauperation in specialized lineages. Tridacna maxima is a plausible modern analog of the lineages that were commonly associated with mass extinctions; it is restricted to a relatively stable biogeographic province, lives in shallow water, is highly specialized, and is associated with reef communities. Our studies show, however, that it is highly polymorphic and heterozygotic, and thus fails to support the depauperate gene-pool hypothesis of mass extinction.

Genomic approaches to understanding heterosis and improving yield of Pacific oysters

According to FAO statistics, the Pacific oyster is the most farmed aquatic organism in the world, so improving growth and yield is worthwhile. Positive correlation of allozyme heterozygosity and growth in oysters, first reported in 1978, was vigorously debated by opposing camps, espousing two classical explanations for heterosis — dominance and overdominance. Crosses between inbred lines of this oyster demonstrate classical heterosis (superior yield of F1 hybrids compared to inbred parents). However, mapping of heterosis genes was initially prevented by distortions of Mendelian segregation ratios similar to those reported for bivalves since 1975. We have now shown that segregation for microsatellite markers is normal in early larvae but becomes distorted by the juvenile stage in a manner consistent with selection against numerous highly deleterious recessive mutations linked to markers. A large genetic load provides a unifying explanation for both distortions of Mendelian segregation ratios and heterosis, according to the dominance theory.