Standish K. Allen

All-triploid Pacific oysters (Crassostrea gigas Thunberg) produced by mating tetraploids and diploids

Abstract To document the reproductive characteristics of tetraploids and determine whether they can be used for triploid production, factorial crosses were made between diploids (D) and tetraploids (T), producing DD, DT, TD and TT groups (female listed first). A normal triploid group was also produced by blocking polar body II with cytochalasin B (3nCB). Survival to spat in TD and DT groups was about the same as in normal diploids, and significantly higher than in the 3nCB and TT groups. As determined by flow cytometry, all surviving oysters from DT and TD crosses were triploids, and only 46% of oysters from the 3nCB group were triploids. TT crosses produced primarily tetraploids despite low survival. At 8 and 10 months of age, triploid oysters from DT and TD groups were 13–51% larger than normal diploids, possibly due to polyploid gigantism. These results suggest that mating tetraploids and diploids is the best method for triploid production, and triploids produced in this way are better suited for aquaculture than those produced by altering meiosis and are ideal for population control.

Growth of American oysters increased by polyploidy induced by blocking meiosis I but not meiosis II

Abstract Triploidy in American oysters ( Crassostrea virginica ) was induced by blocking polar body formation with cytochalasin B. Triploid oysters created by treatment during meiosis I grew faster during the first 3 years of life than did diploid siblings. Triploid oysters created during meiosis II grew at the same rate as their controls. Because heterozygosity was higher in oysters created during meiosis I than in other groups, the increased growth must be due to heterozygosity rather than to triploidy per se.

Evaluation of methods using ray's fluid thioglycollate medium for diagnosis of Perkinsus marinus infection in the eastern oyster, Crassostrea virginica

Abstract Accurate detection and quantification of parasite body burden are critical for understanding many aspects of host-parasite interactions. The standard assay for diagnosing Perkinsus marinus infections in Crassostrea virginica involves incubation of oyster tissue in Rays fluid thioglycollate medium (RFTM), followed by iodine staining and microscopic examination for parasites. The original RFTM tissue assay is destructive and provides only a ranked level of infection intensity. A recent modification provides a technique to enumerate P. marinus after incubation in RFTM and determines total body burden. Application of this technique to hemolymph samples has provided a nondestructive assay. We provide a critical evaluation of these three assays. Data from previous studies were not adequate to critically evaluate performance of the tissue and hemolymph assays. Sensitivity and accuracy of these assays were therefore compared against weight-standardized body burden in Delaware Bay oysters over the course of a year. Determination of total body burden was significantly more sensitive than the other assays. Neither tissue nor hemolymph assays provided accurate estimates of individual infection intensities and were insensitive at low infection levels. The tissue assay was easier to use and slightly more accurate than the hemolymph assay, and both provided reasonable estimates of average infection level in the population. In summary, total body burden assessment is recommended when highly accurate measures of infection intensity or prevalence are necessary, the tissue assay is recommended for monitoring epizootics because of its simplicity and accuracy at the population level, and the hemolymph assay is only recommended when oysters must be kept alive.

Hybridization among Crassostrea species: a review

Abstract Numerous attempts at hybridization in the genus Crassostrea have been reported. Most suffer from one or more of the following: (1) ambiguities in the classification of oysters; (2) contamination of experimental cultures by extraneous gametes or larvae; (3) the absence of control experiments for assessing the quality of gametes as well as larval viabilities; (4) the absence of genetic confirmation of hybrid status. We conclude that to date there is no unequivocal evidence for the existence of viable interspecific hybrids among Crassostrea species. Examples of taxonomic ambiguities that cloud interpretation of hybridization experiments include the frequent misclassification of the Kumamoto oyster ( C. sikamea ) as the Pacific oyster ( C. gigas ), the probably unjustified distinction between C. gigas and the Portuguese oyster C. angulata , and the lumping of C. iredalei with C. rivularis . Low-level contamination of cultures is a common occurrence that may account for many reports of successful hybridization. Experimental controls are essential to properly assess gamete quality and zygote growth and survival, yet are often not included. Finally, few reports provide genetic confirmation that the zygotes produced are actually hybrids. To date, most genetic analyses have demonstrated that putative interspecific hybrid oysters were not hybrids. Use of adequate controls and genetic analysis of parents and offspring are essential to hybridization experiments, and are demonstrated elsewhere.

Large-scale production of triploid oysters, Crassostrea virginica (Gmelin), using “stripped” gametes

Abstract Successful production of triploid oysters requires precise control of the timing of fertilization of eggs. Logistically, using stripped gametes is the most practical method for producing triploids because the time of fertilization can be easily controlled. We examined several factors that may affect the survival and yield of triploid C. virginica from stripped gametes in large scale spawnings. Triploidy was induced by inhibiting polar body II with cytochalasin B. Fourteen replicates containing from 17 to 49 million (M) eggs were treated. Survival and percent triploidy of 48-h-old larvae were not correlated; mean survival was 22% and mean triploidy was 79%. About 72% of 138 spat examined were triploid, indicating that the estimate of triploidy at 48 h was accurate. Overall, 400.8 M eggs yielded about 17.8 triploid spat, an efficiency of 4.4%. All linear regression models incorporating the treatment variables were non-significant. Variability may be random, a reflection of egg quality, or both.

ABNORMAL GAMETOGENESIS AND SEX RATIO IN TRIPLOID SOFT-SHELL CLAMS (MYA ARENARIA)

Triploid soft-shell clams (Mya arenaria) were produced by inhibiting polar body extrusion with cytochalasin B immediately after fertilization. Diploid and triploid clams grown in suspended tray culture were examined histologically during the reproductive season of their second year. Most diploids had matured by the end of May and displayed normal gametogenic development in every individual. Triploids did not mature and most had undeveloped gonads. The few triploid females which developed some oocytes by the end of May had abnormal maturation. Triploids were 77% female as judged by the presence of oocytes; another 16% were female-like and may have been intersexes; the sex of 6% could not be identified because there was no sexual differentiation. Sex determination in this species, based on the sex ratio in triploids, best fits the model of an X:autosome balance mechanism as exemplified in some insect species.

Genetic confirmation of hybridization between Crassostrea gigas (Thunberg) and Crassostrea rivularis (Gould)

Abstract Of the numerous reports of hybridization attempts in the genus Crassostrea, none has been confirmed genetically. Three replicates of a 2×2 factorial mating of Crassostrea gigas×C. rivularis (C. arakiensis) were produced to examine the viability of this cross. Fertilization rate, yield of 48-h-old larvae, and survival of fertilized eggs was lower in the hybrids than pure crosses. All crosses showed similar larval growth rates, except C. rivularis×C. gigas, which grew more slowly. Genetic control of eyed larval size was indicated by the intermediate size of hybrids compared to pure crosses. Spat were obtained from most cultures and were shown by protein electrophoresis to be hybrids. Contaminants were also present, underscoring the need for genetic confirmation in studies on bivalve hybridization.

Inviable hybrids of Crassostrea virginica (Gmelin) with C. rivularis (Gould) and C. gigas (Thunberg)

Abstract Interspecific hybridization may provide important tools for selective breeding programs in oyster culture, especially for enhancement of disease resistance, and may have a bearing on debates concerning the introduction of non-native species. Factorial crosses of C. virginica with C. rivularis (C. arakiensis) and C. gigas were made, producing control and hybrid larvae. Larval survival and growth were documented. After several replicated experiments it became apparent that diploid hybrids were inviable, and so triploid hybrids were also tested and found to be equally inviable. Feeding studies with hybrids were initiated to determine if lack of growth and viability were related to capture of food. Overall, hybrids of C. virginica with C. rivularis and C. gigas can be readily produced, but are inviable after 8–10 days and grow little. With regard to the species examined here, previous reports of successful hybridization should be questioned. Introduction of C. gigas to the native range of C. virginica will not have direct genetic effects on C. virginica .

Gametic incompatibility in wild and cultured populations of the eastern oyster, Crassostrea virginica (Gmelin)

Abstract Fertilization rates were measured in 100 2×2 crosses of oysters ( Crassostrea virginica ) from six hatchery lines and one natural population. Analysis of fertilization by a log-linear model showed significant interaction between sources of gametes for 30 of the crosses. Gametic compatibility indices (GCI), which estimated the rates of fertilization within populations relative to between-population matings, were more often positive than negative, indicating a slight advantage to within-population matings. However, of the 30 significant cases, GCI was positive in 15 cases and negative in 15 cases. A mass spawning of oysters in which aliquots of pooled eggs were fertilized separately by individual males showed a highly significant sire-dam interaction in yield of juveniles, reflecting differences in larval viability as well as sperm-egg interactions. The existence of moderate gametic incompatibility together with sire-dam interaction effects on larval viability may pose problems in oyster breeding programs employing the mass spawning of small numbers of broodstock.

Optimization of triploid induction for the oyster Crassostrea virginica (Gmelin)

Abstract Standard techniques developed for producing triploid oysters ( Crassostrea gigas ) in the northwestern United States cause > 90% mortality of Crassostrea virginica , which is unacceptably high considering the scale of most east coast hatcheries. We examined the effect of cytochalasin B (CB) concentration, timing of treatment after fertilization, and duration of treatment on survival and triploid induction of C. virginica embryos. Survival to D-stage was inversely related to CB concentration and length of treatment. Induction of triploidy was CB-dose dependent. Optimal treatment of 0.25 mg/l CB for 10–15 min beginning when half the embryos were observed to be undergoing meiosis-I yielded mean survival to D-stage of 84% of control values with a mean of 96% triploid production.