Katsuhiko T. Wada

Genetic selection for shell traits in the Japanese pearl oyster, Pinctada fucata martensii

Abstract Selection experiments are currently being conducted on 3-year-old Japanese pearl oysters, Pinctada fucata martensii for shell width and shell convexity, defined as shell width/(shell height + hinge line length + shell width). The third generation was produced in 1981, and offspring were evaluated in 1983 and 1984. Mean shell width was larger for offspring of parents selected for large shell width than in those from parents selected for large shell convexity. Realized heritabilities estimated from regression of the selection response on cumulated selection differential were also larger when selection was based on shell width than when based on shell convexity. Response based on both traits varied every generation and some lines were lost by accident. The variation in selection response is attributed to sampling error and genetic drift since small numbers of parents (6–22 individuals) and offspring were used in each generation.

Detection of induced triploid scallop, Chlamys nobilis, by DNA microfluorometry with DAPI staining

Abstract A simple method of detecting the ploidy levels of shellfish by DNA microfluorometry with DAPI staining was demonstrated. Triploid scallops, Chlamys nobilis, were induced by cytochalasin B treatment (0.5 mg/l) of fertilized eggs, commencing 15 min after insemination for 15 min duration. Ploidy levels were determined at 8–13 months of age by microfluorometry of cells from gill tissue and hemolymph stained with DAPI and excited with UV light. Fluorescence intensity per nucleus was measured using a microphotometer. The ploidy levels determined by microfluorometry were verified by both cytogenetic and flow cytometric assays.

Effect of selection for shell coloration on growth rate and mortality in the Japanese pearl oyster, Pinctada fucata martensii

Abstract Effects of inbreeding on the growth rate and mortality are suggested on the basis of results of comparative growth experiments for a line of the Japanese pearl oyster, Pinctada fucata martensii , selected for white shell color in the prismatic layer. Growth in shell height, whole weight and mortality were measured using one inbred white cross and two hybrid white/brown crosses held in pearl nets under different densities. Analyses were conducted at the juvenile (3–12 months of age) and adult (12–20 months of age) stages. The growth rate was estimated by the differences between measurements at the start and after 4, 5, 6, 8 and 12 months in the juvenile experiment, and after 17 and 20 months in the adult experiment. Growth in whole weight and survival were significantly inferior in the inbred white crosses than in two hybrid crosses. With the exception of one case, there was no observation in which the performance of an inbred cross was significantly superior to that of the two hybrid crosses. There were significant effects of density on growth rate in weight, shell height and mortality.

Genetic variation in wild and hatchery stocks of the black pearl oyster, Pinctada margarififera, from Japan

Abstract The analysis of samples from the first to the third generations of hatchery stocks (G1, G2, G3), of black pearl oysters, P. margaritifera, has been investigated using 18 protein loci to determine the genetic variation compared to a wild sample (WS). A reduction of 17% and 18% in the number of alleles was estimated in G2 and G3, respectively. Seven alleles (of the total of 45 alleles) were not found in the wild sample. The change of selective constraints in hatcheries compared to the wild environment can account for these new alleles. The observed heterozygosities were maintained (Ho: wild=0.237 and hatchery=0.236 to 0.269). Inbreeding depression did not occur in any hatchery sample although an increasing excess of heterozygotes was observed in G2 and G3 samples. This could be explained by selective effects through the choice of broodstock and change of the selective forces in an artificial environment.

Direct comparison of six methods to induce triploidy in bivalves

Abstract Six agents previously reported to induce triploidy in bivalves were compared simultaneously on two separate spawns in the blue mussel, Mytilus galloprovincialis . The agents [cytochalasin B (CB: 1 mg/l), heat (HT: 30°C), calcium (CA: 0.1 M), caffeine (CF: 15 mM), combined calcium and heat (CAHT), and combined caffeine and heat (CFHT)] were applied 20 min after sperm addition, so as to suppress polar body II formation, and left for 15 min. Triploidy was estimated by measuring DNA content in cells from trochophore larvae with microfluorometry. In addition, polar body counts, pronuclei behavior and early larval development are reported. CA was least efficient (4.7–7.5%) for triploid induction. The five other treatments produced on average 86% (CB), 81% (HT, CFHT), 73% (CAHT) and 71% (CF) triploids. The proportion of D-stage larvae at 48 h was reduced by all treatments, being least reduced from CB and most reduced from CAHT. Treatments involving CA and CF resulted in smaller D-stage larvae at 48 h. In all treatments except CB, the proportion of larvae feeding at 96 h was reduced (58–95%) compared to the control group (100%). CB was the most effective overall in producing viable triploid individuals. If regulations inhibit the use of chemicals, heat may be an alternative but viability is reduced.

Ultrastructure of spermatozoa from induced triploid Pacific oyster, Crassostrea gigas

Ultrastructure of spermatozoa produced by artificially induced triploid Pacific oysters, Crassostrea gigas, was studied by transmission and scanning electron microscopy. Spermatozoa from triploids (3n sperm) resembled sperm from diploids (2n sperm) and consisted of an acrosome, nucleus, four mitochondria, and flagellum. The head, acrosome, and flagellum of 3n sperm were significantly (P<0.05) larger than those of 2n sperm. The diameter of the mitochondria was not different between 3n and 2n sperm.

Abnormal Gametogenesis, Male Dominant Sex Ratio, and Sertoli Cell Morphology in Induced Triploid Mussels, Mytilus galloprowincialis

Abstract Gametogenesis of one year-old induced triploid mussels, Mytilus galloprovincialis, was examined histologically and compared to sibling diploid mussels. Histological analysis revealed that triploid mussels developed a number of primary spermatocytes that were arrested at prophase I. Late in the reproductive season, triploid mussels produced an extremely small number of spermatozoa (9/10000 &mgr;m2 gonadal section) compared to diploid mussels (1072/10000 &mgr;m2 gonadal section). All triploid mussels were identified as males, whereas the sex ratio of diploid mussels was almost equal (1.12:1.0, male:female), indicating that sex determination for this species may follow a Z:W model. Sertoli cells in triploid mussels were prominent, had an enlarged cytoplasm, and were easily seen using light microscopy. In comparison, Sertoli cells in diploids were thin and could only be seen by electron microscopy. Sertoli cell hypertrophy in triploid mussels probably reflects their role in eliminating abnormal and degenerating germ cells.

Detection of induced triploidy at different ages for larvae of the Japanese pearl oyster, Pinctada fucata martensii, by microfluorometry with DAPI staining

Abstract Ploidy level of larvae of the Japanese pearl oyster was estimated by DNA microfluorometry with DAPI staining. More than 20 fixed larvae were minced and the cell suspension mounted on glass slides and stained with DAPI. The amount of nuclear DNA was measured by DNA microfluorometry. Four peaks of fluorescence intensity of nuclear DNA were estimated to correspond to diploidy, triploidy and the post-DNA synthesis (G 2 ) stage of diploid and triploid trochophore larvae developed from eggs treated with cytochalasin B (CB, 0.5 mg/l) from 5 to 20 min after insemination (24°C). Percent of triploid plates was estimated to be 65.4% from the observation of metaphase chromosomes. Fluorescence intensity was also measured at different ages in larvae obtained from another batch of eggs treated with CB from 5 to 20 min or low temperature (6–7°C). Measurements were done at 2, 7, 16 and 24 days after hatching. With both treatments, triploid nuclei were detected from peaks in the histogram of fluorescence intensity. However, percent triploid nuclei decreased with larval age. Only a small number of triploid cells was present 24 days after hatching, particularly in low temperature treatment.

Spawning peak occurs during winter in the japanese subtropical population of the pearl oyster, Pinctada fucata fucata (Gould, 1850)

Abstract The pattern of gametogenesis in adult Pinctada fucata fucata from Zamami Island, Okinawa Prefecture (26 °N), Japan, was examined and compared with to data reported for pearl oysters in other Indo-Pacific regions. Gametogenesis was observed in histological preparations of gonad from 232 adults sampled approximately monthly (1992–1993). Maturation was observed throughout the year with a major peak in winter and a minor peak in summer. This is different from the restricted peak in summer reported for pearl oysters ( P. fucata martensii ) in the Japanese northern temperate populations (33–34 °N). A difference in the reproductive cycle between sexes was observed. After the peak of winter spawning only the females entered a resting period during spring (May). From summer to autumn there was a continuous occurrence of mature individuals in both sexes which increased into winter. Differences in reproductive periods among pearl oyster populations may prove useful for breeding programs in the pearl culture industry if environmental requirements for gonadal maturation are genetically influenced.

Genetic variability at four polymorphic loci in Japanese pearl oysters, Pinctada fucata martensii, selected for six generations

Abstract Genetic variability was estimated for four electrophoretically detectable polymorphic loci in four lines of Japanese pearl oysters which had been selected for six generations. One monomorphic locus was observed out of 16 cases analysed, but the number of alleles per locus decreased and minor alleles disappeared in many cases. Observed heterozygosity in the selected stocks was higher in most cases than that of a cultured population which was collected from the same location as the base population. An excess of heterozygotes was observed in many cases. These results are discussed in terms of genetic drift, the number of parents used (6–30 individuals), inbreeding and selection.