Ruihai Yu

Response to selection for fast growth in the second generation of Pacific oyster (Crassostrea gigas)

Mass selection for fast growth was conducted in three Pacific oyster (Crassostrea gigas) stocks from China, Japan and Korea using previously established lines (CS1, JS1, and KS1). To determine whether continuous progress can be achieved by selection for growth, the progeny of three second-generation Pacific oyster lines was selected for shell height and evaluated via a 400-day farming experiment. When harvested at the end of the experiment, the selected crosses of CS2, JS2, and KS2 lines grew by 9.2%, 10.2% and 9.6% larger than the control crosses, respectively. During grow-out stage, the genetic gain of three selected lines was (10.2 ± 1.4)%, (10.4 ± 0.3)%, and (8.4 ± 1.6)%, respectively; and the corresponding realized heritability was 0.457 ± 0.143, 0.312 ± 0.071 and 0.332 ± 0.009, respectively. These results indicated that the selection for fast growth achieved steady progress in the second generation of oyster. Our work provides supportive evidence for the continuity of the Pacific oyster selective breeding program.

Seasonal changes in reproductive activity and biochemical composition of the razor clam Sinonovacula constricta (Lamarck 1818)

Abstract The reproductive cycle and seasonal variations in biochemical composition of the razor clam Sinonovacula constricta (Lamarck) from the Yellow River delta in China were investigated from April 2007 to March 2008 in relation to environmental factors. Histological analysis indicated that the gametogenesis of the S. constricta comprised two phases: a resting phase (February–May) and gametogenesis, including ripeness and spawning, during the rest of the year. Gametogenesis of S. constricta began in June–July and was completed during September–October when food was abundant. A significant decrease in the glycogen content of all tissues both in the female and male during sexual maturation suggests that carbohydrates play the most important role in reproduction of this species. The protein and lipid content increased in association with the gametogenesis in the female gonads, indicating that they are the major organic component of oocytes. Conversely, the protein and lipid decreased in the male gonads and other tissues, demonstrating that they also support reproduction after the glycogen is depleted. The RNA/DNA ratio is a valuable indicator of maturation both in the female and male. The results obtained in this study provide useful information for developing management strategies of S. constricta populations in this area, and for optimizing the hatchery-based seed production.

Seasonal Variations in Biochemical Composition and Reproductive Activity of Venus Clam Cyclina sinensis (Gmelin) from the Yellow River Delta in Northern China in Relation to Environmental Factors

ABSTRACT The seasonal variations in biochemical composition and reproductive activity of the venus clam Cyclina sinensis (Gmelin) from the Yellow River delta in China were investigated from April 2007 to March 2008 in relation to environmental factors. According to histological observations, gametogenesis began in January when the water temperature was low. Gametes matured mainly in June and July when the condition index (CI) and mean oocyte diameter peaked. Spawning occurred primarily in August when the temperature was highest, and coincided with phytoplankton bloom. The CI and oocyte diameter decreased sharply after spawning occurred, as mostly larger, mature gametes have been released. In autumn, the plentiful phytoplankton and higher water temperature were fit for the larvae to grow. Biochemical analysis indicated that gametogenesis took place at the expense of reserves accumulated in the various tissues previously during winter. A slight increase in lipid and protein content in female gonad—visceral mass during sexual maturation was observed, demonstrating that lipid and protein would be accumulated as vitellin in oocytes. Conversely, the lipid and protein content in the male gonad—visceral mass decreased during sexual maturation. The biochemical compositions in the adductor muscle and mantle varied during the study period, suggesting that they could support reproduction and growth. Seasonal variation in the RNA-to-DNA ratio suggests that it cannot reflect the situation of gonad development in this species.

Effects of Salinity on Embryonic Development, Survival, and Growth of Crassostrea hongkongensis

This study examined the effects of salinity on embryonic development, survival, and growth of the Hong Kong oyster Crassostrea hongkongensis. The embryos, larvae, and juveniles of C. hongkongensis were held in artificial seawater at three different salinities (low, 15; medium, 23; and high, 30) to determine the optimum hatchery and nursery conditions for mass production of the seeds. Results showed that the percentage production of straight-hinged larvae from fertilized eggs was significantly lower at the high salinity than at the low- and medium-salinities (P < 0.05). The survival rates of larvae and juveniles differed significantly among the three salinity trials, with the highest survival rate observed at the low salinity (P < 0.05). The shell height of larvae was significantly larger at the low salinity than at the high and medium salinities from days 9 to 15 (P < 0.05), whereas that of juveniles was significantly larger at the low salinity than at the high and medium salinities on day 70 (P < 0.05). These results indicate that the larvae and juveniles of C. hongkongensis are tolerant to a wide range of salinities (15 to 30), but show better growth and survival at relatively low salinities. Thus, it is recommended to use relatively low salinities in hatchery and nursery systems for improved yields of C. hongkongensis.

Transcriptomic analysis of Crassostrea sikamea × Crassostrea angulata hybrids in response to low salinity stress.

Hybrid oysters often show heterosis in growth rate, weight, survival and adaptability to extremes of salinity. Oysters have also been used as model organisms to study the evolution of host-defense system. To gain comprehensive knowledge about various physiological processes in hybrid oysters under low salinity stress, we performed transcriptomic analysis of gill tissue of Crassostrea sikamea ♀ × Crassostrea angulata♂ hybrid using the deep-sequencing platform Illumina HiSeq. We exploited the high-throughput technique to delineate differentially expressed genes (DEGs) in oysters maintained in hypotonic conditions. A total of 199,391 high quality unigenes, with average length of 644 bp, were generated. Of these 35 and 31 genes showed up- and down-regulation, respectively. Functional categorization and pathway analysis of these DEGs revealed enrichment for immune mechanism, apoptosis, energy metabolism and osmoregulation under low salinity stress. The expression patterns of 41 DEGs in hybrids and their parental species were further analyzed by quantitative real-time PCR (qRT-PCR). This study will serve as a platform for subsequent gene expression analysis regarding environmental stress. Our findings will also provide valuable information about gene expression to better understand the immune mechanism, apoptosis, energy metabolism and osmoregulation in hybrid oysters under low salinity stress.

Interspecific hybridization between Crassostrea angulata and C. ariakensis

Interspecific hybridization can generate heterosis, which is proven to be a useful tool in selective breeding programs for oyster culture. Crassostrea angulata and C. ariakensis are two important economic shellfish species in China. We conducted 2 × 2 reciprocal crosses to determine whether these two species can cross-fertilize and their hybrids can hatch, survive and perform heterosis. Fertilization was found symmetrical without delay. The rate of fertilization success of C. angulata ♀ × C. ariakensis ♂ was lower than that of C. ariakensis ♀ × C. angulata ♂, and the success rate of both hybridizations was lower than that of two intraspecific crosses each. During the planktonic period, survival rate of the progeny was lower in the hybrid crosses than in the intraspecific crosses. On day 360, mean shell height of the progeny of C. angulata ♀ × C. angulata ♂ was highest, which was followed by that of C. angulata ♀ × C. ariakensis ♂, C. ariakensis ♀ × C. ariakensis ♂ and C. ariakensis ♀ × C. angulata ♂ in a descending order. Morphology of adults produced by the hybrid crosses was similar to that of C. angulata. Both hybrids underwent normal gonad development and produced mature gametes in the mating season. This study provided new insights into the quantitative traits in interspecific crosses of Crassostrea species, thus being of guidance value for selective breeding of oyster.

Induced metamorphosis in larvae of the veined rapa whelk Rapana venosa using chemical cues

Abstract The larval metamorphosis of the veined rapa whelk Rapana venosa was investigated by exposing competent larvae to the following seven chemical cues at different concentrations and exposure time: EPI (epinephrine), serotonin, L-DOPA (L-3, 4-dihydroxyphenylalanine), GABA (γ-aminobutyric acid), acetylcholine chloride, KCl (potassium chloride), CaCl2 (calcium chloride). Larvae of R. venosa which lost their velum after the veliger stage, crawled on their foot and produced an adult shell were considered to have metamorphosed. Compared with the control treatments, there were significantly higher percentages of larval metamorphosis in the following groups: EPI (10−3 M) at 6 h, L-DOPA (10−3 M, 10−4 M) at 6 h, L-DOPA (10−5 M) at 24 h, GABA (10−5 M) at 24 h, acetylcholine chloride (10−4 M) at 6 h, acetylcholine chloride (10−4 M, 10−6 M, 10−7 M) at 24 h and 5 mM CaCl2 at 24 h. Acetylcholine chloride and CaCl2 were confirmed as active inducers of R. venosa larval metamorphosis with low toxicity. In contrast, EPI, L-DOPA and GABA were less active inducers, showing high mortality rates. Serotonin and KCl had no inductive effects on larval metamorphosis. In conclusion, acetylcholine chloride and CaCl2 are more suitable among the seven chemical cues to be applied to the artificial seeding of R. venosa.

Isolation and characterization of 23 microsatellite loci in the veined rapa whelk (Rapana venosa)

Rapana venosa is a commercially important mollusk species, but has been suffering from severe population decline due to over-exploitation and habitat destruction in China. The first set of 23 polymorphic microsatellite loci was isolated and characterized for population studies. The number of alleles per locus ranged from 3 to 17, and the observed and expected heterozygosities varied from 0.167 to 1.000 and from 0.455 to 0.924, respectively. These microsatellite loci will be useful tools for fisheries management and conservation programme for this species.

Selection of reference genes for expression analysis of Kumamoto and Portuguese oysters and their hybrid

Quantitative real-time polymerase chain reaction (qRT-PCR) is a rapid and reliable technique which has been widely used to quantifying gene transcripts (expression analysis). It is also employed for studying heterosis, hybridization breeding and hybrid tolerability of oysters, an ecologically and economically important taxonomic group. For these studies, selection of a suitable set of housekeeping genes as references is crucial for correct interpretation of qRT-PCR data. To identify suitable reference genes for oysters during low temperature and low salinity stresses, we analyzed twelve genes from the gill tissue of Crassostrea sikamea (SS), Crassostrea angulata (AA) and their hybrid (SA), which included three ribosomal genes, 28S ribosomal protein S5 (RPS5), ribosomal protein L35 (RPL35), and 60S ribosomal protein L29 (RPL29); three structural genes, tubulin gamma (TUBγ), annexin A6 and A7 (AA6 and AA7); three metabolic pathway genes, ornithine decarboxylase (OD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glutathione S-transferase P1 (GSP); two transcription factors, elongation factor 1 alpha and beta (EF1α and EF1β); and one protein synthesis gene (ubiquitin (UBQ). Primers specific for these genes were successfully developed for the three groups of oysters. Three different algorithms, geNorm, NormFinder and BestKeeper, were used to evaluate the expression stability of these candidate genes. BestKeeper program was found to be the most reliable. Based on our analysis, we found that the expression of RPL35 and EF1α was stable under low salinity stress, and the expression of OD, GAPDH and EF1α was stable under low temperature stress in hybrid (SA) oyster; the expression of RPS5 and GAPDH was stable under low salinity stress, and the expression of RPS5, UBQ, GAPDH was stable under low temperature stress in SS oyster; the expression of RPS5, GAPDH, EF1β and AA7 was stable under low salinity stress, and the expression of RPL35, EF1α, GAPDH and EF1β was stable under low temperature stress in AA oyster. Furthermore, to evaluate their suitability, the reference genes were used to quantify six target genes. In conclusion, we have successfully developed primers appropriate for the expression analysis in SS, SA and AA.

Characterization of 57 microsatellite loci for Rapana venosa using genomic next generation sequencing

Abstract A total of 57 polymorphic microsatellite loci were developed for Rapana venosa through 454 sequencing. The 57 loci were characterized in 30 wild individuals. The number of alleles per locus varied from 2 to 14. The observed and expected heterozygosities ranged from 0.100 to 1.000 and 0.127 to 0.919, respectively. Thirty-two loci were in Hardy–Weinberg equilibrium. The microsatellite loci described in this study will facilitate investigation of conservation genetics of this species.