Comparative transcriptomic analyses reveal differences in the responses of diploid and triploid Pacific oysters (Crassostrea gigas) to thermal stress

High temperature is considered to be one of the important causes of mass mortality of diploid and triploid oysters in summer. In order to compare the tolerance adaptability of diploid and triploid oysters to heat stress, the activities of superoxide dismutase activity (SOD), catalase activity (CAT) and contents of malondialdehyde (MDA) and the expression of Hsp70 and metallothionein (MT) genes in diploid and triploid oysters under acute and chronic heat stress were studied. The results showed that the survival rate of diploid oysters was significantly higher than that of triploid oysters under acute and chronic heat stress (p < 0.05). Under acute heat stress, the SOD levels in gills of both diploid and triploid oysters increased significantly from hour 3 (p < 0.05), but there was no significant difference in SOD levels between diploid and triploid oysters at all time points. The SOD level in hepatopancreas of triploid oysters was significantly higher than that of diploid oysters at all time points except 48 h (p < 0.05). The CAT level of diploid and triploid oysters decreased sharply at 48 h and that of diploid oysters was significantly higher than that of triploid oysters (p < 0.05). Under acute heat stress, the increase in MDA content in triploid oysters was significantly higher than that in diploid oysters (p < 0.05). Under acute heat stress, the expression levels of Hsp70 in the gills and hepatopancreas were significantly increased in diploid and triploid oysters (p < 0.05), but increased varies between gills and hepatopancreas. The expression levels of MT in the gills and hepatopancreas were significantly decreased in diploid and triploid oysters (p < 0.05). The results obtained in this study provide physiological and immunological evidence to explain differences in high-temperature tolerance between diploid and triploid oysters and help us better understand the mass mortality of oysters that occurs during high temperatures in summer.

The aim of this study was to evaluate the chemical and nutritional characteristics and quality decay during storage of diploid and triploid Pacific oysters (Crassostrea gigas) reared in Orbetello Lagoon (Grosseto, Italy), a site subject to high variations in environmental parameters. Specimens of diploid (D) and triploid (T) oysters of commercial size and similar age were sampled from an oyster farm (Soc. Coop. GIGAS) in autumn 2005 and in winter, spring and summer 2006. At each sampling date, D (n=60) and T (n=60) oysters were individually weighed, divided into 4 lots, stored under refrigerated conditions (+4°C), and analysed at different times after harvest (1, 3, 7 and 10 days). Oysters were individually weighed again at the different times, measured for maximum length, width and thickness, and opened. The weight of the soft part and shell and the volume of intervalvar liquor were recorded. pH of soft part, gill and mantle colour (CIE L*, a* and b*, Minolta Chromameter) and chemical characteristics (proximate and quantitative fatty acid composition in pooled samples) of soft parts were also analysed. Turbidity of intervalvar liquor was evaluated (by spectrophotometer). A total of 480 specimens (D+T) was studied. For morphological data, the model utilised for the statistical analysis (PROC GLM of SAS Software) included ploidy, season and their interaction as fixed effects; for parameters analysed during storage, the model included ploidy, storage time, season and the interaction between ploidy and storage time. Triploid oysters had greater average (p<0.001) length (102.1 vs 95.7mm), thickness (34.3 vs 28.4mm) and weight (99.3 vs 77.2g) than those diploid. The latter oysters had an inferior market value due to lower economic condition index [thickness/(0.5*(length+width)] (0.38 vs 0.45; p<0.001) and soft part percentage (8.20 vs 12.04%; p<0.001). Differences were also found in chemical characteristics: T showed greater average (p<0.001) dry matter (19.07 vs 14.90%), crude protein (9.47 vs 7.90% w.w.) and total lipid (1.71 vs 0.91%) contents and higher (p<0.01) percentages of C20:5n3 (10.36 vs 8.18%) and PUFAn3 (35.57 vs 28.15%), and lower MUFA (17.72 vs 19.73%; p<0.01) percentage. During storage, T had a higher average weight to length ratio (0.94 vs 0.74; p<0.001), lower weight loss percentage (8.84 vs 10.10%; p<0.05) and higher turbidity of intervalvar liquor (0.754 vs 0.394 absorbance units; p<0.001). T and D differed in colour, T having higher whiteness (L*) and yellowness (b*) of gills and mantle and lower redness (a*) of mantle. Certain parameters reliably reflected changes in oyster quality during storage: percentage weight loss increased significantly from 0% to 12.42% on days 1 and 10, respectively, like pH (days 1-3: 6.19-6.21; day 7: 6.34; day 10: 6.57; p<0.001) while weight/length ratio decreased (from 0.88 to 0.77 on days 1 and 10, respectively; p<0.05). Colour parameters did not change. During storage, trends of parameters were similar in D and T, the interaction between ploidy and storage time never reaching significance. In conclusion, the results confirmed that like D and T oysters from other areas, those from Orbetello Lagoon differ in market and chemical characteristics. The parameters analysed during storage demonstrated the poor average quality of diploid oysters. Further investigations are needed to elucidate the influence of season, which significantly affected almost all the quality parameters analysed, and to highlight the relation between season and quality decay during storage.

Partial or complete sterility is an obvious feature in triploid Pacific oyster (Crassostrea gigas) which contributes to improving rearing performances. Despite the significance of sterility, the molecular mechanism behind it remains elusive and related research was limited. This study focused on six reproduction-related genes and compared their different behavior in gene expression and DNA methylation pattern between triploid and diploid oysters in order to provide more molecular information. The gonadal development of triploid oyster was examined by histology before molecular analysis. Gametogenesis disturbance was observed in triploid oysters at different development stages (stage II and III) with more serious impairment in females. QPCR showed significant gene expression difference between diploid and triploid in two genes: putative Vg and cgER. Gene expression of putative Vg was delayed in triploids while for cgER triploid oyster showed higher expression and the difference was significant at stage III. DNA methylation pattern of these two genes were further investigated by bisulfite sequencing. Between diploid and triploid oysters, no difference was observed in total methylation level but some individual loci showed different patterns: significantly high methylation rate of loci 2284 in cgER was observed in triploid oyster which has a higher expression of this gene. This study indicated that putative Vg and cgER might play a role in partial sterile in triploid C. gigas. Gene expression could be regulated by the methylation pattern at specific individual locus, which deserves equivalent attention as well as total DNA methylation level.

Comparative study between triploid and diploid oysters (Crassostrea gigas) on non-volatile and volatile compounds

Effects of elevated temperature on growth, gametogenesis, physiology, and biochemical composition in diploid and triploid Pacific oysters, Crassostrea gigas Thunberg

Disruption of cell division prevents gametogenesis in triploid Pacific oysters (Crassostrea gigas)

Variance in expression and localization of sex-related genes CgDsx, CgBHMG1 and CgFoxl2 during diploid and triploid Pacific oyster Crassostrea gigas gonad differentiation.

Triploid organisms, in particular oysters, are widely used in marine aquaculture. However, the advantage of triploid oysters compared to diploids has not been proven in all cases. Comparative morphological analysis of Pacific oysters Crassostrea gigas of different ploidy levels allows evaluating the benefits of raising triploids in the Black Sea. First, the morphological characteristics of diploid and triploid Pacific oysters C. gigas farmed in the Black Sea were studied. Allometric ratios of weight (W, g) and shell height (H, mm) of oysters were obtained, which are described by the equations W$_{2n}$ = 4 × 10$^{-4}$ × H$_{2n}$ $^{2.56}$, R2 = 0.86 and W$_{3n}$ = 9 × 10$^{-5}$ × H$_{3n}$ $^{2.90}$, R$^{2}$ = 0.91. Triploids showed positive allometry and had more biomass at smaller shell height than diploid oysters. A clear linear relationship of the height and length of oyster shells was established. Significant differences in linear parameters between 2n and 3n C. gigas farmed in Donuzlav Liman are not seen. Based on these results, it is assumed that the advantage of the growth of 3n mollusks is less evident in farms located at high latitudes. The environmental factors and farm technology have probably affected the growth of oysters to a greater degree than their ploidy.

Studies on triploid oysters in Australia. 2. Growth, condition index gonad area, and glycogen content of triploid and diploid Pacific oysters, Crassostrea gigas, from oyster leases in Tasmania, Australia

Quantification of reproductive effort of the triploid Pacific oyster, Crassostrea gigas raised in intertidal rack and bag oyster culture system off the west coast of Korea during spawning season

Transcriptome analysis reveals polyploidy-related differential gene expression among diploid, triploid, and tetraploid Pacific oysters (Crassostrea gigas) based on growth-related phenotypes

Responses of diploid and triploid Pacific oysters Crassostrea gigas to Vibrio infection in relation to their reproductive status

Three proteins [myosin heavy chain (MHC), filamin-C fragment (FIL-C), and actin 2 (ACT2)] were identified in adductor muscle from diploid and triploid Pacific oysters (Crassostrea gigas) and the relationship between the condition index (CI) and mRNA expression of these genes was investigated, together with the mRNA expression of molluscan insulin-related peptide (MIP), C. gigas insulin receptor-related receptor (CIR), and insulin-like growth factor binding protein complex acid labile subunit (IGFBP-ALS). Monthly changes in the CI were similar to the changes in the tissue weight rate in both groups. ACT2 and MHC mRNA expression was statistically higher in the triploid than the diploid, while FIL-C mRNA expression was significantly higher in the diploid (p<0.05). The MIP, CIR, and IGFBP-ALS mRNA expression of the diploid oysters were all significantly higher in July than in other months (p<0.05). The MIP, CIR, and IGFBP-ALS mRNA expression in the triploid oysters was high in July, but there were no significant differences (p>0.05). Changes in the expression levels of the genes investigated in this study could be used as intrinsic indicators of the annual growth, maturity, and spawning period of cultured diploid and triploid C. gigas in Tongyeong, Korea.

The glycogen content and the activities of two key enzymes in glycogen metabolism, glycogen phosphorylase and glycogen synthetase, in the gonad of diploid and triploid Pacific oysters (Crassostrea gigas) were compared during maturation. The glycogen content in the gonad of diploids decreased with gametogenesis (by 85.7%), but the glycogen content in the gonad of triploids did not vary significantly. Activity of glycogen phosphorylase (GP) in the gonad of diploids decreased with gametogenesis (by 55.5%), while GP activity of triploids did not vary significantly during maturation. Activity of glycogen synthetase (GS) in the gonad of diploids increased slightly with gametogenesis, reaching a peak in June. Activity of GS declined sharply from June to July, which might be due to gonad spawning. GS activity of triploid oysters in spawning time (July and August) was significantly higher than that in other months, which might be explained with a ‘compensating’ mechanism for the higher glycogen content in triploids.

The triploid Pacific oyster, which is produced by mating tetraploid and diploid oysters, is favored by the aquaculture industry because of its better flavor and firmer texture, particularly during the summer. However, tetraploid oyster production is not feasible in all oysters; the development of tetraploid oysters is ongoing in some oyster species. Thus, a method for ploidy verification is necessary for this endeavor, in addition to ploidy verification in aquaculture farms and in the natural environment. In this study, a method for ploidy verification of triploid and diploid oysters was developed using multiplex polymerase chain reaction (PCR) panels containing primers for molecular microsatellite markers. Two microsatellite multiplex PCR panels consisting of three markers each were developed using previously developed microsatellite markers that were optimized for performance. Both panels were able to verify the ploidy levels of 30 triploid oysters with 100% accuracy, illustrating the utility of microsatellite markers as a tool for verifying the ploidy of individual oysters.