Triploid Oysters Research Articles

FINE STRUCTURE OF THE SHELL OF DIPLOID AND TRIPLOID OYSTERS, <i>CRASSOSTREA GIGAS</i> (THUNBERG 1793) (BIVALVIA, OSTREIDAE) REARED IN THE BLACK SEA

The fine structure and chemical composition of the shell growth margin were compared in diploid and triploid oysters, Crassostrea gigas (Thunberg 1793), reared to commercial size in a Crimean marine farm. The diploid oysters were deposited from plankton, whereas the triploid ones were obtained from an Atlantic coast nursery. An electron scanning microscope SEM Hitachi U 3500 with built-in software Oxford Ultin Max 65 for microanalysis was employed in the study. The shell growth margin is shown to consist of two layers: periostracum and prismatic. The periostracum in diploid oysters is smooth and porous, whereas the periostracum of triploid oysters is volumetric and shows longitudinal folds. The prismatic layer of both right and left shell valves consists of prisms surrounded by organic membranes. In contrast to diploid oysters, triploid ones have longer prism facets, their calcite filling is significantly lower than normal, their interprismatic organic membranes are discontinuous and contain calcium carbonate. The proportion of organic matter in diploid oyster shells is significantly higher than that in triploid ones. The factors affecting the fine structure of oyster shells differing in ploidy are discussed.

Effect of ploidy on salinity and temperature tolerance in early life stages of the eastern oyster (Crassostrea virginica)

The U.S. Gulf of Mexico contains the largest remaining wild oyster fishery in the world, but populations have declined in recent decades. A growing interest in off-bottom aquaculture that relies on triploid eastern oysters (Crassostrea virginica) has emerged in the Gulf region, yet these faster growing oysters suffer high mortality as adults during low salinity (<5) events in warmer summer months. The combined effects of low salinity and high temperature stress on early life stages of triploid oysters are unknown. Early life stages are particularly crucial to understand because triploid oysters do not occur naturally and must be reared in hatchery settings, requiring appropriate water conditions to yield the greatest survival and growth. Thus, we tested the effects of different temperatures (28 ºC and 32 ºC) and salinities (5, 10, and 15) on diploid and triploid oysters at three critical production stages: veliger, pediveliger, and spat. Veliger survival was significantly lower for triploids relative to diploid oysters at all experimental conditions, but triploid veligers had faster growth than diploids at 32 ºC and salinity of 15. Pediveliger settlement was not affected by ploidy type and was reduced only at high temperature (32 ºC) and the lowest salinity (5). Diploid spat showed highest survival at 28 ºC and 15 salinity, while triploids survived best at 32 ºC and 15 salinity. Triploid spat attained greater shell height compared to diploids in our 6-day exposures, but growth decreased for both ploidies at lower salinities. At the salinity and temperature levels examined, diploid early life stages performed best at 28 ºC and 15 salinity, whereas triploids were more successful at 32 ºC and 15 salinity. A broader understanding of the combined effects of environmental stressors will improve the success of hatchery production yields and the resulting economic and environmental benefits of the oyster industry.

Transcriptome profiling explores the immune defence mechanism of triploid Pacific oyster (Crassostrea gigas) blood against Vibrio alginolyticus based on protein interaction networks.

Triploid oysters have provided the oyster industry with many benefits, such as fast growth rates, meat quality improvement, and increased oyster production and economic benefits, since the first report on triploid oysters was published. The development of polyploid technology has remarkably increased the output of triploid oysters to meet the increasing demand of consumers for Crassostrea gigas in the past decades. At present, research on triploid oyster has mainly focused on breeding and growth, but studies on the immunity of triploid oysters are limited. According to recent reports, Vibrio alginolyticus is a highly virulent strain that can cause disease and death in shellfish, shrimp, as well as serious economic losses. V. alginolyticus may be a reason why oysters die during summer. Therefore, using V. alginolyticus to explore the resistance and immune defense mechanisms of triploid oysters against pathogens presents practical significance. Transcriptome analysis of gene expression was performed in triploid C. gigas at 12 and 48h after infection with V. alginolyticus, and the respective 2257 and 191 differentially expressed genes (DEGs) were identified. The results of GO and KEGG enrichment analyses showed that multiple significantly enriched GO terms and KEGG signaling pathways are associated with immunity. A protein-protein interaction network was constructed to investigate the interaction relationship of immune-related genes. Finally, we verified the expression situation of 16 key genes using quantitative RT-PCR. This study is the first to use the PPI network in exploring the immune defense mechanism of triploid C. gigas blood to fill the gap in the immune mechanism of triploid oysters and other mollusks, and provide valuable reference for future triploid farming and pathogen prevention and control.

Shells, Gills, and Gonads

The Atlantic oyster of Chesapeake Bay has been the focus of intense economic and ecological pressures. In the 1880s it was the main source for America’s favorite food, and the oyster was overharvested to the point of scarcity. Three scientific discoveries concerning the oyster’s material properties have been paving its way back from the brink of extinction. First, William Keith Brooks studied the embryology of this oyster and showed that its shell served as a necessary part of its life cycle. Second, Roger Newell demonstrated the prodigious water filtration properties of this oyster and linked these properties to its ability to clean the estuary. The discovery of the filtration properties of the oyster was an affordance that enabled the oyster to “partner” with governmental agencies and NGOs who were attempting to restore the bay’s clean water, fish, and birdlife. Third, Standish K. Allen, Ximing Guo, and their colleagues formulated a procedure that enabled the manipulation of oyster development to yield tasty, fast-growing, and disease-resistant triploid oysters. The disease-resistant oysters together with knowledge of the oyster’s life cycle enabled the proliferation of the oyster by conservation groups. The goal of Chesapeake Bay conservation changed from “Save the oyster” to “Plant more oysters; help save the bay.” This paper is part of a special issue entitled “Making Animal Materials in Time,” edited by Laurence Douny and Lisa Onaga.

Analysis of Volatile Compounds and Flavor Fingerprint Using Gas Chromatography-Ion Mobility Spectrometry (GC-IMS) on Crassostrea gigas with Different Ploidy and Gender.

In this study, GC-IMS was used to analyze the volatile component and flavor profiles of Crassostrea gigas individuals of different ploidy and gender. Principal component analysis was used to explore overall differences in flavor profiles, and a total of 54 volatile compounds were identified. The total volatile flavor contents in the edible parts of tetraploid oysters were significantly higher than in diploid and triploid oysters. The concentrations of ethyl (E)-2-butenoate and 1-penten-3-ol were significantly higher in triploid oysters than in diploid and tetraploid oysters. In addition, the volatile compounds propanoic acid, ethyl propanoate, 1-butanol, butanal, and 2-ethyl furan were significantly higher in females than in males. The volatile compounds p-methyl anisole, 3-octanone, 3-octanone, and (E)-2-heptenal were present in higher levels in male than in female oysters. Overall, different ploidy and gender of oysters are connected with different sensory characteristics, providing new insights for understanding the flavor characteristics of oysters.

Identification and Characterization of Infectious Pathogens Associated with Mass Mortalities of Pacific Oyster (Crassostrea gigas) Cultured in Northern China.

The Pacific oyster (Crassostrea gigas) aquaculture industry increased rapidly in China with the introduction and promotion of triploid oysters in recent years. Mass mortalities affecting different life stages of Pacific oysters emerged periodically in several important production areas of Northern China. During 2020 and 2021, we conducted a passive two-year investigation of infectious pathogens linked to mass mortality. Ostreid herpesvirus-1 (OsHV-1) was detected to be associated with mass mortalities of hatchery larvae, but not juveniles and adults in the open sea. Protozoan parasites, such as Marteilia spp., Perkinsus spp. and Bonamia spp. were not detected. Bacterial isolation and identification revealed that Vibrio natriegens and Vibrio alginolyticus were the most frequently (9 out of 13) identified two dominant bacteria associated with mass mortalities. Pseudoalteromonas spp. was identified as the dominant bacteria in three mortality events that occurred during the cold season. Further bacteriological analysis was conducted on two representative isolates of V. natriegens and V. alginolyticus, designated as CgA1-1 and CgA1-2. Multisequence analysis (MLSA) showed that CgA1-1 and CgA1-2 were closely related to each other and nested within the Harveyi clade. Bacteriological investigation revealed faster growth, and more remarkable haemolytic activity and siderophore production capacity at 25 °C than at 15 °C for both CgA1-1 and CgA1-2. The accumulative mortalities of experimental immersion infections were also higher at 25 °C (90% and 63.33%) than at 15 °C (43.33% and 33.33%) using both CgA1-1 and CgA1-2, respectively. Similar clinical and pathological features were identified in samples collected during both naturally and experimentally occurring mortalities, such as thin visceral mass, discolouration, and connective tissue and digestive tube lesions. The results presented here highlight the potential risk of OsHV-1 to hatchery production of larvae, and the pathogenic role of V. natriegens and V. alginolyticus during mass mortalities of all life stages of Pacific oysters in Northern China.

Energetic budget of diploid and triploid eastern oysters during a summer die-off.

Triploid oysters are widely used in off-bottom aquaculture of eastern oysters, Crassostrea virginica. However, farmers of the Gulf of Mexico (GoM) and Atlantic coast estuaries have observed unresolved, late-spring die-offs of triploid oysters, threatening the sustainability of triploid aquaculture. To investigate this, the physiological processes underlying oyster growth (e.g., feeding, respiration) and mortality of one-year-old diploid and triploid oysters were compared in early summer following an uptick in mortality. It was predicted that higher triploid mortality was the result of energetic imbalances (increased metabolic demands and decreased feeding behavior). Oyster clearance rates, percentage of time valves were open, absorption efficiency, oxygen consumption rates (basal and routine), ammonia excretion rate were measured in the laboratory and scope for growth was calculated. In addition, their condition index, gametogenic stage, Perkinsus marinus infection level, and mortality were measured. Mortality of triploids in the laboratory was greater than for diploids, mirroring mortality observed in a related field study. The physiological parameters measured, however, could not explain triploid mortality. Scope for growth, condition index, and clearance rates of triploids were greater than for diploids, suggesting sufficient energy reserves, while all other measurements where similar between the ploidies. It remains to be determined whether mortality could be caused from disruption of energy homeostasis at the cellular level.

Hypoxia and Anoxia Tolerance in Diploid and Triploid Eastern Oysters at High Temperature

Increasing reliance on the use of triploid oysters to support aquaculture production relies on their generally superior growth rate and meat quality over that of diploid oysters. Reports of elevated triploid mortality have generated questions about potential trade-offs between growth and tolerance to environmental stressors. These questions are particularly relevant as climate change, coastal activities, and river management impact water salinity, temperature, nutrients, pH, and oxygen levels within key estuarine oyster growing areas. In particular, the co-occurrence of warm water temperatures and low dissolved oxygen concentration (DO) events are increasingly reported in estuaries, with potentially lethal impacts on sessile, oyster resources. To investigate potential differences in DO tolerance, diploid and triploid market-sized or seed oysters were exposed to continuous normoxia (DO > 5.0 mg L–1), hypoxia (DO < 2.0 mg L–1), and anoxia (DO < 0.5 mg L–1) at 28°C and their mortalities were monitored. The hemolymph of the market-sized oysters was collected to measure cellular and biochemical changes in response to hypoxia and anoxia, whereas their valve movements were also measured. In general, about half of market-sized oysters died within about 1 wk under anoxia (LT50: 5.7–8.9 days) and within about 2 wk under hypoxia (LT50: 11.9–19.4 days) with diploid oysters tending to die faster than triploid oysters. Seed oysters took longer to die than market-sized oysters under both anoxia (LT50: 9.5–12.1 days) and hypoxia (LT50: 21.8–25.0 days) with diploid oysters (LT50: 9.5–11.8 days) dying slightly faster than triploid oysters (LT50: 11.8–12.1 days) under anoxia. Hemolymph pH decreased and plasma calcium and glutathione concentrations increased with decreasing DO, with values under anoxia being different than those under normoxia. Hemocyte density was also lower under anoxia than under either normoxia or hypoxia. Overall, few differences in physiological responses to hypoxia and anoxia were found between diploid and triploid oysters suggesting that ploidy (2N versus 3N) had limited effect on the tolerance and response of eastern oysters to low DO.

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

The quality of diploid oyster (crassostrea gigas) decreased significantly during the breeding period, which reflected the quality advantage of triploid oyster. However, there were few comparative studies on flavor quality characteristics between diploid and triploid oysters. This study compared the non-volatile compounds and volatile compounds between triploid and diploid oysters. These results showed that the percentage of eicosapentaenoic acid (EPA) in diploid oysters (18.68 ± 0.26%) was significantly higher than that in triploid oysters (16.16 ± 1.61%). The content of inosincacid (IMP) in diploid oysters (16.13 ± 0.03 mg/100 g) was significantly lower than that in triploid oysters (22.27 ± 0.41 mg/100 g). Adenosine monophosphate (AMP) content (364.08 ± 0.48 mg/100 g), succinic acid content (1244.51 ± 24.81 mg/100 g), and total free amino acid content (638.34 ± 17.32 mg/100 g) were significantly higher than those of triploid oysters (248.85 ± 2.32 mg/100 g, 420.99 ± 8.76 mg/100 g, 552.71 ± 17.32 mg/100 g, respectively). The main volatile compounds in diploid and triploid Pacific oysters were alcohols, aldehydes, ketones and esters. The volatile compounds content in triploid oysters were higher than those in diploid oysters. This study will provide basic data of Pacific oyster flavor and essential for the flavor pattern of seafood.

Relationship between Condition Index Values and Expression Levels of Gene and Protein in the Adductor Muscle of Diploid and Triploid Oysters Crassostrea gigas

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.

Antioxidant and protective effects of a peptide (VTAL) derived from simulated gastrointestinal digestion of protein hydrolysates of Magallana gigas against acetaminophen-induced HepG2 cells.

Oxidative stress is an automatic mechanism responsible for the commencement and continuance of liver injury. In this study, an antioxidative peptide Val-Thr-Ala-Leu (VTAL) was purified from simulated gastrointestinal digestion of protein hydrolysates of the triploid oyster Magallanagigas. Significant antioxidant activity was identified, as well as a protective effect against acetaminophen (APAP)-induced human liver cancer (HepG2) cells. The results suggested that the antioxidant activity improved in a dose-dependent manner. The highest cell viability (88.105 ± 3.62%) was observed in 15mM APAP-induced cells when treated with 25μg/mL M.gigas peptide [M.g (pep)]. The peptide sequences include hydrophobic amino acids, which could be responsible for its chemoprotective and antioxidant activities. Treatment with M.g (pep) significantly promoted the proliferation of HepG2 cells, thus protecting them against APAP and imbuing them with significant antioxidant capacity. M.g (pep) could be beneficial for treating drug-induced oxidative stress and liver damage. Additionally, M.g (pep) could serve as an alternative to synthetic antioxidant drugs.

Effect of ploidy level on accumulation and depuration of Vibrio parahaemolyticus in Pacific oyster Crassostrea gigas

Triploid cupped oysters represent an advantage over diploid oysters due to their faster growth and their whole year marketable span, especially during the spawning season occurring in the summer. Thus, their commercialization during this warm season could present a risk for human health due to the proliferation of human-pathogenic Vibrio spp. such as Vibrio parahaemolyticus (Vp). The purpose of this study was to evaluate the impact of Crassostrea gigas oyster ploidy on contamination with indigenous Vp in an oyster farm, and on the accumulation and depuration of Vp in laboratory experiments. The study was conducted from May through November 2021 using three batches of diploid oysters and three batches of triploid oysters. We observed that ploidy did not significantly influence contamination by indigenous Vp although the contamination with indigenous Vp trended to be lower in triploids (0.93 log MPN.g−1) in comparison with diploids (3.08 log MPN.g−1) in November. These results could suggest a safer consumption of triploid oysters over diploid oysters in autumn concerning Vp infection risk in the site of this study. Moreover, pathogenic profiles of indigenous Vp varied significantly between July (8% of tdh+ and 100 of trh2+) and November (98% of tdh+ and 0% of trh2+) with no significant difference between ploidies. Ploidy did not significantly influence experimental accumulation or depuration at any time. However, depuration was higher during summer (June to August) than during the other months. In conclusion, our study suggests that Vp levels in C. gigas are not influenced by oyster ploidy. In addition, seasonal variations of indigenous Vp pathogenic profiles and Vp experimental depuration were observed.

Host-microbiota interactions play a crucial role in oyster adaptation to rising seawater temperature in summer

Climate change, represented by rising and fluctuating temperature, induces systematic changes in marine organisms and in their bacterial symbionts. However, the role of host-microbiota interactions in the host's response to rising temperature and the underlying mechanisms are incompletely understood in marine organisms. Here, the symbiotic intestinal microbiota and transcriptional responses between diploid and triploid oysters that displayed susceptible and resistant performance under the stress of rising temperature during a summer mortality event were compared to investigate the host-microbiota interactions. The rising and fluctuating temperatures triggered an earlier onset and higher mortality in susceptible oysters (46.7%) than in resistant oysters (17.3%). Correlation analysis between microbial properties and environmental factors showed temperature was strongly correlated with indices of α-diversity and the abundance of top 10 phyla, indicating that temperature significantly shaped the intestinal microbiota of oysters. The microbiota structure of resistant oysters exhibited more rapid changes in composition and diversity compared to susceptible oysters before peak mortality, indicating that resistant oysters possessed a stronger ability to regulate their symbiotic microbiota. Meanwhile, linear discriminant analysis effect size (LefSe) analysis found that the probiotics Verrucomicrobiales and Clostridiales were highly enriched in resistant oysters, and that potential pathogens Betaproteobacteriales and Acidobacteriales were enriched in susceptible oysters. These results implied that the symbiotic microbiota played a significant role in the oysters' adaptation to rising temperature. Accompanying the decrease in unfavorable bacteria before peak mortality, genes related to phagocytosis and lysozymes were upregulated and the xenobiotics elimination pathway was exclusively expressed in resistant oysters, demonstrating the validity of these immunological functions in controlling proliferation of pathogens driven by rising temperature. Compromised immunological functions might lead to proliferation of pathogens in susceptible oysters. This study might uncover a conserved mechanism of adaptation to rising temperature in marine invertebrates from the perspective of interactions between host and symbiotic microbiota.

Genetic improvement of aquaculture performance for tetraploid Pacific oysters, Crassostrea gigas: A case study of four consecutive generations of selective breeding

Tetraploid oysters play an important role in the global oyster industry, especially as the core germplasm for producing triploid oysters. About 30 years ago, the tetraploid Pacific oyster was successfully produced, but their genetic improvement is poorly understood. To further understand the biology of tetraploid Pacific oyster, the viability, growth, ploidy and fertility of these progenies were evaluated through four consecutive generations of mass selection. The viability of progeny has not been well improved, which is mainly affected by the quality of gametes. The growth traits of these progenies were significantly improved through mass selection over generations, similar to those of diploid oysters previous reported. The ploidy-level of tetraploid progeny differentiated and transformed into triploids, diploids, aneuploids, or mosaics in the early stage of gametogensis. With the increase of generations, the proportion of tetraploid progeny increased significantly. After three generations of oyster height selection, the proportion of tetraploid reached more than 90%. Like diploid oysters, tetraploid progeny can undergo gonadal development to produce gametes and perform normal reproductive functions. Since the fourth generation progenies have medium-high heritability, further genetic improvement is feasible. This study provides theoretical basis and practical experience for the improvement of tetraploid oyster lines.

Physiological and gene expression responses of diploid and triploid Pacific oyster ( Crassostrea gigas ) to heat acclimation

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.

Investigation of the role of endogenous miRNAs in determining sterility in triploid Pacific oysters (Crassostrea gigas)

Triploid oysters generate fewer gametes than diploid oysters and exhibit varying degrees of gonad development. The expression of related genes, such as those involved in gametogenesis and energy metabolism, may influence reproductive potential. In addition, microRNAs (miRNAs) are important regulators of gene expression. However, the contribution of miRNAs has not yet been assessed in sterile triploid Pacific oysters (Crassostrea gigas). We therefore obtained extensive miRNA and gene expression data to identify potentially critical miRNA-mRNA interactions that are linked to sterility in triploid C. gigas. Here, we found miR-263-x to be significantly downregulated in the gonads of sterile females. The upregulated target genes were associated with lipid droplet formation, which revealed the likely involvement of nutrient accumulation during gonadal development as regulated by miR-263-x in female sterile triploids. In males, the upregulated miRNAs (miR-1992-y and miR-2001-x) were shown to regulate spermatogenesis-related gene expression, including flagellar formation, and cell proliferation and migration. Our results suggest that energy redistribution regulated by miRNAs may be critical for gonad development in females, indicating that sterile triploids undergo a significant reduction in gamete number and show an increased accumulation of nutrients in the gonads. Our findings further indicate that decreased expression of genes related to spermatogenesis regulated by miRNAs is important in gonadal dysgenesis in sterile triploids. These results shed light on the epigenetic regulatory mechanisms governing sterility in triploid bivalves.

Male triploid oysters of Crassostrea gigas exhibit defects in mitosis and meiosis during early spermatogenesis.

The Pacific oyster, Crassostrea gigas, is a successive irregular hermaphrodite mollusk which has an annual breeding cycle. Oysters are naturally diploid organisms, but triploid oysters have been developed for use in shellfish aquaculture, with the aim of obtaining sterile animals with commercial value. However, studies have shown that some triploid oysters are partially able to undergo gametogenesis, with numerous proliferating cells closed to diploids (3n alpha) or a partial one with an accumulation of locked germ cells (3n beta). The aim of our study therefore was to understand the regulation of spermatogenesis in both groups of triploid oysters (alpha and beta) from the beginning of spermatogenesis, during mitosis and meiosis events. Our results demonstrate that the reduced spermatogenesis in triploids results from a deregulation of the development of the germinal lineage and the establishment of the gonadal tract led by a lower number of tubules. Morphological cellular investigation also revealed an abnormal condensation of germ cell nuclei and the presence of clear patches in the nucleoplasm of triploid cells, which were more pronounced in beta oysters. Furthermore, studies of molecular and cellular regulation showed a downregulation of mitotic spindle checkpoint in beta oysters, resulting in disturbance of chromosomal segregation, notably on spindle assembly checkpoint involved in the binding of microtubules to chromosomes. Taken together, our results suggest that the lower reproductive ability of triploid oysters may be due to cellular and molecular events such as impairment of spermatogenesis and disruptions of mitosis and meiosis, occurring early and at various stages of the gametogenetic cycle.

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

Triploid oysters with their large size and consistent meat quality have become a major component of the oyster aquaculture industry worldwide. It has been observed that triploid Pacific oysters (Crassostrea gigas) showed high levels of variation and sex-specific differences in sterility. Here, we analyzed mitosis and meiosis during gametogenesis in triploid Pacific oysters and the expression of key cell cycle regulatory proteins, namely members of the cyclin family (A, B, D, E). We found that in female C. gigas, expression of cyclin D and E in gonad of triploids were higher than those in diploids, while expression of cyclin A and B were significantly suppressed in the gonads of triploids. To test whether mitosis of gonia might be arrested at G1/S or S/G2 phase in triploids, we compared EdU labeling in gonadal cells of triploid and diploid oysters. The results showed a similar number of EdU-labelled cells in gonads of triploid and diploid females, indicating that gonia in triploids successfully entered the S-phase as diploids. In male triploids, gonadal expression of the four cyclin genes at the mature stage were similar to that in male diploids at the growth stage with numerous spermatogenic cells. The number of EdU-labelled cells in triploid male gonads at the mature stage was significantly more than that in diploids, suggesting more proliferating cells in triploid gonads in males. Collectively, these data inidate that the sterility of triploid females is likely caused by the failure of germ cells to enter G2-phase during mitosis. In addition, we speculate that gonadal development of triploid males was arrested at the growth stage and defective spermatogenesis might be associated with disruption of meiosis.

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

Triploid oysters often have higher growth rates and survival rates than diploid oysters. However, some studies have observed the opposite patterns. Triploid oysters have also been observed to show reduced resistance to stress and disease compared with diploid oysters. We compared the transcriptional patterns of diploid and triploid Pacific Oysters (Crassostrea gigas) in response to acute and prolonged thermal stress. The survival rate of triploid C. gigas was lower than that of diploid oysters after prolonged heat stress at 28 °C. A total of 779 and 396 differentially expressed genes (DEGs) were detected in diploid and triploid oysters exposed to acute heat stress, respectively, and the number of DEGs was 418 in diploids and 483 in triploids after prolonged heat stress. Functional enrichment analysis revealed that the DEGs were significantly enriched in protein processing in the endoplasmic reticulum pathway, NF-kappa B signaling pathway, NOD-like receptor signaling pathway, and TNF signaling pathway in both diploid and triploid C. gigas under heat stress. Genes encoding translation initiation factors, including MKNK1, eIF4G3, eIF2a kinase 1, and eIF2a kinase 3, were differentially expressed under acute heat stress in diploid oysters. The number of differentially expressed HSP and IAP genes was greater in diploids than in triploids under acute heat stress, and the expression levels of most of these HSP and IAP genes were higher in diploid oysters than in triploid oysters. Overall, our findings revealed differences in the response of diploid and triploid C. gigas to thermal stress. Triploid C. gigas might be less capable of rapidly modifying the inflammatory response and apoptosis in response to heat stress compared with diploid C. gigas. Different genetic lines of diploid and tetraploid oysters should be used to produce triploid oysters with increased stress resistance.

In situ dynamics of Vibrio parahaemolyticus and Vibrio vulnificus in water, sediment and triploid Crassostrea virginica oysters cultivated in floating gear.

To understand spatial-temporal distribution of Vibrio parahaemolyticus and Vibrio vulnificus in triploid Crassostrea virginica in off-bottom aquaculture. Oysters, sediments and water were seasonally collected in Georgia, USA. V. parahaemolyticus and V. vulnificus were quantified with tlh/tdh/trh and vvhA genes, respectively. No tdh/trh genes were detected. Highest concentrations of tlh gene were observed in summer sediments>oysters>water (105 /g, 104 /g, 103 /ml). VvhA concentrations were similar in sediments and oysters but never exceeded ≥3×101 /ml in water. Concentrations of tlh and vvhA genes correlated to temperature and turbidity, respectively, which along with their different spatial distribution indicated different environmental drivers. In oysters, ratios of the tlh and vvhA to 16S rRNA gene have increased from 0 to 10-1 and 10-2 in summer, while these ratios in water and sediments were lower by 2-3 orders of magnitude. Dynamics of tlh and vvhA concentrations and abundances suggested enrichment of V. parahaemolyticus and V. vulnificus by off-bottom triploid oysters in summer resulting in their abundance by far exceeding that in water. This first report on enrichment of Vibrio pathogens in triploid oysters with no direct contact to sediments reveals a threat to human health suggesting their monitoring in triploid off-bottom C. virginica aquaculture.