Exploring Release, Isomerization, and Absorption of Cypermethrin in Pacific Oysters (Crassostrea gigas) with Different Processing Methods during In Vivo Digestion: Insights from a Gastrointestinal Tract Quantitative Tracing Method.

Cypermethrin (CP) is a neurotoxic insecticide found accumulated in oysters, one of the most commonly consumed seafoods, posing potential health risks to the human body. We designed a gastrointestinal tracing method allowing for accurate quantification of the propulsion of chyme and further established the mouse in vivo digestion model to explore the behavior of CP in the digestion of raw, steamed, and roasted oysters. The results showed that bioaccumulation of CP in oysters may be accompanied by the biotransformation of CP. Thermal processing decreased both the CP content in oysters and its bioaccessibility. The small intestine is the main site for CP digestion and absorption. The cis-isomers of CP might finally accumulate in the body at a higher ratio and further become the predominant configuration for toxic effects. Taken together, the study contributes to the risk assessment of the dietary exposure of CP from aquatic products.

Vertical transfer of microplastics in nearshore water by cultured filter-feeding oysters.

Microplastics (MPs) are widely distributed in the sea, but the vertical transfer of MPs by marine organisms in coastal area is still poorly understood. In this study, we used laser direct infrared (LDIR) spectroscopy to determine the number and characteristics of MPs deposited by cultured oyster Crassostrea gigas and further compared the differences between MPs of natural deposit and biodeposit in field environments. The amounts of MPs found in the biodeposit of cultured oysters were 3.54 times greater than that in the natural deposition. The polymer types of biodeposit MPs also differed from those of natural deposition. It was estimated that a single oyster can deposit 15.88 MPs per day, which is a figure much higher than the initial results, and hotspots of MPs deposition may be formed within the oyster aquaculture area. We used generalized linear mixed model (GLMM) to further infer the sources of MPs in sediments and found that distance to shore, cultured zone and urban center were important predictors of MPs abundance in sediments of aquaculture area. The above results suggest that cultured bivalves have an important capacity for MPs biodeposition and will further change the vertical distribution pattern of MPs in coastal environments.

Molecular structural modification of myofibrillar protein from oyster (Crassostrea gigas) with oligosaccharides for improving its gel properties.

Glycation is a promising approach to enhance protein gel characteristics in the food industry. The impact of oyster myofibrillar protein (MP) being glycosylated with six oligosaccharides (dextran [Dex]-1 kDa, 5 kDa, 6 kDa, and 10 kDa, xylan [Xyla], and xyloglucan [Xyg]) on structural properties, aggregation behavior and gel properties was investigated in this study. The findings demonstrated that oligosaccharides significantly increased the glycation degree of MP by forming a stable tertiary conformation, increasing the contents of the disulfide bond and hydrogen bonds. Additionally, particle sizes decreased and solubility increased after glycation, improving the gel's strength, water-holding capacity, thermal stability, elastic modulus, and ordered network layout. It was determined that MP-Dex 5 had the best gel properties. The gel strength and water holding capacity of MP-Dex 5 increased by 70.59% and 32.27%, respectively. Molecular dynamics simulations results showed van der Waals energy and electrostatic interactions favor myosin binding to Dex or Xyla units. This study will provide insights into the relationship between molecular structure, aggregation behavior and gel property of oyster MP-oligosaccharide couples, and expand the application of oyster MP in food gels.

Abundance of microplastics at and near a shellfish aquaculture farm: An eastern oyster (Crassostrea virginica) transplant study.

Microplastics (MP) have repeatedly been found in commercially cultured species of bivalves. There are concerns regarding the amount of MP released into the environment by aquaculture activities, and questions regarding possible higher MP loads in farm-grown shellfish compared to levels in shellfish collected from recreational beds. To explore this concept, seawater, aquaculture gear, and eastern oysters (Crassostrea virginica) were sampled from an aquaculture site in Niantic Bay, CT, USA, and a 2-week transplantation experiment was performed in which oysters were transplanted between the aquaculture site and a plastic-free cage off the dock at the University of Connecticut-Avery Point campus. The digestive gland-stomach complex (gut) was dissected from the oysters and MP were extracted from the adjacent seawater and oyster gut samples using previously validated extraction methods. Extensive quality assurance and control measures were taken to reduce MP contamination. Particles in all samples were isolated, imaged under a stereomicroscope, and characterized (size, shape, polymer) using ImageJ software and micro-Fourier transform infrared spectroscopy. Water samples contained 0-0.3 MP/L and oyster gut samples contained 0-1.3 MP/g wet weight indicating very low concentrations of MP at the farm (0-2 MP/individual) or away from the farm (0-3 MP/individual). Aquaculture gear in this area is not contributing to MP ingestion in farmed oysters or elevated MP levels in the surrounding water.

Comparative Analysis of the Biochemical Composition, Amino Acid, and Fatty Acid Contents of Diploid, Triploid, and Tetraploid Crassostrea gigas.

Tetraploid oysters are artificially produced oysters that do not exist in nature. The successful breeding of 100% triploid oysters resolved the difficulties of traditional drug-induced triploids, such as the presence of drug residues and a low triploid induction rate. However, little is known concerning the biochemical composition and nutrient contents of such tetraploids. Therefore, we investigated compositional differences among diploid, triploid, and tetraploid Crassostrea gigas as well as between males and females of diploids and tetraploids. The findings indicated that glycogen, EPA, ∑PUFA, and omega-3 contents were significantly higher in triploid oysters than in diploids or tetraploids; tetraploid oysters had a significantly higher protein content, C14:0, essential amino acid, and flavor-presenting amino acid contents than diploids or triploids. For both diploid and tetraploids, females had significantly higher levels of glutamate, methionine, and phenylalanine than males but lower levels of glycine and alanine. In addition, female oysters had significantly more EPA, DHA, omega-3, and total fatty acids, a result that may be due to the fact that gonadal development in male oysters requires more energy to sustain growth, consumes greater amounts of nutrients, and accumulates more proteins. With these results, important information is provided on the production of C. gigas, as well as on the basis and backing for the genetic breeding of oysters.

Comparative transcriptome analysis reveals molecular damage associated with cryopreservation in Crassostrea angulata D-larvae rather than to cryoprotectant exposure.

BACKGROUND: The Portuguese oyster Crassostrea angulata, a bivalve of significant economic and ecological importance, has faced a decline in both production and natural populations due to pathologies, climate change, and anthropogenic factors. To safeguard its genetic diversity and improve reproductive management, cryopreservation emerges as a valuable strategy. However, the cryopreservation methodologies lead to some damage in structures and functions of the cells and tissues that can affect post-thaw quality. Transcriptomics may help to understand the molecular consequences related to cryopreservation steps and therefore to identify different freezability biomarkers. This study investigates the molecular damage induced by cryopreservation in C. angulata D-larvae, focusing on two critical steps: exposure to cryoprotectant solution and the freezing/thawing process. RESULTS: Expression analysis revealed 3 differentially expressed genes between larvae exposed to cryoprotectant solution and fresh larvae and 611 differentially expressed genes in cryopreserved larvae against fresh larvae. The most significantly enriched gene ontology terms were "carbohydrate metabolic process", "integral component of membrane" and "chitin binding" for biological processes, cellular components and molecular functions, respectively. Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified the "neuroactive ligand receptor interaction", "endocytosis" and "spliceosome" as the most enriched pathways. RNA sequencing results were validate by quantitative RT-PCR, once both techniques presented the same gene expression tendency and a group of 11 genes were considered important molecular biomarkers to be used in further studies for the evaluation of cryodamage. CONCLUSIONS: The current work provided valuable insights into the molecular repercussions of cryopreservation on D-larvae of Crassostrea angulata, revealing that the freezing process had a more pronounced impact on larval quality compared to any potential cryoprotectant-induced toxicity. Additionally, was identify 11 genes serving as biomarkers of freezability for D-larvae quality assessment. This research contributes to the development of more effective cryopreservation protocols and detection methods for cryodamage in this species.

CgSHIP2 negatively regulates the mRNA expressions of CgIL-17s in response to Vibrio splendidus stimulation in Crassostrea gigas.

SH2 domain containing inositol polyphosphate5-phosphatase-2 (SHIP2) is a member of the 5-phosphatase family, acting as a vital negative regulator of immune response in vertebrates. In the present study, a SHIP2 homologue (designed as CgSHIP2) was identified from Pacific oyster, Crassostrea gigas. There was a SH2 domain, an IPPc domain and a SAM domain in CgSHIP2. The mRNA transcripts of CgSHIP2 were widely expressed in all the tested tissues with the highest expression in haemolymph. The mRNA expressions of CgSHIP2 in haemocytes increased significantly at 6, 12, 48 and 72 h after Vibrio splendidus stimulation. The positive green signals of CgSHIP2 protein were mainly located in cytoplasm of haemocytes. After the expression of CgSHIP2 was inhibited by RNA interference, the mRNA transcripts of interleukin 17s (CgIL-17-1, CgIL-17-2, CgIL-17-3 and CgIL-17-6) in the haemocytes increased significantly at 24 h after V. splendidus stimulation, which were 8.15-fold (p < 0.001), 3.44-fold (p < 0.05), 2.15-fold (p < 0.01) and 4.63-fold (p < 0.05) compared with that in NC-RNAi group, respectively. Obvious branchial swelling and cilium shedding in gills were observed in CgSHIP2-RNAi group at 24 h after V. splendidus stimulation. The results suggested that CgSHIP2 played an important role in controlling inflammatory response induced by bacteria in oysters.

Temperature effects on plasmalogen profile and quality characteristics in Pacific oyster (Crassostrea gigas) during depuration.

The quality of Pacific oyster (Crassostrea gigas) can be affected by many factors during depuration, in which temperature is the major element. In this study, we aim to determine the quality and plasmalogen changes in C. gigas depurated at different temperatures. The quality was significantly affected by temperature, represented by varying survival rate, glycogen content, total antioxidant capacity, alkaline phosphatase activity between control and stressed groups. Targeted MS analysis demonstrated that plasmalogen profile was significantly changed during depuration with PUFA-containing plasmalogen species being most affected by temperature. Proteomics analysis and gene expression assay further verified that plasmalogen metabolism is regulated by temperature, specifically, the plasmalogen synthesis enzyme EPT1 was significantly downregulated by high temperature and four plasmalogen-related genes (GPDH, PEDS, Pex11, and PLD1) were transcriptionally regulated. The positive correlations between the plasmalogen level and quality characteristics suggested plasmalogen could be regarded as a quality indicator of oysters during depuration.

Intra-decadal increase in globally-spread Magallana gigas in southern California estuaries.

Introduction and establishment of non-indigenous species (NIS) has been accelerated on a global scale by climate change. NIS Magallana gigas' (formerly Crassostrea gigas') global spread over the past several decades has been linked to warming waters, specifically during summer months, raising the specter of more spread due to predicted warming. We tracked changes in density and size distribution of M. gigas in two southern California, USA bays over the decade spanning 2010-2020 using randomly placed quadrats across multiple intertidal habitats (e.g., cobble, seawalls, riprap) and documented density increases by 2.2 to 32.8 times at 7 of the 8 sites surveyed across the two bays. These increases in density were coincident with 2-4° C increases in median monthly seawater temperature during summer months, consistent with global spread of M. gigas elsewhere. Size frequency distribution data, with all size classes represented across sites, suggest now-regular recruitment of M. gigas. Our data provide a baseline against which to compare future changes in density and abundance of a globally-spread NIS of significant concern.

Interfacial effects of perfluorooctanoic acid and its alternative hexafluoropropylene oxide dimer acid with polystyrene nanoplastics on oxidative stress, histopathology and gut microbiota in Crassostrea hongkongensis oysters.

The increasing interfacial impacts of polystyrene nanoplastics (PS) and per- and polyfluoroalkyl substances (PFAS) complex aquatic environments are becoming more evident, drawing attention to the potential risks to aquatic animal health and human seafood safety. This study aims to investigate the relative impacts following exposure (7 days) of Crassostrea hongkongensis oysters to the traditional PFAS congener, perfluorooctanoic acid (PFOA) at 50 µg/L, and its novel alternative, hexafluoropropylene oxide dimer acid (HFPO-DA), also known as GenX at 50 µg/L, in conjunction with fluorescent polystyrene nanoplastics (PS, 80 nm) at 1 mg/L. The research focuses on assessing the effects of combined exposure on oxidative stress responses and gut microbiota in the C. hongkongensis. Comparing the final results of PS + GenX (PG) and PS + PFOA (PF) groups, we observed bioaccumulation of PS in both groups, with the former causing more pronounced histopathological damage to the gills and intestines. Furthermore, the content of antioxidant enzymes induced by PG was higher than that of PF, including Superoxide Dismutase (SOD), Catalase (CAT), Glutathione Reductase (GR) and Glutathione Peroxidase (GSH). Additionally, in both PG and PF groups, the expression levels of several immune-related genes were significantly upregulated, including tnfα, cat, stat, tlr-4, sod, and ß-gbp, with no significant difference between these two groups (p > 0.05). Combined exposure induced significant changes in the gut microbiota of C. hongkongensis at its genus level, with a significant increase in Legionella and a notable decrease in Endozoicomonas and Lactococcus caused by PG. These shifts led to beneficial bacteria declining and pathogenic microbes increasing. Consequently, the microbial community structure might be disrupted. In summary, our findings contribute to a deeper understanding of the comparative toxicities of marine bivalves under combined exposure of traditional and alternative PFAS.

Morphological and Molecular Analysis Identified a Subspecies of Crassostrea ariakensis (Fujita, 1913) along the Coast of Asia.

Crassostrea ariakensis (Fujita, 1913) is one of the most important economic and ecological oysters that is naturally distributed along the coast of Asia, separated by the Yangtze River estuary. They are usually compared as different populations, while there is no consensus on whether C. ariakensis in northern and southern areas should be considered as two species or subspecies. Here, we analyzed morphological characteristics, COI, 16s rRNA, mitogenome sequences, and species delimitation analysis (ASAP and PTP) to resolve the intraspecific taxonomic status of the C. ariakensis. Phylogenetic and ASAP analysis highlight that C. ariakensis was divided into N-type and S-type. PTP was unable to differentiate between the two types of C. ariakensis. The divergence time of N-type and S-type C. ariakinsis is estimated to be 1.6 Mya, using the relaxed uncorrelated lognormal clock method. Additionally, significant morphological differences exist between the two groups in terms of the adductor muscle scar color. Despite these differences, the COI (0.6%) and 16S rRNA (0.6%) genetic distance differences between N-type and S-type C. ariakensis has not yet reached the interspecific level. These results suggest that N-type and S-type C. ariakensis should be treated as different subspecies and renamed as C. ariakensis ariakensis subsp. nov and C. ariakensis meridioyangtzensis subsp. nov.

CgADAR1 involved in regulating the synthesis of interferon-like protein in Crassostrea gigas.

Adenosine deaminases acting on RNA 1 (ADAR1) is a dsRNA adenosine (A)-to-inosine (I) editing enzyme that regulates the innate immune response against virus invasion. In the present study, a novel CgADAR1 was identified from the oyster Crassostrea gigas. The open reading frame (ORF) of CgADAR1 was of 3444 bp encoding a peptide of 1147 amino acid residues with two Zα domains, one dsRNA binding motif (DSRM) and one RNA adenosine deaminase domain (ADEAMc). The mRNA transcripts of CgADAR1 were detected in all the examined tissues, with higher expression levels in mantle and gill, which were 7.11-fold and 4.90-fold (p < 0.05) of that in labial palp, respectively. The mRNA transcripts of CgADAR1 in haemocytes were significantly induced at 24 h and 36 h after Poly (A: U) stimulation, which were 6.03-fold (p < 0.01) and 1.37-fold (p < 0.001) of that in control group, respectively. At 48 h after Poly (A:U) stimulation, the mRNA expression of CgRIG-Ⅰ, CgIRF8 and CgIFNLP significantly increased, which were 4.36-fold (p < 0.001), 1.82-fold (p < 0.05) and 1.92-fold (p < 0.05) of that in control group. After CgADAR1 expression was inhibited by RNA interference (RNAi), the mRNA expression levels of CgMDA5, CgRIG-Ⅰ, CgTBK1, CgIRF8 and CgIFNLP were significantly increased, which were 11.88-fold, 11.51-fold, 2.22-fold, 2.85-fold and 2.52-fold of that in control group (p < 0.001), and the phosphorylation level of CgTBK1 was also significantly increased. These results suggested that CgADAR1 played a regulation role in the early stages of viral infection by inhibiting the synthesis of interferon-like protein.

The Pacific oyster reproduction is affected by early-life exposure to environmental pesticide mixture: A multigenerational study.

Pesticides threat marine organisms worldwide. Among them, the Pacific oyster is a bivalve mollusc model in marine ecotoxicology. A large body of literature already stated on the multiple-scale effects pesticides can trigger in the Pacific oyster, throughout its life cycle and in a delayed manner. In particular, reproductive toxicity is of major concern because of its influence on population dynamics. However, past studies mostly investigated pesticide reprotoxicity as a direct effect of exposure during gametogenesis or directly on gametes and little is known about the influence of an early embryo exposure on the breed capacity. Therefore, we studied delayed and multigenerational consequences through gametogenesis features (i.e. sex ratio, glycogen content, gene expression) and reproductive success in two consecutive oyster generations (F0 and F1) exposed to an environmentally-relevant pesticide mixture (sum nominal concentration: 2.85 µg.L-1) during embryo-larval development (0-48 h post fertilization, hpf). In the first generation, glycogen content increased in exposed individuals and the expression of some gametogenesis target genes was modified. The reproductive success measured 48 hpf was higher in exposed individuals. A multigenerational influence was observed in the second generation, with feminisation, acceleration of gametogenesis processes and the sex-specific modification of glycogen metabolism in individuals from exposed parents. This study is the first to highlight the delayed effects on reproduction induced by an early exposure to pesticides, and its multigenerational implications in the Pacific oyster. It suggests that environmental pesticide contamination can have impacts on the recruitment and the dynamics of natural oyster populations exposed during their embryo-larval phase.

Comparison of the effects of reef and anthropogenic soundscapes on oyster larvae settlement.

Settlement is a critical period in the life cycle of marine invertebrates with a planktonic larval stage. For reef-building invertebrates such as oysters and corals, settlement rates are predictive for long-term reef survival. Increasing evidence suggests that marine invertebrates use information from ocean soundscapes to inform settlement decisions. Sessile marine invertebrates with a planktonic stage are particularly reliant on environmental cues to direct them to ideal habitats. As gregarious settlers, oysters prefer to settle amongst members of the same species. It has been hypothesized that oyster larvae from species Crassostrea virginica and Ostrea angasi use distinct conspecific oyster reef sounds to navigate to ideal habitats. In controlled laboratory experiments we exposed Pacific Oyster Magallana gigas larvae to anthropogenic sounds from conspecific oyster reefs, vessels, combined reef-vessel sounds as well as off-reef and no speaker controls. Our findings show that sounds recorded at conspecific reefs induced higher percentages of settlement by about 1.44 and 1.64 times compared to off-reef and no speaker controls, respectively. In contrast, the settlement increase compared to the no speaker control was non-significant for vessel sounds (1.21 fold), combined reef-vessel sounds (1.30 fold), and off-reef sounds (1.18 fold). This study serves as a foundational stepping stone for exploring larval sound feature preferences within this species.

The c.503A>G polymorphism in ZIP1-II of Pacific oyster Crassostrea gigas associated with zinc content.

The Pacific oyster Crassostrea gigas is renowned for its high zinc content, but the significant variation among individuals diminishes its value as a reliable source of zinc supplementation. The Zrt/Irt-like protein 1 (ZIP1), a pivotal zinc transporter that facilitates zinc uptake in various organisms, plays crucial roles in regulating zinc content. In the present study, polymorphisms of a ZIP1 gene in C. gigas (CgZIP1-II) were investigated, and their association with zinc content was evaluated through preliminary association analysis in 41 oysters and verification analysis in another 200 oysters. A total of 17 single nucleotide polymorphisms (SNPs) were identified in the exonic region of CgZIP1-II gene, with c.503A>G significantly associated with zinc content. Protein sequence and structure prediction showed that c.503A>G caused a p.Met110Val nonsynonymous mutation located in the metal-binding region of CgZIP1-II, which could influence its affinity for zinc ions, thereby modulating its zinc transport functionality. These results indicate the potential influence of CgZIP1-II polymorphisms on zinc content and provide candidate markers for selecting C. gigas with high zinc content.

miRNA-seq provides novel insight into the response to hyper- and hypo- salinity acclimation in Crassostrea hongkongensis.

The Hong Kong oyster, Crassostrea hongkongensis, is a significant bivalve species with economic importance. It primarily inhabits the estuarine intertidal zones in southern China, making it susceptible to salinity fluctuations. Consequently, investigating the molecular mechanisms governing salinity regulation in C. hongkongensis is essential. In this study, we conducted miRNA-seq on C. hongkongensis to compare miRNA expression differences under varying salinities (5‰, 25‰, and 35‰). The miRNA sequencing revealed 51 known miRNAs and 95 novel miRNAs across nine small RNA libraries (S5, S25, and S35). Among these miRNAs, we identified 6 down-regulated differentially expressed (DE) miRNAs in response to hypo-salinity stress (5‰), while 1 up-regulated DE miRNA and 5 down-regulated DE miRNAs were associated with hyper-salinity stress (35‰). Additionally, we predicted 931 and 768 potential target genes for hypo- and hyper-salinity stress, respectively. Functional gene annotation indicated that the target genes under hypo-salinity stress were linked to vesicle-mediated transport and metal ion binding. Conversely, those under hyper-salinity stress were primarily involved in signal transduction and metabolic processes. These findings have provided insights into the regulatory role of miRNAs, their potential target genes and associated pathways in oyster hypo- and hyper-salinity stress, which establish a foundation for future studies on the roles of miRNAs in salinity acclimation mechanisms in C. hongkongensis.

In silico structural features of the CgNR5A: CgDAX complex and its role in regulating gene expression of CYP target genes in Crassostrea gigas.

Contamination of aquatic environments has been steadily increasing due to human activities. The Pacific oyster Crassostrea gigas has been used as a key species in studies assessing the impacts of contaminants on human health and the aquatic biome. In this context, cytochrome P450 (CYPs) play a crucial role in xenobiotic metabolism. In vertebrates many of these CYPs are regulated by nuclear receptors (NRs) and little is known about the NRs role in C. gigas. Particularly, the CgNR5A represents a homologue of SF1 and LRH-1 found in vertebrates. Members of this group can regulate genes of CYPs involved in lipid/steroid metabolism, with their activity regulated by other NR, called as DAX-1, generating a NR complex on DNA response elements (REs). As C. gigas does not exhibit steroid biosynthesis pathways, CgNR5A may play other physiological roles. To clarify this issue, we conducted an in silico investigation of the interaction between CgNR5A and DNA to identify potential C. gigas CYP target genes. Using molecular docking and dynamics simulations of the CgNR5A on DNA molecules, we identified a monomeric interaction with extended REs. This RE was found in the promoter region of 30 CYP genes and also the NR CgDAX. When the upstream regulatory region was analyzed, CYP2C39, CYP3A11, CYP4C21, CYP7A1, CYP17A1, and CYP27C1 were mapped as the main genes regulated by CgNR5A. These identified CYPs belong to families known for their involvement in xenobiotic and lipid/steroid metabolism. Furthermore, we reconstructed a trimeric complex, previously proposed for vertebrates, with CgNR5A:CgDAX and subjected it to molecular dynamics simulations analysis. Heterotrimeric complex remained stable during the simulations, suggesting that CgDAX may modulate CgNR5A transcriptional activity. This study provides insights into the potential physiological processes involving these NRs in the regulation of CYPs associated with xenobiotic and steroid/lipid metabolism.

Expression and functional analysis of Fushi Tarazu transcription factor 1 (FTZ-F1) in the regulation of steroid hormones during the gonad development of Fujian Oyster, Crassostrea angulata.

Crassostrea angulata, a major shellfish cultivated in Southern China, has experienced a notable surge in commercial value in recent years. Understanding the molecular mechanisms governing their reproductive processes holds significant implications for advancing aquaculture practices. In this study, we cloned the orphan nuclear receptor gene, Fushi Tarazu transcription factor 1 (FTZ-F1), of C. angulata and investigated its functional role in the gonadal development. The full-length cDNA of FTZ-F1 spans 2357 bp and encodes a protein sequence of 530 amino acids. Notably, the amino acid sequence of FTZ-F1 in C. angulata shares remarkable similarity with its homologues in other species, particularly in the DNA-binding region (>90%) and ligand-binding region (>44%). In C. angulata, the highest expression level of FTZ-F1 was observed in the ovary, exhibiting more than a 200-fold increase during the maturation stage compared to the initiation stage (P < 0.001). Specifically, FTZ-F1 was mainly expressed in the follicular cells surrounding the oocytes of C. angulata. Upon inhibiting FTZ-F1 gene expression in C. angulata through RNA interference (RNAi), a substantial reduction in the expression of genes involved in the synthesis of sex steroids in the gonads, including 3ß-HSD, Cyp17, and follistatin, was observed. In addition, estradiol (E2) and testosterone (T) levels also showed a decrease upon FTZ-F1 silencing, resulting in a delayed gonadal development. These results indicate that FTZ-F1 acts as a steroidogenic factor, participating in the synthesis and regulation of steroid hormones and thus playing an important role in the reproductive and endocrine systems within oysters.

Mitochondrial responses to constant and cyclic hypoxia depend on the oxidized fuel in a hypoxia-tolerant marine bivalve Crassostrea gigas.

Sessile benthic organisms like oysters inhabit the intertidal zone, subject to alternating hypoxia and reoxygenation (H/R) episodes during tidal movements, impacting respiratory chain activities and metabolome compositions. We investigated the effects of constant severe hypoxia (90 min at ~ 0% O2 ) followed by 10 min reoxygenation, and cyclic hypoxia (5 cycles of 15 min at ~ 0% O2 and 10 min reoxygenation) on isolated mitochondria from the gill and the digestive gland of Crassostrea gigas respiring on pyruvate, palmitate, or succinate. Constant hypoxia suppressed oxidative phosphorylation (OXPHOS), particularly during Complex I-linked substrates oxidation. It had no effect on mitochondrial reactive oxygen species (ROS) efflux but increased fractional electron leak (FEL). In mitochondria oxidizing Complex I substrates, exposure to cyclic hypoxia prompted a significant drop after the first H/R cycle. In contrast, succinate-driven respiration only showed significant decline after the third to fifth H/R cycle. ROS efflux saw little change during cyclic hypoxia regardless of the oxidized substrate, but Complex I-driven FEL tended to increase with each subsequent H/R cycle. These observations suggest that succinate may serve as a beneficial stress fuel under H/R conditions, aiding in the post-hypoxic recovery of oysters by reducing oxidative stress and facilitating rapid ATP re-synthesis. The impacts of constant and cyclic hypoxia of similar duration on mitochondrial respiration and oxidative lesions in the proteins were comparable indicating that the mitochondrial damage is mostly determined by the lack of oxygen and mitochondrial depolarization. The ROS efflux in the mitochondria of oysters was minimally affected by oxygen fluctuations indicating that tight regulation of ROS production may contribute to robust mitochondrial phenotype of oysters and protect against H/R induced stress.

Transcriptomic investigation and biomarker discovery for zinc response in oysters Crassostrea gasar.

In an era of unprecedented industrial and agricultural growth, metal contamination in marine environments is a pressing concern. Sentinel organisms such as the mangrove oyster Crassostrea gasar provide valuable insights into these environments' health. However, a comprehensive understanding of the molecular mechanisms underlying their response to metal exposure remains elusive. To address this gap, we reanalyzed the 454-sequencing data of C. gasar, utilizing an array of bioinformatics workflow of CDTA (Combined De Novo Transcriptome Assembly) to generate a more representative assembly. In parallel, C. gasar individuals were exposed to two concentrations of zinc (850 and 4500 µg L-1 Zn) for 48 h to understand their molecular responses. We utilized Trinotate workflow for the 11,684-CDTA unigenes annotation, with most transcripts aligning with the genus Crassostrea. Our analysis indicated that 67.3% of transcript sequences showed homology with Pfam, while 51.4% and 54.5%, respectively had GO and KO terms annotated. We identified potential metal pollution biomarkers, focusing on metal-related genes, such as those related to the GSH biosynthesis (CHAC1 and GCLC-like), to zinc transporters (ZNT2-like), and metallothionein (MT-like). The evolutionary conservation of these genes within the Crassostrea genus was assessed through phylogenetic analysis. Further, these genes were evaluated by qPCR in the laboratory exposed oysters. All target genes exhibited significant upregulation upon exposure to Zn at both 850 and 4500 µg L-1, except for GCLC-like, which showed upregulation only at the higher concentration of 4500 µg L-1. This result suggests distinct activation thresholds and complex interactions among these genes in response to varying Zn concentrations. Our study provides insights into the molecular responses of C. gasar to Zn, adding valuable tools for monitoring metal pollution in marine ecosystems using the mangrove oyster as a sentinel organism.