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.

Transcriptomic and Physiological Analysis Reveal Melanin Synthesis-Related Genes and Pathways in Pacific Oysters (Crassostrea gigas).

Shell color is one of the shell traits of molluscs, which has been regarded as an economic trait in some bivalves. Pacific oysters (Crassostrea gigas) are important aquaculture shellfish worldwide. In the past decade, several shell color strains of C. gigas were developed through selective breeding, which provides valuable materials for research on the inheritance pattern and regulation mechanisms of shell color. The inheritance patterns of different shell colors in C. gigas have been identified in certain research; however, the regulation mechanism of oyster pigmentation and shell color formation remains unclear. In this study, we performed transcriptomic and physiological analyses using black and white shell oysters to investigate the molecular mechanism of melanin synthesis in C. gigas. Several pigmentation-related pathways, such as cytochrome P450, melanogenesis, tyrosine metabolism, and the cAMP signaling pathway were found. The majority of differentially expressed genes and some signaling molecules from these pathways exhibited a higher level in the black shell oysters than in the white, especially after L-tyrosine feeding, suggesting that those differences may cause a variation of tyrosine metabolism and melanin synthesis. In addition, the in vitro assay using primary cells from mantle tissue showed that L-tyrosine incubation increased cAMP level, gene and protein expression, and melanin content. This study reveals the difference in tyrosine metabolism and melanin synthesis in black and white shell oysters and provides evidence for the potential regulatory mechanism of shell color in oysters.

Effects of salinity on pre- and post-fertilization developmental events in the mangrove oyster Crassostrea rhizophorae (GUILDING, 1828).

The mangrove oyster Crassostrea rhizophorae is identified as a potentially valuable species for tropical aquaculture, however, information on the physiological mechanisms of reproduction under laboratory conditions for this species is limited. This study investigated the effects of salinity at different concentrations (15, 20, 25, 30, 35, and 40 g/L) on the induction of germinal vesicle breakdown (GVBD) of oocytes obtained through stripping, the release of polar bodies (PB1 and PB2), and the larval development of the mangrove oyster. The results revealed a relationship between salinity and the percentage of GVBD, with the most effective range being 30-40 g/L within the hydration time frame between 70 and 120 min. The release of 50 % of PB1 was detected within this salinity range, while for the release of 50 % of PB2, the saline treatments of 35 and 40 g/L showed the best results. Overall, the salinity range of 30-40 g/L is suggested as the most suitable of polyploidy induction methodologies through the retention of PB1 or PB2. Regarding larval hatching, while salinities between 25 and 40 g/L presented similar percentages, at 15 g/L no hatching was observed. This study demonstrated that salinity is a key factor in early pre- and post-fertilization stages for the successful reproduction of mangrove oyster in hatcheries and that the percentages of oocyte maturation and artificial fertilization can be optimized by adjusting salinity.

MicroRNA transcriptome analysis reveals the immune regulatory mechanism of Crassostrea hongkongesis against Vibrio harveyi infection.

MicroRNAs (miRNAs) are small non-coding RNA molecules that modulate target-genes expression and play crucial roles in post-transcriptional regulation and immune system regulation. The Hong Kong oyster (Crassostrea hongkongesis), as the main marine aquaculture shellfish in the South China Sea, not only has high economic and ecological value, but also is an ideal model for conducting research on pathogen host interaction. Vibrio harveyi, a Gram negative luminescent marine bacterium, is widely distributed in coastal water environments and can cause large-scale death of C. hongkongesis. However, little in formation is available on the immune regulatory mechanisms of C. hongkongesis infected with V. harveyi. Therefore, we performed microRNA transcriptome analysis for elucidating the immunoregulation mechanism of C. hongkongesis infected with V. harveyi. The results show that a total of 308468208 clean reads and 288371159 clean tags were obtained. 222 differentially expressed miRNAs were identified. A total of 388 target genes that were differentially expressed and negatively correlated with miRNA expression were predicted by 222 DEmiRs. GO enrichment analysis of 388 DETGs showed that they were mainly enriched in the immune-related term of membrane-bounded vesicle, endocytic vesicle lumen, antigen processing and presentation of exogenous peptide antigen via MHC class I, antigen processing and presentation of peptide antigen via MHC class I, and other immune-related term. KEGG enrichment analysis showed that DETGs were mainly enriched in the Complement and coagulation cascades, Herpes simplex virus 1 infection, Bacterial invasion of epithelial cells, Antigen processing and presentation and NOD-like receptor signaling pathway. The 16 key DEmiRs and their target genes form a regulatory network for seven immune-related pathways. These results suggest that V. harveyi infection induces a complex miRNA response with wide-ranging effects on immune gene expression in the C. hongkongesis. This study explored the immune response of C. hongkongesis to V. harveyi infection at the level of miRNAs, which provides new ideas for the healthy culture and selective breeding of C. hongkongesis.

Structural and functional characterization of an egg-laying hormone signaling system in a lophotrochozoan - The pacific oyster (Crassostrea gigas).

The egg-laying hormones (ELHs) of gastropod mollusks were characterized more than forty years ago. Yet, they have remained little explored in other mollusks. To gain insights into the functionality of the ELH signaling system in a bivalve mollusk - the oyster Crassostrea gigas, this study investigates the processing of its ELH precursor (Cragi-ELH) by mass spectrometry. Some of the ELH mature peptides identified in this study were subsequently investigated by nuclear magnetic resonance and shown to adopt an extended alpha-helix structure in a micellar medium mimicking the plasma membrane. To further characterize the ELH signaling system in C. gigas, a G protein-coupled receptor phylogenetically related to ecdysozoan diuretic hormone DH44 and corticotropin-releasing hormone (CRH) receptors named Cragi-ELHR was also characterized functionally and shown to be specifically activated by the two predicted mature ELH peptides and their N-terminal fragments. Both Cragi-ELH and Cragi-ELHR encoding genes were mostly expressed in the visceral ganglia (VG). Cragi-ELH expression was significantly increased in the VG of both fully mature male and female oysters at the spawning stage. When the oysters were submitted to a nutritional or hyposaline stress, no change in the expression of the ligand or receptor genes was recorded, except for Cragi-ELHR only during a mild acclimation episode to brackish water. These results suggest a role of Cragi-ELH signaling in the regulation of reproduction but not in mediating the stress response in our experimental conditions.

Adaptive response of triploid Fujian oyster (Crassostrea angulata) to nanoplastic stress: Insights from physiological, metabolomic, and microbial community analyses.

Triploid Fujian oyster (Crassostrea angulata) is crucial to aquaculture and coastal ecosystems because of its accelerated growth and heightened resilience against environmental stressors. In light of the increasing prevalence of nanoplastic pollution in the ocean, understanding its potential impact on this organism, particularly its adaptive responses, is of paramount importance. Despite this, the effects of nanoplastic pollution on the physiology of C. angulata remain largely unexplored. In this study, we explored the responses of triploid Fujian oysters to nanoplastic stress during a 14-day exposure period, employing an integrative methodology that included physiological, metabolomic, and 16S rRNA sequencing analyses. Our results demonstrate that the oysters exhibit a strong adaptive response to nanoplastic exposure, characterized by alterations in enzyme activity, metabolic pathways, and microbial community composition, indicative of an adaptive recovery state as opposed to a disordered state. Oysters subjected to elevated nanoplastic levels exhibited adaptive responses primarily by boosting the activity of the antioxidant enzyme catalase and elevating the levels of antioxidants such as adenosine, 3-(4-hydroxyphenyl)pyruvate, D-sorbitol, d-mannose, and unsaturated fatty acids, as well as the functional amino acids l-proline and l-lysine. Nanoplastic treatment also resulted in increased activity of succinate dehydrogenase, a key component of energy metabolism, and increased contents of intermediate metabolites or products of energy metabolism, such as adenosine monophosphate, adenosine, guanosine, creatine, and thiamine. Nanoplastic treatment led to an increase in the abundance of certain advantageous genera of gut bacteria, specifically Phaeobacter and Nautella. The observed adaptive response of triploid Fujian oysters to nanoplastic stress provides valuable insights into the mechanisms underpinning resilience in marine bivalves.

Employing molecular, chemical and physiological techniques using Crassostrea virginica to assess ecosystem health along coastal South Carolina and North Carolina, United States.

Natural and anthropogenic environmental impacts can introduce contaminants into sensitive habitats, threatening ecosystems and human health. Consistent monitoring of coastal areas provides critical environmental assessment data. Sediments and Eastern Oyster (Crassostrea virginica) tissues were collected at fourteen South Carolina (SC) and four North Carolina (NC) sites as part of the National Oceanic and Atmospheric Administration's Mussel Watch environmental monitoring program. Cellular and molecular techniques were employed to measure C. virginica stress response, specifically, Lipid Peroxidation (LPx), Glutathione (GSH), and qPCR techniques. Gene specific primers targeted for detecting oxidative stress and cellular death were developed in C. virginica to gauge response to current environmental conditions using gill and hepatopancreas (HP) tissue. In order to validate gene specific markers as additional assessment tools, a 96 h zinc (Zn) laboratory exposure was performed. Cellular biomarker data revealed tissue specific responses. Hepatopancreas data showed C. virginica exhibited stress through the lipid peroxidation assay amongst sampling sites, however, response was managed through glutathione detoxification. Gill tissue data had significantly lower levels of cellular biomarker response compared to hepatopancreas. Molecular biomarkers targeting these cellular stress pathways through qPCR analysis show upregulation of Metallothionein in hepatopancreas and gill tissue with a concurrent > 2-fold upregulation in the detoxification marker Superoxide Dismutase (SOD) at three NC sites. SC sites displayed higher stress levels through LPx assays and down-regulation in GPx gene activity. Laboratory zinc exposure revealed no significance in cellular biomarker results, however, molecular data showed gills responding to zinc treatment through upregulation of Metallothionein, SOD and Cathepsin L, indicating an acute response in gills. Collectively, chemical, cellular and molecular methods clarify sentinel stress response of biological impacts and aid in evaluating environmental health in coastal ecosystems. This combined methodological approach provides a detailed analysis of environmental conditions and improves land-use management decisions.

A bone morphogenetic protein regulates the shell formation of Crassostrea gigas under ocean acidification.

Bone morphogenetic proteins (BMPs) are key factors controlling osteoblast differentiation, which have been proved to be involved in the hard tissue formation of marine mollusks. In the present study, a member of BMPs gene (CgBMP7) was identified from Pacific oyster Crassostrea gigas (C. gigas) with the aim to understand its possible role in the regulation of shell formation under ocean acidification (OA) conditions. The open reading frame (ORF) of CgBMP7 was of 1254 bp encoding a polypeptide of 417 amino acids. The deduced amino acid sequence of CgBMP7 was comprised of one signal peptide, one prodomain and one TGF-ß domain, which shared 21.69%-61.10% identities with those from other species. The mRNA transcript of CgBMP7 was ubiquitously expressed in all the tested tissues of adult oysters with a higher expression level in mantle, notably highest in the middle fold (MF) of the three folds of mantle. The expression level of bone morphogenetic protein type I receptor (CgBMPR1B) mRNA was also highest in the MF and up-regulated dramatically post recombinant BMP7 protein (rCgBMP7) stimulation. After the blockage of BMPR1B with inhibitor LDN193189 (LDN), the mRNA expression level and phosphorylation level of CgSmad1/5/8 in mantle were decreased, and the mRNA expression levels of CgCaM and Cgengrailed-1 were down-regulated significantly. During the oysters were exposed to acidified seawater for weeks, the expression levels of CgBMP7, CgBMPR1B and CgSmad1/5/8 in the MF decreased significantly (p < 0.01) at the 4th week, and CgCaM and Cgengrailed-1 also exhibited the same variable expression patterns as CgBMP7. In addition, the growth of shell in the treatment group (pH 7.8) was slower than that in the control group (pH 8.1). These results collectively indicated that BMP7 was able to trigger the BMPR-Smad signaling pathway and involved in controlling the formation of oyster calcified shell under OA conditions.

Structural and expression analysis of the dopamine receptors reveals their crucial roles in regulating the insulin signaling pathway in oysters.

Dopamine performs its critical role upon binding to receptors. Since dopamine receptors are numerous and versatile, understanding their protein structures and evolution status, and identifying the key receptors involved in the modulation of insulin signaling will provide essential clues to investigate the molecular mechanism of neuroendocrine regulating the growth in invertebrates. In this study, seven dopamine receptors were identified in the Pacific oysters (Crassostrea gigas) and were classified into four subtypes according to their protein secondary and tertiary structures, and ligand-binding activities. Of which, DR2 (dopamine receptor 2) and D(2)RA-like (D(2) dopamine receptor A-like) were considered the invertebrate-specific type 1 and type 2 dopamine receptors, respectively. Expression analysis indicated that the DR2 and D(2)RA-like were highly expressed in the fast-growing oyster "Haida No.1". After in vitro incubation of ganglia and adductor muscle with exogenous dopamine and dopamine receptor antagonists, the expression of these two dopamine receptors and ILPs (insulin-like peptides) was also significantly affected. Dual-fluorescence in situ hybridization results showed that D(2)RA-like and DR2 were co-localized with MIRP3 (molluscan insulin-related peptide 3) and MIRP3-like (molluscan insulin-related peptide 3-like) in the visceral ganglia, and were co-localized with ILP (insulin-like peptide) in the adductor muscle. Furthermore, the downstream components of dopamine signaling, including PKA, ERK, CREB, CaMKK1, AKT, and GSK3ß were also significantly affected by the exogenous dopamine and dopamine receptor antagonists. These findings confirmed that dopamine might affect the secretion of ILPs through the invertebrate-specific dopamine receptors D(2)RA-like and DR2, and thus played crucial roles in the growth regulation of the Pacific oysters. Our study establishes the potential regulatory relationship between the dopaminergic system and insulin-like signaling pathway in marine invertebrates.

Triploid Pacific oysters exhibit stress response dysregulation and elevated mortality following heatwaves.

Polyploidy has been suggested to negatively impact environmental stress tolerance, resulting in increased susceptibility to extreme climate events. In this study, we compared the genomic and physiological response of diploid (2n) and triploid (3n) Pacific oysters (Crassostrea gigas) to conditions present during an atmospheric heatwave that impacted the Pacific Northwestern region of the United States in the summer of 2021. Climate stressors were applied either singly (single stressor; elevated seawater temperature, 30°C) or in succession (multiple stressor; elevated seawater temperature followed by aerial emersion at 44°C), replicating conditions present within the intertidal over a tidal cycle during the event. Oyster mortality rate was elevated within stress treatments with respect to the control and was significantly higher in triploids than diploids following multiple stress exposure (36.4% vs. 14.8%). Triploids within the multiple stressor treatment exhibited signs of energetic limitation, including metabolic depression, a significant reduction in ctenidium Na+ /K+ ATPase activity, and the dysregulated expression of genes associated with stress response, innate immunity, glucose metabolism, and mitochondrial function. Functional enrichment analysis of ploidy-specific gene sets identified that biological processes associated with metabolism, stress tolerance, and immune function were overrepresented within triploids across stress treatments. Our results suggest that triploidy impacts the transcriptional regulation of key processes that underly the stress response of Pacific oysters, resulting in downstream shifts in physiological tolerance limits that may increase susceptibility to extreme climate events that present multiple environmental stressors. The impact of chromosome set manipulation on the climate resilience of marine organisms has important implications for domestic food security within future climate scenarios, especially as triploidy induction becomes an increasingly popular tool to elicit reproductive control across a wide range of species used within marine aquaculture.

Comparative biochemical and molecular responses of biotransformation and antioxidant systems in three species of Crassostrea (Sacco, 1897) oysters exposed to chrysene.

Chrysene (CHR) is among the most persistent polycyclic aromatic hydrocarbons (PAH) in water and a priority compound for pollutants monitoring, due to its carcinogenic, mutagenic and genotoxic potential. Aquatic animals exposed to CHR may present alterations of biomarkers involved in the biotransformation and oxidative stress-related parameters. The aim of this study was to investigate differences in antioxidant and biotransformation (phase I and II) systems of Crassostrea gigas, C. gasar and C. rhizophorae and its effects resulting from CHR exposure. Adult oysters of these species were exposed to 10 µg L-1 of CHR for 24 h and 96 h. In gills, the transcripts CYP1-like, CYP2-like, CYP2AU1-like, GSTO-like, MGST-like, SULT-like were evaluated after 24 h of exposure. The activity of SOD, CAT, GPx, GR and G6PDH were analyzed in gills and digestive glands after 96 h of exposure. CHR bioaccumulated in tissues. Differences in the remaining levels of CHR in water after 96 h were observed in aquaria containing C. gigas or C. gasar oysters and may be associated to the different filtration rates between these species. Downregulate of biotransformation genes were observed in gills of C. gasar (CYP2AU1-like and GSTO-like) and C. rhizophorae (CYP1-like1, CYP2-like, MGST-like and SULT-like), suggesting that biotransformation responses may be species-specific. Differential activity of antioxidant enzymes were observed in gills and digestive gland of oysters exposed to CHR. Biochemical responses suggested that C. gigas and C. gasar are more responsive to CHR. Differential responses observed among the three Crassostrea species can be related to evolutionary differences, ecological niches and adaptation to environment.

A MASP-like functions as PRR to regulate the mRNA expressions of inflammatory factors in the Pacific oyster Crassostrea gigas.

Mannose-binding lectin-associated serine protease (MASP) is a type of central serine protease in the complement lectin pathway. In the present study, a MASP-like was identified from the Pacific oyster Crassostrea gigas, defined as CgMASPL-2. The cDNA sequence of CgMASPL-2 was of 3399 bp with an open reading frame of 2757 bp and encoded a polypeptide of 918 amino acids containing three CUB domains, an EGF domain, two IG domains, and a Tryp_SPC domain. In the phylogenetic tree, CgMASPL-2 was firstly clustered with Mytilus californianus McMASP-2-like, and then assigned into the invertebrate branch. CgMASPL-2 shared similar domains with M. californianus McMASP-2-like and Littorina littorea LlMReM1. CgMASPL-2 mRNA was expressed in all the tested tissues with the highest expression in haemolymph. CgMASPL-2 protein was mainly distributed in the cytoplasm of haemocytes. The mRNA expression of CgMASPL-2 increased significantly in haemocytes after Vibrio splendidus stimulation. The recombinant 3 × CUB-EGF domains of CgMASPL-2 displayed binding activities to diverse polysaccharides (lipopolysaccharide, peptidoglycan and mannose) and microbes (Staphylococcus aureus, Micrococcus luteus, Pichia pastoris, Vibrio anguillarum, V. splendidus and Escherichia coli). In anti-CgMASPL-2 treated oysters, the mRNA expressions of CgIL17-1 and CgIL17-2 in haemocytes decreased significantly after V. splendidus stimulation. The results indicated that CgMASPL-2 could directly sense microbes and regulate the mRNA expressions of inflammatory factors.

Species-specific CgCaspase-Cg-5 in the pacific oyster induces haemocyte apoptosis by regulating the mRNA expression of apoptosis-related genes in the early stage of immune response.

Caspases are cysteinyl aspartate-specific proteinases, playing critical roles in apoptotic pathway to induce apoptosis and inflammatory response. In this study, the expanded repertoire of Caspases was revealed in the Pacific oyster Crassostrea gigas, and a total of 30 Caspases were identified from the genomic and stress-induced transcriptomic databases of the Pacific oyster. They were clustered into CgCaspase-2/9, CgCaspase-8/10, CgCaspase-3/6/7, CgCaspase-Cg, and CgCaspase-L. CgCaspase-Cg subgroup was found to be specifically expanded after a positive selection in oyster with average Ka/Ks of 0.50. The mRNA expression of CgCaspase-Cg-5 was found to be obviously induced against various bacterial and viral stimulations or environmental stresses. The relative expression level of CgCaspase-Cg-5 in haemocytes increased and reached the peak at 6 h after Vibrio splendidus stimulation, which was 5.57-fold of that in the control group (p < 0.01). In the oysters whose CgCaspase-Cg-5 expression was knocked down, the mRNA expression of apoptosis-related genes including CgBcl2, CgBax, CgCaspase3 and CgCaspase9 changed significantly at 12 h after V. splendidus stimulation. The expression of CgBax, CgCaspase3 and CgCaspase9 decreased, which was 0.64-fold (p < 0.05), 0.53-fold (p < 0.05) and 0.62-fold (p < 0.01), while the expression of CgBcl2 increased, which was 2.81-fold (p < 0.01) of that in the EGFP-dsRNA group, respectively. Meanwhile, the apoptotic rate of haemocytes (1.90 ± 0.71%) significantly decreased compared to that in the EGFP-dsRNA group (5.40 ± 0.72%) (p < 0.05), and the histological damages of widened cell spacing, gill filament swelling and loose cytoplasm were observed in the CgCaspase-Cg-5-knockdown oysters after V. splendidus stimulation. Collectively, CgCaspase-Cg subgroup was specifically expanded in oyster and some bivalve species, and species-specific CgCaspase-Cg-5 regulated the mRNA expression of the apoptosis-related genes to induce haemocyte apoptosis in the early stage of immune response. This provided insight into the evolutionary and functional characteristics of Caspase repertoire in the Pacific oyster and highlighted the important role of CgCaspase-Cg-5 in the response to pathogen infection and environmental stresses.

Effects of cadmium and zinc on gene expression of novel molecular biomarkers in the mangrove oyster Crassostrea gasar.

Metal contamination impacts various aquatic species, and mollusk bivalves are appropriate sentinel organisms in coastal pollution assessment. Metal exposure can disrupt homeostasis, alter gene expression, and harm cellular processes. However, organisms have evolved mechanisms to regulate metal ions and counteract their toxicity. This study examined the effect of acute cadmium (Cd) and zinc (Zn) on metal-related gene expression in gills of Crassostrea gasar following 24 and 48 h of laboratory exposure. We focused on Zn transport, metallothionein (MT), glutathione (GSH) biosynthesis, and calcium (Ca) transporter genes to understand the underlying Cd and Zn-accumulating mechanisms that prevent metal toxicity. Our findings revealed increased Cd and Zn levels in oyster gills, with significantly higher accumulation after 48 h. C. gasar accumulated high Cd concentrations even in scarce conditions and increased Zn levels, suggesting a strategy to cope with toxicity. While no significant gene expression differences were observed after 24 h, the increased metal accumulation after 48 h led to upregulation of CHAC1, GCLC, ZnT2, and MT-like genes in oysters exposed to Cd, and increased ZnT2-like expression following exposure to higher Cd/Zn mixtures. We found evidence of oysters may mobilize metal-related genes to mitigate Cd-induced toxicity by both chelating metals and/or reducing their intracellular concentrations. The observed genes upregulation also indicates their sensitivity to changes in metal bioavailability. Overall, this study offers insights into oyster mechanisms for coping with metal toxicity and suggests ZnT2, MT, CHAC1, and GCLC-like as molecular biomarkers for monitoring aquatic metal pollution using C. gasar as sentinel species.

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 48 h 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.

An IRF2BP member (CgIRF2BP) involved in negative regulation of CgIFNLP expression in oyster Crassostrea gigas.

The IRF2BP family of transcription regulators act as corepressor molecules by inhibiting both enhancer-activated and basal transcription involving in many biological contexts. In the present study, an IRF2BP homologue (CgIRF2BP) was identified from oyster C. gigas. Its open reading frame is of 1809 bp encoding a polypeptide of 602 amino acids, which contains an IRF-2BP1_2 domain and a RING domain. The mRNA transcripts of CgIRF2BP were detected in all tested tissues with highest level in haemocytes (28.99-fold of that in mantle, p < 0.05). After poly (I:C) stimulation, the expression level of CgIRF2BP was significantly down-regulated at 3 h (0.50-fold of that in control group, p < 0.001) and gradually increased from 6 h to 48 h (2.69-fold of that in control group, p < 0.01). The recombinant protein of CgIRF2BP (rCgIRF2BP) showed high affinity to both rCgIRF1 and rCgIRF8 with Kd value of 1.02 × 10-7 and 2.09 × 10-7, respectively. In CgIRF2BP-RNAi oysters, the mRNA expression of CgIFNLP, CgMx1, CgViperin and CgIFI44L were significantly increased after poly (I:C) stimulation, which were 2.88 (p < 0.01), 1.83 (p < 0.05), 2.47 (p < 0.05), and 1.99-fold (p < 0.01) of that in EGFP group, respectively. These findings suggested that CgIRF2BP negatively regulated CgIFNLP expression by binding with CgIRF1 and CgIRF8.

Transcription factor CgPOU3F4-like regulates expression of pheomelanin synthesis related gene CgB-aat1 in the Pacific oyster (Crassostrea gigas).

Previous study has found that b (0, +) -type amino acid transporter 1 (CgB-aat1) plays an essential role on mantle pigmentation in the Pacific oyster Crassostrea gigas. However, the molecular regulation of CgB-aat1 gene expression remains unclear. Herein, three POU domain family members, CgPOU2F1, CgPOU3F4-like and CgPOU4F3-X1 were characterized and they all had POUs and HOX domains, respectively, which were important in transcriptional regulation. CgPOU3F4-like gene expression was the highest in mantle edge. Subsequently, the dual-luciferase reporter result showed that the core regulatory region of CgB-aat1 gene was from -632 to -350 bp of promoter. In transient co-transfection assays, the strongest activity was activated only by CgPOU3F4-like, suggesting CgPOU3F4-like was a valid transcriptional activator of CgB-aat1 gene promoter. And the structural integrity of CgPOU3F4-like was essential for its activation function. In addition, site directed mutagenesis assay was applied to detect three key binding sites between CgPOU3F4-like and core region of CgB-aat1 gene promoter, and this interaction was verified by ChIP test. Furthermore, CgPOU3F4-like knockdown by RNA interference led to obvious decreases in CgB-aat1 and cystathionine beta-synthase (CgCbs) expressions at both mRNA and protein levels. Collectively, these results indicate that CgPOU3F4-like positively regulate CgB-aat1 gene expression and it may be a critical upstream transcriptional regulation factor in pheomelanin synthesis in C. gigas.

An ATP-binding cassette transporter G2 (CgABCG2) regulates the haemocyte proliferation by modulating the G1/S phase transition of cell cycle in oyster Crassostrea gigas.

ATP-binding cassette transporter G2 (ABCG2) is a half-transporter of the G subfamily in ATP-binding cassette transporters (ABC transporter), which is involved in the regulation of multidrug-resistant, cell cycle, and cell proliferation. In the present study, a homologue of ABCG2 (named as CgABCG2) with the conserved AAA domain and ABC2 membrane domain was identified from the Pacific oyster Crassostrea gigas. The open reading frame (ORF) of CgABCG2 was of 1956 bp encoding a predicted polypeptide of 652 amino acids, which shared 56.7%-65.7% sequence similarities with previously identified ABCG2s from other animals. The mRNA transcripts of CgABCG2 were detected in all the tested tissues with higher expression levels in gonad and haemocytes (19.31-fold and 11.23-fold of that in adductor muscle respectively, p < 0.05). CgABCG2 was mainly distributed on the cell membrane of the haemocytes with a partial distribution in the cytoplasm and nucleus. After Vibrio splendidus stimulation, the mRNA expression level of CgABCG2 in haemocytes was significantly up-regulated at 3 h and 6 h, which was 5.22-fold and 8.60-fold (p < 0.05) of that in control, respectively. After the expression of CgABCG2 was interfered by RNAi, the number of cells with EdU positive signals was reduced in both haemocytes and the potential hematopoietic sites. And the mRNA expression level of CgPCNA, CgGATA3, CgRunx, CgSCL and CgC-kit decreased significantly (p < 0.05), which were about 0.66-, 0.37-, 0.32-, 0.50-, and 0.50-fold of that in the negative control group, respectively. While the mRNA expression level of CgCDK2 increased significantly (1.84-fold to that in control, p < 0.05) and that of stem cell-related factor CgSOX2 did not change significantly in the si-CgABCG2 oysters. Moreover, the cell cycle of haemocytes was detected by flow cytometry, which was arrested at G0/G1 phase in the si-CgABCG2 oysters. All the results collectively suggested that CgABCG2 might involve the proliferation of haemocytes by regulating the expression of haematopoiesis related transcription factors and the G1/S phase transition of the cell cycle in oyster C. gigas.

Involvement of B-aat1 and Cbs in regulating mantle pigmentation in the Pacific oyster (Crassostrea gigas).

BACKGROUND: Shell color formation is an important physiological process in bivalves, the molecular genetic basis has potential application in bivalve aquaculture, but there is still remaining unclear about this issue. The cystine/glutamate transporter (Slc7a11) and cystathionine beta-synthase (Cbs) are integral genes in pheomelanin synthesis pathway, which is vital to skin pigmentation. METHODS AND RESULTS: Here, the sequences of b (0, +) -type amino acid transporter 1 (B-aat1) and Cbs in Pacific oyster (Crassostrea gigas) (CgB-aat1, CgCbs) were characterized. Phylogenetically, the deduced amino acid sequences of CgB-aat1 and CgCbs both possessed conserved features. Genes were both ubiquitously expressed in six tested tissues with more abundant expression level in central mantle. Besides, the polyclonal antibodies of CgB-aat1, CgCbs, CgTyr, and CgTyrp2 were successfully prepared. Immunofluorescence analysis revealed that CgB-aat1 and CgCbs proteins were both expressed in gill rudiments of eyed-larvae and concentrated mainly in cytoplasm of epithelial cell and nerve axons in mantle. Additionally, after CgB-aat1 or CgCbs silencing, expressions at mRNA and protein levels of CgB-aat1 and CgCbs involved in pheomelanin synthesis were significantly suppressed, and CgTyr, CgTyrp1 and CgTyrp2 related to eumelanin synthesis were also down-regulated but no apparent differences, respectively. Moreover, micrographic examination found less brown-granules at mantle edge in CgB-aat1 interference group. CONCLUSION: These results implied that pheomelanin synthesis was possible induced by CgB-aat1-CgTyr-CgCbs axis, and it played an essential role on mantle pigmentation in the oysters. These findings provide the useful genetic knowledge and enrich the physiological information for the shell color formation in bivalve aquaculture.

Identification of the Immunoglobulin E Epitope of Arginine Kinase, an Important Allergen from Crassostrea angulata.

Arginine kinase (AK) was identified as an allergen in Crassostrea angulata. However, little information is available about its epitopes. In this study, AK from C. angulata was registered to the World Health Organization/International Union of Immunological Societies allergen nomenclature committee to be named as Cra a 2. The immunoglobulin G/immunoglobulin E-binding capacity of Cra a 2 was significantly reduced after chemical denaturation treatment. Further, eight linear mimotopes and five conformational mimotopes of Cra a 2 were obtained using phage panning. In addition to six linear epitopes that have been identified, two linear epitopes were verified by a synthetic peptide, of which L-Cra a 2-2 was conserved in shellfish. Four conformational epitopes were verified by site-directed mutation, among which mutation of C-Cra a 2-1 affected the structure and reduced the immunoreactivity of Cra a 2 most significantly. Overall, the identified epitopes may lay a foundation for the development of hypoallergenic oyster products through food processing.