Inhibitors of LAMP used to detect Tenacibaculum sp. strain Pbs-1 associated with black-spot shell disease in Akoya pearl oysters, and additives to reduce the effect of the inhibitors.

Black-spot shell disease is an unresolved disease that decreases pearl quality and threatens pearl oyster survival. In previous studies, the bacterium Tenacibaculum sp. strain Pbs-1 was isolated from diseased Akoya pearl oysters Pinctada fucata, and a rapid, specific, and sensitive loop-mediated isothermal amplification (LAMP) assay for detecting this pathogen was established. This technology has considerable potential for routine diagnosis of strain Pbs-1 in oyster hatcheries and/or pearl farms; therefore, it is vital to identify substances in environmental samples that might inhibit LAMP and to find additives that can reduce the inhibition. In this study, we investigated the effects of six chemicals or proteins, otherwise known as conventional PCR inhibitors, on LAMP, using the DNA of strain Pbs-1 as template: humic acid, urea, iron (III) chloride hexahydrate, melanin, myoglobin, and Ethylenediamine-N,N,N',N'-tetraacetic acid, disodium salt, dihydrate (EDTA; pH 6.5). Next, to reduce the effects of identified inhibitors, we tested the addition of bovine serum albumin (BSA) or T4 gene 32 protein (gp32) to the LAMP assay. When 50 ng of DNA template was used, 4 ng/µL of humic acid, 0.05% melanin, and 10 mM of EDTA (pH 6.5) inhibited the LAMP reaction, whereas myoglobin, urea, and FeCl3 had no effect. When 50 pg of DNA template was used, 4 ng/µL of humic acid, 0.05% melanin, 4 µg/µL of myoglobin, 10 µg/µL of urea, and 10 mM of EDTA inhibited the LAMP reaction. Thus, it was shown that the gene-amplification inhibitory effect of melanin, humic acid, and urea could be reduced by adding BSA or gp32 to the LAMP reaction mixture. This technique could be applied as part of a protocol to prevent mass mortalities of pearl oysters; moreover, the results enhance our knowledge about substances that inhibit LAMP and methods to reduce the inhibition, which have rarely been reported.

The role of Pm-miR-184-3p in regulating the immune response in the pearl oyster Pinctada fucata martensii.

microRNAs are a class of non-coding RNAs with post-transcriptional regulatory functions in eukaryotes. In our previous study, miR-184-3p was identified in the hemocyte transcriptome of Pinctada fucata martensii (Pm-miR-184-3p), and its expression was shown to be up-regulated following transplantation surgery; however, its role in regulating transplantation immunity has not yet been clarified. Here, the role of Pm-miR-184-3p in regulating the immune response of P. f. martensii was studied. The expression of Pm-miR-184-3p increased following the stimulation of pathogen-associated molecular patterns, and Pm-miR-184-3p overexpression increased the activity of antioxidant-related enzymes, such as superoxide dismutase and catalase. Transcriptome analysis obtained 1096 differentially expressed genes (DEGs) after overexpression of Pm-miR-184-3p, and these DEGs were significantly enriched in conserved pathways such as the Cell cycle pathway and NF-kappa B signaling pathway, as well as GO terms including base excision repair, cell cycle, and DNA replication, suggesting that Pm-miR-184-3p could enhance the inflammation process. Target prediction and dual luciferase analysis revealed that pro-inflammatory related genes Pm-TLR3 and Pm-FN were the potential target of Pm-miR-184-3p. We speculate that Pm-miR-184-3p may utilize negative regulation of target genes to delay the activation of corresponding immune pathways, potentially preventing excessive inflammatory responses and achieving a delicate balance within the organism. Overall, Pm-miR-184-3p play a key role in regulating cellular responses to transplantation. Our findings provide new insights into the immune response of P. f. martensii to transplantation.

Identification and functional characterization of a superoxide dismutase (CuZnSOD) from Pinctada fucata martensii.

Copper/zinc superoxide dismutase (Cu/Zn-SOD) can effectively eliminate reactive oxygen species (ROS)，avoid damage from O2 to the body, and maintain O2 balance. In this study, multi-step high-performance liquid chromatography (HPLC), combined with Mass Spectrometry (MS), was used to isolate and identify Cu/Zn-SOD from the serum of Pinctada fucata martensii (P. f. martensii) and was designated as PmECSOD. With a length of 1864 bp and an open reading frame (ORF) of 1422 bp, the cDNA encodes a 473 amino acid protein. The PmECSOD transcript was detected in multiple tissues by quantitative real-time PCR (qRT-PCR), with its highest expression level being in the gills. Additionally, the temporal expression of PmECSOD mRNA in the hemolymph was highest at 48 h after in vivo stimulation with Escherichia coli and Micrococcus luteus. The results from this study provide a valuable base for further exploration of molluscan innate immunity and immune response.

A novel birnavirus identified as the causative agent of summer atrophy of pearl oyster (Pinctada fucata (Gould)).

The Akoya pearl oyster (Pinctada fucata (Gould)) is the most important species for pearl cultivation in Japan. Mass mortality of 0-year-old juvenile oysters and anomalies in adults, known as summer atrophy, have been observed in major pearl farming areas during the season when seawater temperatures exceed about 20 °C since 2019. In this study, we identified a novel birnavirus as the pathogen of summer atrophy and named it Pinctada birnavirus (PiBV). PiBV was first presumed to be the causative agent when it was detected specifically and frequently in the infected oysters in a comparative metatranscriptomics of experimentally infected and healthy pearl oysters. Subsequently, the symptoms of summer atrophy were reproduced by infection tests using purified PiBV. Infection of juvenile oysters with PiBV resulted in an increase in the PiBV genome followed by the atrophy of soft body and subsequent mortality. Immunostaining with a mouse antiserum against a recombinant PiBV protein showed that the virus antigen was localized mainly in the epithelial cells on the outer surface of the mantle. Although the phylogenetic analysis using maximum likelihood method placed PiBV at the root of the genus Entomobirnavirus, the identity of the bi-segmented, genomic RNA to that of known birnaviruses at the full-length amino acid level was low, suggesting that PiBV forms a new genus. The discovery of PiBV will be the basis for research to control this emerging disease.

cAMP-Mediated CREM-MITF-TYR Axis Regulates Melanin Synthesis in Pacific Oysters.

Colorful shells in bivalves are mostly caused by the presence of biological pigments, among which melanin is a key component in the formation of shell colours. Cyclic adenosine monophosphate (cAMP) is an important messenger in the regulation of pigmentation in some species. However, the role of cAMP in bivalve melanogenesis has not yet been reported. In this study, we performed in vitro and in vivo experiments to determine the role of cAMP in regulating melanogenesis in Pacific oysters. Besides, the function of cAMP-responsive element modulator (CREM) and the interactions between CREM and melanogenic genes were investigated. Our results showed that a high level of cAMP promotes the expression of melanogenic genes in Pacific oysters. CREM controls the expression of the MITF gene under cAMP regulation. In addition, CREM can regulate melanogenic gene expression, tyrosine metabolism, and melanin synthesis. These results indicate that cAMP plays an important role in the regulation of melanogenesis in Pacific oysters. CREM is a key transcription factor in the oyster melanin synthesis pathway, which plays a crucial role in oyster melanin synthesis through a cAMP-mediated CREM-MITF-TYR axis.

Effects of titanium dioxide nanoparticle exposure on the gut microbiota of pearl oyster (Pinctada fucata martensii).

With the advancement of nanotechnology and the growing utilization of nanomaterials, titanium dioxide (TiO2) has been released into aquatic environments, posing potential ecotoxicological risks to aquatic organisms. In this study, the toxicological effects of TiO2 nanoparticles were investigated on the intestinal health of pearl oyster (Pinctada fucata martensii). The pearl oysters were subjected to a 14-day exposure to 5-mg/L TiO2 nanoparticle, followed by a 7-day recovery period. Subsequently, the intestinal tissues were analyzed using 16S rDNA high-throughput sequencing. The results from LEfSe analysis revealed that TiO2 nanoparticle increased the susceptibility of pearl oysters to potential pathogenic bacteria infections. Additionally, the TiO2 nanoparticles led to alterations in the abundance of microbial communities in the gut of pearl oysters. Notable changes included a decrease in the relative abundance of Phaeobacter and Nautella, and an increase in the Actinobacteria, which could potentially impact the immune function of pearl oysters. The abundance of Firmicutes and Bacteroidetes, as well as the expression of genes related to energy metabolism (AMPK, PK, SCS-1, SCS-2, SCS-3), were down-regulated, suggesting that TiO2 nanoparticles exposure may affect the digestive and energy metabolic functions of pearl oysters. Furthermore, the short-term recovery of seven days did not fully restore these levels to normal. These findings provide crucial insights and serve as an important reference for understanding the toxic effects of TiO2 nanoparticles on bivalves.

Substantial gene expression shifts during larval transitions in the pearl oyster Pinctada margaritifera.

Early development stages in marine bivalve are critical periods where larvae transition from pelagic free-life to sessile mature individuals. The successive metamorphosis requires the expression of key genes, the functions of which might be under high selective pressure, hence understanding larval development represents key knowledge for both fundamental and applied research. Phenotypic larvae development is well known, but the underlying molecular mechanisms such as associated gene expression dynamic and molecular cross-talks remains poorly described for several nonmodel species, such as P. margaritifera. We designed a whole transcriptome RNA-sequencing analysis to describe such gene expression dynamics following four larval developmental stages:  d-shape, Veliger, Umbo and Eye-spot. Larval gene expression and annotated functions drastically diverge. Metabolic function (gene expression related to lipid, amino acid and carbohydrate use) is highly upregulated in the first development stages, with increasing demand from  d-shape to umbo. Morphogenesis and larval transition are partly ordered by Thyroid hormones and Wnt signaling. While larvae shells show some similar characteristic to adult shells, the cause of initialization of biomineralization differ from the one found in adults. The present study provides a global overview of Pinctada margaritifera larval stages transitioning through gene expression dynamics, molecular mechanisms and ontogeny of biomineralization, immune system, and sensory perception processes.

Phosphoproteomics changes due to allograft-induced stress responses of Pinctada fucata martensii.

Protein phosphorylation modifications are post-translational modifications (PTMs) that play important roles in signal transduction and immune regulation. Implanting a spherical nucleus into a recipient shellfish is critical in marine pearl aquaculture. Protein phosphorylation may be important in the immune responses of Pinctada fucata martensii after nucleus implantation, but their involvement in regulation remains unclear. Here, phosphoproteomics of P. f. martensii gill tissues was conducted 12 h after nuclear implantation using label-free data-independent acquisition (DIA) with LC-MS/MS. Among the 4024 phosphorylated peptides with quantitative information, 181 were up-regulated and 148 were down-regulated. Functional enrichment analysis of these differentially expressed phosphorylated proteins (DEPPs) revealed significant enrichment in functions related to membrane trafficking, exosomes, cytoskeleton, and signal transduction mechanisms. Further, 16 conserved motifs were identified among the DEPPs, including the RSphP, SphP, RSphA, RSphE, PTphP, and ATphP motifs that were significantly conserved, and which may be related to specific kinase recognition. Parallel response monitoring (PRM) analysis validated the abundances of 12 DEPPs from the proteomics, indicating that the phosphoproteomics analyses were robust. 12 DEPPs were selected from the proteomics results through Quantitative real-time PCR (qPCR) technology, and verification analysis was conducted at the gene level. The study suggests that kinases such as MAPKs, Akt, and CK2 may regulate the phosphorylation of related proteins following nuclear implantation. Furthermore, the important signaling pathways of Rap 1, IL-17A, and NF-κB, which are influenced by phosphorylated or dephosphorylated proteins, are found to be involved in this response. Overall, this study revealed the protein phosphorylation responses after nucleus implantation in P. f. martensii, helping to elucidate the characteristics and mechanisms of immune regulation responses in P. f. martensii, in addition to promoting a further understanding of protein phosphorylation modification functions in P. f. martensii.

Transcriptome and exosome proteome analyses provide insights into the mantle exosome involved in nacre color formation of pearl oyster Pinctada fucata martensii.

Color polymorphisms in molluscan shells play an important economic in the aquaculture industry. Among bivalves, shell color diversity can reflect properties such as growth rate and tolerance. In pearl oysters, the nacre color of the donor is closely related to the pearl color. Numerous genes and proteins involved in nacre color formation have been identified within the exosomes of the mantle. In this study, we analyzed the carotenoids present in the mantle of gold- and silver-lipped pearl oysters, identifying capsanthin and xanthophyll as crucial pigments contributing to coloration. Transcriptome analysis of the mantle revealed several differentially expressed genes (DEGs) involved in color formation, including ferric-chelate reductase, mantle genes, and larval shell matrix proteins. We also isolated and identified exosomes from the mantles of both gold- and silver-lipped strains of the pearl oyster Pinctada fucata martensii, revealing the extracellular transition mechanism of coloration-related proteins. From these exosomes, we obtained a total of 1223 proteins, with 126 differentially expressed proteins (DEPs) identified. These proteins include those associated with carotenoid metabolism and Fe(III) metabolism, such as apolipoproteins, scavenger receptor proteins, ß,ß-carotene-15,15'-dioxygenase, ferritin, and ferritin heavy chains. This study may provide a new perspective on the nacre color formation process and the pathways involved in deposition within the pearl oyster P. f. martensii.

Effect of elevated temperature, sea water acidification, and phenanthrene on the expression of genes involved in the shell and pearl formation of economic pearl oyster (Pinctada radiata).

Our study aims to examine the effect of some stressors on the gene expression levels of shell matrix proteins in a pearl oyster. Oysters were exposed to the different combinations of the temperature, pH, and phenanthrene concentration is currently measured in the Persian Gulf and the predicted ocean warming and acidification for 28 days. The expression of all the studied genes was significantly downregulated. Time and temperature had the greatest effects on the decreases in n19 and n16 genes expression, respectively. Aspein and msi60 genes expression were highly influenced by pH. Pearlin was affected by double interaction temperature and phenanthrene. Moreover, a correlation was observed among the expression levels of studied genes. This study represents basic data on the relationship between mRNA transcription genes involved in the shell and pearl formation and climate changes in pollutant presence conditions and acclimatizing mechanism of the oyster to the future scenario as well.

Whole-genome bisulfite sequencing reveals the function of DNA methylation in the allotransplantation immunity of pearl oysters.

Introduction: In the pearl culture industry, a major challenge is the overactive immunological response in pearl oysters resulting from allotransplantation, leading to shell-bead rejection and death. To better understand the molecular mechanisms of postoperative recovery and the regulatory role of DNA methylation in gene expression, we analyzed the changes in DNA methylation levels after allotransplantation in pearl oyster Pinctada fucata martensii, and elucidated the regulatory function of DNA methylation in promoter activity of nicotinic acetylcholine receptor (nAChR) gene. Methods: We constructed nine DNA methylomes at different time points after allotransplantation and used bisulfite genomic sequencing PCR technology (BSP) to verify the methylation status in the promoter of nAChR. We performed Dual luciferase assays to determine the effect of the dense methylation region in the promoter on transcriptional activity and used DNA pull-down and mass spectrometry analysis to assess the capability of transcription factor binding with the dense methylation region. Result: The DNA methylomes reveal that CG-type methylation is predominant, with a trend opposite to non-CG-type methylation. Promoters, particularly CpG island-rich regions, were less frequently methylated than gene function elements. We identified 5,679 to 7,945 differentially methylated genes (DMGs) in the gene body, and 2,146 to 3,385 DMGs in the promoter at each time point compared to the pre-grafting group. Gene ontology and pathway enrichment analyses showed that these DMGs were mainly associated with "cellular process", "Membrane", "Epstein-Barr virus infection", "Notch signaling pathway", "Fanconi anemia pathway", and "Nucleotide excision repair". Our study also found that the DNA methylation patterns of the promoter region of nAChR gene were consistent with the DNA methylomics data. We further demonstrated that the dense methylation region in the promoter of nAChR affects transcriptional activity, and that the methylation status in the promoter modulates the binding of different transcription factors, particularly transcriptional repressors. Conclusion: These findings enhance our understanding of the immune response and regulation mechanism induced by DNA methylation in pearl oysters after allotransplantation.

Toll-like receptor may be involved in acquired immune response in pearl oyster Pinctada fucata.

The increasing experimental evidence suggests that there are some forms of specific acquired immunity in invertebrates, in which Toll-like receptors (TLRs) play vital roles in activating innate and adaptive immunity and have been comprehensively investigated in mammalian species. Yet, the immune mechanisms underlying TLR mediation in mollusks remain obscure. In this study, we identified a TLR13 gene in the pearl oyster Pinctada fucata for the first time and named it PfTLR13 which consists of a 5'-untranslated terminal region (5'-UTR) of 543 bp, an open reading frame (ORF) of 2667 bp, and a 3'-UTR of 729 bp. We found that PfTLR13 mRNA was expressed in all tissues examined, with the highest level in the gills. The expression of PfTLR13 in the gills of oysters exposed to Vibrio alginolyticus or pathogen-associated molecular patterns (PAMPs) (including LPS, PGN, and poly(I:C)) was significantly higher than in the control group. Interestingly, the immune response to the first stimulation was weaker than the response to the second stimulation, suggesting that the primary stimulation may lead to immune priming of TLR in pearl oysters, similar to acquired immunity in vertebrates. Furthermore, we found that PfTLR13 expression was differentially associated with allograft and xenograft in the pearl oyster P. fucata, with the highest expression levels observed at 12 h post-allograft and 24 h post-xenograft. Overall, our findings provide new insights into the immune mechanisms underlying TLR mediation in mollusks and suggest that PfTLR13 may play a crucial role in the specific acquired immunity of pearl oysters.

Purification and Characterization of the Lecithin-Dependent Thermolabile Hemolysin Vhe1 from the Vibrio sp. Strain MA3 Associated with Mass Mortality of Pearl Oyster (Pinctada fucata).

In a previous study, we isolated a Vibrio sp. strain MA3 and its virulence factor, a hemolysin encoded by vhe1. This strain is associated with mass mortalities of the pearl oyster Pinctada fucata. In the present study, the vhe1 gene from strain MA3 was cloned and its encoded product was purified and characterized. Our results show that the vhe1 gene encodes a protein of 417 amino acids with an estimated molecular mass of 47.2 kDa and a pI of 5.14. The deduced protein, Vhe1, was found to contain the conserved amino acid sequence (GDSL motif) of the hydrolase/esterase superfamily and five conserved blocks characteristic of SGNH hydrolases. A BLAST homology search indicated that Vhe1 belongs the lecithin-dependent hemolysin/thermolabile hemolysin (LDH/TLH) family. In activity analyses, the optimal temperature for both the hemolytic and phospholipase activities of Vhe1 was 50 °C. Vhe1 hemolytic activity and phospholipase activity were highest at pH 8.5 and pH 8.0, respectively. However, both enzymatic activities sharply decreased at high temperature (> 50 °C) and pH < 7.0. Compared with previously reported hemolysins, Vhe1 appeared to be more thermal- and pH-labile. Both its hemolytic activity and phospholipase activity were significantly inhibited by CuCl2, CdCl2, ZnCl2, and NiCl2, and slightly inhibited by MnCl2 and CoCl2. Vhe1 showed higher phospholipase activity toward medium-chain fatty acids (C8-C12) than toward shorter- and longer-chain fatty acids. These results accumulate knowledge about the LDH/TLH of V. alginolyticus, which detailed characterization has not been reported, and contribute to solving of the mass mortality of pearl oyster.

The first reporting of prevalence Vibrio species and expression of HSP genes in rayed pearl oyster (Pinctada radiata) under thermal conditions.

The main goal of the present study was to evaluate the influence of thermal exposure on Vibrio population and HSP genes expression (HSP 90, HSP70, and HSP20) in rayed pearl oyster (P. radiata). To this end, the oysters were reared for 30 days at temperatures of 22 °C (control), 25 °C, 27 °C, and 29 °C. The results showed that five dominate Vibrio strains including Vibrio hepatarius, V. harveyi, V. alginolyticus, V. parahaemolyticus, and V. rotiferianus were identified. The highest population of V. parahaemolyticus, V. alginolyticus, and V. harveyi, was found in 29οC group. According to real-time PCR, mantle exhibited the highest expression levels of HSP20, HSP70, and HSP90 genes. A higher level of HSP20 expression was observed at high temperatures (25 °C, 27 °C, and 29 °C) in the gonad and mantle compared to the control group (22 °C) while decrease in HSP90 expression level was recorded in 25 °C, 27 °C, and 29 °C groups. HSP20 expression level in adductor muscle was remarkably down-regulated in 27 °C and 29 °C groups. In this tissue, HSP70 was detected at highest levels in the 29οC group. In mantle, HSP90 gene expression was lowest at 22 °C water temperature. Several Vibrio strains have been identified from pearl Gulf oyster that haven't been previously reported. The identification of dominant Vibrio species is essential for epidemiological management strategies to control and prevent Vibrio outbreaks in pearl oyster farms. The expression pattern of HSP genes differs in rayed pearl oyster tissues due to differences in their thermal tolerance capability and physiological and biological characteristics. The present study provides useful molecular information for the ecological adaptation of rayed pearl oysters after exposure to different temperature levels.

Short-term in vitro exposure of Pinctada imbricata's haemocytes to quaternium-15: Exploring physiological and cellular responses.

Since the 2000 s, the pearl oyster Pinctada imbricata (Röding, 1798) has become established along the transitional waterways of the "Capo Peloro Lagoon" natural reserve, where it is now abundant due to its adaptability to different hydrological, climatic, environmental, and pollution conditions. This study aims to evaluate haemocyte immune-mediated responses in vitro to quaternium-15, a common pollutant in aquatic ecosystems. Cell viability and phagocytosis activity decreased when exposed to 0.1 or 1 mg L- 1 of quaternium-15. Moreover, decreasing phagocytosis was confirmed by gene expression modulation of actin, involved in cytoskeleton rearrangement. Effects on oxidative stress-related genes were also assessed (Cat, MnSod, Zn/CuSod, GPx). The qPCR data revealed alterations in antioxidant responses through gene dose- and time-dependent modulation. This study presents insights into the physiological responses and cellular mechanisms of P. imbricata haemocytes to environmental stressors, indicating that this species is useful as a novel bioindicator for future toxicological studies.

miR-10a-3p Participates in Nacre Formation in the Pearl Oyster Pinctada fucata martensii by Targeting NPY.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression via the recognition of their target messenger RNAs. MiR-10a-3p plays an important role in the process of ossification. In this study, we obtained the precursor sequence of miR-10a-3p in the pearl oyster Pinctada fucata martensii (Pm-miR-10a-3p) and verified its sequence by miR-RACE technology, and detected its expression level in the mantle tissues of the pearl oyster P. f. martensii. Pm-nAChRsα and Pm-NPY were identified as the potential target genes of Pm-miR-10a-3p. After the over-expression of Pm-miR-10a-3p, the target genes Pm-nAChRsα and Pm-NPY were downregulated, and the nacre microstructure became disordered. The Pm-miR-10a-3p mimic obviously inhibited the luciferase activity of the 3' untranslated region of the Pm-NPY gene. When the interaction site was mutated, the inhibitory effect disappeared. Our results suggested that Pm-miR-10a-3p participates in nacre formation in P. f. martensii by targeting Pm-NPY. This study can expand our understanding of the mechanism of biomineralization in pearl oysters.

Reproductive condition of the black-lip pearl oyster Pinctada margaritifera during the lunar phase.

This study analyzed the relationship between the lunar phase and the reproductive cycle of Pinctada margaritifera inhabiting Weno Island, Chuuk Lagoon, Micronesia. We measured indicators of maturity (gonadosomatic index [GSI] and sexual maturation-related genes) and investigated changes in the gonadal maturity stages (GMS) of P. margaritifera over lunar cycle. GSI was higher around the full moon. GMS of P. margaritifera were classified as the early gametogenesis stage, ripe and spawning stage, and spent and degenerating stage. A large percentage of oysters was observed in the ripe and spawning stage at the first quarter moon in female and the full moon in male as well as in the spent and degenerating stages at the third quarter moon in both sexes. In addition, the expression of doublesex- and mab-3-related transcription factor 2 (DMRT2) in the male P. margaritifera black-lip pearl oyster was the highest during the full and third quarter moon phases, whereas no difference in expression was observed with the lunar phase in females. In contrast, the expression of vitellogenin (VTG) was the highest in female P. margaritifera during the first and third quarters. No difference in expression was observed according to the lunar phase in males. The results suggest that the lunar phase directly affects the expression of sexually mature gonads in P. margaritifera black-lip pearl oyster.

Integrated transcriptomic and metabolomic analysis sheds new light on adaptation of Pinctada fucata martensii to short-term hypoxic stress.

Analyses of the transcriptome and metabolome were conducted to clarify alterations of key genes and metabolites in pearl oysters following exposure to short-term hypoxic treatment. We totally detected 209 DEGs between the control and hypoxia groups. Enrichment analysis indicated the enrichment of GO terms including "oxidation-reduction process", "ECM organization", "chaperone cofactor-dependent protein refolding", and "ECM-receptor interaction" KEGG pathway by the DEGs. In addition, between the two groups, a total of 28 SDMs were identified, which were implicated in 13 metabolic pathways, such as "phenylalanine metabolism", "D-amino acid metabolism", and "aminoacyl-tRNA biosynthesis". Results suggest that pearl oysters are exposed to oxidative stress and apoptosis under short-term hypoxia. Also, pearl oysters might adapt to short-term hypoxic treatment by increasing antioxidant activity, modulating immune and biomineralization activities, maintaining protein homeostasis, and reorganizing the cytoskeleton. The results of our study help unveil the mechanisms by which pearl oysters respond adaptively to short-term hypoxia.

Genome-wide association study analysis to resolve the key regulatory mechanism of biomineralization in Pinctada fucata martensii.

Biomineralization-controlled exo-/endoskeleton growth contributes to body growth and body size diversity. Molluscan shells undergo ectopic biomineralization to form the exoskeleton and biocalcified "pearl" involved in invading defence. Notably, exo-/endoskeletons have a common ancestral origin, but their regulation and body growth are largely unknown. This study employed the pearl oyster, Pinctada fucata marntensii, a widely used experimental model for biomineralization in invertebrates, to perform whole-genome resequencing of 878 individuals from wild and breeding populations. This study characterized the genetic architecture of biomineralization-controlled growth and ectopic biomineralization. The insulin-like growth factor (IGF) endocrine signal interacted with ancient single-copy transcription factors to form the regulatory network. Moreover, the "cross-phylum" regulation of key long noncoding RNA (lncRNA) in bivalves and mammals indicated the conserved genetic and epigenetic regulation in exo-/endoskeleton growth. Thyroid hormone signal and apoptosis regulation in pearl oysters affected ectopic biomineralization in pearl oyster. These findings provide insights into the mechanism underlying the evolution and regulation of biomineralization in exo-/endoskeleton animals and ectopic biomineralization.

The combined effects of temperature and salinity on the digestion and respiration metabolism of Pinctada fucata.

The combined effects of temperature and salinity on the digestion and respiration metabolism of Pinctada fucata were evaluated via response surface methodology and box-benhnken design under laboratory condition. Results indicated that the primary and secondary effects of salinity and temperature had significant effects on amylase (AMS) of P. fucata (P < 0.05)., The digestive enzyme reached the maximum activity when temperature was 26 °C. The AMS and trypsin (TRYP) increased at first, and then decreased with increasing temperature. The Lipase (LPS) was positively correlated with either salinity or temperature. Salinity had no significant effect on TRYP as a primary effect (P > 0.05), but had a significant effect on TRYP as a secondary effect (P < 0.01). These effects were completely opposite to the effect of temperature on pepsin (PEP) as primary and secondary effects. The combined effects of salinity and temperature on AMS, TRYP and PEP were significant (P < 0.01), but had no significant effect on LPS (P > 0.05). The primary, secondary and interaction effects of salinity had significant effects on NKA (Na+-K+-ATPase) of P. fucata (P < 0.05), and NKA presented a U-shaped distribution with increasing salinity. The quadratic and interactive effects of temperature had a significant effect on AKP (P < 0.05), and AKP showed a U-shaped distribution with increasing temperature. Lactate dehydrogenase (LDH) activity decreased at first, and then increased when temperature and salinity changed from 20 to 30 °C and 23-33 ‰, respectively. The expression of GPX gene affected by temperature in gills may be delayed compared with that in hepatopancreas, and its expression is tissue-specific. The appropriate digestion and respiratory metabolism index models were established under the combined temperature and salinity conditions. The optimization results showed that the optimal combination of temperature and salinity was 26.288 °C/28.272‰. The desirability was 0.832. Results from the present study will provide a theoretical reference for shellfish culture affected by environmental interactions and the establishment of related index models.