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.

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.

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.

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.

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.

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.

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.

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.

Development and evaluation of a rapid, specific, and sensitive loop-mediated isothermal amplification assay to detect Tenacibaculum sp. strain pbs-1 associated with black-spot shell disease in Akoya pearl oysters.

Black-spot shell disease decreases pearl quality and threatens pearl oyster survival. Establishment of a rapid, specific, and sensitive assay to detect Tenacibaculum sp. strain Pbs-1 associated with black-spot shell disease is of commercial importance. We developed a rapid, specific, and highly sensitive loop-mediated isothermal amplification (LAMP) assay to detect Tenacibaculum sp. Pbs-1 in Akoya pearl oysters Pinctada fucata. A set of five specific primers (two inner, two outer, and a loop) were designed based on the 16S-23S internal spacer region of strain Pbs-1. The optimum reaction temperature was 63 °C, and concentrations of the inner and loop primers were 1.4 and 1.0 µM, respectively. The LAMP product can be detected using agarose gel electrophoresis, and the color change in the reaction tube can be detected visually (by the naked eye) following the addition of malachite green. Our assay proved to be specific for strain Pbs-1, with no cross-reactivity with five other species of Tenacibaculum. The detection limit of the LAMP assay at 35 min is 50 pg, and at 60 min it is 5 fg. We evaluated the LAMP assay using diseased and healthy pearl oysters. The results demonstrate the suitability and simplicity of this test for rapid field diagnosis of strain Pbs-1.

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.

A high-quality, haplotype-phased genome reconstruction reveals unexpected haplotype diversity in a pearl oyster.

Homologous chromosomes in the diploid genome are thought to contain equivalent genetic information, but this common concept has not been fully verified in animal genomes with high heterozygosity. Here we report a near-complete, haplotype-phased, genome assembly of the pearl oyster, Pinctada fucata, using hi-fidelity (HiFi) long reads and chromosome conformation capture data. This assembly includes 14 pairs of long scaffolds (>38 Mb) corresponding to chromosomes (2n = 28). The accuracy of the assembly, as measured by an analysis of k-mers, is estimated to be 99.99997%. Moreover, the haplotypes contain 95.2% and 95.9%, respectively, complete and single-copy BUSCO genes, demonstrating the high quality of the assembly. Transposons comprise 53.3% of the assembly and are a major contributor to structural variations. Despite overall collinearity between haplotypes, one of the chromosomal scaffolds contains megabase-scale non-syntenic regions, which necessarily have never been detected and resolved in conventional haplotype-merged assemblies. These regions encode expanded gene families of NACHT, DZIP3/hRUL138-like HEPN, and immunoglobulin domains, multiplying the immunity gene repertoire, which we hypothesize is important for the innate immune capability of pearl oysters. The pearl oyster genome provides insight into remarkable haplotype diversity in animals.

Comprehensive analysis of the lncRNAs, mRNAs, and miRNAs implicated in the immune response of Pinctada fucata martensii to Vibrio parahaemolyticus.

Non-coding RNAs (ncRNAs) have been implicated in a variety of biological processes. However, most ncRNAs are of unknown function and are as-yet unannotated. The immune-related functions of ncRNAs in the pearl oyster Pinctada fucata martensii were explored based on transcriptomic differences in the expression levels of long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and messenger RNAs (mRNAs) in the hemocytes of P.f. martensii after challenge by the pathogenic bacterium Vibrio parahaemolyticus. Across the challenged and control pearl oysters, 144 miRNAs and 14,571 lncRNAs were identified. In total, 13,375 ncRNAs were differentially expressed between the challenged and control pearl oysters; in the challenged pearl oysters as compared to the controls, 15 miRNAs and 5147 lncRNAs were upregulated, while 51 miRNAs and 8162 lncRNAs were downregulated. The sequencing results were validated using quantitative real-time polymerase chain reaction (qRT-PCR) analysis. GO and KEGG pathway analysis showed that genes targeted by the differentially expressed ncRNAs were associated with the vascular endothelial growth factor (VEGF) signaling pathway and the nuclear factor kappa-B (NF-κB) signaling pathway. An lncRNA-mRNA-miRNA network that was developed based on the transcriptomic results of this study suggested that lncRNAs may compete with miRNAs for mRNA binding sites. This study may provide a useful framework for the detection of additional novel ncRNAs, as well as new insights into the pathogenic mechanisms underlying the response of P.f. martensii to V. parahaemolyticus.

Lysine acetylation plays a role in the allograft-induced stress response of the pearl oyster (Pinctada fucata martensii).

Implanting a spherical nucleus into a recipient oyster is a critical step in artificial pearl production using the pearl oyster Pinctada fucata martensii. However, little is known about the role of post-translational modifications (PTMs) in the response of the pearl oyster to this operation. Lysine acetylation, a highly conserved PTM, may be an essential adaptive strategy to manage multiple biotic or abiotic stresses. We conducted the first lysine acetylome analysis of the P. f. martensii gill 12 h after nucleus implantation, using tandem mass tags (TMT) labeling and Kac affinity enrichment. We identified 2443 acetylated sites in 1301 proteins, and 1511 sites on 895 proteins were quantitatively informative. We found 25 conserved motifs from all of the identified lysine sites, particularly motifs Kac H, Kac S, and Kac Y were strikingly conserved, of which Kac Y, Kac H, Y Kac, Kac K, Kac *K, Kac R, and Kac F which have been observed in other species and are therefore highly conserved. We identified 58 sites that were significantly differently acetylated in P. f. martensii in response to allograft (|fold change|>1.2, P-value ≤ 0.05); 38 newly acetylated and 20 deacetylated. According to GO functional analysis, subcellar location, and KOG classIfication, these proteins were divided into four categories: cytoskeleton, response to stimulus, metabolism, and other. The differentially acetylated proteins (DAPs) enriched pathways include aminoacyl-tRNA biosynthesis, salmonella infection, and longevity regulating pathway-worm-Caenorhabditis elegans (nematode). Parallel reaction-monitoring (PRM) validation of the differential acetylation of 10 randomly selected differentially acetylated sites from the acetylome analysis. These results indicated that our acetylome analysis results were sufficiently reliable and reproducible. These results provide an essential resource for in-depth exploration of the stress responses and adaptation mechanisms associated with lysine acetylation in marine invertebrates and P. f. martensii.