Search

Heat shock transcription factor 1 (HSF1) is an important activator of innate immunity in the response of invertebrates to pathogen invasion. In the present study, a HSF1 was characterized in Pacific oyster Magallana gigas (MgHSF1), and its association of single nucleotide polymorphisms with susceptibility/resistance of oyster to Halomonas sp. 7T were investigated. MgHSF1 shared highly conserved DNA binding and acetylation sites with other organisms. The MgHSF1 mRNA showed high expression in haemocytes, and was significantly induced by lipopolysaccharide stimulation. The single nucleotide polymorphisms (SNPs) within promoter region of MgHSF1 gene from two oyster populations (the bacterial-resistant population and the common population) were investigated to analyze their association with bacterial resistance. Nine out of 14 SNPs including -543 A/G, -494 C/T, -488 T/A, -476 G/A, -336 C/T, -335 T/C, -334 C/T, -307 T/C and -268 T/C were found to be associated with bacterial resistance. Moreover, haplotypes TCTCGA was associated with bacterial resistance. The results threw lights on the molecular mechanisms of different oyster populations' resistance to bacterial diseases which suggested that the increased bacterial resistance of bacterial-resistant population was associated with the higher expression of MgHSF1. Meanwhile, the nine genotypes (-543 G/G, -494 T/T, -488 A/A, -476 A/A, -336 T/T, -335 C/C, -334 T/T, -307 T/T, and -268 C/C) and one haplotype (TCTCGA) could be used as potential markers for oyster selection breeding with higher bacterial resistance., (Copyright © 2025 Elsevier Ltd. All rights reserved.)

Background: The genus Magallana represents the most commercially important oysters globally. Due to phenotypic plasticity, members of Magallana genus are often confused morphologically and therefore their taxonomical as well as conservation efforts are limited. Therefore, we aimed to sequence the complete mitochondrial genome (mitogenome) of Magallana gryphoides to explore its phylogenetic relationship., Methods and Results: By employing the next-generation sequencing, the complete mitochondrial genome (mitogenome) of M. gryphoides was sequenced for the first time in the present study. Results suggest that the mitogenome of M. gryphoides is of 18,313 bp in length (accession number OR177662). The mitogenome contains 13 protein-coding genes including atp8 and 26 tRNAs with 3 new suppressor tRNAs as well as 2 rRNAs. The absence of rrnL splitting is unique among Crassostreinae oysters and the positions of trnG, trnV, as well as nad2 genes in the mitogenome of M. gryphoides are significantly altered. Thus, the phylogenetic analysis conducted in the present study indicates a close relationship of M. gryphoides with the native Indian oyster M. bilineata and another Asian oyster M. iredalei., Conclusion: Data of the present study suggest a close relationship of M. gryphoides with the native Indian oyster M. bilineata and another Asian oyster M. iredalei. These results provide a new insight into phylogenetic relationship and may help to place the correct taxonomical status of these species that are often confused in the Ostreidae taxonomy due to phenotypic plasticity., Competing Interests: Declarations. Ethical approval: All the experiments meet ethical guidelines and adheres to the legal requirements of the study country. The Departmental Doctoral committee, Department of Zoology, University of Kerala, approved this research as part of the Ph.D program of the principal author, Vineetha V.S (Order No. Ac.EVI/563/2022/UOK dated 22.01.2022). Competing interests: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature B.V.)

Amplicon panels using genotyping by sequencing methods are now common, but have focused on characterizing SNP markers. We investigate how microhaplotype (MH) discovery within a recently developed Pacific oyster (Magallana gigas) amplicon panel could increase the statistical power for relationship assignment. Trios (offspring and two parents) from three populations in a newly established breeding program were genotyped on a 592 locus panel. After processing, 92% of retained amplicons contained polymorphic MH variants and 85% of monomorphic SNP markers contained MH variation. The increased allelic richness resulted in substantially improved power for relationship assignment with much lower estimated false positive rates. No substantive differences in assignment accuracy occurred between SNP and MH datasets, but using MHs increased the separation in log-likelihood values between true parents and highly related potential parents (aunts and uncles). A high number of Mendelian incompatibilities among trios were observed, likely due to null alleles. Further development of a MH panel, including removing loci with high rates of null alleles, would enable high-throughput genotyping by reducing panel size and therefore cost for Pacific oyster research and breeding programs., Competing Interests: Conflict of interest: BJGS is affiliated with Sutherland Bioinformatics. The author has no competing financial interests to declare., (Published by Oxford University Press on behalf of The Genetics Society of America 2024.)

Clock genes regulate physiological and metabolic processes by responding to changes in environmental light and temperature, and genetic variations in these genes may facilitate environmental adaptation, offering opportunities for resilience to climate change. However, the genetic and molecular mechanisms remain unclear in marine organisms. In this study, we investigated the role of a key clock gene, the circadian locomotor output cycles kaput ( Clock ), in thermal adaptation using DNA affinity purification sequencing (DAP-Seq) and RNA interference (RNAi)-based transcriptome analysis. In cold-adapted Crassostrea gigas and warm-adapted Crassostrea angulata , Clock was subject to environmental selection and exhibited contrasting expression patterns. The transcriptome analysis revealed 2054 differentially expressed genes (DEGs) following the knockdown of the Clock expression, while DAP-Seq identified 150,807 genes regulated by Clock , including 5273 genes located in promoter regions. The combined analyses identified 201 overlapping genes between the two datasets, of which 98 were annotated in public databases. These 98 genes displayed distinct expression patterns in C. gigas and C. angulata under heat stress, which were potentially regulated by Clock , indicating its role in a molecular regulatory network that responds to heat stress. Notably, a heat-shock protein 70 family gene ( Hsp12b ) and a tripartite motif-containing protein ( Trim3 ) were significantly upregulated in C. angulata but showed no significant changes in C. gigas , further highlighting their critical roles in thermal adaptation. This study preliminarily constructs a thermal regulatory network involving Clock , providing insights into the molecular mechanisms of clock genes in thermal adaptation., Competing Interests: All authors declare that they have no competing interests.

Pacific oysters face recurring outbreaks of Pacific Oyster Mortality Syndrome (POMS), a polymicrobial multifactorial disease. Although this interaction is increasingly understood, the role of epigenetics (e.g., DNA methylation) appears to be of fundamental importance because of its ability to shape oyster resistance/susceptibility and respond to environmental triggers, including infections. In this context, we comprehensively characterized basal (no infection) and POMS-induced changes in the methylome of resistant and susceptible oysters, focusing on the gills and mantle. Our analysis identified differentially methylated regions (DMRs) that revealed distinct methylation patterns uniquely associated with the susceptible or resistant phenotypes in each tissue. Enrichment analysis of genes bearing DMRs highlighted that these epigenetic changes were specifically linked to immunity, signaling, metabolism, and transport. Notably, 31 genes with well-known immune functions were differentially methylated after POMS, with contrasting methylation patterns between the phenotypes. Based on the methylome differences between phenotypes, we identified a set of candidate epibiomarkers that could characterize whether an oyster is resistant or susceptible (1998 candidates) and whether a site has been exposed to POMS (164 candidates). Overall, the findings provide a deeper understanding of the molecular interactions between oysters and POMS infection, opening new questions about the broader implications of epigenetic mechanisms in host-pathogen dynamics and offering promising strategies for mitigating the impacts of this devastating disease. Beyond its biological aspects, this study provides insights into potential epigenetic biomarkers for POMS disease management and targets for enhancing oyster health and productivity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier B.V. All rights reserved.)

The influence of sex and heredity on DNA methylation in the somatic tissues of mice has been well-documented, with similar hereditary effects reported in honeybees. However, the extent to which these factors affect DNA methylation in molluscan somatic tissues remains poorly understood. In this study, we investigated genomic DNA methylation patterns in the adductor muscle of two genetically distinct oyster strains using whole-genome bisulfite sequencing (WGBS). Our analysis identified significant differences in DNA methylation between sexes, with females exhibiting a global reduction compared to males. Furthermore, approximately half of the differentially methylated sites between the two parental strains were conserved in their offspring. Regions with differential methylation in parents typically exhibited intermediate methylation levels in the F1 progeny, whereas consistently methylated regions in parents maintained similar methylation levels in their progeny. These findings suggest that offspring DNA methylation is strongly influenced by parental methylation profiles, highlighting its potential role in sexual determination in oysters., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

DM9 domain-containing protein (DM9CP) as pattern recognition molecule is involved in regulating the inflammation-related signaling pathway in invertebrates. In the present study, a DM9CP with two tandem DM9 repeats (designated as CgDM9CP-8) was identified from Crassostrea gigas. The mRNA transcript of CgDM9CP-8 was the highest in haemocytes. Recombinant CgDM9CP-8 was able to bind various PAMPs (MAN, PGN and LPS) and microbes (Vibrio splendidus, Staphylococcus aureus, Bacillus subtilis, Pichia pastoris, and Yarrowia lipolytica). CgDM9CP-8 protein was distributed in haemocytes and cell-free haemolymph. After lipopolysaccharide (LPS) or V. splendidus stimulation, the mRNA expressions of CgDM9CP-8 in haemocytes were significantly up-regulated at 3 and 6 h, respectively. rCgDM9CP-8 exhibited binding activity to V. splendidus and E. coli, inhibiting their growth in vitro. CgDM9CP-8 was able to bind CgMASPL-1 both in vivo and in vitro to lead to complement CgC3 cleavage. In addition, in CgDM9CP-8-RNAi oysters, the mRNA expressions of interleukin 17s (CgIL17s) in haemocytes decreased significantly after V. splendidus stimulation. These results suggested that CgDM9CP-8 functioned as a pattern recognition and antibacterial molecule to regulate the mRNA expressions of CgIL17s through the activation of CgC3 in C. gigas., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier B.V. All rights reserved.)

Alternative splicing (AS) of pre-mRNA is a crucial mechanism that regulates the expression of genes involved in sex determination and differentiation. Despite its importance, AS has been rarely characterized in molluscs. In this study, PacBio Iso-Seq was employed to obtain full-length transcriptome and unveil AS patterns of gonads in the Pacific oyster Crassostrea gigas. A total of 24,783 AS events were identified across 6259 genes, with many enriched in phosphorylation-related processes. Splicing factors were found to drive a high frequency of AS events in gonads. Significant sex-based differences in isoform abundance and the incidence of AS events were observed. Comparative analysis of mature female and male gonads revealed a subset of overlapping differential alternative splicing genes and differentially expressed genes enriched in processes related to microtubule function and cell motility. In addition, the expression levels of sex-biased genes were found correlated with their isoform number in both female and male gonads. A novel isoform of Dmrt1 was identified with male specific expression in mature gonads. This study provides the first comprehensive understanding of full-length transcriptome and AS patterns in molluscan gonads, shedding light on the post-transcriptional regulatory mechanisms underlying sex determination and differentiation in molluscs and potentially across other animals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Maintaining genetic diversity in cultured shellfish can be challenging due to high variance in individual reproductive success, founder effects, and rapid genetic drift, but is important to retain adaptive potential and avoid inbreeding depression. To support broodstock management and selective breeding in cultured Pacific oysters (Crassostrea (Magallana) gigas), we developed an amplicon panel targeting 592 genomic regions and SNP variants with an average of 50 amplicons per chromosome. Target SNPs were selected based on elevated observed heterozygosity or differentiation in Pacific oyster populations in British Columbia, Canada. The use of the panel for parentage applications was evaluated using multiple generations of oysters from a breeding program on Vancouver Island, Canada (n = 181) and families selected for Ostreid herpesvirus-1 resistance from the Molluscan Broodstock Program in Oregon, USA (n = 136). Population characterization was evaluated using wild, naturalized, farmed, or hatchery oysters sampled throughout the Northern Hemisphere (n = 189). Technical replicates showed high genotype concordance (97.5%; n = 68 replicates). Parentage analysis found suspected pedigree and sample handling errors, demonstrating the panel's value for quality control in breeding programs. Suspected null alleles were identified and found to be largely population dependent, suggesting population-specific variation impacting target amplification. Null alleles were identified using existing data without the need for pedigree information, and once they were removed, assignment rates increased to 93.0 and 86.0% of possible assignments in the two breeding program datasets. A pipeline for analyzing the amplicon sequence data from sequencer output, amplitools, is also provided., Competing Interests: Conflicts of interest B.J.G.S. is affiliated with Sutherland Bioinformatics. The author has no competing financial interests to declare. Some authors affiliated with ThermoFisher Scientific have potential conflicts considering that the AgriSeq Targeted Genotyping by Sequencing solutions and associated Oligo panels that were designed and validated in the study are offered by ThermoFisher Scientific. However, the selection of markers, and data analysis was primarily conducted by other authors. The authors declare that the research was conducted in a scientific manner without any commercial considerations that could be construed as potential conflict of interest and further declare no other conflicts of interest. The other authors declare no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Recent advances in genetic manipulation such as triploid breeding and artificial selection, have rapidly emerged as valuable hatchery methodologies for enhancing seafood stocks. The Pacific oyster Magallana gigas is a leading aquaculture species worldwide and key ecosystem engineer that has received particular attention in this field of science. In light of the growing recognition of the ecological effects of intraspecific variation, oyster polyploids provide a valuable opportunity to assess whether intraspecific diversity affects physiological responses to environmental stressors. While the responses of diploid and triploid oysters to climate change have been extensively investigated, research on their sensitivity to environmental pollution remains scarce. Here, we assess whether genotypic (i.e., ploidy) variation within Magallana gigas affects physiological responses to microplastic pollution. We show that diploid and triploid M. gigas have similar clearance rates and ingest similar amounts of microplastics under laboratory-controlled condition. In addition, they exhibited similar heart rates after prolonged exposure to microplastic leachates. Our findings suggest that intraspecific variations within M. gigas ploidy does not affect oyster responses to microplastic pollution. However, regardless of ploidy, our work highlights significant adverse effects of microplastic leachates on the heart rate of M. gigas and provides evidence of microplastic ingestion in the laboratory., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This study exposed adult Sydney rock oysters, of either sex or both, to the synthetic estrogen 17α-ethinylestradiol (EE2) at 50 ng/L for 21 days, followed by an examination of developmental endpoints and transcriptomic responses in unexposed larvae. Reduced survival was observed at 1 day post-fertilisation (dpf) in larvae from bi-parental exposure (F T M T ). Motile larvae at 2 dpf were fewer from maternal (F T M C ), paternal (F C M T ), and F T M T exposures. Additionally, shell length at 7 dpf decreased in larvae from F T M C and F T M T parents. RNA sequencing (RNA-seq) revealed 1064 differentially expressed genes (DEGs) in 1-dpf larvae from F T M T parents, while fewer DEGs were detected in larvae from F T M C and F C M T parents, with 258 and 7, respectively. GO and KEGG analyses showed significant enrichment of DEGs in diverse terms and pathways, with limited overlap among treatment groups. IPA results indicated potential inhibition of pathways regulating energy production, larval development, transcription, and detoxification of reactive oxygen species in F T M T larvae. qRT-PCR validation confirmed significant downregulation of selected DEGs involved in these pathways and relevant biological processes, as identified in the RNA-seq dataset. Overall, our results suggest that the intergenerational toxicity of EE2 is primarily maternally transmitted, with bi-parental exposure amplifying these effects., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: G R MacFarlane reports financial support was provided by Australian Research Council. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The molluscan family Ostreidae, commonly known as oysters, is an important molluscan group due to its economic and ecological importance. In recent years, an abundance of genomic data of Ostreidae species has been generated and available in public domain. However, there is still a lack of a high-efficiency database platform to store and distribute these data with comprehensive tools. In this study, we developed an oyster genome database (OysterDB) to consolidate oyster genomic data. This database includes eight oyster genomes and 208,923 protein-coding gene annotations. Bioinformatic tools, such as BLAST and JBrowse, are integrated into the database to provide a user-friendly platform for homologous sequence searching, visualization of genomes, and screen for candidate gene information. Moreover, OysterDB will be continuously updated with ever-growing oyster genomic resources and facilitate future studies for comparative and functional genomic analysis of oysters ( http://oysterdb.com.cn/ )., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Heat shock protein 70 (HSP70), the most prominent and well-characterized stress protein in animals, plays an important role in assisting animals in responding to various adverse conditions. In the present study, a total of 113 HSP70 gene family members were identified in the updated genome of Magallana gigas (designated MgHSP70) (previously known as Crassostrea gigas). There were 75, 12, 11, and 8 HSP70s located in the cytoplasm, nucleus, mitochondria, and endoplasmic reticulum, respectively, and 7 HSP70s were located in both the nucleus and cytoplasm. Among 113 MgHSP70 genes, 107 were unevenly distributed in 8 chromosomes of M. gigas with the greatest number in chromosome 07 (61 genes, 57.01%). The MgHSP70 gene family members were mainly assigned into five clusters, among which the HSPa12 subfamily underwent lineage-specific expansion, consisting of 89 members. A total of 68 MgHSP70 genes (60.18%) were tandemly duplicated and formed 30 gene pairs, among which 14 gene pairs were under strong positive selection. In general, the expression of MgHSP70s was tissue-specific, with the highest expression in labial palp and gill and the lowest expression in adductor muscle and hemocytes. There were 35, 31, and 47 significantly upregulated genes at 6, 12, and 24 h after heat shock treatment (28 °C), respectively. The expression patterns of different tandemly duplicated genes exhibited distinct characteristics after shock treatment, indicating that these genes may have different functions. Nevertheless, genes within the same tandemly duplicated group exhibit similar expression patterns. Most of the tandemly duplicated HSP70 gene pairs showed the highest expression levels at 24 h. This study provides a comprehensive description of the MgHSP70 gene family in M. gigas and offers valuable insights into the functions of HSP70 in the mollusc adaptation of oysters to environmental stress., Competing Interests: Declarations of interest Chuanyan Yang and Linsheng Song report that financial support was provided by the National Natural Science Foundation of China and the Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai). Lingling Wang reports that financial support was provided by Dalian High Level Talent Innovation Support Programs. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Background: The ecology and biology of oysters (Ostreidae) across the tropics is poorly understood. Morphological plasticity and shared characteristics among oysters have resulted in the misidentification of species, creating challenges for understanding basic species-specific biological information that is required for restoration and aquaculture. Genetic barcoding has proven essential for accurate species identification and understanding species geographic ranges. To reduce the costs of molecular species identification we developed multiplex assays using the cytochrome c oxidase subunit I (COI or cox1) barcoding gene for the rapid identification of five species of oysters within the genus Saccostrea that are commonly found in Queensland, Australia: Saccostrea glomerata, Saccostrea lineage B, Saccostrea lineage F, Saccostrea lineage G, and Saccostrea spathulata (lineage J)., Results: Multiplex assays were successful in species-specific amplification of targeted species. The practical application of these primers was tested on wild spat collected from a pilot restoration project in Moreton Bay, Queensland, with identified species (S. glomerata, lineage B and lineage G) validated by Sanger sequencing. DNA sampling by extraction of oyster pallial fluid was also tested on adult oysters collected from the Noosa estuary in Queensland to assess whether oysters were able to be identified non-destructively. DNA concentrations as low as 1 ng/ μL still amplified in most cases, allowing for identification, and mortality at 6 weeks post pallial fluid collection was low (3 out of 104 sampled oysters)., Conclusion: These multiplex assays will be essential tools for species identification in future studies, and we successfully demonstrate their practical application in both restoration and aquaculture contexts in Queensland. The multiplex assays developed in this study outline easily replicable methods for the development of additional species-specific primer sets for the rapid identification of other species of Saccostrea found across the Indo-Pacific, which will be instrumental in unravelling the taxonomic ambiguities within this genus in tropical regions., (© 2024. The Author(s).)

16S rRNA short amplicon sequencing-based microbiota profiling has been thought of and suggested as a feasible method to assess food safety. However, even if a comprehensive microbial information can be obtained by microbiota profiling, it would not be necessarily sufficient for all circumstances. To prove this, the feasibility of the most widely used V3-V4 amplicon sequencing method for food safety assessment was examined here. We designed a pathogen ( Vibrio parahaemolyticus ) contamination and/or V. parahaemolyticus -specific phage treatment model of raw oysters under improper storage temperature and monitored their microbial structure changes. The samples stored at refrigerator temperature (negative control, NC) and those that were stored at room temperature without any treatment (no treatment, NT) were included as control groups. The profiling results revealed that no statistical difference exists between the NT group and the pathogen spiked- and/or phage treated-groups even when the bacterial composition was compared at the possible lowest-rank taxa, family/genus level. In the beta-diversity analysis, all the samples except the NC group formed one distinct cluster. Notably, the samples with pathogen and/or phage addition did not form each cluster even though the enumerated number of V. parahaemolyticus in those samples were extremely different. These discrepant results indicate that the feasibility of 16S rRNA short amplicon sequencing should not be overgeneralized in microbiological safety assessment of food samples, such as raw oyster.

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 29 ο C 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 29 ο C 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., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The N 6 -methylation of RNA adenosines (N 6 -methyladenosine, m 6 A) is an important regulator of gene expression with critical implications in vertebrate and insect development. However, the developmental significance of epitranscriptomes in lophotrochozoan organisms remains unknown. Using methylated RNA immunoprecipitation sequencing (MeRIP-seq), we generated transcriptome-wide m 6 A-RNA methylomes covering the entire development of the oyster from oocytes to juveniles. Oyster RNA classes display specific m 6 A signatures, with messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) exhibiting distinct profiles and being highly methylated compared to transposable element (TE) transcripts. Epitranscriptomes are dynamic and correspond to the chronological steps of development (cleavage, gastrulation, organogenesis, and metamorphosis), with minimal mRNA and lncRNA methylation at the morula stage followed by a global increase. mRNA m 6 A levels are correlated with transcript levels, and shifts in methylation profiles correspond to expression kinetics. Differentially methylated transcripts cluster according to embryo-larval stages and bear the corresponding developmental functions (cell division, signal transduction, morphogenesis, and cell differentiation). The m 6 A level of TE transcripts is also regulated and peaks during the gastrulation. We demonstrate that m 6 A-RNA methylomes are dynamic and associated with gene expression regulation during oyster development. The putative epitranscriptome implication in the cleavage, maternal-to-zygotic transition, and cell differentiation in a lophotrochozoan model brings new insights into the control and evolution of developmental processes., (Copyright © 2022 Beijing Institute of Genomics, Chinese Academy of Sciences and Genetics Society of China. Published by Elsevier B.V. All rights reserved.)

As the second largest phylum in the zoological kingdom next to arthropods, the mechanism of gonadal differentiation in mollusca is quite complex. Currently, although much has been carried out on gonadal differentiation in the Pacific oyster, there is still unknown information that needs to be further explored. Here, analysis of the Foxl2 and Dmrt1l expression in samples at different development periods of male and female gonads as well as in annual gonad samples revealed that Log 10 (Foxl2/Dmrt1l) values were an effective method for sex identification in oysters. In differentiated gonadal tissue, Log 10 (Foxl2/Dmrt1l) values greater than 2 were females and less than 1 for males. Subsequent sequential sampling of the same individuals verified that Log 10 (Foxl2/Dmrt1l) values greater than 2 for resting gonads would develop as females and less than 1 would develop as males in the future. Relative expression analysis of Foxl2 and Dmrt1l in the annual samples revealed a negative correlation between Log 10 (Foxl2) and Log 10 (Dmrt1l). Double fluorescence reporter validation results showed that DMRT1L protein was able to bind the Foxl2 promoter and repress its activity with a weak dosage effect. Antagonism between Dmrt1l and Foxl2 is therefore not restricted to vertebrates, and the competing regulatory networks are of great significance in the maintenance of gonadal sex in oysters after sexual differentiation. This study provides novel ideas and insights into the study of early gonadal differentiation in the adult oyster., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Due to translocation heterozygosity for all chromosomes in the cell complement, the oyster plant (Tradescantia spathacea) forms a complete meiotic ring. It also shows Rabl-arrangement at interphase, featured by polar centromere clustering. We demonstrate that the pericentromeric regions of the oyster plant are homogenized in concert by three subtelomeric sequences: 45S rDNA, (TTTAGGG)n motif, and TSrepI repeat. The Rabl-based clustering of pericentromeric regions may have been an excellent device to combine the subtelomere-pericentromere sequence migration (via inversions) with the pericentromere-pericentromere DNA movement (via whole arm translocations) that altogether led to the concerted homogenization of all the pericentromeric domains by the subtelomeric sequences. We also show that the repetitive sequence landscape of interstitial chromosome regions contains many loci consisting of Arabidopsis-type telomeric sequence or of TSrepI repeat, and it is extensively heterozygous. However, the sequence arrangement on some chromosomal arms suggest segmental inversions that are fully or partially homozygous, a fact that could be explained if the inversions started to create linkages already in a bivalent-forming ancestor. Remarkably, the subterminal TSrepI loci reside exclusively on the longer arms that could be due to sharing sequences between similarly-sized chromosomal arms in the interphase nucleus. Altogether, our study spotlights the supergene system of the oyster plant as an excellent model to link complex chromosome rearrangements, evolution of repetitive sequences, and nuclear architecture., (© 2022. The Author(s).)

The accumulation of organic and inorganic components in sediments leads to a deterioration in the environment and an imbalance in the coastal ecosystem. Currently, capping is the most effective technology for remediating polluted sediment and restoring ecosystems. A microcosm experiment was designed using pyrolyzed oyster shell (POS). These were mixed in with coastal sediment or added as a capping layer. The results showed that POS effectively decreased pollutants, including PO 4 -P and NH 4 -N. Metagenomics analysis was performed using 16S rRNA gene sequencing and the most abundant phyla identified in the POS treated and untreated sediments were Proteobacteria, followed by Firmicutes, Bacteroidetes, Chloroflexi, Fusobacteria, Nitrospirae, and Spirochaetes. The relative abundance of Proteobacteria members of the Class Gammaproteobacteria significantly increased, but Deltaproteobacteria gradually decreased throughout the experiment in POS-covered sediment. This suggests that the POS effectively promoted a shift from anaerobic to facultative anaerobic or aerobic microbial communities in the sediment. Dominant species of facultative anaerobic or microaerophilic bacteria from the order Chromatiales and phylum Nitrospirae were observed in the POS-covered sediment. Based on these study results, it can be concluded that POS is an effective covering material for sediment remediation and restores the microbial communities in sediments., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The Olympia oyster ( Ostrea lurida ) of the Puget Sound suffered a dramatic population crash, but restoration efforts hope to revive this native species. One overlooked variable in the process of assessing ecosystem health is association of bacteria with marine organisms and the environments they occupy. Oyster microbiomes are known to differ significantly between species, tissue type, and the habitat in which they are found. The goals of this study were to determine the impact of field site and habitat on the oyster microbiome and to identify core oyster-associated bacteria in the Puget Sound. Olympia oysters from one parental family were deployed at four sites in the Puget Sound both inside and outside of eelgrass ( Zostera marina ) beds. Using 16S rRNA gene amplicon sequencing of the oyster gut, shell, and surrounding seawater and sediment, we demonstrate that gut-associated bacteria are distinct from the surrounding environment and vary by field site. Furthermore, regional differences in the gut microbiota are associated with the survival rates of oysters at each site after 2 months of field exposure. However, habitat type had no influence on microbiome diversity. Further work is needed to identify the specific bacterial dynamics that are associated with oyster physiology and survival rates. IMPORTANCE This is the first exploration of the microbial colonizers of the Olympia oyster, a native oyster species to the West Coast, which is a focus of restoration efforts. The patterns of differential microbial colonization by location reveal microscale characteristics of potential restoration sites which are not typically considered. These microbial dynamics can provide a more holistic perspective on the factors that may influence oyster performance.

Teneurin C-terminal associated peptide (TCAP) is an ancient bioactive peptide that is highly conserved in metazoans. TCAP administration reduces cellular and behavioral stress in vertebrate and urochordate models. There is little information for invertebrates regarding the existence or function of a TCAP. This study used the Sydney rock oyster (SRO) as a molluscan model to characterize an invertebrate TCAP, from molecular gene analysis to its physiological effects associated with hemocyte phagocytosis. We report a single teneurin gene (and 4 teneurin splice variants), which encodes a precursor with TCAP that shares a vertebrate-like motif, and is similar to that of other molluscan classes (gastropod, cephalopod), arthropods and echinoderms. TCAP was identified in all SRO tissues using western blotting at 1-2 different molecular weights (~22 kDa and ~37kDa), supporting precursor cleavage variation. In SRO hemolymph, TCAP was spatially localized to the cytosol of hemocytes, and with particularly high density immunoreactivity in granules. Based on 'pull-down' assays, the SRO TCAP binds to GAPDH, suggesting that TCAP may protect cells from apoptosis under oxidative stress. Compared to sham injection, the intramuscular administration of TCAP (5 pmol) into oysters modulated their immune system by significantly reducing hemocyte phagocytosis under stress conditions (low salinity and high temperature). TCAP administration also significantly reduced hemocyte reactive oxygen species production at ambient conditions and after 48 h stress, compared to sham injection. Transcriptomic hemocyte analysis of stressed oysters administered with TCAP demonstrated significant changes in expression of genes associated with key metabolic, protective and immune functions. In summary, this study established a role for TCAP in oysters through modulation of physiological and molecular functions associated with energy conservation, stress and cellular defense., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Abramov, Suwansa-ard, da Silva, Wang, Dove, O’Connor, Parker, Lovejoy, Cummins and Elizur.)

Background: This study aimed to assess the correlation between Norovirus (NoV), diarrhea, and raw oysters from the eastern coastal areas of Yantai, Shandong, China., Methods: Marine oysters were selected from the three aquatic markets in Laishan district, Yantai City, in March 2019. Meanwhile, 100 fecal samples were collected from patients with diarrhea from the same areas during the same period. Nucleic acids were extracted from these samples and detected by employing reverse transcription polymerase chain reaction (RT-PCR) for NoV GI/GII. The VP1 gene of the coat protein of NoV was amplified by semi-nested RT-PCR and sequenced. Sequence comparison of VP1 was performed with BioEdit software, and the evolutionary tree was constructed with Mega7.0 software., Results: Of the 151 oysters, 42 (27.8%) were positive for NoV. Among them, 32 (21.2%) were GII-positive, 10 (6.6%) were GI-positive, and one GI VP1 sequence was obtained in the oyster samples. Of 100 fecal samples from patients with diarrhea, 38 were GII-positive and 17 were GI-positive. Totally, 19 GII VP 1 sequences and eight GI VP 1 sequences were obtained. Two G1 VP 1 sequences in two fecal samples showed 98.7% nucleotide sequence identity and 99.1% amino acid sequence identity G1 VP 1 acquired in the oyster sample., Conclusions: The results suggest that oysters may be responsible for the spread of NoV in Yantai, Shandong province, China., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sun, Niu, Jin, Gao, Wang and Ma.)

Background: The diversified aquaporin (AQP) family that was derived from gene duplication and subsequent functional differentiation play critical roles in multiple physiological processes and in adaptation to the dynamic environments during the evolutionary process. Oysters are a group of bivalve fauna in Mollusca that were widely distributed around the world and show extraordinary adaptation to harsh environments. However, knowledge is lacking with the diversity and evolution of the AQP family in oysters, even in molluscs., Results: Here, we performed a comprehensive analysis of the AQP family in three geographical isolated oyster species that are native to different environments. Genome distribution and phylogenetic analysis revealed that the expansion of the AQP family in oysters were attributed to tandem duplication. Synteny analysis indicated that large-scale inversions lead to the independent duplication or deletion of the AQPs after speciation. As a consequence, these independent duplication events contributed to the diversification of the AQP family in different oysters. Pore pattern analysis suggested that the duplicated AQPs in oysters were highly diversified in inner surface profiles, implying the subsequent functional differentiation. The comparison conducted based on the transcriptome data demonstrated that the functional differentiated AQP family members in oysters may play critical roles in maintaining the balance between the stationary homeostasis and dynamic environments., Conclusions: Our observation provides evidence for the correlation between the duplicated and functional differentiated AQP family and the adaptation to stationary life under dynamic environments in oysters. Additionally, it also broadens our knowledge of the evolution of AQP family in molluscs., (© 2022. The Author(s).)

Background: The microbiome affects the health of plants and animals, including humans, and has many biological, ecological, and evolutionary consequences. Microbiome studies typically rely on sequencing ribosomal 16S RNA gene fragments, which serve as taxonomic markers for prokaryotic communities; however, for eukaryotic microbes this approach is compromised, because 18S rRNA gene sequences from microbial eukaryotes are swamped by contaminating host rRNA gene sequences., Results: To overcome this problem, we developed CRISPR-Cas Selective Amplicon Sequencing (CCSAS), a high-resolution and efficient approach for characterizing eukaryotic microbiomes. CCSAS uses taxon-specific single-guide RNA (sgRNA) to direct Cas9 to cut 18S rRNA gene sequences of the host, while leaving protistan and fungal sequences intact. We validated the specificity of the sgRNA on ten model organisms and an artificially constructed (mock) community of nine protistan and fungal pathogens. The results showed that > 96.5% of host rRNA gene amplicons were cleaved, while 18S rRNA gene sequences from protists and fungi were unaffected. When used to assess the eukaryotic microbiome of oyster spat from a hatchery, CCSAS revealed a diverse community of eukaryotic microbes, typically with much less contamination from oyster 18S rRNA gene sequences than other methods using non-metazoan or blocking primers. However, each method revealed taxonomic groups that were not detected using the other methods, showing that a single approach is unlikely to uncover the entire eukaryotic microbiome in complex communities. To facilitate the application of CCSAS, we designed taxon-specific sgRNA for ~16,000 metazoan and plant taxa, making CCSAS widely available for characterizing eukaryotic microbiomes that have largely been neglected., Conclusion: CCSAS provides a high-through-put and cost-effective approach for resolving the eukaryotic microbiome of metazoa and plants with minimal contamination from host 18S rRNA gene sequences. Video Abstract., (© 2021. The Author(s).)

One of the major impacts of climate change has been the marked rise in global temperature. Recently, we demonstrated that high temperatures (1-week exposure) disrupt prooxidant-antioxidant homeostasis and promote cellular apoptosis in the American oyster. In this study, we evaluated the effects of seasonal sea surface temperature (SST) on tissue morphology, extrapallial fluid (EPF) conditions, heat shock protein-70 (HSP70), dinitrophenyl protein (DNP, an indicator of reactive oxygen species, ROS), 3-nitrotyrosine protein (NTP, an indicator of RNS), catalase (CAT), superoxide dismutase (SOD) protein expressions, and cellular apoptosis in gills and digestive glands of oysters collected on the southern Texas coast during the winter (15 °C), spring (24 °C), summer (30 °C), and fall (27 °C). Histological observations of both tissues showed a notable increase in mucus production and an enlargement of the digestive gland lumen with seasonal temperature rise, whereas biochemical analyses exhibited a significant decrease in EPF pH and protein concentration. Immunohistochemical analyses showed higher expression of HSP70 along with the expression of DNP and NTP in oyster tissues during summer. Intriguingly, CAT and SOD protein expressions exhibited significant upregulation with rising seasonal temperatures (15 to 27 °C), which decreased significantly in summer (30 °C), leaving oysters vulnerable to oxidative and nitrative damage. qRT-PCR analysis revealed a significant increase in HSP70 mRNA levels in oyster tissues during the warmer seasons. In situ TUNNEL assay showed a significant increase in apoptotic cells in seasons with high temperature. These results suggest that elevated SST induces oxidative/nitrative stress through the overproduction of ROS/RNS and disrupts the antioxidant system which promotes cellular apoptosis in oysters., (© 2021. Cell Stress Society International.)

Oysters (Bivalvia: Ostreidae Rafinesque, 1815) live in the intertidal and shallow subtidal areas worldwide. Despite their long evolutionary histories, abundant fossil records, global distribution, and ecological significance, a systematic time-dependent biogeographical analysis of this family is still lacking. Using combined mitochondrial (COI and 16S rRNA) and nuclear (18S rRNA, 28S rRNA, H3 and ITS2) gene makers for 80% (70/88) of the recognized extant Ostreidae, we reconstructed the global phylogenetic and biogeographical relationships throughout the evolutionary history of oysters. The result provided a holistic view of the origin, migration and dispersal patterns of Ostreidae. The phylogenetic results and fossil evidence indicated that Ostreidae originated from the circum-Arctic region in the Early Jurassic. The widening of the Atlantic Ocean and changes in the Tethys Ocean further facilitated their subsequent diversification during the Cretaceous and the Palaeogene periods. In particular, Crassostrea and Saccostrea exhibited relatively low dispersal abilities and their major diversifications were consistent with the tectonic events. Environmental adaptations and reproductive patterns, therefore, should play key roles in the formation of oyster distribution patterners, rather than the dispersal ability of their planktonic larvae. The diversity dynamics inferred by standard phylogenetic are consistent with the fossil record, however, further systematic classification, especially for fossil genus Ostrea, would enhance our understanding on extant and fossil oysters. The present study of the historical biogeography of oysters provides new insights into the evolution and speciation of oysters. Our findings also provide a foundation for the assessment of evolutionary patterns and ecological processes in intertidal and inshore life., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Many marine ectotherms, especially those inhabiting highly variable intertidal zones, develop high phenotypic plasticity in response to rapid climate change by modulating gene expression levels. Herein, we examined the regulatory architecture of heat-responsive gene expression plasticity in oysters using expression quantitative trait loci (eQTL) analysis. Using a backcross family of Crassostrea gigas and its sister species Crassostrea angulat a under acute stress, 56 distant regulatory regions accounting for 6-26.6% of the gene expression variation were identified for 19 heat-responsive genes. In total, 831 genes and 164 single nucleotide polymorphisms (SNPs) that could potentially regulate expression of the target genes were screened in the eQTL region. The association between three SNPs and the corresponding target genes was verified in an independent family. Specifically, Marker13973 was identified for heat shock protein (HSP) family A member 9 ( HspA9 ). Ribosomal protein L10a ( RPL10A ) was detected approximately 2 kb downstream of the distant regulatory SNP. Further, Marker14346-48 and Marker14346-85 were in complete linkage disequilibrium and identified for autophagy-related gene 7 ( ATG7 ). Nuclear respiratory factor 1 ( NRF1 ) was detected approximately 3 kb upstream of the two SNPs. These results suggested regulatory relationships between RPL10A and HSPA9 and between NRF1 and ATG7 . Our findings indicate that distant regulatory mutations play an important role in the regulation of gene expression plasticity by altering upstream regulatory factors in response to heat stress. The identified eQTLs provide candidate biomarkers for predicting the persistence of oysters under future climate change scenarios.

Hox and ParaHox genes encode transcription factors with similar expression patterns in divergent animals. The Pdx (Xlox) homeobox gene, for example, is expressed in a sharp spatial domain in the endodermal cell layer of the gut in chordates, echinoderms, annelids and molluscs. The significance of comparable gene expression patterns is unclear because it is not known if downstream transcriptional targets are also conserved. Here, we report evidence indicating that a classic transcriptional target of Pdx1 in vertebrates, the insulin gene, is a likely direct target of Pdx in Pacific oyster adults. We show that one insulin-related gene, cgILP, is co-expressed with cgPdx in oyster digestive tissue. Transcriptomic comparison suggests that this tissue plays a similar role to the vertebrate pancreas. Using ATAC-seq and ChIP, we identify an upstream regulatory element of the cgILP gene which shows binding interaction with cgPdx protein in oyster hepatopancreas and demonstrate, using a cell culture assay, that the oyster Pdx can act as a transcriptional activator through this site, possibly in synergy with NeuroD. These data argue that a classic homeodomain-target gene interaction dates back to the origin of Bilateria.

The wellbeing of marine organisms is connected to their microbiome. Oysters are a vital food source and provide ecological services, yet little is known about how climate change such as ocean acidification and warming will affect their microbiome. We exposed the Sydney rock oyster, Saccostrea glomerata, to orthogonal combinations of temperature (24, 28 °C) and pCO 2 (400 and 1000 μatm) for eight weeks and used amplicon sequencing of the 16S rRNA (V3-V4) gene to characterise the bacterial community in haemolymph. Overall, elevated pCO 2 and temperature interacted to alter the microbiome of oysters, with a clear partitioning of treatments in CAP ordinations. Elevated pCO 2 was the strongest driver of species diversity and richness and elevated temperature also increased species richness. Climate change, both ocean acidification and warming, will alter the microbiome of S. glomerata which may increase the susceptibility of oysters to disease., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Genomic selection has been widely used in terrestrial animals but has had limited application in aquaculture due to relatively high genotyping costs. Genomic information has an important role in improving the prediction accuracy of breeding values, especially for traits that are difficult or expensive to measure. The purposes of this study were to (i) further evaluate the use of genomic information to improve prediction accuracies of breeding values from, (ii) compare different prediction methods (BayesA, BayesCπ and GBLUP) on prediction accuracies in our field data, and (iii) investigate the effects of different SNP marker densities on prediction accuracies of traits in the Portuguese oyster ( Crassostrea angulata ). The traits studied are all of economic importance and included morphometric traits (shell length, shell width, shell depth, shell weight), edibility traits (tenderness, taste, moisture content), and disease traits ( Polydora sp. and Marteilioides chungmuensis ). A total of 18,849 single nucleotide polymorphisms were obtained from genotyping by sequencing and used to estimate genetic parameters (heritability and genetic correlation) and the prediction accuracy of genomic selection for these traits. Multi-locus mixed model analysis indicated high estimates of heritability for edibility traits; 0.44 for moisture content, 0.59 for taste, and 0.72 for tenderness. The morphometric traits, shell length, shell width, shell depth and shell weight had estimated genomic heritabilities ranging from 0.28 to 0.55. The genomic heritabilities were relatively low for the disease related traits: Polydora sp. prevalence (0.11) and M. chungmuensis (0.10). Genomic correlations between whole weight and other morphometric traits were from moderate to high and positive (0.58-0.90). However, unfavourably positive genomic correlations were observed between whole weight and the disease traits (0.35-0.37). The genomic best linear unbiased prediction method (GBLUP) showed slightly higher accuracy for the traits studied (0.240-0.794) compared with both BayesA and BayesCπ methods but these differences were not significant. In addition, there is a large potential for using low-density SNP markers for genomic selection in this population at a number of 3000 SNPs. Therefore, there is the prospect to improve morphometric, edibility and disease related traits using genomic information in this species.

Background: Homeobox-containing genes encode crucial transcription factors involved in animal, plant and fungal development, and changes to homeobox genes have been linked to the evolution of novel body plans and morphologies. In animals, some homeobox genes are clustered together in the genome, either as remnants from ancestral genomic arrangements, or due to coordinated gene regulation. Consequently, analyses of homeobox gene organization across animal phylogeny provide important insights into the evolution of genome organization and developmental gene control, and their interaction. However, homeobox gene organization remains to be fully elucidated in several key animal ancestors, including those of molluscs, lophotrochozoans and bilaterians., Results: Here, we present a high-quality chromosome-level genome assembly of the Hong Kong oyster, Magallana hongkongensis (2n = 20), for which 93.2% of the genomic sequences are contained on 10 pseudomolecules (~ 758 Mb, scaffold N50 = 72.3 Mb). Our genome assembly was scaffolded using Hi-C reads, facilitating a larger scaffold size compared to the recently published M. hongkongensis genome of Peng et al. (Mol Ecol Resources, 2020), which was scaffolded using the Crassostrea gigas assembly. A total of 46,963 predicted gene models (45,308 protein coding genes) were incorporated in our genome, and genome completeness estimated by BUSCO was 94.6%. Homeobox gene linkages were analysed in detail relative to available data for other mollusc lineages., Conclusions: The analyses performed in this study and the accompanying genome sequence provide important genetic resources for this economically and culturally valuable oyster species, and offer a platform to improve understanding of animal biology and evolution more generally. Transposable element content is comparable to that found in other mollusc species, contrary to the conclusion of another recent analysis. Also, our chromosome-level assembly allows the inference of ancient gene linkages (synteny) for the homeobox-containing genes, even though a number of the homeobox gene clusters, like the Hox/ParaHox clusters, are undergoing dispersal in molluscs such as this oyster.

Background: Albino mutations are commonly observed in the animal kingdom, including in bivalves. In the black-lipped pearl oyster Pinctada margaritifera, albino specimens are characterized by total or partial absence of colouration resulting in typical white shell phenotype expression. The relationship of shell colour with resulting cultured pearl colour is of great economic interest in P. margaritifera, on which a pearl industry is based. Hence, the albino phenotype provides a useful way to examine the molecular mechanisms underlying pigmentation., Results: Whole transcriptome RNA-sequencing analysis comparing albino and black wild-type phenotypes at three stages over the culture cycle of P. margaritifera revealed a total of 1606, 798 and 187 differentially expressed genes in whole juvenile, adult mantle and pearl sac tissue, respectively. These genes were found to be involved in five main molecular pathways, tightly linked to known pigmentation pathways: melanogenesis, calcium signalling pathway, Notch signalling pathway, pigment transport and biomineralization. Additionally, significant phenotype-associated SNPs were selected (N = 159), including two located in the Pif biomineralization gene, which codes for nacre formation. Interestingly, significantly different transcript splicing was detected between juvenile (N = 1366) and adult mantle tissue (N = 313) in, e.g., the tyrosinase Tyr-1 gene, which showed more complex regulation in mantle, and the Notch1 encoding gene, which was upregulated in albino juveniles., Conclusion: This multiple RNA-seq approach provided new knowledge about genes associated with the P. margaritifera albino phenotype, highlighting: 1) new molecular pathways, such as the Notch signalling pathway in pigmentation, 2) associated SNP markers with biomineraliszation gene of interest like Pif for marker-assisted selection and prevention of inbreeding, and 3) alternative gene splicing for melanin biosynthesis implicating tyrosinase.

Molluscs, the largest marine phylum, display extraordinary shell diversity and sophisticated biomineral architectures. However, mineral-associated biomolecules involved in biomineralization are still poorly characterised. We report the first comprehensive structural and biomolecular study of Spondylus gaederopus, a pectinoid bivalve with a peculiar shell texture. Used since prehistoric times, this is the best-known shell of Europe's cultural heritage. We find that Spondylus microstructure is very poor in mineral-bound organics, which are mostly intercrystalline and concentrated at the interface between structural layers. Using high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) we characterized several shell protein fractions, isolated following different bleaching treatments. Several peptides were identified as well as six shell proteins, which display features and domains typically found in biomineralized tissues, including the prevalence of intrinsically disordered regions. It is very likely that these sequences only partially represent the full proteome of Spondylus, considering the lack of genomics data for this genus and the fact that most of the reconstructed peptides do not match with any known shell proteins, representing consequently lineage-specific sequences. This work sheds light onto the shell matrix involved in the biomineralization in spondylids. Our proteomics data suggest that Spondylus has evolved a shell-forming toolkit, distinct from that of other better studied pectinoids - fine-tuned to produce shell structures with high mechanical properties, while limited in organic content. This study therefore represents an important milestone for future studies on biomineralized skeletons and provides the first reference dataset for forthcoming molecular studies of Spondylus archaeological artifacts., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Segments of the genome enriched in repetitive sequences still present a challenge and are omitted in genome assemblies. For that reason, the exact composition of DNA sequences underlying the heterochromatic regions and the active centromeres are still unexplored for many organisms. The centromere is a crucial region of eukaryotic chromosomes responsible for the accurate segregation of genetic material. The typical landmark of centromere chromatin is the rapidly-evolving variant of the histone H3, CenH3, while DNA sequences packed in constitutive heterochromatin are associated with H3K9me3-modified histones. In the Pacific oyster Crassostrea gigas we identified its centromere histone variant, Cg-CenH3, that shows stage-specific distribution in gonadal cells. In order to investigate the DNA composition of genomic regions associated with the two specific chromatin types, we employed chromatin immunoprecipitation followed by high-throughput next-generation sequencing of the Cg-CenH3- and H3K9me3-associated sequences. CenH3-associated sequences were assigned to six groups of repetitive elements, while H3K9me3-associated-ones were assigned only to three. Those associated with CenH3 indicate the lack of uniformity in the chromosomal distribution of sequences building the centromeres, being also in the same time dispersed throughout the genome. The heterochromatin of C. gigas exhibited general paucity and limited chromosomal localization as predicted, with H3K9me3-associated sequences being predominantly constituted of DNA transposons.

Macrophage migration inhibitory factor (MIF) dynamically connects innate and adaptive immune systems in vertebrate animals, allowing highly orchestrated systemic responses to various insults. The occurrence of MIF-like genes in non-vertebrate organisms suggests its origin from an ancestral metazoan gene, whose function is still a matter of debate. In the present work, by analyzing available genomic and transcriptomic data from bivalve mollusks, we identified 137 MIF-like sequences, which were classified into three types, based on phylogeny and conservation of key residues: MIF, D-DT, and the lineage-specific type MDL. Comparative genomics revealed syntenic conservation of homologous genes at the family level, the loss of D-DT in the Ostreidae family as well as the expansion of MIF-like genes in the Mytilidae family, possibly underpinning the neofunctionalization of duplicated gene copies. In M. galloprovincialis, MIF and one D-DT were mostly expressed in haemocytes and mantle rim of untreated animals, while D-DT paralogs often showed very limited expression, suggesting an accessory role or their persistence as relict genes., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The metal-binding protein metallothionein (MT) is widely used as a biomarker of metal contamination. In this study, we cloned a MT gene (sgMT) from the Sydney rock oyster Saccostrea glomerata. The gene encodes a MT-I protein with a classical αβ domain structure and is expressed as two transcripts resulting from alternative polyadenylation. The gene promoter contains two putative metal-responsive elements (MREs) which are known to be required for metal-inducible transcription. A specific and efficient qPCR assay was developed to quantify sgMT mRNA expression. Further, we assessed whether prior metal exposure history influences sgMT mRNA expression upon subsequent metal exposure. Oysters with varying prior metal exposure histories (contaminated and reference) were exposed to Cu, Cd and Zn. Expression of sgMT generally increased with metal dose, and oysters with an elevated past metal exposure history exhibited higher sgMT expression under Cd and Zn stress, representing a potential acclimatory response to prior metal exposure., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: The black-lip rock oyster (Saccostrea echinata) has considerable potential for aquaculture throughout the tropics. Previous attempts to farm S. echinata failed due to an insufficient supply of wild spat; however, the prospect of hatchery-based aquaculture has stimulated renewed interest, and small-scale farming is underway across northern Australia and in New Caledonia. The absence of knowledge surrounding the population genetic structure of this species has raised concerns about the genetic impacts of this emerging aquaculture industry. This study is the first to examine population genetics of S. echinata and employs both mitochondrial cytochrome c oxidase subunit I gene (COI) and single nucleotide polymorphism (SNP) markers., Results: The mitochondrial COI data set included 273 sequences of 594 base pair length, which comprised 74 haplotypes. The SNP data set included 27,887 filtered SNPs for 272 oysters and of these 31 SNPs were identified as candidate adaptive loci. Data from the mitochondrial COI analyses, supports a broad tropical Indo-Pacific distribution of S. echinata, and showed high haplotype and nucleotide diversities (0.887-1.000 and 0.005-0.008, respectively). Mitochondrial COI analyses also revealed a 'star-like' haplotype network, and significant and negative neutrality tests (Tajima's D = - 2.030, Fu's Fs = - 25.638, P < 0.001) support a recent population expansion after a bottleneck. The SNP analyses showed significant levels of population subdivision and four genetic clusters were identified: (1) the Noumea (New Caledonia) sample location; (2) the Bowen (north Queensland, Australia) sample location, and remaining sample locations in the Northern Territory, Australia (n = 8) were differentiated into two genetic clusters. These occurred at either side of the Wessel Islands and were termed (3) 'west' and (4) 'east' clusters, and two migrant individuals were detected between them. The SNP data showed a significant positive correlation between genetic and geographic distance (Mantel test, P < 0.001, R 2  = 0.798) and supported isolation by distance. Three candidate adaptive SNPs were identified as occurring within known genes and gene ontology was well described for the sex peptide receptor gene., Conclusions: Data supports the existence of genetically distinct populations of S. echinata, suggesting that management of wild and farmed stocks should be based upon multiple management units. This research has made information on population genetic structure and connectivity available for a new aquaculture species.

The release of hatchery-propagated fish and shellfish is occurring on a global scale, but the genetic impacts of these practices are often not fully understood and rarely monitored. Slow recovery of depleted eastern oyster populations in the Chesapeake Bay, USA has prompted a hatchery-based restoration program focused in the Choptank River, Maryland consisting of the mass release of hatchery-produced juveniles from local, wild broodstock. To evaluate potential genetic effects of this program, we (1) examined changes in genetic diversity (allelic richness, heterozygosity) and the effective number of breeders (N b ) over the hatchery production cycle with microsatellite-based parentage of natural, mass- and controlled-spawned cohorts, and (2) compared genetic diversity and effective population size (N e ) of a restored reef to wild source populations. Mass-spawned cohorts showed high variance in reproductive contribution, particularly among males, leading to a 45% average reduction in N b from spawning adult numbers and higher relatedness-lower magnitude reductions in heterozygosity and significant reductions in allelic richness were also observed. While controlled-spawns (single-male fertilizations of pooled eggs) reduced male variance, overall reproductive variance (V k ) remained high. Finally, oysters sampled from a restored reef displayed comparable N e , genetic diversity, and relatedness to samples from wild populations, with no significant genetic differentiation among them. Overall, the hatchery-based results and initial field-based population genetic analyses suggest that despite reductions in diversity from parents to offspring owing to high V k , enhancement with rotated, wild broodstock appears to have maintained genetic diversity in a restored reef population compared to proximal wild populations.

IκB kinases (IKKs) play critical roles in innate immunity through signal-induced activation of the key transcription factors nuclear factor-κB (NF-κB) and interferon regulatory factors (IRFs). However, studies of invertebrate IKK functions remain scarce. In this study, we performed phylogenetic analysis of IKKs and IKK-related kinases encoded in the Pacific oyster genome. We then cloned and characterized the oyster IKK α / β -2 gene. We found that oyster IKKα/β-2, a homolog of human IKKα/IKKβ, responded to challenge with lipopolysaccharide (LPS), peptidoglycan (PGN), and polyinosinic-polycytidylic acid [poly(I:C)]. As a versatile immune molecule, IKKα/β-2 activated the promoters of NF- κ B, TNF α, and IFN β, as well as IFN-stimulated response element (ISRE)-containing promoters, initiating an antibacterial or antiviral immune state in mammalian cells. Importantly, together with the cloned oyster IKKα/β-1, we investigated the signal transduction pathways mediated by these two IKKα/β proteins. Our results showed that IKKα/β-1 and IKKα/β-2 could interact with the oyster TNF receptor-associated factor 6 (TRAF6) and that IKKα/β-2 could also bind to the oyster myeloid differentiation factor 88 (MyD88) protein directly, suggesting that oyster IKKα/βs participate in both RIG-I-like receptor (RLR) and Toll-like receptor (TLR) signaling for the reception of upstream immune signals. The fact that IKKα/β-1 and IKKα/β-2 formed homodimers by interacting with themselves and heterodimers by interacting with each other, along with the fact that both oyster IKKα/β proteins interacted with NEMO protein, indicates that oyster IKKα/βs and the scaffold protein NEMO form an IKK complex, which may be a key step in phosphorylating IκB proteins and activating NF-κB. Moreover, we found that oyster IKKα/βs could interact with IRF8, and this may be related to the IKK-mediated activation of ISRE promotors and their involvement in the oyster "interferon (IFN)-like" antiviral pathway. Moreover, the expression of oyster IKKα/β-1 and IKKα/β-2 may induce the phosphorylation of IκB proteins to activate NF-κB. These results reveal the immune function of oyster IKKα/β-2 and establish the existence of mollusk TLR and RLR signaling mediated by IKKα/β proteins for the first time. Our findings should be helpful in deciphering the immune mechanisms of invertebrates and understanding the development of the vertebrate innate immunity network.

Background: Computing centrality is a foundational concept in social networking that involves finding the most "central" or important nodes. In some biological networks defining importance is difficult, which then creates challenges in finding an appropriate centrality algorithm., Results: We instead generalize the results of any k centrality algorithms through our iterative algorithm MATRIA, producing a single ranked and unified set of central nodes. Through tests on three biological networks, we demonstrate evident and balanced correlations with the results of these k algorithms. We also improve its speed through GPU parallelism., Conclusions: Our results show iteration to be a powerful technique that can eliminate spatial bias among central nodes, increasing the level of agreement between algorithms with various importance definitions. GPU parallelism improves speed and makes iteration a tractable problem for larger networks.

Shellfish, including oysters, often cause allergic reactions in children and adults. Oysters are inevitably consumed because of its delicacy and nutritional benefit, leading to frequent occurrence of severe clinical symptoms observed in patients with oyster hypersensitivity. We aimed to identify the immunodominant epitopes of oyster tropomyosin and crucial amino acids for IgE binding, which will help us to further understand the immunochemical characteristics of Cra g 1. The potential epitopes were predicted by immunoinformatics tools and the resultant immunodominant epitopes were identified by inhibition ELISA with pooled sera and individual serum from oyster allergic patients. Surprisingly, homologous substitution of multiple amino acids led to obviously decrease affinity of IgE antibodies, but this manner did not abrogate binding completely. Five major linear epitopes were evenly distributed on the surface of homology-based Cra g 1 model and hydrophilic residues appeared to be the most important for IgE binding. These results not only offer a better understanding of the molecular mechanism of interaction between Cra g 1 and oyster-specific IgE but also have significance in clinical diagnosis and immunotherapy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

β-carotene-15,15-dioxygenase is an enzyme involved in carotenoid metabolism to catalyze oxidative cleavage of β-carotene at its central double bond to two molecules of retinal in intestinal cells of vertebrate. In this study, we cloned and characterized β-carotene-15,15-dioxygenase in pearl oyster Pinctada fucata martensii (PmβCDOX). The full length of PmβCDOX gene was 1802 bp, including 1554 bp of the open reading frame (ORF) that encoded 517 amino acids, a 5'UTR of 134 bp and a 3' UTR of 114 bp. PmβCDOX was expressed at various tissues with highest level in hepatopancreas. Eighteen and fifteen single nucleotide polymorphisms (SNPs) were separately obtained in the exon and promoter of PmβCDOX. Eight SNPs (six SNPs in the exon and two SNPs in the promoter region) were significantly associated to total carotenoid content (TCC) (P < .05). The eight SNPs of significantly associated TCC were divided three haploblocks. Haplotypes CCTT had larger TCC than other haplotypes. The present results suggest that PmβCDOX is involved in carotenoid metabolism in pearl oyster. Our study will be helpful for development gene marker in selective breeding programs for TCC trait of the species., (Copyright © 2018. Published by Elsevier Inc.)

Bivalves are frequently exposed to salinity and temperature fluctuations in the estuary. This study explored the molecular effect of these fluctuations by exposing Sydney rock oysters, (Saccostrea glomerata), native to Australia, to either low salinity, elevated temperature or a combined salinity and temperature stress. Following the exposures, RNA-Seq was carried out on the collected oyster tissues. Differential transcript analysis resulted in a total of 1473, 1232 and 2571 transcripts, which were differentially expressed in S. glomerata exposed to low salinity (10 ppt), elevated temperature (30 °C) or the combined stressor (15 ppt and 30 °C), respectively, when compared to control oysters. All stress treatments had some effect on molecular processes such as innate immune response or respiration, with overall the strongest effects seen in S. glomerata exposed to the combined stressor. Additionally, most transporters putatively involved in osmoregulation were found to be suppressed in response to the combined stressor and the low salinity exposure. This study provides insight into the oyster's responses to both, single and dual stressors commonly found in an estuarine environment., (Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.)

Oysters are keystone species in estuarine ecosystems and are of substantial economic value to fisheries and aquaculture worldwide. Contending with disease and environmental stress are considerable challenges to oyster culture. Here we report a draft genome of the Sydney Rock Oyster, Saccostrea glomerata, an iconic and commercially important species of edible oyster in Australia known for its enhanced resilience to harsh environmental conditions. This is the second reference genome to be reported from the family Ostreidae enabling a genus-level study of lophotrochozoan genome evolution. Our analysis of the 784-megabase S. glomerata genome shows extensive expansions of gene families associated with immunological non-self-recognition. Transcriptomic analysis revealed highly tissue-specific patterns of expression among these genes, suggesting a complex assortment of immune receptors provide this oyster with a unique capacity to recognize invading microbes. Several gene families involved in stress response are notably expanded in Saccostrea compared with other oysters, and likely key to this species' adaptations for improved survival higher in the intertidal zone. The Sydney Rock Oyster genome provides a valuable resource for future research in molluscan biology, evolution and environmental resilience. Its close relatedness to Crassostrea will further comparative studies, advancing the means for improved oyster agriculture and conservation.

The paired-box 3 ( Pax3 ) is a transcription factor and it plays an important part in melanin synthesis. In this study, a new Pax3 gene was identified from Pteria penguin ( Röding , 1798 ) ( P. penguin ) by RACE-PCR (rapid-amplification of cDNA ends-polymerase chain reaction) and its effect on melanin synthesis was deliberated by RNA interference (RNAi). The cDNA of PpPax3 was 2250 bp long, containing an open reading fragment of 1365 bp encoding 455 amino acids. Amino acid alignment and phylogenetic tree showed PpPax3 shared the highest (69.2%) identity with Pax3 of Mizuhopecten yessoensis . Tissue expression profile showed that PpPax3 had the highest expression in mantle, a nacre-formation related tissue. The PpPax3 silencing significantly inhibited the expression of PpPax3 , PpMitf , PpTyr and PpCdk2 , genes involved in Tyr -mediated melanin synthesis, but had no effect on PpCreb2 and an increase effect on PpBcl2 . Furthermore, the PpPax3 knockdown obviously decreased the tyrosinase activity, the total content of eumelanin and the proportion of PDCA (pyrrole-2,3-dicarboxylic acid) in eumelanin, consistent with influence of tyrosinase ( Tyr ) knockdown. These data indicated that PpPax3 played an important regulating role in melanin synthesis by Tyr pathway in P. penguin .

Ostreid oysters (the 'true oysters') represent a large and commercially important family of bivalve molluscs. Several species, such as the Pacific oyster (Magallana gigas), the American oyster (Crassostrea virginica), the European oyster (Ostrea edulis) and the Sydney rock oyster (Saccostrea glomerata), are currently farmed at a large scale. However a number of other species may also be suitable for commercial-scale aquaculture. One such species is the 'black-lip oyster', a large Saccostrea species of uncertain taxonomic affinity found in northern Australia. Here, phylogenetic analysis of the COI gene places this oyster within a clade identified in a previous study of Japanese Saccostrea species, 'Saccostrea lineage J'. To facilitate comparisons between this oyster and the better-studied S. glomerata, de novo transcriptomes were generated from larval stages and adult tissues of both species. Patterns of orthology indicated an expansion of repetitive elements within Saccostrea genomes when compared to M. gigas and C. virginica, which may be reflected in increased evolutionary rates and/or genome sizes. The generation of high-quality transcriptomes for these two commercially relevant oysters provides a valuable resource for gene identification and comparison of molecular processes in these and other mollusc species., Competing Interests: The author has declared that no competing interests exist.

Color is one of the most important factors determining the commercial value of pearls. Pinctada fucata is a well-known pearl oyster producing high-quality Akoya pearls. Phenotypic variation in amount of yellow pigmentation produces white and yellowish pearls. It has been reported that polymorphism of yellow pigmentation of Akoya pearls is genetically regulated, but the responsible gene(s) has remained unknown. Here, we prepared pearl sac pairs formed in the same recipient oyster but coming from donor oysters that differ in their color. These two pearl sacs produced pearls with different yellowness even in the same recipient oyster. Yellow tone of produced pearls was consistent with shell nacre color of donor oysters from which mantle grafts were prepared, indicating that donor oysters strongly contribute to the yellow coloration of Akoya pearls. We also conducted comparative RNA-seq analysis and retrieved several candidate genes involved in the pearl coloration. Whole gene expression patterns of pair sacs were not grouped by pearl color they produced, but grouped by recipient oysters in which they were grown, suggesting that the number of genes involved in the yellow coloration is quite small, and that recipient oyster affects gene expression of the majority of genes in the pearl sac.

The relatively conserved sequences of signal peptides and proregions that antimicrobial peptides (AMPs) contain have been successfully used to search for and identify novel AMPs from databases within the same lineages of fish and amphibians and across different animal classes. If such an approach is applicable to invertebrate species such as oyster has not yet been tested so far. In this study, we found a cDNA from the digestive gland of the oyster Magallana gigas, designated Mgdefdg, which contains two exons interspaced by one intron. Mgdefdg coded for a protein with features characteristic of defensins. The mature peptide had the cysteine-stabilized α-helix/β-sheet motif (CSαβ) and the consensus pattern C-X 5-6 -C-X 3 -C-X 4-6 -C-X 3-4 -C-X 7-8 -C-X-C-X 2 -C forming potential disulfide linkages C1-C5, C2-C6, C3-C7 and C4-C8 in the predicted tertiary structure. Functional assays revealed that recombinant mature MgDefdg (rmMgDefdg) was able to kill the Gram-negative bacterium Aeromonas hydrophila and the Gram-positive bacterium Staphylococcus aureus, and to induce bacterial membrane/cytoplasmic damage. ELISA showed that rmMgDefdg had high affinity to both A. hydrophila and S. aureus as well as the microbe-associated molecular pattern molecules LPS and LTA. Moreover, rmMgDefdg was capable of causing bacterial membrane permeabilization and depolarization, and intracellular ROS increase. Additionally, rmMgDefdg was not cytotoxic to human red blood cells and murine RAW264.7 cells. Taken together, our results indicate that MgDefdg is a previously uncharacterized defensin with membrane selectivity towards bacterial cells. It also shows that the use of conserved sequences of signal peptides of defensins can be an effective tool to identify potential defensins across different animal genera in invertebrates., (Copyright © 2018. Published by Elsevier Ltd.)

Marine pearl production is directly influenced by the growth speed of Pinctada fucata martensii. However, the slow growth rate of this organism remains the main challenge in aquaculture production. Epidermal growth factor receptor (EGFR), an important receptor of tyrosine kinases in animals, plays versatile functions in development, growth and tissue regeneration. In this study, we described the characteristic and function of an EGFR gene identified from P. f. martensii (PmEGFR). PmEGFR possesses a typical EGFR structure and is expressed in all studied tissues, with the highest expression level in adductor muscle. PmEGFR expression level is significantly higher in the fast-growing group than that in the slow-growing one. Correlation analysis represents that shell height and shell weight show positive correlation with PmEGFR expression (p < 0.05), and total weight and tissue weight exhibit positive correlation with it (p < 0.01). This study indicates that PmEGFR is a valuable functional gene associated with growth traits.