Functional Analysis of ß-Carotene Oxygenase 2 (BCO2) Gene in Yesso Scallop (Patinopecten yessoensis).

Carotenoids are essential nutrients for humans and animals, and carotenoid coloration represents an important meat quality parameter for many farmed animals. Increasingly, studies have demonstrated that vertebrate carotenoid cleavage oxygenases (CCOs) are essential enzymes in carotenoid metabolism and are therefore potential candidate genes for improving carotenoid deposition. However, our understanding of carotenoid bioavailability and CCOs functions in invertebrates, particularly marine species, is currently quite limited. We previously identified that a CCO homolog, PyBCO-like 1, was the causal gene for carotenoid coloration in the 'Haida golden scallop', a variety of Yesso scallop (Patinopecten yessoensis) characterized by carotenoid enrichment. Here, we found that another CCO-encoding gene named PyBCO2 (ß-carotene oxygenase 2) was widely expressed in P. yessoensis organs/tissues, with the highest expression in striated muscle. Inhibiting BCO2 expression in P. yessoensis through RNA interference led to increased carotenoid (pectenolone and pectenoxanthin) deposition in the striated muscle, and the color of the striated muscle changed from white to light orange. Our results indicate that PyBCO2 might be a candidate gene used for improving carotenoid content in normal Yesso scallops, and also in 'Haida golden scallops'.

Water stratification alters phytoplankton assemblages in scallop farming waters of the North Yellow Sea in China.

As evaluation indicators of the primary productivity, the phytoplankton biomass and community structure are of great significance to the fishery industry, which can be driven by ocean currents, nutrients and water stratification. In the present study, the characteristics of phytoplankton assemblages in different water layers of a typical Yesso scallop farming area in Zhangzi Island, the North Yellow Sea were investigated from March 2021 to January 2022. According to the vertical distribution of temperature, water stratification was observed from June to August (stratification period), and disappeared in March, October and the following January with vertical homogeneity (mixing period). 18S rRNA gene sequencing results revealed that Pyrrophyta was the most dominant phylum during the sampling period, with high gene proportions in the stratification (63.36%) and mixing periods (77.35%). The gene proportion of Bacillariophyta in the stratification period was 5.44%, which was significantly lower than that in the mixing period of 8.93% (p < 0.05). Moreover, Pseudo-nitzschia, a toxin-producing taxon affiliated with Bacillariophyta, exhibited a significantly higher proportion in the stratification period than in the mixing period. During the stratification period, a number of toxin-producing taxa such as Pseudo-nitzschia and Karlodinium were enriched in the bottom layer, which was 1.29-fold and 1.37-fold of that in the surface layer, respectively. Redundancy analysis showed that phosphate and water temperature were major environmental factors driving the vertical distribution of phytoplankton assemblages. The phosphate (0.11 µM) and silicate (2.09 µM) concentrations in the surface layer approached the minimum threshold for phytoplankton growth, and the stoichiometric limitation of phosphate was detected in the surface and middle layers. Collectively, these results indicated that the decreased proportion ratio of Bacillariophyta to Pyrrophyta and unfavorable community composition of Bacillariophyta for scallops were observed during summer, which might result from the phosphate limitation driven by water stratification. The results will further our understanding of the dynamics of phytoplankton communities under the background of intensifying ocean stratification and provide ecological guidance for mollusc mariculture.

Quantitative proteomic analysis reveals the molecular mechanism of the Yesso scallop (Patinopecten yessoensis) in response to Polydora infection.

The Yesso scallop is a large and ancient molluscan group with great economic value; however, it has recently suffered severe cases of Polydora infection. Polydora parasitizes the shells of scallops, badly damaging shell structures and affecting growth and mortality. To investigate the molecular mechanism of Yesso scallops' response to Polydora infection, proteomic profiling changes in the mantle tissues of Polydora-infected (diseased) and healthy scallops were systematically analysed by tandem mass tags (TMT) labelling technology in this study. A total of 519 differentially expressed proteins (DEPs) were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed most innated immune-related functions and pathways were significantly downregulated in diseased scallops, except the phagocytosis pathway, indicating an important role of phagocytosis in response to Polydora infection. DEPs involved in the phagocytosis pathway were associated with phagocytic receptor recognition, phagosome biogenesis and pathogen degradation, and they were further verified by quantitative real-time PCR. The results elucidate the molecular components of phagocytosis in molluscs for the first time. Polydora can be encapsulated by melanization with an obvious appearance in shells; indeed, melanization-related DEPs were upregulated in diseased scallops. Inhibition of apoptosis and nervous modulation may be also involved in the response mechanism, with some highly associated proteins significantly differentially expressed. Finally, a protein-protein interaction network was constructed to provide a global view of the interaction relationships of the DEPs. The study predicts the molecular response mechanism of Yesso scallops to Polydora infection, and lays a theoretical foundation for Polydora disease control.

Effects of microbial community and disease resistance against Vibrio splendidus of Yesso scallop (Patinopecten yessoensis) fed supplementary diets of tussah immunoreactive substances and antimicrobial peptides.

This study was conducted to investigate the effects of dietary supplementation of tussah immunoreactive substances (TIS) and antimicrobial peptides (AMPs) on microbial community and resistance against Vibrio splendidus of Yesso scallop Patinopecten yessoensis. Scallops were fed with the basal diets supplemented with TIS (T group), AMPs (A group), or both of the two (TA group). After the feeding trial, the microbial community changes were evaluated, and the challenge test with V. splendidus was conducted, as well as the immune parameters and digestive enzyme activities were determined. The results revealed that the TA group was more capable of modulating the bacterial community composition of scallops by increasing the potentially beneficial bacteria and suppressing the pathogenic microorganism during the feeding trial. After injection, the cumulative mortality rate in TA group was notably lower than others. In addition, the TA group showed better digestive and immune parameters involved in digestive capacity, phagocyte function, phosphatase-responsiveness, and oxidation resistance. These results collectively confirmed that dietary TIS and AMPs in diet could effectively modulate the microflora structure and improve disease resistance against V. splendidus of scallop, and the positive effects were more obvious when dietary supplementation of them in combination.

Genome-wide DNA methylation profile changes associated with shell colouration in the Yesso scallop (Patinopecten yessoensis) as measured by whole-genome bisulfite sequencing.

BACKGROUND: Mollusca, a phylum of highly rich species, possess vivid shell colours, but the underlying molecular mechanism remains to be elucidated. DNA methylation, one of the most common epigenetic modifications in eukaryotes, is believed to play a vital role in various biological processes. However, analysis of the effects of DNA methylation on shell colouration has rarely been performed in molluscs, limiting the current knowledge of the molecular mechanism of shell colour formation. RESULTS: In the present study, to reveal the role of epigenetic regulation in shell colouration, WGBS, the "gold standard" of DNA methylation analysis, was first performed on the mantle tissues of Yesso scallops (Patinopecten yessoensis) with different shell colours (brown and white), and DNA methylomes at single-base resolution were generated. About 3% of cytosines were methylated in the genome of the Yesso scallop. A slight increase in mCG percentage and methylation level was found in brown scallops. Sequence preference of nearby methylated cytosines differed between high and low methylation level sites and between the brown- and white-shelled scallops. DNA methylation levels varied among the different genomic regions; all the detected regions in the brown group exhibited higher methylation levels than the white group. A total of 41,175 DMRs (differentially methylated regions) were detected between brown and white scallops. GO functions and pathways associated with the biosynthesis of melanin and porphyrins were significantly enriched for DMRs, among which several key shell colour-related genes were identified. Further, different correlations between mRNA expression levels and DNA methylation status were found in these genes, suggesting that DNA methylation regulates shell colouration in the Yesso scallop. CONCLUSIONS: This study provides genome-wide DNA methylation landscapes of Yesso scallops with different shell colours, offering new insights into the epigenetic regulatory mechanism underlying shell colour.

NKL-24: A novel antimicrobial peptide derived from zebrafish NK-lysin that inhibits bacterial growth and enhances resistance against Vibrio parahaemolyticus infection in Yesso scallop, Patinopecten yessoensis.

The extensive use of antibiotics in aquaculture has resulted in the prevalence of antibiotic-resistant bacteria and, consequently, new antibacterial strategies or drugs with clear modes of action are urgently needed. Antimicrobial peptides (AMPs) are currently widely considered as alternatives to antibiotics in the treatment of infections in aquatic animals. In this study, we aimed to evaluate the effects of NKL-24, a truncated peptide derived from zebrafish NK-lysin, against Yesso scallop (Patinopecten yessoensis) pathogen, Vibrio parahaemolyticus. The results showed that NKL-24 had a potent antibacterial effect against V. parahaemolyticus via a membrane active cell-killing mechanism. The in vitro study showed that sub-lethal levels of NKL-24 obviously reduced bacterial swimming ability and downregulated the transcription of the selected genes associated with V. parahaemolyticus virulence. Studies on NKL-24 biosafety in hemocytes and in Yesso scallop have shown no adverse effects from this peptide. Bacteria challenge test results demonstrated that NKL-24 significantly decreased the mortality and inhibited bacterial growth in the scallop infected with V. parahaemolyticus, while further in vivo examination revealed that NKL-24 could enhance non-specific immune parameters. Moreover, NKL-24 was capable of modulating a series of V. parahaemolyticus-responsive genes in the scallop. These results suggest the protective action of NKL-24 against V. parahaemolyticus and the potential of this peptide as a promising candidate for aquaculture applications.

Genome-wide identification, characterisation and expression analysis of the ALAS gene in the Yesso scallop (Patinopecten yessoensis) with different shell colours.

Porphyrins, one of the most common shell pigments, are by-products of the haem pathway. 5-Aminolaevulinate synthase (ALAS) is the first and rate-limiting enzyme in this pathway and has been well studied in vertebrate species. However, the function of ALAS in shell colouration has been poorly studied in molluscs, which are renowned for their colourful shells. In the present study, an ALAS gene, named PyALAS, was identified through whole-genome scanning in the Yesso scallop (Patinopecten yessoensis), an economically and evolutionarily important bivalve species in which the shell colour represents polymorphism. Two conserved domains were detected in the PyALAS protein sequence, including a Preseq-ALAS domain and a 5-ALAS domain, confirming the identification of PyALAS. Phylogenetic analysis of the ALAS proteins among various invertebrate and vertebrate species revealed a high consistency between the molecular evolution of ALAS and the species taxonomy. PyALAS was ubiquitously expressed in most adult tissues of the Yesso scallop. The left mantle expressed a significantly higher level of PyALAS than the right side in brown scallops, whereas there was no significant difference in white scallops. Significantly different expression levels of PyALAS was also detected between the two different shell colour strains. These data indicate that PyALAS plays an important role in shell colouration in Yesso scallops and the present study provides new insights into the molecular mechanism of shell colouration in molluscs.

The differences of bacterial communities in the tissues between healthy and diseased Yesso scallop (Patinopecten yessoensis).

The tissues of marine invertebrates are colonized by species-rich microbial communities. The dysbiosis of host's microbiota is tightly associated with the invertebrate diseases. Yesso scallop (Patinopecten yessoensis), one of the most important maricultured scallops in northern China, has recently suffered massive summer mortalities, which causes huge production losses. The knowledge about the interactions between the Yesso scallop and its microbiota is important to develop the strategy for the disease prevention and control. In the present study, the bacterial communities in hemolymph, intestine, mantle and adductor muscle were compared between the healthy and diseased Yesso scallop based on the high-throughput sequencing of 16S rRNA gene. The results indicated obvious difference of the composition rather than the diversity of the bacterial communities between the healthy and diseased Yesso scallop. Vibrio, Francisella and Photobacterium were found to overgrow and dominate in the mantle, adductor muscle and intestine of the diseased scallops, respectively. The prediction of bacterial community metagenomes and the variations of KEGG pathways revealed that the proportions of the pathways related with neurodegenerative diseases and carbohydrate metabolism both increased significantly in the mantle and hemolymph of the diseased scallops. The abundance of the metabolism pathways including carbohydrate metabolism, lipid metabolism and amino acid metabolism decreased significantly in the intestine of diseased scallops. The results suggested that the changes of bacterial communities might be closely associated with the Yesso scallop's disease, which was helpful for further investigation of the pathogenesis as well as prevention and control of the disease in Yesso scallop.

Histological and Expression Differences Among Different Mantle Regions of the Yesso Scallop (Patinopecten yessoensis) Provide Insights into the Molecular Mechanisms of Biomineralization and Pigmentation.

The molecular mechanisms of shell formation and pigmentation are issues of great interest in molluscan studies due to the unique physical and biological properties of shells. The Yesso scallop, Patinopecten yessoensis, is one of the most important maricultural bivalves in Asian countries, and its shell color shows polymorphism. To gain more information about the underlying mechanisms of shell formation and pigmentation, this study presents the first analyses of histological and transcriptional differences between different mantle regions of the Yesso scallop, which are thought to be responsible for the formation of different shell layers. The results showed major microstructural differences between the edge and central mantles, which were closely associated with their functions. Different biomineralization-related GO functions, which might participate in the formation of different shell layers, were significantly enriched in the different mantle regions, indicating the different molecular functions of the two mantle regions in shell formation. The melanogenesis pathway, which controls melanin biosynthesis, was the most significantly enriched pathway in the DEGs between the two mantle regions, indicating its important role in shell pigmentation. Tyr, the key and rate-limiting gene in melanogenesis, was expressed at a remarkably high level in the central mantle, while the upstream regulatory genes included in melanogenesis were mainly upregulated in the edge mantle, suggesting the different molecular functions of the two mantle regions in shell pigmentation.

Transcriptomics analysis revealing candidate genes and networks for sex differentiation of yesso scallop (Patinopecten yessoensis).

BACKGROUND: The Yesso scallop, Patinopecten (Mizuhopecten) yessoensis, is a commercially important bivalve in the coastal countries of Northeast Asia. It has complex modes of sex differentiation, but knowledge of the mechanisms underlying this sex determination and differentiation is limited. RESULTS: In this study, the gonad tissues from females and males at three developmental stages were used to investigate candidate genes and networks for sex differentiation via RNA-Req. A total of 901,980,606 high quality clean reads were obtained from 18 libraries, of which 417 expressed male-specific genes and 754 expressed female-specific genes. Totally, 10,074 genes differentially expressed in females and males were identified. Weighted gene co-expression network analysis (WGCNA) revealed that turquoise and green gene modules were significantly positively correlated with male gonads, while coral1 and black modules were significantly associated with female gonads. The most important gene for sex determination and differentiation was Pydmrt 1, which was the only gene discovered that determined the male sex phenotype during early gonadal differentiation. Enrichment analyses of GO terms and KEGG pathways revealed that genes involved in metabolism, genetic and environmental information processes or pathways are sex-biased. Forty-nine genes in the five modules involved in sex differentiation or determination were identified and selected to construct a gene co-expression network and a hypothesized sex differentiation pathway. CONCLUSIONS: The current study focused on screening genes of sex differentiation in Yesso scallop, highlighting the potential regulatory mechanisms of gonadal development in P. yessoensis. Our data suggested that WCGNA can facilitate identification of key genes for sex differentiation and determination. Using this method, a hypothesized P. yessoensis sex determination and differentiation pathway was constructed. In this pathway, Pydmrt 1 may have a leading function.

Genome-wide identification, characterization and expression analysis of the MITF gene in Yesso scallops (Patinopecten yessoensis) with different shell colors.

The microphthalmia-associated transcription factor (MITF) is the center of the regulator network of melanin synthesis in vertebrates. However, the role of MITF in shell color formation is poorly studied in mollusks. In the present study, an MITF gene, PyMITF, was first identified at the whole-genome level in Yesso scallop (Patinopecten yessoensis), an evolutionarily and economically important species, the shell color of which shows polymorphism. The PyMITF is a large gene spanning ~37 kb in the genome with 7 introns and 8 exons. A basic helix-loop-helix leucine zipper (bHLH-LZ) domain was detected in the PyMITF protein sequence, which can bind the canonical E-box sequence in the promoter region of the downstream genes. Phylogenetic analysis of the MITFs among vertebrates and invertebrates revealed that the molecular evolution of MITFs was consistent with the species taxonomy. Different expression levels of PyMITF were detected among different shell color strains, indicating the important role of PyMITF involved in shell pigmentation. Besides, PyMITF was expressed at a significantly higher level in the central mantle than that in the edge mantle, proving the participation of the central mantle in shell color formation in molecular level for the first time. The work provides valuable information for the molecular mechanism study of shell color formation.

FOXL2 and DMRT1L Are Yin and Yang Genes for Determining Timing of Sex Differentiation in the Bivalve Mollusk Patinopecten yessoensis.

Sex determination and differentiation have long been a research hotspot in metazoans. However, little is known about when and how sex differentiation occurs in most mollusks. In this study, we conducted a combined morphological and molecular study on sex differentiation in the Yesso scallop Patinopecten yessoensis. Histological examination on gonads from 5- to 13-month-old juveniles revealed that the morphological sex differentiation occurred at 10 months of age. To determine the onset of molecular sex differentiation, molecular markers were screened for early identification of sex. The gonadal expression profiles of eight candidate genes for sex determination or differentiation showed that only two genes displayed sexually dimorphic expression, with FOXL2 being abundant in ovaries and DMRT1L in testes. In situ hybridization revealed that both of them were detected in germ cells and follicle cells. We therefore developed LOG10(DMRT1L/FOXL2) for scallop sex identification and confirmed its feasibility in differentiated individuals. By tracing its changes in 5- to 13-month-old juveniles, molecular sex differentiation time was determined: some scallops differentiate early in September when they are 7 months old, and some do late in December when they are 10 months old. Two kinds of coexpression patterns were found between FOXL2 and DMRT1L: expected antagonism after differentiation and unexpected coordination before differentiation. Our results revealed that scallop sex differentiation co-occurs with the formation of follicles, and molecular sex differentiation is established prior to morphological sex differentiation. Our study will assist in a better understanding of the molecular mechanism underlying bivalve sex differentiation.

Genome-wide identification and expression profiling of the SOX gene family in a bivalve mollusc Patinopecten yessoensis.

SOX family is composed of transcription factors that play vital roles in various developmental processes. Comprehensive understanding on evolution of the SOX family requires full characterization of SOX genes in different phyla. Mollusca is the second largest metazoan phylum, but till now, systematic investigation on the SOX family is still lacking in this phylum. In this study, we conducted genome-wide identification of the SOX family in Yesso scallop Patinopecten yessoensis and profiled their tissue distribution and temporal expression patterns in the ovaries and testes during gametogenesis. Seven SOX genes were identified, including SOXB1, B2, C, D, E, F and H, representing the first record in protostomes with SOX members identical to that proposed to exist in the last common ancestor of chordates. Genomic structure analysis identified relatively conserved exon-intron structures, accompanied by intron insertion. Quantitative real-time PCR analysis revealed possible involvement of scallop SOX in various functions, including neuro-sensory cell differentiation, hematopoiesis, myogenesis and gametogenesis. This study represents the first systematic characterization of SOX gene family in Mollusca. It will assist in a better understanding of the evolution and function of SOX family in metazoans.

A Genome-Wide Association Study Identifies the Genomic Region Associated with Shell Color in Yesso Scallop, Patinopecten yessoensis.

The shell color polymorphism widely exists in economic shellfish, which not only results in a better visual perception but also shows great value as an economic trait for breeding. Small numbers of reddish-orange shell Yesso scallops, Patinopecten yessoensis, were found in cultured populations compared to the brown majority. In this study, a genome-wide association study was conducted to understand the genetic basis of shell color. Sixty-six 2b-RAD libraries with equal numbers of reddish-orange and brown shell individuals were constructed and sequenced using the Illumina HiSeq 2000 platform. A total of 322,332,684 high-quality reads were obtained, and the average sequencing depth was 18.4×. One genomic region on chromosome 11 that included 239 single-nucleotide polymorphisms (SNPs) was identified as significantly associated with shell color. After verification by high-resolution melting in another population, two SNPs were selected as specific loci for reddish-orange shell color. These two SNPs could be used to improve the selective breeding progress of true-breeding strains with complete reddish-orange scallops. In addition, within the significantly associated genomic region, candidate genes were identified using marker sequences to search the draft genome of Yesso scallop. Three genes (LDLR, FRIS, and FRIY) with known functions in carotenoid metabolism were identified. Further study using high-performance liquid chromatography proved that the relative level of carotenoids in the reddish-orange shells was 40 times higher than that in the brown shells. These results suggested that the accumulation of carotenoids contributes to the formation of reddish-orange shells.

Transcriptome Sequencing and Comparative Analysis of Ovary and Testis Identifies Potential Key Sex-Related Genes and Pathways in Scallop Patinopecten yessoensis.

Bivalve mollusks have fascinatingly diverse modes of reproduction. However, research investigating sex determination and reproductive regulation in this group of animals is still in its infancy. In this study, transcriptomes of three ovaries and three testes of Yesso scallop were sequenced and analyzed. Transcriptome comparison revealed that 4394 genes were significantly different between ovaries and testes, of which 1973 were ovary-biased (upregulated in the ovaries) and 2421 were testis-biased. Crucial sex-determining genes that were previously reported in vertebrates and putatively present in bivalves, namely FOXL2, DMRT, SOXH, and SOXE, were investigated. The genes all possessed conserved functional domains and were detected in the gonads. Except for PySOXE, the other three genes were significantly differentially expressed between the ovaries and testes. PyFOXL2 was ovary-biased, and PyDMRT and PySOXH were testis-biased, suggesting that these three genes are likely to be key candidates for scallop sex determination/differentiation. Furthermore, GO and KEGG enrichment analyses were conducted for both ovary- and testis-biased genes. Interestingly, both neurotransmitter transporters and GABAergic synapse genes were overrepresented in the ovary-biased genes, suggesting that neurotransmitters, such as GABA and glycine, are likely to participate in scallop ovary development. Our study will assist in better understanding of the molecular mechanisms underlying bivalve sex determination and reproductive regulation.

An SCD gene from the Mollusca and its upregulation in carotenoid-enriched scallops.

Carotenoids are a diverse group of red, orange, and yellow pigments that act as vitamin A precursors and antioxidants. Animals can only obtain carotenoids through their diets. Amongst the carotenoids identified in nature, over one third are of marine origin, but current research on carotenoid absorption in marine species is limited. Bivalves possess an adductor muscle, which is normally white in scallops. However, a new variety of Yesso scallop (Patinopecten yessoensis), the 'Haida golden scallop', can be distinguished by its adductor muscle's orange colour, which is caused by carotenoid accumulation. Studying the genes related to carotenoid accumulation in this scallop could benefit our understanding of the mechanisms underlying carotenoid absorption in marine organisms, and it could further improve scallop breeding for carotenoid content. Stearoyl-CoA desaturase (SCD) is the rate-limiting enzyme in the production of monounsaturated fatty acids, which enhance carotenoid absorption. Here, the full-length cDNA and genomic DNA sequences of the SCD gene from the Yesso scallop (PySCD) were obtained. The PySCD gene consisted of four exons and three introns, and it contained a 990-bp open reading frame encoding 329 amino acids. It was ubiquitously expressed in adult tissues, embryos and larvae of both white Yesso scallops and 'Haida golden' scallops. Although the expression pattern of PySCD in both types of scallops was similar, significantly more PySCD transcripts were detected in the 'Haida golden' scallops than in the white scallops. Elevated PySCD expression was found in tissues including the adductor muscle, digestive gland, and gonad, as well as in veliger larvae. This study represents the first characterisation of an SCD gene from the Mollusca. Our data imply that PySCD functions in multiple biological processes, and it might be involved in carotenoid accumulation.