Search

Exosomes are extracellular vesicles secreted by diverse cell under normal or abnormal physiological conditions, which could carry a range of bioactive molecules and play significant roles in biological processes, such as intercellular communication and immune response. In the current study, a preliminary study was performed to investigate the exosomal shuttle protein in Chlamys farreri (designated as CfesPro) and to predict the potential function of exosomes in scallop innate immunity. The serum derived exosomes (designated as CfEVs) were obtained from lipopolysaccharide (LPS)-stimulated C. farreri and untreated ones. After confirmation and characterization by transmission electron microscopy (TEM), nano-HPLC-MS/MS spectrometry was performed on CfEVs using a label-free quantitative method. Totally 2481 exosomal shuttle proteins were identified in CfEVs proteomic data, which included many innate immune related proteins. GO and KOG functional annotation showed that CfesPro participated in cellular processes, metabolism reactions, signaling transductions, immune responses and so on. Moreover, 1421 proteins in CfesPro were enriched to 324 pathways by KEGG analysis, including several immune-related pathways, such as autophagy, apoptosis and lysosome pathway. Meanwhile, eight autophagy-related proteins were initially identified in CfesPro, indicating that CfEVs had a potential role with autophagy. All these findings showed that CfEVs were involved in C. farreri innate immune defenses. This research would enrich the protein database of marine exosomes and provide a basis for the exploration of immune defense systems in marine invertebrates.

Genome miniaturization drives key evolutionary innovations of adaptive traits in vertebrates, such as the flight evolution of birds. However, a similar evolutionary process remains poorly understood in invertebrates. Derived from the second-largest animal phylum, scallops are a special group of bivalve molluscs and acquire the evolutionary novelty of the swimming lifestyle, providing excellent models for investigating the coordinated genome and lifestyle evolution. Here, we show for the first time that genome sizes of scallops exhibit a generally negative correlation with locomotion activity. To elucidate the co-evolution of genome size and swimming lifestyle, we focus on the Asian moon scallop (Amusium pleuronectes) that possesses the smallest known scallop genome while being among scallops with the highest swimming activity. Whole-genome sequencing of A. pleuronectes reveals highly conserved chromosomal macrosynteny and microsynteny, suggestive of a highly contracted but not degenerated genome. Genome reduction of A. pleuronectes is facilitated by significant inactivation of transposable elements, leading to reduced gene length and elevated expression of energy-producing gene pathways and the decrease of copy number and expression level of biomineralization genes. Similar evolutionary changes of relevant pathways are also observed for bird genome reduction with flight evolution. The striking mimicry of genome miniaturization underlying the evolution of bird flight and scallop swimming unveils the potentially common, pivotal role of genome size fluctuation in the evolution of novel lifestyles in the animal kingdom.

Lysozyme is relevant to the innate immune system as a vital protein for crustaceans. In the present study, we cloned and characterized a novel c-type lysozyme gene (LvLYZ) from the Pacific white shrimp (Litopenaeus vannamei). The obtained full-length cDNA of LvLYZ was 990 bp and contained an open reading frame of 693 bp. Its deduced amino acid sequence consisted of 230 amino acids (aa) with a 17 aa signal peptide at the N-terminal and 130 aa functional domains. The multiple sequence alignment (MSA) indicated that the typical active sites in LvLYZ were similarly conserved as c-type lysozymes from other species. The transcription of LvLYZ appeared in all detected tissues and had relatively higher expression levels in hemocytes, hepatopancreas, gill and intestine. The mRNA expression profiles of LvLYZ were up-regulated in hemocyte and hepatopancreas post the stimulation of Vibrio parahaemolyticus or white spot syndrome virus (WSSV), respectively. The recombinant protein of LvLYZ (rLvLYZ) exhibited antibacterial activities against various microbes, including Escherichia coli, Vibrio splendidus, Micrococcaus luteus, Vibrio parahaemolyticus and Staphylococcus aureus. These results indicated that LvLYZ could cope with bacteria in L. vannamei and may play a significant role in immune response against invading pathogens.

The papilla number is one of the most economically important traits of sea cucumber in the China marketing trade. However, the genetic basis for papilla number diversity in holothurians is still scarce. In the present study, we conducted genome-wide association studies (GWAS) for the trait papilla number of sea cucumbers utilizing a set of 400,186 high-quality SNPs derived from 200 sea cucumbers. Two significant trait-associated SNPs that passed Bonferroni correction (P PATS1 and the genic region of EIF4G, which were reported to play a pivotal role in cell growth and proliferation. The fine-mapping regions around the top two lead SNPs provided precise causative loci/genes related to papilla formation and cellular activity, including PPP2R3C, GBP1, and BCAS3. Potential SNPs with P PATS1, PPP2R3C, and EIF4G that near or cover the two lead SNPs was conducted in papilla tissue of TG (Top papilla number group) and BG (Bottom papilla number group) by qRT-PCR. We found the significantly higher expression profile of PATS1 (3.34-fold), PPP2R3C (4.90-fold), and EIF4G (4.23-fold) in TG, implying their potential function in papilla polymorphism. The present results provide valuable information to decipher the phenotype differences of the papilla trait and will provide a scientific basis for selective breeding in sea cucumbers.

Soft-bodied slow-moving sea creatures such as sea stars and sea cucumbers lack an adaptive immune system and have instead evolved the ability to make specialized protective chemicals (glycosylated steroids and triterpenes) as part of their innate immune system. This raises the intriguing question of how these biosynthetic pathways have evolved. Sea star saponins are steroidal, while those of the sea cucumber are triterpenoid. Sterol biosynthesis in animals involves cyclization of 2,3-oxidosqualene to lanosterol by the oxidosqualene cyclase (OSC) enzyme lanosterol synthase (LSS). Here we show that sea cucumbers lack LSS and instead have two divergent OSCs that produce triterpene saponins and that are likely to have evolved from an ancestral LSS by gene duplication and neofunctionalization. We further show that sea cucumbers make alternate sterols that confer protection against self-poisoning by their own saponins. Collectively, these events have enabled sea cucumbers to evolve the ability to produce saponins and saponin-resistant sterols concomitantly.

Carotenoids are essential nutrients for humans and animals, and carotenoid content has become an important trait to evaluate the nutritional value of many cultured animals. Marine animals provide humans with diverse carotenoids, and developing carotenoid-enriched varieties has been the focus of marine animal breeding. Understanding the molecular mechanism of carotenoid deposition could benefit marine animal breeding for carotenoid content improvement. In the present study, transcriptomic analysis of adductor muscle was performed between Yesso scallop (Patinopecten yessoensis) with white muscle (WM) and carotenoid-enriched orange muscle (OM). A total of 683 differentially expressed genes (DEGs) were identified, with 302 and 381 genes being up- and down-regulated in OM scallop. Gene co-expression network analysis identified four carotenoid accumulation−related modules, including three up-regulated modules and one down-regulated module. The genes in up-regulated modules mainly participate in the pathways of translation and transcription (MEgreen), immune system (MElightyellow), and lipid metabolism (MEpink), while the down-regulated module is mainly enriched with genes involved in various metabolic pathways (MEturquoise). As the causal gene responsible for muscle coloration in scallop, PyBCO-like 1 is the hub gene of MEturquoise and showed strong connectivity with NR2F1A, a transcriptional factor involved in the regulation of retinoic acid. In addition, the up-regulated DEGs, including WDR3, RPP29, TBL3, RIOK2, and NOB1 from “ribosome biogenesis”, HSP70s and HSP702Bs from “antigen processing and presentation”, and ACOX1 from “PPAR signaling pathway” were identified as hub genes, indicating the potential regulatory role of these genes and pathways in response to carotenoid accumulation. Our data contribute to a deeper understanding of the regulatory and response mechanisms of carotenoid accumulation in marine animals.

Plectropomus leopardus, as known as leopard coral grouper, is a valuable marine fish that has gradually been bred artificially. To promote future conservation, molecular breeding, and comparative studies, we generated an improved high-quality chromosomal-level genome assembly of leopard coral grouper using Nanopore long-reads, Illumina short reads, and the Hi-C sequencing data. The draft genome is 849.74 Mb with 45 contigs and N50 of 35.59 Mb. Finally, a total of 846.49 Mb corresponding to 99.6% of the contig sequences was anchored to 24 pseudo-chromosomes using Hi-C technology. A final set of 25,965 genes is annotated after manual curation of the predicted gene models, and BUSCO analysis yielded a completeness score of 99.5%. This study significantly improves the utility of the grouper genome and provided a reference for the study of molecular breeding, genomics and biology in this species.

It is of vital importance to understand the population structure, dissect the genetic bases of performance traits, and make proper strategies for selection in breeding programs. However, there is no single webserver covering the specific needs in aquaculture. We present Aquaculture Molecular Breeding Platform (AMBP), the first web server for genetic data analysis in aquatic species of farming interest. AMBP integrates the haplotype reference panels of 18 aquaculture species, which greatly improves the accuracy of genotype imputation. It also supports multiple tools to infer genetic structures, dissect the genetic architecture of performance traits, estimate breeding values, and predict optimum contribution. All the tools are coherently linked in a web-interface for users to generate interpretable results and evaluate statistical appropriateness. The webserver supports standard VCF and PLINK (PED, MAP) files, and implements automated pipelines for format transformation and visualization to simplify the process of analysis. As a demonstration, we applied the webserver to Pacific white shrimp and Atlantic salmon datasets. In summary, AMBP constitutes comprehensive resources and analytical tools for exploring genetic data and guiding practical breeding programs. AMBP is available at http://mgb.qnlm.ac.

External tagging methods can aid in the research of leopard coral grouper (Plectropomus leopardus) in terms of its spatio-temporal behavior at population and individual scales. However, due to the strong exclusion ability and the damage to the body wall of P. leopardus, the retention rate of traditional invasive tagging methods is low. To develop a non-invasive identification method for P. leopardus, we adopted a multiscale image processing method based on matched filters with Gaussian kernels and partial differential equation (PDE) multiscale hierarchical decomposition with the deep convolutional neural network (CNN) models VGG19 and ResNet50 to extract shape and texture image features of individuals. Then based on image features, we used three classifiers Random forest (RF), support vector machine (SVM), and multilayer perceptron (MLP)) for individual recognition on sequential images of P. leopardus captured for 50 days. The PDE, ResNet50 and MLP combination obtained a maximum accuracy of 0.985 ± 0.045 on the test set. For individual temporal tracking recognition, feature extraction and model training were performed using images taken in 1-20 days. The classifier could achieve an accuracy of 0.960 ± 0.049 on the test set consisting of images collected in the periods of 20-50 days. The results show that CNNs with the PDE decomposition can effectively and accurately identify P. leopardus.

The thioredoxin-like protein exists widely, in various organisms, as a regulator of redox homeostasis. In this study, the full-length cDNA of a thioredoxin-like protein gene from rotifer Brachionus plicatilis (designated as BpTXNL) was obtained by 5' rapid amplification of cDNA end (RACE) technology. The complete cDNA of BpTXNL was 1111 bp, and contained a 5' untranslated region (UTR) of 69 bp, a 3' UTR of 163 bp with a polyadenylate additional signal and a polyadenylation site (PAS), and an open reading frame (ORF) of 878 bp, encoding 292 amino acids. The calculated molecular weight and the theoretical isoelectric point (pI) of the deduced BpTXNL peptide were 32.7 kDa and 4.97, respectively. The deduced protein sequence of BpTXNL contained a thioredoxin domain with the conserved redox-active site at

Background Gonadal development is driven by a complex genetic cascade in vertebrates. However, related information remains limited in molluscs owing to the long generation time and the difficulty in maintaining whole life cycle in the lab. The dwarf surfclam Mulinia lateralis is considered an ideal bivalve model due to the short generation time and ease to breed in the lab. Results To gain a comprehensive understanding of gonadal development in M. lateralis, we conducted a combined morphological and molecular analysis on the gonads of 30 to 60 dpf. Morphological analysis showed that gonad formation and sex differentiation occur at 35 and 40–45 dpf, respectively; then the gonads go through gametogenic cycle. Gene co-expression network analysis on 40 transcriptomes of 35–60 dpf gonads identifies seven gonadal development-related modules, including two gonad-forming modules (M6, M7), three sex-specific modules (M14, M12, M11), and two sexually shared modules (M15, M13). The modules participate in different biological processes, such as cell communication, glycan biosynthesis, cell cycle, and ribosome biogenesis. Several hub transcription factors including SOX2, FOXZ, HSFY, FOXL2 and HES1 are identified. The expression of top hub genes from sex-specific modules suggests molecular sex differentiation (35 dpf) occurs earlier than morphological sex differentiation (40–45 dpf). Conclusion This study provides a deep insight into the molecular basis of gonad formation, sex differentiation and gametogenesis in M. lateralis, which will contribute to a comprehensive understanding of the reproductive regulation network in molluscs.

Leucine-rich repeat (LRR)-only proteins have been proved to be involved in the innate immune responses as they could mediate protein-protein or protein-ligand interactions. In the present study, a novel LRR-only protein (LvLRRop-1) was identified and characterized from Pacific white shrimp Litopenaeus vannamei. The complete cDNA sequence of LvLRRop-1 contains an open reading frame (ORF) of 1488 bp, which encoded a polypeptide of 495 amino acids with a predicted molecular mass of 55.67 kDa and a calculated theoretical isoelectric point of 6.435. There are five LRR motifs, six LRR_TYP motifs in the protein sequence of LvLRRop-1 with consensus signature sequences of LxxLxxLxLxxNxL. The LvLRRop-1 mRNA transcripts could be detected in all the tested tissues, including eyestalk, gill, gonad, heart, hemocytes, hepatopancreas, intestine, muscle, nerve and stomach, especially highest in hemocytes and hepatopancreas. The mRNA transcripts of LvLRRop-1 increased within the first 6 h in hemocytes and hepatopancreas after Vibrio parahaemolyticus or white spot syndrome virus (WSSV) challenges. The recombinant LvLRRop-1 could bind four typical pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), peptidoglycan (PGN), glucan (GLU) and polycytidine-polycytidylic acid (poly IC), in a dose-dependent manner, and inhibit the growth of bacteria Micrococcus luteus. These data indicated that LvLRRop-1 could play a pivotal role in the innate immune response of shrimps as a kind of pattern recognition receptor (PRR).

Bivalve molluscs are filter-feeding organisms that can accumulate paralytic shellfish toxins (PST) through ingesting toxic marine dinoflagellates. While the effects of PST accumulation upon the physiology of bivalves have been documented, the underlying molecular mechanism remains poorly understood. In this study, transcriptomic analysis was performed in the gills of Zhikong scallop (

The element ratio of Mg/Ca is a primary proxy in studying the composition of seashells, and laser-induced breakdown spectroscopy (LIBS) is a practical technique for elemental analysis. In this work, we tried to apply LIBS to investigate the Mg/Ca ratio in cultured seashells of two scallop species ( Chlamys farreri and Patinopecten yessoensis). It was found that LIBS was capable of tracking Mg/Ca variation on the cross section of the scallop shell ( Chlamys farreri), and the variation trend was well verified by the measurement of SEM-EDS. Based on this, LIBS detection was carried out for Mg/Ca investigation on another species of the scallop shell ( Patinopecten yessoensis), and the intensity ratio of Mg/Ca presented a similar variation to δ18O measurement under mass spectroscopy. The result also indicated that the LIBS technique is appropriate in the Mg/Ca investigation for scallop shells, and in cultured scallops, the Mg/Ca ratio would be equally useful for tracking temperature variations. In the future, LIBS is expected to be developed as a diagnostic tool in seashell cultivation, and more elemental compositions would be involved in this kind of investigation.

Temperature is an important factor affecting the growth, development and survival of marine organisms. A short episode of high temperature has been proven to be a severe threat to sustainable shellfish culture. Zhikong scallop (

Interferon regulatory factors (IRFs) are a family of transcription factors that control many facets during innate and adaptive immune responses. Vertebrate IRFs play important roles in regulating the expression of interferons (IFNs) and IFN-stimulated genes, while only limited studies were conducted on invertebrate IRFs. In the present study, four IRF family genes (CfIRF1, CfIRF1-like, CfIRF2, and CfIRF8) were identified from Zhikong scallop (Chlamys farreri) through whole-genome scanning. CfIRFs contain a highly conserved N-terminal DNA-binding domain and a variable C-terminal regulatory domain. CfIRFs were constitutively expressed during development as well as in adult tissues, especially in hepatopancreas, hemolymph, gill, and mantle. In hemolymph, qRT-PCR analysis revealed that CfIRF1, CfIRF1-like, and CfIRF2 were significantly upregulated in response to Vibrio anguillarum infection, and their encoding proteins could translocate into nucleus. Dual-luciferase reporter assay on CfIRF1, CfIRF1-like, and CfIRF2 showed that these three proteins were capable to induce a strong activation of ISRE promoters. Notably, in comparison with CfIRF1 and CfIRF1-like, CfIRF2 showed the most sensitive responses in coping with V. anguillarum, and consistently, CfIRF2 exhibited the most significant activation on ISRE. This study would provide valuable information for the innate immune roles of the IRF gene family in bivalve molluscs.

Vasa (

Secretome is involved in almost all physiological, developmental, and pathological processes, but to date there is still a lack of highly-efficient research strategy to comprehensively study the secretome of invertebrates. Adhesive secretion is a ubiquitous and essential physiological process in aquatic invertebrates with complicated protein components and unresolved adhesion mechanisms, making it a good subject for secretome profiling studies. Here we proposed a computational pipeline for systematic profiling of byssal secretome based on spatiotemporal transcriptomes of scallop. A total of 186 byssus-related proteins (BRPs) were identified, which represented the first characterized secretome of scallop byssal adhesion. Scallop byssal secretome covered almost all of the known structural elements and functional domains of aquatic adhesives, which suggested this secretome-profiling strategy had both high efficiency and accuracy. We revealed the main components of scallop byssus (including EGF-like domain containing proteins, the Tyr-rich proteins and 4C-repeats containing proteins) and the related modification enzymes primarily contributing to the rapid byssus assembly and adhesion. Spatiotemporal expression and co-expression network analyses of BRPs suggested a simultaneous secretion pattern of scallop byssal proteins across the entire region of foot and revealed their diverse functions on byssus secretion. In contrast to the previously proposed "root-initiated secretion and extension-based assembly" model, our findings supported a novel "foot-wide simultaneous secretion and in situ assembly" model of scallop byssus secretion and adhesion. Systematic analysis of scallop byssal secretome provides important clues for understanding the aquatic adhesive secretion process, as well as a common framework for studying the secretome of non-model invertebrates.

Exosomes are 30-150 nm-sized extracellular vesicles of endocytic origin that are released into the extracellular environment and play roles in cell-cell communication. Accumulating research achievements demonstrated that exosomes could act as innate immune effectors that contribute to the host defense mechanism. To better understand the immune functions of exosomes in Crassostrea gigas against bacterial stimulation, the iTRAQ LC-MS/MS approach was applied to identifying differentially expressed proteins (DEPs) of exosomes in oyster post Staphylococcus aureus and Vibrio splendidus stimulation. A total of 9467 unique peptides corresponding to 1634 proteins were identified. Among them, 99 proteins were upregulated and 152 were downregulated after S. aureus infection. After V. splendidus infection, 431 proteins were identified as differentially abundant, including 76 that were upregulated and 355 were downregulated. Several proteins related to apoptosis, including E3 ubiquitin-protein ligase, eukaryotic translation initiation factor 3, and protein kinase C delta type were found up-regulated in the S. aureus stimulation group, indicating that the apoptosis process was involved in the response to S. aureus stimulation. Thirty up-regulated and 123 down-regulated proteins were identified as differentially abundant after both bacterial stimuli. Among them, some proteins related to the actin-myosin cytoskeleton process were down-regulated, indicating that phagocytosis may be inhibited in both bacterial stimuli. This study would enrich the C. gigas proteome database and provide information for further understanding the immune functions of oyster exosomes against bacterial infection.

The Pacific white shrimp Litopenaeus vannamei is among the top aquatic species of commercial importance around the world. Over the last four decades, the breeding works of L. vannamei have been carried out intensively and have generated multiple strains with improved production and performance traits. However, signatures of domestication and artificial selection across the L. vannamei genome remain largely unexplored. In the present study, we conducted whole genomic resequencing of 180 Pacific white shrimps from two artificially selective breeds and four market-leading companies. A total of 37 million single nucleotide polymorphisms (SNPs) were identified with an average density of 22.5 SNPs/Kb across the genome. Ancestry estimation, principal component analysis, and phylogenetic inference have all revealed the obvious stratifications among the six breeds. We evaluated the linkage disequilibrium (LD) decay in each breed and identified the genetic variations driven by selection. Pairwise comparison of the fixation index (Fst) and nucleotide diversity (θπ) has allowed for mining the genomic regions under selective sweep in each breed. The functional enrichment analysis revealed that genes within these regions are mainly involved in the cellular macromolecule metabolic process, proteolysis, structural molecule activity, structure of the constituent ribosome, and responses to stimulus. The genome-wide SNP datasets provide valuable information for germplasm resources assessment and genome-assisted breeding of Pacific white shrimps, and also shed light on the genetic effects and genomic signatures of selective breeding.

The glutathione S-transferases (GSTs) are a superfamily of enzymes that function in cellular protection against toxic substances and oxidative stress. Bivalves could accumulate high concentration of paralytic shellfish toxins (PSTs) from harmful algae. To understand the possible involvement of GSTs in protecting bivalves during PST accumulation and metabolism, the GST genes were systemically analyzed in two cultured scallops, Azumapecten farreri and Mizuhopecten yessoensis, which were reported for PST deposition during harmful algae bloom. A total of 35 and 37 GSTs were identified in A. farreri (AfGSTs) and M. yessoensis (MyGSTs) genome, respectively, and the expansion of the sigma class from the cytosolic subfamily was observed. In both scallop species, sigma class GSTs showed higher expression than other members. The high GSTs expression was detected mainly during/after larvae stages and in the two most toxic organs, hepatopancreas and kidney. After ingesting PST-producing dinoflagellates, all the regulated AfGSTs in the hepatopancreas were from the sigma class, but with opposite regulation pattern between Alexandrium catenella and A. minutum exposure. In scallop kidneys, where PSTs transformed into higher toxicity, more AfGSTs were regulated than in the hepatopancreas, and most of them were from the sigma class, with similar regulation pattern between A. catenella and A. minutum exposure. In M. yessoensis exposed to A. catenella, MyGST-σ2 was the only up-regulated MyGST in both hepatopancreas and kidney. Our results suggested the possible diverse function of scallop GSTs and the importance of sigma class in the defense against PSTs, which would contribute to the adaptive evolution of scallops in marine environments.

The transient larva-bearing biphasic life cycle is the hallmark of many metazoan phyla, but how metazoan larvae originated remains a major enigma in animal evolution. There are two hypotheses for larval origin. The ‘larva-first’ hypothesis suggests that the first metazoans were similar to extant larvae, with later evolution of the adult-added biphasic life cycle; the ‘adult-first’ hypothesis suggests that the first metazoans were adult forms, with the biphasic life cycle arising later via larval intercalation. Here, we investigate the evolutionary origin of primary larvae by conducting ontogenetic transcriptome profiling for Mollusca—the largest marine phylum characterized by a trochophore larval stage and highly variable adult forms. We reveal that trochophore larvae exhibit rapid transcriptome evolution with extraordinary incorporation of novel genes (potentially contributing to adult shell evolution), and that cell signalling/communication genes (for example, caveolin and innexin) are probably crucial for larval evolution. Transcriptome age analysis of eight metazoan species reveals the wide presence of young larval transcriptomes in both trochozoans and other major metazoan lineages, therefore arguing against the prevailing larva-first hypothesis. Our findings support an adult-first evolutionary scenario with a single metazoan larval intercalation, and suggest that the first appearance of proto-larva probably occurred after the divergence of direct-developing Ctenophora from a metazoan ancestor. Evolutionary analysis of transcriptomes across Metazoa supports a scenario of ancestral adult stages and a single intercalation event at the origin of larvae.

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), are crucial sensors with a conserved structure in cytoplasm, inducing the production of cytokines, chemokines and host restriction factors which mediate a variety of intracellular activities to interfere with distinct PAMPs (pathogen-associated molecular patterns) for eliminating pathogens in innate immune system. Although RLR genes have been investigated in most vertebrates and some invertebrates, the systematic identification and characterization of RLR genes have not been reported in scallops. In this study, four RLR genes (PY-10413.4, PY-10413.5, PY-443.7 and PY-443.8, designated PyRLRs) were identified in Yesso scallop (Patinopecten yessoensis) through whole-genome scanning through in silico analysis, including two pairs of tandem duplicate genes located on the same scaffold (PY-10413.4 and PY-10413.5, PY-443.7 and PY-443.8, respectively). Phylogenetic and protein structural analyses were performed to determine the identities and evolutionary relationships of these genes. The expression profiles of PyRLRs were determined in all developmental stages, in healthy adult tissues, and in mantles that simulated ocean acidification (OA) exposure (pH = 6.5 and 7.5) at different time points (3, 6, 12 and 24 h). Spatiotemporal expression patterns suggested the functional roles of PyRLRs in all stages of development and growth of the scallop. Regulation expressions revealed PY-10413.4 and PY-10413.5 with one or two CARD(s) (caspase activation and recruitment domain) were up-regulated expressed at most time points, whereas PY-443.8 and PY-10413.4 without CARD were significantly down-regulated at each time points, suggesting functional differentiations in the two pairs of PyRLRs based on the structural differences in response to OA. Collectively, this study demonstrated gene duplication of RLR family genes and provide primary analysis for versatile roles in the response of the bivalve innate immune system to OA challenge.

Graphical abstract, Highlights • Genome-wide characterization of GH families is conducted for Mollusca. • GH9, GH10, GH18 and GH20 families are remarkably expanded in molluscs. • The wide adoption of CBMs likely facilitates the hydrolysis of polysaccharides. • Hepatopancreas is the main organ for the prominent expression of GH families. • Functional divergence of GH families possibly contributes to their adaptive roles., The hydrolysis of sugar-containing compounds by glycoside hydrolases (GHs) plays essential roles in many major biological processes, but to date our systematic understanding of the functional diversity and evolution of GH families remains largely limited to a few well-studied terrestrial animals. Molluscs represent the largest marine phylum in the animal kingdom, and many of them are herbivorous that utilize algae as a main nutritional source, making them good subjects for studying the functional diversity and adaptive evolution of GH families. In the present study, we conducted genome-wide identification and functional and evolutionary analysis of all GH families across major molluscan lineages. We revealed that the remarkable expansion of the GH9, GH10, GH18 and GH20 families and the wide adoption of carbohydrate-binding modules in molluscan expanded GH families likely contributed to the efficient hydrolysis of marine algal polysaccharides and were involved in the consolidation of molluscan algae-feeding habits. Gene expression and network analysis revealed the hepatopancreas as the main organ for the prominent expression of approximately half of the GH families (well corresponding to the digestive roles of the hepatopancreas) and key or hub GHs in the coexpression gene network with potentially diverse functionalities. We also revealed the evolutionary signs of differential expansion and functional divergence of the GH family, which possibly contributed to lineage-specific adaptation. Systematic analysis of GH families at both genomic and transcriptomic levels provides important clues for understanding the functional divergence and evolution of GH gene families in molluscs in relation to their algae-feeding biology.

The cysteine aspartic acid-specific protease (caspase) family is distributed across vertebrates and invertebrates, and its members are involved in apoptosis and response to cellular stress. The Zhikong scallop (

Cathepsin L is widely found in eukaryotes and prokaryotes, and it plays important roles in innate immunity. In the present study, we cloned two cathepsin L genes (designated as MmCTSL1 and MmCTSL2, respectively) from Asiatic hard clam (Meretrix meretrix). The complete sequence of MmCTSL1 cDNA contained a 5' untranslated region (UTR) of 31 bp, a 3' UTR of 228 bp with a poly (A) tail, and an open reading frame (ORF) of 1005 bp encoding 334 amino acids with predicted molecular weight of 37.5 kDa and theoretical isoelectric point of 5.27, and contained a signal peptide (from M1 to A16), a protease inhibitor I29 family domain (from W27 to F87), and a papain family cysteine protease domain (from L118 to T333). The complete sequence of MmCTSL2 cDNA contained a 5' UTR of 50 bp, a 3' UTR of 162 bp with a poly (A) tail, and an ORF of 996 bp encoding a polypeptide of 331 amino acids with predicted molecular weight of 36.8 kDa and theoretical isoelectric point of 7.07. It contained a signal peptide (from M1 to A16), a protease inhibitor I29 family domain (from W30 to F89), and a papain family cysteine protease domain (from L115 to T330). Real-time quantitative PCR analysis demonstrated that MmCTSL1 and MmCTSL2 were widely expressed in all the tested tissues, including adductor muscle, foot, gill, hemocytes, hepatopancreas and mantle, with the highest mRNA expression level in hepatopancreas and hemocytes, respectively. After Vibrio splendidus challenge, the mRNA expression levels of MmCTSL1 and MmCTSL2 in hemocytes and hepatopancreas were both significantly up-regulated with different expression profiles. In hemocytes, the expression levels of MmCTSL1 and MmCTSL2 reached their respective peaks (3.4-fold and 13.0-fold compared with the control, respectively) at 12 h after bacterial challenge, and MmCTSL2 responds earlier than MmCTSL1. In hepatopancreas, the expression levels of MmCTSL1 and MmCTSL2 reached their respective peaks at 6 h (9.0-fold compared with the control) and 24 h (2.8-fold compared with the control) after bacterial challenge, meaning that MmCTSL1 responds earlier than MmCTSL2. At the same time, whether in hepatopancreas or hemocytes, MmCTSL1 persist for a while after the bacterial challenge peak, while MmCTSL2 would quickly return to the initial level after the bacterial challenge peak. These results indicate that cathepsin L may be involved in the immune process of hard clam against V. splendidus with different potential roles.

Anti-lipopolysaccharide factors (ALFs) are a family of common innate immune effectors in crustaceans, and they exhibit broad spectrum antimicrobial activity. In this study, we identified and characterized five novel ALF genes (designated as LvALF1-5) from the Pacific white shrimp (Litopenaeus vannamei) to investigate their potential immune functions. The amino acid sequence alignments showed that LvALFs contained two conserved cysteine residues, a hydrophobic N-terminal region, and the conserved signature sequence W(T/K)CPG(S)WT(A). They all shared high similarity with previously reported ALFs and were clearly novel members of the ALF family. The mRNA transcripts of LvALFs were most highly expressed in hemocytes and the hepatopancreas. After shrimp were stimulated with Vibrio parahaemolyticus or white spot syndrome virus, expression of the LvALFs was significantly induced in hemocytes and the hepatopancreas with various expression profiles. Recombinant proteins of LvALFs exhibited potent bacteriostatic activity in vitro. Together, these results suggest that LvALF1-5 participate in the immune response of Pacific white shrimp against invading pathogens.

Background Oysters inhabit in the intertidal zone and may be suffered from environmental stresses, which can increase the production of reactive oxygen species (ROS), resulting in mass mortality. Superoxide dismutases (SODs) protect oysters from ROS damage through different mechanisms compared with vertebrates. However, the molecular and functional differentiation in oyster SODs were rarely analyzed. Result In this study, a total of 13, 13, 10, and 8 candidate SODs were identified in the genome of Crassostrea gigas, Crassostrea virginica, Crassostrea hongkongensis, and Saccostrea glomerata respectively. The domain composition, gene structure, subcellular locations, conserved ligands, and cis-elements elucidated the SODs into five groups (Mn-SODs, Cu-only-SODs, Cu/Zn ion ligand Cu/Zn-SOD with enzyme activity, Zn-only-SODs, and no ligand metal ions Cu/Zn-SODs). For single domain Cu/Zn-SODs, only one cytosolic Cu/Zn-SOD (cg_XM_034479061.1) may conserve enzymatic activity while most extracellular Cu/Zn-SOD proteins appeared to lose SOD enzyme activity according to conserved ligand amino acid analysis and expression pattern under biotic and abiotic stress in C. gigas. Further, multi-domain-SODs were identified and some of them were expressed in response to biotic and abiotic stressors in C. gigas. Moreover, the expression patterns of these genes varied in response to different stressors, which may be due to the cis-elements in the gene promoter. Conclusion These findings revealed the most extracellular Cu/Zn-SOD proteins appeared to lose SOD enzyme activity in oysters. Further, our study revealed that only one cytosolic Cu/Zn-SOD (cg_XM_034479061.1) may conserve enzymatic activity of SOD. Moreover, the expression patterns of these genes varied in response to different stressors, which may be due to the cis-elements in the promoter. This study provides important insights into the mechanisms through which oysters adapt to harsh intertidal conditions, as well as potential biomarkers of stress response in related species.

Many marine organisms are generally poikilotherms, making seawater temperature one of the most important environmental factors affecting gonadal sex differentiation. Mollusca is the second-largest animal phylum with diverse reproductive systems, but studies on the impact of temperature on sex differentiation are limited to a few sequential hermaphrodites. By combining morphological and molecular analyses, we investigated the effect of temperature on gonadal sex differentiation of a commercially important gonochoristic scallop

Transient receptor potential (TRP) channel plays a significant role in mediating various sensory physiological functions. It is widely present in the vertebrate and invertebrate genomes and can be activated by multiple compounds, messenger molecules, temperature, and mechanical stimulation. Mollusks are the second largest phylum of the animal kingdom and are sensitive to environmental factors. However, the molecular underpinnings through which mollusks sense and respond to environmental stimulus are unknown. In this study, we systematically identified and characterized 17 TRP channels (C.FA TRPs, seven subfamilies) in the genome of the Zhikong scallop (Chlamys farreri). All C.FA TRPs had six transmembrane structures (TM1–TM6). The sequences and structural features of C.FA TRPs are highly conserved with TRP channels of other species. Spatiotemporal expression profiling suggested that some C.FA TRPs participated in the early embryonic development of scallops and the sensory process of adult tissues. Notably, the expression of C.FA TRPM3 continuously increased during developmental stages and was highest among all C.FA TRPs. C.FA TRPC-α was specifically expressed in eyes, which may be involved in light transmission of scallop eyes. Under high temperature stress, C.FA TRPA1 and C.FA TRPA1-homolog upregulated significantly, which indicated that the TRPA subfamily is the thermoTRPs channel of scallops. Our results provided the first systematic study of TRP channels in scallops, and the findings will provide a valuable resource for a better understanding of TRP evolution and function in mollusks.

The increasing sea temperature caused by global warming has led to serious death of Zhikong scallop (Chlamys farreri) and improving its thermal tolerance has become an active research area in scallop aquaculture industry. Gene transcriptional coactivator p15 (PC4) plays pivotally multi-faced roles in most vertebrates and some invertebrates, but the systematic identification and characterization of PC4 genes have less been reported in scallops. In this study, 15 PC4 genes (CfPC4s) were identified in Zhikong scallop through whole-genome scanning, including two pairs of tandem duplicate genes located in the same scaffold (CF-19495.9 and CF-19495.10, CF-6819.1 and CF-6819.2). Protein structural and phylogenetic analyses were performed to verify identities and evolutionary relationships of these genes. Spatiotemporal expression patterns were determined at different development stages and in healthy adult tissues, as well as expression regulations in selected tissues (mantles, gills, hemocytes and hearts) after high temperatures challenge (27 °C) with different durations (3 h, 6 h, 12 h, 24 h, 3 d, 6 d, 15 d and 30 d). Spatiotemporal expressions of CfPC4s were ubiquitous but exhibited different patterns, suggesting the functional roles of CfPC4s in all stages of growth and development of the scallop. Expression regulations of CfPC4s and their functional related factors (TFIIA, TFIID, TFIIH and RNAPII) in pre-initiation complex (PIC) in various tissues displayed up- and/or down-regulated responses at different time points, showing time- and/or tissue-dependent expression patterns with function allocation upon different thermal durations. Collectively, this study demonstrated that gene allocation of CfPC4s provided implications for deciphering thermal response in Zhikong scallop and potentially helped in developing strategies for long-term healthy sustainable Zhikong scallop culture.

To consolidate the genetic, physical, and cytogenetic maps of scallop (Patinopecten yessoensis), we constructed a molecular cytogenetic map by localizing 84 fosmid clones that contain different SNP markers from 19 linkage groups (LGs) using fluorescence in situ hybridization (FISH). Among these 84 SNP-anchored clones, 56 clones produced specific and stable signals on one pair of chromosomes. Dual-color FISH assigned 19 LGs to their corresponding chromosomes with 38 SNP-anchored clones as probes. Among these 19 LGs, 17 LGs were assigned to their corresponding one pair of chromosomes, while two clones containing SNPs from LG10 and LG19 were located on two different pairs of chromosomes separately. The orientation of 7 LGs was corrected according to the chromosome location of SNPs within the same LG. In addition, a probe panel of SNP-anchored clones was developed to identify each chromosome of P. yessoensis. The molecular cytogenetic map will facilitate molecular breeding in scallop and enable comparative studies on chromosome evolution of bivalve mollusk.

Inbreeding often causes a decline in biological fitness, known as inbreeding depression. In genetics study, inbreeding coefficient f gives the proportion by which the heterozygosity of an individual is reduced by inbreeding. With the development of high-throughput sequencing, researchers were able to perform deep approaches to investigate which genes are affected by inbreeding and reveal some molecular underpinnings of inbreeding depression. As one commercially important species, Yesso scallop Patinopecten yessoensis confront the same dilemma of inbreeding depression. To examine how inbreeding affects gene expression, we compared the transcriptome of two experimentally selfing families with inbreeding coefficient f reached 0.5 as well as one natural population (f ≈ 0) of P. yessoensis. A total of 24 RNA-Seq libraries were constructed using scallop adductor muscle, and eventually 676.56 M (96.85%) HQ reads were acquired. Based on differential gene analysis, we were able to identify nine common differentially expressed genes (DEGs) across the top-ranked 30 DEGs in both selfing families in comparation with the natural population. Remarkable, through weighted gene co-expression network analysis (WGCNA), five common DEGs were found enriched in the most significant inbreeding related functional module M14 (FDR = 1.64E-156), including SREBP1, G3BP2, SBK1, KIAA1161, and AATs-Glupro. These five genes showed significantly higher expression in self-bred progeny. Suggested by the genetic functional analysis, up-regulated SREBP1, G3BP2, and KIAA1161 may suggest a perturbing lipid metabolism, a severe inframammary reaction or immune response, and a stress-responsive behavior. Besides, the significant higher SBK1 and AATs-Glupro may reflect the abnormal cellular physiological situation. Together, these genetic aberrant transcriptomic performances may contribute to inbreeding depression in P. yessoensis, deteriorating the stress tolerance and survival phenotype in self-bred progeny. Our results would lay a foundation for further comprehensive understanding of bivalve inbreeding depression, which may potentially benefit the genetic breeding for scallop aquaculture.

Aureococcus anophagefferens caused brown tides for three consecutive years from2009 to2011 in the coastal waters of Qinhuangdao, China, with numerous, widespread ecological and economic impact on ecosystems. To understand the population dynamics of nanoplankton during the brown tides, sequences of the V9 region of the 18S rDNA gene, used as a marker, were analyzed by Illumina sequencing to assess nanoplankton biomass, and real-time fluorescence quantitative PCR was performed to analyze spatial variation in the 18S rDNA copy concentrations of nanoplankton off the Qinhuangdao coast in July,2011. The results showed that A. anophagefferens and Minutocellus polymorphus were the dominant species in the local phytoplankton community during the brown tide in July2011. The highest 18S rDNA copy concentrations of A. anophagefferens and M. polymorphus were detected at stations SHG and FN, respectively. The central area most strongly affected by the brown tide migrated southward from2011 to2013. Redundancy analysis (RDA) showed that the decreasing NOx concentration might provide suitable nutrient conditions for the A. anophagefferens outbreak. During the brown tide caused by A. anophagefferens, other phytoplankton, such as diatoms, cryptophytes, chlorophytes, dinoflagellates and other flagellates, could co-occur with it. For zooplankton, due to less selective feeding behavior, Amoebozoa was the most abundant zooplankton at station SHG, while Ciliophora was the most abundant zooplankton at other stations for its more selective feeding.

Brown tides caused by Aureococcus anophagefferens have occurred frequently in the coastal waters of Qinhuangdao since 2009. The Changli coast is the area most strongly affected by the brown tides, which result in significant economic losses and serious ecological harm. To understand the effects of brown tides on nanoplankton diversity and biomass, samples were collected from May to July in 2012 at station XKK, which is approximately 0.2 nautical miles off the Changli coast. The V9 region of the 18S rDNA gene was used as a marker and was analyzed by Illumina sequencing to assess the nanoplankton diversity and biomass, and real-time fluorescence quantitative PCR was performed to analyze the temporal variations in the copy concentrations of the 18S rDNA of nanoplankton. The results showed that nanoplankton diversity, as determined by the Shannon index, decreased from May to June and increased in July. Illumina sequencing analysis revealed that the plankton community differed among the samples from May to July, and over this period, the dominant phytoplankton groups changed from pelagophytes to chlorophytes. Sufficient nutrients and low grazing pressure by zooplankton and scallops accelerated the growth of small-celled phytoplankton. A. anophagefferens was the most dominant phytoplankton species, with an 18S rDNA maximum copy concentration detected on 26 June, followed by Micromonas sp. and Bathycoccus prasinos, with maximum copy concentrations observed on 3 July. Ciliophora, Arthropoda and Cercozoa were the dominant taxa of the local zooplankton community. The Ciliophora and Arthropoda biomasses decreased, and the Cercozoa biomass increased when the copy concentration of A. anophagefferens peaked on 26 June. We suspect that A. anophagefferens had a significantly negative effect on the growth of Ciliophora and Arthropoda and could be ingested by Cercozoa to promote its proliferation. The small and low-abundance plankton that are likely missed in microscopic and clone library analyses could be detected using Illumina sequencing and qPCR, which provide an effective method for a comprehensive analysis of the plankton community.

As lipid microconstituents mainly of plant origin, carotenoids are essential nutrients for humans and animals, and carotenoid coloration represents an important meat quality parameter for many farmed animals. Currently, the mechanism of carotenoid bioavailability in animals is largely unknown mainly due to the limited approaches applied, the shortage of suitable model systems and the restricted taxonomic focus. The mollusk Yesso scallop (Patinopecten yessoensis) possessing orange adductor muscle with carotenoid deposition, provides a unique opportunity to research the mechanism underlying carotenoid utilization in animals. Herein, through family construction and analysis, we found that carotenoid coloration in scallop muscle is inherited as a recessive Mendelian trait. Using a combination of genomic approaches, we mapped this trait onto chromosome 8, where PyBCO-like 1 encoding carotenoid oxygenase was the only differentially expressed gene between the white and orange muscles (FDR = 2.75E-21), with 11.28-fold downregulation in the orange muscle. Further functional assays showed that PyBCO-like 1 is capable of degrading β-carotene, and inhibiting PyBCO-like 1 expression in the white muscle resulted in muscle coloration and carotenoid deposition. In the hepatopancreas, which is the organ for digestion and absorption, neither the scallop carotenoid concentration nor PyBCO-like 1 expression were significantly different between the two scallops. These results indicate that carotenoids could be taken up in both white- and orange-muscle scallops and then degraded by PyBCO-like 1 in the white muscle. Our data suggest that PyBCO-like 1 is the essential gene for carotenoid metabolism in scallop muscle, and its downregulation leads to carotenoid deposition and muscle coloration.

Vasa (Ddx4, DEAD box polypeptide 4), an extremely specific marker of germ cells in vivo, is an ATP-dependent RNA helicase that plays an essential role in germ cell development and gametogenesis. However, the expression and function information about this gene in groupers remains lacking. Here, vasa homolog termed Plvasa was isolated and identified Plvasa as a putative germ cell marker in the leopard coral grouper, (Plectropomus leopardus). Results indicated that Plvasa contained 17 exons in the genomic sequence and 9 conserved motifs of the DEAD-box protein by sequence analysis. The sequence comparison, phylogenetic analyses and synteny analyses showed that Plvasa was homologous with other teleosts. Additionally, the expression of Plvasa was significantly higher in gonads than in other tissues in adult individuals (p < 0.05). Further, the distribution of Plvasa revealed that it was only expressed in the germ cells, such as spermatids, germline stem cells and oocytes at different stages, and could not be detected in the somatic cells of gonads. The current study verified that the Plvasa gene is a valuable molecular marker of germ cells in leopard coral grouper, which potentially plays an important role in investigating the genesis and development of teleost germ cells.

In selective breeding, a central parameter in summarizing the proportion of variance due to genetics for the purpose of predicting gains from selection is realized heritability (h2). We applied the genome-wide complex trait analysis (GCTA) method to genome-wide SNP data obtained by 2b-RAD reduced-representation genotyping and phenotypic data for four traits (shell length, shell height, shell width and whole wet weight) with the aim of establishing the heritability for growth in Zhikong scallop (Chlamys farreri) in a selective breeding program. The GCTA-based heritabilities of 0.42 (S.E. 0.09) for shell length, 0.47 (S.E. 0.07) for shell height, 0.54 (S.E. 0.11) for shell width and 0.28 (S.E. 0.03) for whole wet weight, which were estimated with uncommon SNPs (26,471 SNPs with MAF >2%), were close to and strongly correlated (r = 0.957) with traditional estimates of realized heritability and in the moderate-to-high range, in line with values previously obtained for growth rates in bivalves. hGCTA2 with 20,000 and 10,000 SNPs were very close to estimates with uncommon SNPs and appeared relatively robust to SNP number. Removing causal SNPs had little effect on obtaining reliable estimates of hGCTA2, suggesting that causal SNPs is not necessary for accurate estimates of h2. Chromosome-wise heritability estimates suggested that the genetic contribution to growth complex traits is scattered across the genome and driven by many loci with small effect rather than a few causal loci with a large effect. These results indicate that the genome-wide complex trait analysis method may be useful for estimating realized heritability for growth in Zhikong scallop with SNPs obtained by reduced-representation genotyping approaches, which is less expensive and faster than full-genome sequencing for non-model species growing in natural environments.

Microbiome samples with low microbial biomass or severe DNA degradation remain challenging for amplicon-based or whole-metagenome sequencing approaches. Here, we introduce 2bRAD-M, a highly reduced and cost-effective strategy which only sequences ~ 1% of metagenome and can simultaneously produce species-level bacterial, archaeal, and fungal profiles. 2bRAD-M can accurately generate species-level taxonomic profiles for otherwise hard-to-sequence samples with merely 1 pg of total DNA, high host DNA contamination, or severely fragmented DNA from degraded samples. Tests of 2bRAD-M on various stool, skin, environmental, and clinical FFPE samples suggest a successful reconstruction of comprehensive, high-resolution microbial profiles. Supplementary Information The online version contains supplementary material available at 10.1186/s13059-021-02576-9.