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Abstract Background Bivalves represent a vital economic resource in aquaculture for their high productivity and extensive market demand. Growth is one of the most important and desired aquaculture traits for bivalves, regulated by multiple levels, notably intricate metabolic processes. However, the understanding of the metabolic profiles that influence bivalve growth is limited, particularly from a multi-tissue perspective. Results In this study, metabolic profiles of multiple tissues of Chlamys farreri with different growth performance were systematically investigated by ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Through comparing the metabolic variation between fast-growing (FG) scallops and slow-growing (SG) scallops, 613, 509, 105, and 192 significantly different metabolites (SDMs) were identified in the mantle, gill, adductor muscle, and digestive gland, respectively. Growth-related metabolic pathways including sphingolipid metabolism, fatty acid biosynthesis, and ABC transporter pathway, along with 11 SDMs associated with growth traits were identified in all four tissues, implying they were involved in the growth of multiple tissues in scallops. Tissue-specific metabolic profiling indicated that sulfur-containing amino acid metabolism in the mantle potentially contributed to shell growth, while the gill synergistically participated with the mantle through various metabolic processes, such as tyrosine metabolism, glycine, serine, and threonine metabolism and melanogenesis; energy metabolism was crucial for adductor muscle growth; and nutrients digestion and absorption in the digestive gland were linked to scallop growth. Conclusions Our results represent the first comprehensive analysis of the crucial pathways and metabolites associated with the growth of C. farreri, offering valuable insights for future bivalve aquaculture production.

Abstract Background The leopard coral grouper (Plectropomus leopardus) is a commercially valuable tropical marine fish species known to be sensitive to low temperatures. A comprehensive understanding of the molecular mechanisms governing its response to acute cold stress is of great importance. However, there is a relative scarcity of fundamental research on low-temperature tolerance in the leopard coral grouper. Methods In this study, a cooling and rewarming experiment was conducted on 6-month-old leopard coral groupers. Within 24 h, we decreased the ambient temperature from 25 °C to 13 °C and subsequently allowed it to naturally return to 25 °C. During this process, a comprehensive investigation of serum hormone levels, enzyme activity, and brain transcriptome analysis was performed. Results P. leopardus displayed a noticeable adaptive response to the initial temperature decrease by temporarily reducing its life activities. Our transcriptome analysis revealed that the differentially expressed genes (DEGs) were primarily concentrated in crucial pathways including the blood-brain barrier (BBB), inflammatory response, and coagulation cascade. In situ hybridization of claudin 15a (cldn15a), a key gene for BBB maintaining, further confirmed that exposure to low temperatures led to the disruption of the blood-brain barrier and stimulated a pronounced inflammatory reaction within the brain. Upon rewarming, there was a recovery of BBB integrity accompanied by the persistence of inflammation within the brain tissue. Conclusions Our study reveals the complex interactions between blood-brain barrier function, inflammation, and recovery in P. leopardus during short-term temperature drops and rewarming. These findings provide valuable insights into the physiological responses of this species under cold stress conditions.

Abstract Background The genetic basis underlying spawning abilities in the Pacific white shrimp, Penaeus vannamei, remains largely unexplored. To investigate genetic variations potentially related to reproductive performance, a systematic bioinformatic analysis was conducted to identify structural variations (SVs) with different polymorphic spectra in P. vannamei with high fertility (HF) and low fertility (LF). Results A total of 2,323 and 1,859 SV events were identified exclusively in the HF and LF groups, respectively. These SVs were mapped to 277 genes in the HF group and 231 genes in the LF group. Gene Ontology (GO) enrichment analysis based on SNPs (single nucleotide polymorphism) and SVs revealed several neural-related processes, suggesting the importance of neural regulation in reproduction. Notably, we identified a set of promising genes, including Cttn, Spast, Ppp4c, Spire1, Lhcgr, and Ftz-f1, which may enhance fertility in shrimp. Conclusion In conclusion, this study is the first to establish a link between SVs and reproductive traits in P. vannamei. The promising genes discovered have the potential to serve as crucial markers for enhancing reproductive traits through targeted genotyping.

Abstract The “hologenome” concept is an increasingly popular way of thinking about microbiome-host for marine organisms. However, it is challenging to track hologenome dynamics because of the large amount of material, with tracking itself usually resulting in damage or death of the research object. Here we show the simple and efficient holo-2bRAD approach for the tracking of hologenome dynamics in marine invertebrates (i.e., scallop and shrimp) from one holo-2bRAD library. The stable performance of our approach was shown with high genotyping accuracy of 99.91% and a high correlation of r > 0.99 for the species-level profiling of microorganisms. To explore the host-microbe association underlying mass mortality events of bivalve larvae, core microbial species changed with the stages were found, and two potentially associated host SNPs were identified. Overall, our research provides a powerful tool with various advantages (e.g., cost-effective, simple, and applicable for challenging samples) in genetic, ecological, and evolutionary studies.

Background: The Pacific white shrimp is one of the world’s most economically significant aquatic species, being one of the top three species cultured globally. However, the increasing incidence of diseases such as acute hepatopancreatic necrosis disease and hepatopancreatic microsporidia has led to a serious decline in shrimp production and severe economic losses. With the increasing demand for pathogen detection in shrimp farms, rapid DNA extraction technology has become more sophisticated. In this study, a rapid and crude method of extracting genomic DNA from shrimp muscle and hepatopancreas using Chelex-100 was established. Results: DNA was successfully extracted from muscle and hepatopancreatic tissues using both the Chelex-100 method and commercial kits. The internal reference genes of shrimp were successfully amplified via PCR and real-time PCR using the obtained DNA samples. Moreover, a field assay was successfully conducted using real-time PCR and real-time enzymatic recombinase amplification (real-time ERA), indicating that the quality of the DNA extracted using Chelex-100 is sufficient for use in conjunction with nucleic acid amplification to detect pathogens in shrimps. Conclusions: Chelex-100 is an efficient method for extracting DNA from shrimp muscle or hepatopancreas tissues, with a short extraction time, high extraction efficiency, and simple operation, making it appropriate for use in the detection of pathogens in shrimp.How to cite: Yang H, Zhou Q, Hu J, et al. A Chelex-100-based rapid DNA extraction method and its application in the detection of shrimp pathogens. Electron J Biotechnol 2024;70. https://doi.org/10.1016/j.ejbt.2024.04.004.

Nitrite exposure has become a significant concern in the aquaculture industry, posing a severe threat to aquatic animals such as shrimp. While studies have reported the adverse effects of nitrite on shrimp growth, the part played by the gut microbiota in shrimp mortality resulting from nitrite exposure is poorly understood. Here, the effects of nitrite on shrimp gut bacterial community were investigated using 16S rRNA amplicon sequencing, bacterial isolation, genomic analysis, and infection experiments. Compared to the control_healthy group, changes in the bacterial composition of the nitrite_dead group were associated with reduced abundance of specific beneficial bacteria and increased abundance of certain pathogenic bacteria. Notably, members of the Photobacterium genus were found to be significantly enriched in the nitrite_dead group. Genomic analysis of a representative Photobacterium strain (LvS-8n3) revealed a variety of genes encoding bacterial toxins, including hemolysin, adhesin, and phospholipase. Furthermore, it was also found that LvS-8n3 exhibits strong pathogenicity, probably due to its high production of pathogenic factors and the ability to utilize nitrite for proliferation. Therefore, the proliferation of pathogenic Photobacterium species appears pivotal for driving shrimp mortality caused by nitrite exposure. These findings provide novel insights into the disease mechanism in shrimp under conditions of environmental change.

Utilizing image-based computer vision techniques, many high-throughput phenotyping methods have been employed to capture intricate growth trait characteristics, offering reliable estimates of phenotypic traits crucial for breeding programs. In this study, we explored the application of partial differential equation (PDE)-based level set approaches to introduce image-based body area percentage (IBAP) as a novel growth trait in Plectropomus leopardus, as a substitution for the traditional growth trait body weight (BW). Assessing the genetic parameters essential for robust growth trait improvement in P. leopardus breeding programs, we estimated SNP-based heritability for IBAP and BW using a comprehensive set of SNPs (673,039 SNPs with MAF >2%). Results revealed heritability estimates of 0.515 (S.E. 0.06) for IBAP and 0.542 (S.E. 0.06) for BW. Moreover, strong phenotypic and genetic correlations of 0.812 (S.E. 0.001) and 0.903 (S.E 0.021) between IBAP and BW, respectively, underscored the potential for IBAP as a surrogate trait of BW for the genetic improvement of P. leopardus. We established a linear regression model of IBAP and BW (y ​= ​−730 ​+ ​1700×, (R2 ​= ​0.71)), after rigorous assessments of linearity, normality, and homoscedasticity, to confirm model fit. Evaluation of breeding value prediction accuracies using two linear models (rr-GBLUP and Bayes B) and a non-linear (RKHS) model demonstrated the superior performance of RKHS across IBAP and BW. Exploring the impact of varied marker densities for SNP selection on genomic prediction accuracy for IBAP and BW demonstrated a threshold of 10,000 SNPs for maximal model accuracy. These findings provide essential reference information and methodological groundwork for leveraging image-based traits in P. leopardus breeding endeavors, facilitating more efficient and precise genetic improvement programs.

Abstract Background Due to its enormous biomass, Antarctic krill (Euphausia superba) plays a crucial role in the Antarctic Ocean ecosystem. In recent years, Antarctic krill has found extensive application in aquaculture, emerging as a sustainable source of aquafeed with ideal nutritional profiles. However, a comprehensive study focused on the detailed effects of dietary Antarctic krill on aquaculture animals, especially farmed marine fishes, is yet to be demonstrated. Results In this study, a comparative experiment was performed using juvenile P. leopardus, fed with diets supplemented with Antarctic krill (the krill group) or without Antarctic krill (the control group). Histological observation revealed that dietary Antarctic krill could reduce lipid accumulation in the liver while the intestine exhibited no obvious changes. Enzyme activity measurements demonstrated that dietary Antarctic krill had an inhibitory effect on oxidative stress in both the intestine and the liver. By comparative transcriptome analysis, a total of 1,597 and 1,161 differentially expressed genes (DEGs) were identified in the intestine and liver, respectively. Functional analysis of the DEGs showed multiple enriched terms significantly related to cholesterol metabolism, antioxidants, and immunity. Furthermore, the expression profiles of representative DEGs, such as dhcr7, apoa4, sc5d, and scarf1, were validated by qRT-PCR and fluorescence in situ hybridization. Finally, a comparative transcriptome analysis was performed to demonstrate the biased effects of dietary Antarctic krill and astaxanthin on the liver of P. leopardus. Conclusions Our study demonstrated that dietary Antarctic krill could reduce lipid accumulation in the liver of P. leopardus, enhance antioxidant capacities in both the intestine and liver, and exhibit molecular-level improvements in lipid metabolism, immunity, and antioxidants. It will contribute to understanding the protective effects of Antarctic krill in P. leopardus and provide insights into aquaculture nutritional strategies.

Target of rapamycin complex 1 (TORC1) is a key regulator of metabolism in eukaryotes across multiple pathways. Although TORC1 has been extensively studied in vertebrates and some invertebrates, research on this complex in scallops is limited. In this study, we identified the genes encoding TORC1 complex subunits in the scallop Argopecten irradians irradians through genome-wide in silico scanning. Five genes, including TOR, RAPTOR, LST8, DEPTOR, and PRAS40, that encode the subunits of TORC1 complex were identified in the bay scallop. We then conducted structural characterization and phylogenetic analysis of the A. i. irradians TORC1 (AiTORC1) subunits to determine their structural features and evolutionary relationships. Next, we analyzed the spatiotemporal expressions of AiTORC1-coding genes during various embryo/larvae developmental stages and across different tissues in healthy adult scallops. The results revealed stage- and tissue-specific expression patterns, suggesting diverse roles in development and growth. Furthermore, the regulation of AiTORC1-coding genes was examined in temperature-sensitive tissues (the mantle, gill, hemocyte, and heart) of bay scallops exposed to high-temperature (32 °C) stress over different durations (0 h, 6 h, 12 h, 24 h, 3 d, 6 d, and 10 d). The expression of AiTORC1-coding genes was predominantly suppressed in the hemocyte but was generally activated in the mantle, gill, and heart, indicating a tissue-specific response to heat stress. Finally, functional validation was performed using the TOR inhibitor rapamycin to suppress AiTORC1, leading to an enhanced catabolism, a decreased antioxidant capacity, and a significant reduction in thermotolerance in bay scallops. Collectively, this study elucidates the presence, structural features, evolutional relationships, expression profiles, and roles in antioxidant capacity and metabolism regulation of AiTORC1 in the bay scallop, providing a preliminary understanding of its versatile functions in response to high-temperature challenges in marine mollusks.

Heat stress resulting from global climate change has been demonstrated to adversely affect growth, development, and reproduction of marine organisms. The Zhikong scallop (Chlamys farreri), an important economical mollusk in China, faces increasing risks of summer mortality due to the prolonged heat waves. The heart, responsible for transporting gas and nutrients, is vital in maintaining homeostasis and physiological status in response to environmental changes. In this study, the effect of heat stress on the cardiac function of C. farreri was investigated during the continuous 30-day heat stress at 27 °C. The results showed the heart rate of scallops increased due to stress in the initial phase of high temperature exposure, peaking at 12 h, and then gradually recovered, indicating an acclimatization at the end of the experiment. In addition, the levels of catalase (CAT), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC) exhibited an initial increase followed by recovery in response to heat stress. Furthermore, transcriptome analysis of the heart identified 3541 differentially expressed genes (DEGs) in response to heat stress. Subsequent GO and KEGG enrichment analysis showed that these genes were primarily related to signal transduction and oxidative stress, such as the phosphatidylinositol signaling system, regulation of actin cytoskeleton, MAPK signaling pathway, FoxO signaling pathway, etc. In addition, two modules were identified as significant responsive modules according to the weighted gene co-expression network analysis (WGCNA). The upregulation of key enzymes within the base excision repair and gap junction pathways indicated that the heart of C. farreri under heat stress enhanced DNA repair and maintained cellular integrity. In addition, the variable expression of essential signaling molecules and cytoskeletal regulators suggested that the heart of C. farreri modulated cardiomyocyte contraction, intracellular signaling, and heart rate through complex regulation of phosphorylation and calcium dynamics in response to heat stress. Collectively, this study enhances our understanding of cardiac function and provides novel evidence for unraveling the mechanism underlying the thermal response in mollusks.

Filter-feeding bivalves could accumulate paralytic shellfish toxins (PSTs) produced by harmful dinoflagellates through diet. Despite that bivalves are resistant to these neurotoxins due to possessing PST-resistant sodium channel, exposure to PSTs-producing dinoflagellates impair bivalve survival. We hypothesized that ingesting PSTs-producing dinoflagellates may influence the gut microbiota, and then the health of bivalves. To test this idea, we compared the gut microbiota of the scallop Patinopecten yessoensis, after feeding with PST-producing or non-toxic dinoflagellates. Exposure to PSTs-producing dinoflagellates resulted in a decline of gut microbial diversity and a disturbance of community structure, accompanied by a significant increase in the abundance and richness of pathogenic bacteria, represented by Vibrio. Moreover, network analysis demonstrated extensive positive correlations between pathogenic bacteria abundances and PSTs concentrations in the digestive glands of the scallops. Furthermore, isolation of a dominant Vibrio strain and its genomic analysis revealed a variety of virulence factors, including the tolC outer membrane exporter, which were expressed in the gut microbiota. Finally, the infection experiment demonstrated scallop mortality caused by the isolated Vibrio strain; further, the pathogenicity of this Vibrio strain was attenuated by a mutation in the tolC gene. Together, these findings demonstrated that the PSTs may affect gut microbiota via direct and taxa-specific interactions with opportunistic pathogens, which proliferate after transition from seawater to the gut environment. The present study has revealed novel mechanisms towards deciphering the puzzles in environmental disturbances-caused death of an important aquaculture species.

Fish exhibit a broad spectrum of colors and patterns facilitated by specialized cells known as chromatophores. The vibrant coloration of fish, controlled by complex genetic and environmental interactions, serves critical roles in ecological functions such as mating, predation, and camouflage. This diversity not only makes fish an invaluable model for exploring the molecular mechanisms of pigmentation but also significantly impacts their economic value within the aquaculture industry, where color traits can drive marketability and breeding choices. This review delves into the sophisticated biological processes governing fish pigmentation and discusses their applications in enhancing aquaculture practices. By exploring the intersection of genetic regulation, environmental influences, and advanced breeding techniques, this review highlights both the scientific understanding and practical applications of fish coloration, providing a bridge between basic biological research and its application in commercial aquaculture.

Abstract Microbial lysis of dimethylsulfoniopropionate (DMSP) is a key step in marine organic sulfur cycling and has been recently demonstrated to play an important role in mediating interactions between bacteria, algae, and zooplankton. To date, microbes that have been found to lyse DMSP are largely confined to free‐living and surface‐attached bacteria. In this study, we report for the first time that a symbiont (termed “Rhodobiaceae bacterium HWgs001”) in the gill of the marine scallop Argopecten irradians irradians can lyse and metabolize DMSP. Analysis of 16S rRNA gene sequences suggested that HWgs001 accounted for up to 93% of the gill microbiota. Microscopic observations suggested that HWgs001 lived within the gill tissue. Unlike symbionts of other bivalves, HWgs001 belongs to Alphaproteobacteria rather than Gammaproteobacteria, and no genes for carbon fixation were identified in its small genome. Moreover, HWgs001 was found to possess a dddP gene, responsible for the lysis of DMSP to acrylate. The enzymatic activity of dddP was confirmed using the heterologous expression, and in situ transcription of the gene in scallop gill tissues was demonstrated using reverse‐transcription PCR. Together, these results revealed a taxonomically and functionally unique symbiont, which represents the first‐documented DMSP‐metabolizing symbiont likely to play significant roles in coastal marine ecosystems.

Abstract The dwarf surf clam, Mulinia lateralis, is considered as a model species for bivalves because of its rapid growth and short generation time. Recently, successful breeding of this species for multiple generations in our laboratory revealed its acquisition of adaptive advantages during artificial breeding. In this study, 310 individuals from five different generations were genotyped with 22,196 single nucleotide polymorphisms (SNPs) with the aim of uncovering the genetic basis of their adaptation to laboratory conditions. Results revealed that M. lateralis consistently maintained high genetic diversity across generations, characterized by high observed heterozygosity (Ho: 0.2733–0.2934) and low levels of inbreeding (Fis: −0.0244–0.0261). Population analysis indicated low levels of genetic differentiation among generations of M. lateralis during artificial breeding (Fst

Abstract 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.

Genome miniaturization drives key evolutionary innovations of adaptive traits in vertebrates, such as the flight evolution of birds. However, whether similar evolutionary processes exist in invertebrates remains poorly understood. 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, elevated expression of genes involved in energy-producing pathways, and decreased copy numbers and expression levels of biomineralization-related 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.

Highlights Mulinia lateralis reaches sexual maturation within 2 months, facilitating comprehensive studies of gonadal development. A combined morphological and molecular analysis reveals the timing of gonad formation, sex differentiation and gametogenesis. WGCNA on 40 transcriptomes identifies key modules and molecular drivers of clam gonadal development. SOX2, FOXZ, HSFY, FOXL2 and HES1 are hub transcription factors for gonadal development of Mulinia lateralis.

Targeted genotyping is an extremely powerful approach for the detection of known genetic variations that are biologically or clinically important. However, for non-model organisms, large-scale target genotyping in a cost-effective manner remains a major challenge. To address this issue, we present an ultrahigh-multiplex, in-solution probe array-based high-throughput diverse marker genotyping (HD-Marker) approach that is capable of targeted genotyping of up to 86 000 loci, with coverage of the whole gene repertoire, in what is a 27-fold and six-fold multiplex increase in comparison with the conventional Illumina GoldenGate and original HD-Marker assays, respectively. We perform extensive analyses of various ultrahigh-multiplex levels of HD-Marker (30 k-plex, 56 k-plex, and 86 k-plex) and show the power and excellent performance of the proposed method with an extremely high capture rate (about 96%) and genotyping accuracy (about 96%). With great advantages in terms of cost (as low as 0.0006 USD per genotype) and high technical flexibility, HD-Marker is a highly efficient and powerful tool with broad application potential for genetic, ecological, and evolutionary studies of non-model organisms.

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.

The CpG oligodeoxynucleotides (CpG ODNs) reportedly possess the capacity to strengthen immunity in mammals. This experiment was conducted to evaluate the impact of dietary supplementation with 17 types of CpG ODNs on intestinal microbiota diversity, antioxidant capacity, and immune-related gene expression profiles of the shrimp Litopenaeus vannamei. Diets including 50 mg kg-1 CpG ODNs wrapped in egg whites were prepared and divided into 17 different groups, with 2 control groups (normal feed and feed with egg whites). These CpG ODNs supplemented diets and the control diets were fed to L. vannamei (5.15 ± 0.54 g) three times daily at 5%-8% shrimp body weight for three weeks. The results of consecutive detection of intestinal microbiota by 16S rDNA sequencing indicated that 11 of the 17 types of CpG ODNs significantly enhanced intestinal microbiota diversity, increased the populations of several probiotic bacteria, and activated possible mechanisms relevant to diseases. The immune-related genes expression and antioxidant capacity in hepatopancreas further demonstrated that the 11 types of CpG ODNs effectively improved the innate immunity of shrimp. Additionally, histology results showed that the CpG ODNs in the experiment did not damage the tissue structure of hepatopancreas. The results suggest that CpG ODNs could be used as a trace supplement to improve the intestinal health and immunity of shrimp.

Abstract 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.

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.

Abstract 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.

Mollusca is the second largest animal phylum and represents one of the most evolutionarily successful animal groups. Mulinia lateralis, a small bivalve, is a promising model organism to facilitate studies of mollusc development. However, because of the lack of published genomic and transcriptomic resources, integrated research on the formation of larval shells in this species, which is a representative developmental process of molluscs and of great importance for larva survival, is hindered. In this study, the blastula, gastrula, trochophore larva, and D-shaped larva of M. lateralis were utilized for generating a comprehensive full-length transcriptome through Pacific BioSciences (PacBio) isoform sequencing (Iso-seq) and Illumina RNA-Seq. A total of 238,919 full-length transcripts with an average length of 3,267 bp and 121,424 annotated genes were obtained. Illumina RNA-Seq data analysis showed that 4,512, 10,637, and 17,829 differentially expressed genes (DEGs) were obtained between the two adjacent developmental stages. Functional annotation and enrichment analysis revealed the specific function of genes in shell biomineralization during different developmental stages. Twelve genes that may be involved in the formation of the larval shell of M. lateralis were identified, including insoluble shell matrix protein-encoding gene 1 (ISMP1), ISMP2, ISMP5, chitin synthase, tyrosinase, chitin-binding protein, collagen and pu14 involved in shell matrix deposition, and carbonic anhydrase, solute carrier family 4 member 8 (slc4a8), EF-hand, and a calmodulin coding gene C-2442 participated in ion transportation. In addition, calcium ion binding function, calcium signaling pathway, and endocrine and other factor-regulated calcium reabsorption pathways were significantly enriched. Weighted gene correlation network analysis (WGCNA) identified two modules related to biomineralization and larval shell formation, and slc4a8 and ring finger protein 41 (rnf41) were key hub genes that may be involved in this process. Moreover, it could be implied that the process of ion transport occurs earlier than the deposition of the shell matrix. This work provided a clear view of the transcriptome for M. lateralis and will be valuable in elucidating the mechanisms of larval shell formation as well as other developmental processes in molluscs.

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.

Paralytic shellfish toxins (PST) could be accumulated in bivalves and cause safety problems. To protect public health, bivalves are examined for PST contamination before entering the market, usually by high-performance liquid chromatography (HPLC) or LC-tandem mass spectrometry (LC-MS/MS) in the lab, which needs PST standards not all available and is time-consuming for large sample sizes. To detect PST toxicity in bivalves rapidly and sensitively, a biomarker gene is highly demanded, but the related study is very limited. In this study, we fed a commercially important bivalve, Patinopecten yessoensis, with the PST-producing dinoflagellate Alexandrium catenella. After 1, 3, and 5 days of exposure, both PST concentrations and toxicity levels in the digestive gland continuously increased. Transcriptome analysis revealed that the differentially expressed genes were significantly enriched in oxidation-reduction process, which included the cytochrome P450 genes (CYPs), type I iodothyronine deiodinase (IOD1s), peroxidasin (PXDN), and acyl-Coenzyme A oxidase 1 (ACOX1) at day 1 and a superoxide dismutase (SOD) at day 5, highlighting the crucial roles of these genes in response to oxidative stress induced by PST. Among the 33 continuously upregulated genes, five showed a significant correlation between gene expression and PST concentration, with the highest correlation present in PyC1QL4-1, the gene encoding Complement C1Q-like protein 4, C1QL4. In addition, the correlation between PyC1QL4-1 expression and PST toxicity was also the highest. Further analysis in another aquaculture scallop (Chlamys farreri) indicated that the expression of CfC1QL4-1, the homolog of PyC1QL4-1, also exhibited significant correlations with both PST toxicity and concentration. Our results reveal the gene expression responses of scallop digestive glands to PST-producing algae and indicate that the C1QL4-1 gene might be a potential biomarker for PST monitoring in scallops, which may provide a convenient way for the early warning and sensitive detection of PST contamination in the bivalves.

The COMMD (copper metabolism gene MuRR1 domain) gene family, highly conserved among multicellular eukaryotic organisms, plays important roles in a variety of biological processes, ranging from copper homeostasis, ionic transport, protein trafficking, NF-κB-mediated transcription, and cell proliferation. However, systematic identification, spatiotemporal expression, and stress-responsive patterns of COMMD genes remain obscure in molluscs. Here, we analyzed the characteristics of the COMMD gene family in four bivalve molluscs based on both genome and extensive transcriptomic resources. Firstly, we investigated the genomic signatures, functional domains, and phylogenetic relationships, and ten single-copy members were identified in Yesso scallop (Patinopecten yessoensis), Zhikong scallop (Chlamys farreri), Pacific oyster (Crassostrea gigas), and dwarf surf clam (Mulinia lateralis), respectively. Strong purifying selection was revealed for COMMD4. Higher expressions of most COMMDs were observed in the hepatopancreas, besides which a different tissue preference of COMMDs’ expression was found among four bivalves. Moreover, in the dwarf surf clam, the responses of COMMD members under stresses were found more sensitive in the hepatopancreas than in the gill, and MlCOMMD9 and MlCOMMD4 might be the good candidate stress indicator genes respectively for copper ion stress and V. Anguillarum infection. Our study would contribute to a better understanding for the evolution of the COMMD gene family and provide valuable information for their innate immune roles in bivalve molluscs.

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.

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.

Cilia are organelles located on the surface of eukaryotic cells and play important roles in numerous physiological and developmental processes. Cilia- and flagella-associated proteins (CFAP family) are well known due to their close relation to the assembly and functionalization of both cilia and flagella. In vertebrates, Cfap206 is reported to function during motile cilia assembly, and its abolished expression could lead to abnormal ciliary beating and decreased sperm motility. However, there is scarcely any information on the function of Cfap206 in marine invertebrates. Here, through using an ideal shellfish research model, the dwarf surf calm Mulinia lateralis, we explored the role of Cfap206 during embryonic ciliogenesis as well as during male gametogenesis. In trochophore larvae, effective knocking down or knocking out of Cfap206 was detected respectively through RNAi and CRISPR/Cas9 technology, showing an obviously decreased number of cilia. During the period of gonadal maturation, we managed to deliver the dsRNA of Cfap206 to male individuals for 14 days through carrier vector feeding. Significant suppression of Cfap206 was observed, together with impaired sperm motility and aberrant sperm tail assembly. The present study provided valuable information on gene function exploration methods in M. lateralis and further enriched the understanding of Cfap206 on ciliogenesis in marine invertebrates. Furthermore, by revealing the relationship between Cfap206 and spermatozoa flagellum, it is possible to generate male sterile populations in hermaphroditic marine organisms to improve crossbreeding efficiency.

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.

Dwarf surf clam, Mulinia lateralis, is widely considered as a model species for bivalves. The development of a standard culture system could greatly promote the production of high-quality individuals, yet information on the culture conditions for M. lateralis spat is still limited. This study aims to determine the suitable microalgae diet, temperature, and salinity for M. lateralis spat culture. The typically fed microalgae species, including Chlorella pyrenoidesa (Cp), Platymonas helgolandica (Ph), Dunaliella salina (Ds), Nitzschia closterium (Nc), and Chaetoceros muelleri (Cm), could be taken up by M. lateralis spat, and their filtration rates on Cp, Nc, and Cm were higher than those on Ds and Ph. For the entire spat culture, all diet trials showed similar survival percentages, while the mono-specific diet Cp exhibited the highest growth rate, suggesting that Cp was the optimal microalgae species for M. lateralis spat. Through simultaneously maximizing the growth and survival of spat, the optimal microalgae concentration and stocking density were 5 × 104 cells ml–1 and 400–600 individuals m–2 for 30–40-day-old spat as well as 1 × 105 cells ml–1 and 400–600 individuals m–2 for 40–50-day-old spat, respectively. In addition, the spat had higher growth rates and survival percentages at the temperature of 20–22°C and salinity of 22–25 ppt. The results of this study provide a basis for further culture of M. lateralis spat, and the optimized conditions will be of great significance for the construction of the standard culture system of M. lateralis.

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 Patinopecten yessoensis in the field and under laboratory conditions. Based on the relative expression of FoxL2 and Dmrt1L in the gonads of 6- to 12 month-old scallops, we found the scallops start to differentiate at 7 months old in September when the seawater temperature was 21°C. To eliminate the effect of factors other than temperature on sex differentiation, we compared the gonadal development of juvenile scallops at different temperatures (21, 16 and 11°C) under laboratory conditions. After 50 days of treatment, the 11°C group contain more germ cell types, and have higher sex differentiation rates than the 21°C group. But no obvious sex bias was observed. These results suggest that high temperature (21°C) inhibits sex differentiation, whereas low temperature (11°C) accelerates sex differentiation by 2 months for this cold-water species. It also supports juvenile P. yessoensis is gonochoristic rather than protandrous hermaphroditic. Our study addresses for the first time an environmental influence associated with genetic controls on scallop sex differentiation. It will facilitate a better understanding of how environmental factors affect gonadal development in poikilotherms, especially in the less studied molluscs.

Viral diseases have seriously restricted the healthy development of aquaculture, and decapod iridescent virus 1 (DIV1) has led to heavy losses in the global shrimp aquaculture industry. Due to the lack of effective treatment, early detection and regular monitoring are the most effective ways to avoid infection with DIV1. In this study, a novel real-time quantitative recombinase polymerase amplification (qRPA) assay and its instrument-free visualization improvement were described for the rapid detection of DIV1. Optimum primer pairs, suitable reaction temperatures, and probe concentrations of a DIV1-qRPA assay were screened to determine optimal reaction conditions. Then, its ability to detect DIV1 was evaluated and compared with real-time quantitative polymerase chain reactions (qPCRs). The sensitivity tests demonstrated that the limit of detection (LOD) of the DIV1-qRPA assay was 1.0 copies μL−1. Additionally, the presentation of the detection results was improved with SYBR Green I, and the LOD of the DIV1-RPA-SYBR Green I assay was 1.0 × 103 copies μL−1. Both the DIV1-qRPA and DIV1-RPA-SYBR Green I assays could be performed at 42 °C within 20 min and without cross-reactivity with the following: white spot syndrome virus (WSSV), Vibrio parahaemolyticus associated with acute hepatopancreatic necrosis disease (VpAHPND), Enterocytozoon hepatopenaei (EHP), and infectious hypodermal and hematopoietic necrosis virus (IHHNV). In conclusion, this approach yields rapid, straightforward, and simple DIV1 diagnoses, making it potentially valuable as a reliable tool for the detection and prevention of DIV1, especially where there is a paucity of laboratory equipment.

Molluscan shell color has consistently drawn attention for its abundant diversity and commercial use in shellfish breeding projects. Recently, two new strains of bay scallop (Argopecten irradians irradians) with different shell colors as marked phenotypic traits have been artificially bred to improve their economic values; however, the inheritance mechanism of their shell pigmentation is still unclear. In this study, a genome-wide association study (GWAS) was conducted to determine the genetic basis of shell color in bay scallops utilizing 29,036 high-quality single-nucleotide polymorphisms (SNPs) derived from 80 purple-red (PP) and 80 black-brown (BP) shell color individuals. The result of the GWAS showed that 469 SNPs (p

Zhikong scallop (Chlamys farreri) is a bivalve species with broad economic and biological value, and an essential species of aquaculture in North China. Recently, efforts have been made to improve knowledge of genome, genetics, and cytogenetics, which is devoted to develop the molecular breeding project for the scallop. In this study, we constructed a cytogenetic map and identified all chromosomes of C. farreri using fluorescence in situ hybridization (FISH). A total of 100 Bacterial Artificial Chromosome (BAC) clones and 27 fosmid clones, including 58 microsatellite marker-anchored BAC clones, 4 genes-anchored BAC clones, 38 random BAC clones, 22 repetitive sequences-anchored fosmid clones, and 5 gene-anchored fosmid clones, were tested as probes, and 69 of them produced specific and stable signal on one pair of chromosomes. Then, multiple co-hybridizations were conducted to distinguish all the submetacentric and subtelocentric chromosomes with similar morphology by the abovementioned chromosome-specific markers. On this basis, a cytogenetic map of C. farreri containing 69 clones was constructed by co-hybridization and karyotype analysis. The markers covered all 19 pairs of chromosomes, and the average number of markers on each chromosome was 3.6. The cytogenetic map provides a platform for genetic and genomic analysis of C. farreri, which facilitates the molecular breeding project of C. farreri and promotes the comparative studies of chromosome evolution in scallops and even bivalves.

The increasing sea temperature caused by global warming has resulted in severe mortalities in maricultural scallops. Therefore, improving thermal tolerance has become an active research area in the scallop farming industry. Bay scallop (Argopecten irradians irradians) was introduced into China in 1982 and has developed into a vast aquaculture industry in northern China. To date, genetic studies on thermal tolerance in bay scallops are limited, and no systematic screening of thermal tolerance-related loci or genes has been conducted in this species. In the present study, we conducted a genome-wide association study (GWAS) for thermal tolerance using the Arrhenius break temperature (ABT) indicators of 435 bay scallops and 38,011 single nucleotide polymorphism (SNP) markers. The GWAS identified 1,906 significant thermal tolerance-associated SNPs located in 16 chromosomes of bay scallop. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that 638 genes were enriched in 42 GO terms, while 549 annotated genes were enriched in aggregation pathways. Additionally, the SNP (15-5091-20379557-1) with the lowest P value was located in the transcriptional coactivator p15 (PC4) gene, which is involved in regulating DNA damage repair and stabilizing genome functions. Further analysis in another population identified two new thermal tolerance-associated SNPs in the first coding sequence of PC4 in bay scallops (AiPC4). Moreover, AiPC4 expression levels were significantly correlated (r = 0.675–0.962; P < 0.05) with the ABT values of the examined bay scallops. Our data suggest that AiPC4 might be a positive regulator of thermal tolerance and a potential candidate gene for molecular breeding in bay scallop aiming at thermal tolerance improvement.

Mitogen-activated protein kinase (MAPK) cascades are fundamental signal transduction modules in all eukaryotic organisms, participating growth and development, as well as stress response. In the present study, three MAPK genes were successfully identified from the genome of Chlamys farreri, respectively, named CfERK1/2, CfJNK, and Cfp38, and only one copy of ERK, JNK, and p38 were detected. Domain analysis indicated that CfMAPKs possessed the typical domains, including S_TKc, Pkinase, and PKc_like domain. Phylogenetic analysis showed that three CfMAPKs of MAPK subfamilies exists in the common ancestor of vertebrates and invertebrates. All CfMAPKs specifically expressed during larval development and in adult tissues, and the expression level of CfERK1/2 and Cfp38 was apparently higher than that of CfJNK. Under heat stress, the expression of CfERK1/2 and Cfp38 were significantly downregulated and then upregulated in four tissues, while the expression of CfJNK increased in all tissues; these different expression patterns suggested a different molecular mechanism of CfMAPKs for bivalves to adapt to temperature changes. The diversity of CfMAPKs and their specific expression patterns provide valuable information for better understanding of the functions of MAPK cascades in bivalves.

Abstract Background Reverse transcription quantitative PCR (RT-qPCR) is widely used for gene expression analysis in various organisms. Its accuracy largely relies on the stability of reference genes, making reference gene selection a vital step in RT-qPCR experiments. However, previous studies in mollusks only focused on the reference genes widely used in vertebrates. Results In this study, we conducted the transcriptome-wide identification of reference genes in the bivalve mollusk Mizuhopecten yessoensis based on 60 transcriptomes covering early development, adult tissues and gonadal development. A total of 964, 1210 and 2097 candidate reference genes were identified, respectively, resulting in a core set of 568 genes. Functional enrichment analysis showed that these genes are significantly overrepresented in Gene Ontology (GO) terms or Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to ribosomes, energy production, etc. Six genes (RS23, EF1A, NDUS4, SELR1, EIF3F, and OLA1) were selected from the candidate genes for RT-qPCR validation, together with 6 commonly used reference genes (ACT, CYTC, HEL, EF1B, GAPDH and RPL16). Stability analyses using geNorm, NormFinder and the comparative delta-Ct method revealed that the new candidate reference genes are more stable than the traditionally used genes, and ACT and CYTC are not recommended under either of the three circumstances. There was a significant correlation between the Ct of RT-qPCR and the log2(TPM) of RNA-Seq data (Ct = − 0.94 log2(TPM) + 29.67, R2 = 0.73), making it easy to estimate the Ct values from transcriptome data prior to RT-qPCR experiments. Conclusion Our study represents the first transcriptome-wide identification of reference genes for early development, adult tissues, and gonadal development in the Yesso scallop and will benefit gene expression studies in other bivalve mollusks.

Filter-feeding bivalves can accumulate paralytic shellfish toxins (PST) produced by toxic microalgae, which may induce oxidative stress and lipid peroxidation. Peroxisomal acyl-coenzyme A oxidases (ACOXs) are key enzymes functioning in maintaining redox and lipid homeostasis, but their roles in PST response in bivalves are less understood. Herein, a total of six and six ACOXs were identified in the Chlamys farreri and Patinopecten yessoensis genome, respectively, and the expansion of ACOX1s was observed. Gene expression analysis revealed an organ/tissue-specific expression pattern in both scallops, with all ACOXs being predominantly expressed in the two most toxic organs, digestive glands and kidneys. The regulation patterns of scallop ACOXs after exposure to different PST-producing algaes Alexandrium catenella (ACDH) and A. minutum (AM-1) were revealed. After ACDH exposure, more differentially expressed genes (DEGs) were identified in C. farreri digestive glands (three) and kidneys (five) than that in P. yessoensis (two), but the up-regulated DEGs showed similar expression patterns in both species. In C. farreri, three DEGs were found in both digestive glands and kidneys after AM-1 exposure, with two same CfACOX1s being acutely and chronically induced, respectively. Notably, these two CfACOX1s also showed different expression patterns in kidneys between ACDH (acute response) and AM-1 (chronic response) exposure. Moreover, inductive expression of CfACOXs after AM-1 exposure was observed in gills and mantles, and all DEGs in both tissues were up-regulated and their common DEGs exhibited both acute and chronic induction. These results indicate the involvement of scallop ACOXs in PST response, and their plasticity expression patterns between scallop species, among tissues, and between the exposure of different PST analogs.

C1Q (Complement 1Q) is an important recognition molecule in the immunological complement system, which could also be putatively involved in the stress responses induced by endotoxins or exotoxins, potentially through detoxification processes. Marine bivalves are well adapted to highly complex aquatic environments with various stressors. As filter feeders, they have to cope with highly potent microalgae-derived neurotoxins, such as paralytic shellfish toxin (PSTs). Zhikong scallops, Chlamys farreri, are commercially important bivalve with the remarkable ability to accumulate PSTs. Exploring the C1Qs related to PST accumulation in C. farreri could benefit our understanding of the adaptations of bivalve species. In the present study, we systematically analyzed C1Q genes in C. farreri. In total, 97 CfC1Q genes mainly from the expanded C1Q-B subfamily were identified, from which the C1QL, C1QTNF, and C1QDC1 members in C. farreri were revealed to be under positive selection. Spatiotemporal expression analysis revealed that most CfC1QLs and CfC1QDC1s were highly expressed during the post-umbo stage and in hepatopancreas, while most CfC1QTNF members were highly expressed after the creeping larva stage and in mantle. The hepatopancreas and kidney in C. farreri are two toxin-rich organs with the highest concentrations of PSTs, acting as major “centers” for toxin accumulation and transformation, respectively. Therefore, after feeding the scallops with PST-producing dinoflagellates Alexandrium minutum and Alexandrium catenella, we determined the expression patterns of CfC1Qs in these two organs. In kidney, more than 85% of CfC1QLs and CfC1QDC1s showed drastic up-regulation with both diets. However, among these members with significant induction, a different response manner was detected after feeding with A. minutum and A. catenella, respectively as acute and chronic response patterns. In comparison, far fewer CfC1Qs showing significant up-regulation in hepatopancreas with both toxic diets and only mild regulation pattern could be found. This organ-, toxin-, and time-dependent genetic regulation of CfC1Qs may contribute to the virulence difference on account of the tissue-specific or dinoflagellate-specific different toxin analogs composition, implying the possible involvement of CfC1Qs in PST transport and homeostasis. Our findings imply the functional diversity of scallop C1Q genes in coping with PST accumulation, which might be developed as potential molecular indicators for monitoring toxin accumulation in edible mollusks or provide insight into the lineage-specific adaptation of scallops for dealing with microalgal toxin challenges.

Simultaneous or functional hermaphrodites possessing both ovary and testis at the same time are good materials for studying sexual development. However, previous research on sex determination and differentiation was mainly conducted in gonochoristic species and studies on simultaneous hermaphrodites are still limited. In this study, we conducted a combined morphological, endocrine and molecular study on the gonadal development of a hermaphroditic scallop Argopecten irradians aged 2–10 month old. Morphological analysis showed that sex differentiation occurred at 6 months of age. By examining the dynamic changes of progesterone, testosterone and estradiol, we found testosterone and estradiol were significantly different between the ovaries and testes almost throughout the whole process, suggesting the two hormones may be involved in scallop sex differentiation. In addition, we identified two critical sex-related genes FoxL2 and Dmrt1L, and investigated their spatiotemporal expression patterns. Results showed that FoxL2 and Dmrt1L were female- and male-biased, respectively, and mainly localized in the germ cells and follicular cells, indicating their feasibility as molecular markers for early identification of sex. Further analysis on the changes of FoxL2 and Dmrt1L expression in juveniles showed that significant sexual dimorphic expression of FoxL2 occurred at 2 months of age, earlier than that of Dmrt1L. Moreover, FoxL2 expression was significantly correlated with estradiol/testosterone ratio (E2/T). All these results indicated that molecular sex differentiation occurs earlier than morphological sex differentiation, and FoxL2 may be a key driver that functions through regulating sex steroid hormones in the scallop. This study will deepen our understanding of the molecular mechanism underlying sex differentiation and development in spiralians.

Members of the testis-specific serine/threonine kinases (Tssk) family play critical roles in spermatogenesis in vertebrates. But in mollusks, research on Tssk family is still lagging. In this study, we systematically identified Tssk family based on the genomic and transcriptomic data from a commercially important scallop Argopecten irradians and detected the spatiotemporal expression in adult gonads. Five members were identified, with the gene length varying from 1,068 to 10,729 bp and the protein length ranging from 294 to 731 aa. All the Tssks possess a serine/threonine protein kinase catalytic (S_TKc) domain. Phylogenetic analysis revealed existence of four homologs of vertebrate Tssk1/2, Tssk3, Tssk4, Tssk5, and absence of Tssk6 in the scallop. The remaining gene (Tssk7) formed an independent clade with Tssks of other mollusks and arthropods, indicating that it may be a new member of Tssk family unique to protostomes. By investigating the expression of Tssks in four developmental stages of testes and ovaries, we found all five Tssks were primarily expressed in mature testis. In situ hybridization experiment revealed the five Tssks were localized in the spermatids and spermatozoa. The testis-predominant expression of Tssk family suggests Tssks may play pivotal roles in spermiogenesis in the scallop. Our study provides basic information on the characteristics and expression profiles of Tssk family of A. irradians. To our knowledge, it represents the first comprehensive analysis of Tssk family in mollusks.

Inhibitors of apoptosis proteins (IAPs) are conserved regulators involved in cell cycle, cell migration, cell death, immunity and inflammation, should be due to the fact that they can assist with the ability to cope with different kinds of extrinsic or intrinsic stresses. Bivalve molluscs are well adapted to highly complex marine environments. As free-living filter feeders that may take toxic dinoflagellates as food, bivalves can accumulate and put up with significant levels of paralytic shellfish toxins (PSTs). PSTs absorption and accumulation could have a deleterious effect on bivalves, causing negative impact on their feeding and digestion capabilities. In the present study, we analyzed IAP genes (PyIAPs) in Yesso scallop (Patinopecten yessoensis), a major fishery and aquaculture species in China. Forty-seven PyIAPs from five sub-families were identified, and almost half of the PyIAP genes were localized in clusters on two chromosomes. Several sites under positive selection was revealed in the significantly expanded sub-families BIRC4 and BIRC5. After exposure to PST-producing dinoflagellates, Alexandrium catenella, fourteen PyIAPs showed significant responses in hepatopancreas and kidney, and more than eighty-five percent of them were from the expanded sub-families BIRC4 and BIRC5. The regulation pattern of PyIAPs was similar between the two tissues, with more than half exhibited expression suppression within three days after exposure. In contrast to hepatopancreas, more acute changes of PyIAPs expression could be detected in kidney, suggesting the possible involvement of these PyIAPs in tissue-specific PST tolerance. These findings also imply the adaptive expansion of bivalve IAP genes in response to algae derived biotoxins.

The bay scallop (Argopecten irradians irradians) is one of the most important shellfish species in China. Since their introduction into China, only mass selection has been used in bay scallop breeding. With its gradual expansion and shortage of mate selection, population homozygosity increased, and fitness decreased. To investigate the effects of inbreeding and provide reference for improving breeding strategies and mating management, the variance components of the growth traits of the bay scallop were decomposed with genomic relationship matrices. The results indicated that the genetic variations in shell height and length were mainly accounted for by the additive effects. The genetic variation in shell width was mainly caused by dominance or dominance-by-dominance epistasis. The genetic variation in body weight was accounted for by dominance. No significant directional dominances were detected for all growth traits. Cross-validation for genomic prediction showed that including insignificant inbreeding in the genomic prediction model is not necessary, and we suggest that the genomic prediction model should be optimized with both likelihood ratio tests and cross-validation before utilization in practice.

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 (Chlamys farreri), a shellfish with broad economic and biological value in North China, has frequently experienced heat stress in summer in recent years. To understand the effects of heat stress on shellfish, the metabolism of C. farreri was analyzed after exposure to 27 °C for either 6 h or 30 d. After 6 h of heat stress exposure, a total of 326 and 264 significantly different metabolites (SDMs) were identified in gill and mantle tissues, respectively. After 30 d of heat stress exposure, a total of 381 and 341 SDMs were found in the gill and mantle tissues, respectively. These SDMs were mainly related to the metabolism of amino acids, carbohydrates, lipids and nucleotides. A decline in pyruvic acid, and an increase in citric acid and fumaric acid in the gills and mantle of C. farreri indicated an alteration in energy metabolism, which may be attributed to increased ATP production in order to overcome the heat stress. Among the SDMs, 33 metabolites, including pyruvic acid, glycine and citric acid, were selected as potential biomarkers for heat stress response in C. farreri. In addition, a decline in glutamine and β-Alanine levels indicated oxidative stress in C. farreri exposed to heat, as well as an increase in the total antioxidant capacity (T-AOC). Our findings suggested C. farreri have the potential to adapt to heat stress by regulating energy metabolism and antioxidant capacity.

The deep-sea limpet Bathyacmaea lactea, inhibited in cold seeps of the South China Sea, belongs to the most primitive gastropod groups, the Patellogastropoda (true limpets). Here, we report the complete mitochondrial genome of B. lactea, which is 18,446 bp in length and contains 13 protein-coding genes, 2 rRNAs, and 22 tRNA genes. The mitochondrial genome of B. lactea enriches the molecular resources for further understanding deep-sea molluskan adaptation and Gastropoda evolution.