Zhikong Scallop Chlamys Farreri Research Articles

IKK2 modulates the innate immune response of Zhikong scallop (Chlamys farreri) to bacterial and viral stimuli through the MyD88-dependent Toll-like receptor signaling pathway

Members of the inhibitor of nuclear factor kappa-B kinase (IKK) family are pivotal components of the innate immune signaling pathway responsible for regulating immune and inflammatory responses. Nevertheless, the various roles that IKKs play in the physiology of invertebrates, particularly marine mollusks, remain largely unexplored. In this study, an IKK gene was cloned from the Zhikong scallop, Chlamys farreri, and denoted as CfIKK2. CfIKK2 belongs to the TANK-binding kinase 1 (TBK1)/IKKε family. Real-time quantitative reverse transcription-polymerase chain reaction analysis showed that CfIKK2 was expressed in various scallop tissues and that its transcript levels substantially increased under different pathogenic stress conditions (including lipopolysaccharide, peptidoglycan, poly(I:C), acute viral necrosis virus, and Vibrio challenge). Co-immunoprecipitation experiments revealed that CfIKK2 interacts with MyD88 (a crucial adaptor in the Toll-like receptor signaling pathway) proteins through its C-terminal TBK1 coiled-coil domain 1. Moreover, CfIKK2 has been shown to interact with itself. In HEK293T cells, CfIKK2 overexpression resulted in an augmentation of mitogen-activated protein kinase phosphorylation. Furthermore, overexpression of CfIKK2 resulted in the activation of interferon reporter genes. In summary, our findings offer valuable insights into the mechanisms through which CfIKK2 responds to stress generated by pathogen-associated molecular patterns, participates in Toll-like receptor signaling, and ultimately activates some of the key cytokines involved in innate immunity in C. farreri. As a critical signaling molecule and immunoregulatory factor, CfIKK2 plays a vital role in the innate immune system of the Zhikong scallop.

Genome-wide investigation of the TGF-β superfamily in scallops.

Transforming growth factor β (TGF-β) superfamily genes can regulate various processes, especially in embryogenesis, adult development, and homeostasis. To understand the evolution and divergence patterns of the TGF-β superfamily in scallops, genome-wide data from the Bay scallop (Argopecten irradians), the Zhikong scallop (Chlamys farreri) and the Yesso scallop (Mizuhopecten yessoensis) were systematically analysed using bioinformatics methods. Twelve members of the TGF-β superfamily were identified for each scallop. The phylogenetic tree showed that these genes were grouped into 11 clusters, including BMPs, ADMP, NODAL, GDF, activin/inhibin and AMH. The number of exons and the conserved motif showed some differences between different clusters, while genes in the same cluster exhibited high similarity. Selective pressure analysis revealed that the TGF-β superfamily in scallops was evolutionarily conserved. The spatiotemporal expression profiles suggested that different TGF-β members have distinct functions. Several BMP-like and NODAL-like genes were highly expressed in early developmental stages, patterning the embryonic body plan. GDF8/11-like genes showed high expression in striated muscle and smooth muscle, suggesting that these genes may play a critical role in regulating muscle growth. Further analysis revealed a possible duplication of AMH, which played a key role in gonadal growth/maturation in scallops. In addition, this study found that several genes were involved in heat and hypoxia stress in scallops, providing new insights into the function of the TGF-β superfamily. Characteristics of the TGF-β superfamily in scallops were identified, including sequence structure, phylogenetic relationships, and selection pressure. The expression profiles of these genes in different tissues, at different developmental stages and under different stresses were investigated. Generally, the current study lays a foundation for further study of their pleiotropic biological functions in scallops.

Development and evaluation of a haplotype reference panel of Zhikong scallop (Chlamys farreri) for genotype imputation

Single nucleotide polymorphisms (SNPs) across the genome are remarkably useful resources for investigating the genome architecture, studying the genetic basis of complex traits and implementation of genomic selection. However, information on high-density SNPs is unavailable for most aquaculture species owing to the high costs of whole genome sequencing. Genotype imputation is an attractive approach for obtaining large number of genomic variants at a low cost. In this study, the first haplotype reference panel was constructed for Chlamys farreri, an economically important mollusk species and a major contributor to aquaculture in China. A total of 174 scallops from six populations in the main scallop culture area were selected for whole-genome resequencing. More than 87.47 million SNPs were identified and 3,968,417 SNPs genome-wide were selected for constructing the haplotype reference panel for C. farreri. The number of SNPs per chromosome ranged from 125,000 to 279,981 with an average spacing of 242 bases. Several factors were evaluated based on the imputation performance of the reference panel. The imputation accuracies of 19 chromosomes ranged from 0.91 to 0.95, and there were no significant differences among them (p > 0.05). The imputation accuracies of populations could reach 0.95, 0.93, and 0.90 for ZY, PR, and CS respectively, which confirmed a positive relationship between the kinship within the population and the imputation accuracy. The imputation accuracies increased with sequencing depth and plateaued at 0.5×, reaching accuracies of 0.91–0.93 for artificially breeding populations. Taken together, we constructed a haplotype reference panel for C. farreri, which can provide a cost-effective way to extend the limited number of genotypes for boosting the power of downstream analyses such as genome-wide association studies and genomic selection.

Tissue-specific antioxidative response and metabolism of paralytic shellfish toxins in scallop (Chlamys farreri) mantle with Alexandrium dinoflagellate exposure

Bivalves show remarkable capacity to acclimate paralytic shellfish toxins (PSTs) produced by dinoflagellates, severely affecting fishery industry and public health. Here, transcriptomic response to PSTs-producing dinoflagellate (Alexandrium minutum) was investigated in Zhikong scallop (Chlamys farreri) mantle. The PSTs accumulated in C. farreri mantle continually increased during the 15 days exposure, with “oxidation-reduction” genes induced compared to the control group at the 1st and 15th day. Through gene co-expression network analysis, 16 PSTs-responsive modules were enriched with up- or down-regulated genes. The concentration of GTXs, major PSTs in A. minutum and accumulated in scallops, was correlated with the up-regulated magenta module, enriching peroxisome genes as the potential mantle-specific PSTs biomarker. Moreover, Hsp70B2s were inhibited throughout the exposure, which together with the expanded neurotransmitter transporter SLC6As, may play essential roles on neurotransmitter homeostasis in scallop mantle. These results paved the way for a comprehensive understanding of defensive mechanism and homeostatic response in scallop mantle against PSTs.

Offshore Wind Farms in South Korea: A Potential Site for Scallop Culture

Owing to the constant wind generated by the vast ocean, energy production from offshore wind farms (OWFs) plays an important role in the expansion of renewable energy. However, areas close to large wind farms are often left unutilized, and aquaculture farmers find it difficult to efficiently utilize these unoccupied spaces due to limited information showing the feasibility of utilization of OWFs as potential scallop culture sites. To analyze whether the two scallop species Zhikong scallop (Chlamys farreri) and bay scallop (Argopecten irradians) can be grown at OWFs of Gochang and Buan, Jeollabuk-do, Republic of Korea, the growth characteristics of the two scallop species were analyzed and compared with those grown at the Tongyeong Megacosm Test Station. The results clearly showed that the growth of scallops at the OWF was significantly lower with respect to the shell lengths, height, width, and weight, compared to those grown at the megacosm station. However, scallops grown at the OWF still showed consistent growth in parallel with those grown at the megacosm test station. Yet, there was a species-specific mortality rate between the two sites. In addition, our results suggest that temperature may be a key determinant of the growth of C. farreri and A. irradians. Overall, this study contributes to establishing a foundation for the stable and continuous farming of marine bivalves (e.g., clams, oysters, mussels, and scallops) in OWF areas.

Assessment of germplasm resource and detection of genomic signature under artificial selection of Zhikong scallop (Chlamys farreri)

Zhikong scallop (Chlamys farreri) is an economically important bivalve species and a major contributor to aquaculture in China as it is nutrient-dense and has a large adductor muscle. Farming and artificial breeding of this species mainly have relied mainly upon natural collected seed to be moved onto farms or be artificially bred. Hence, it is vital to determine the current status of C. farreri germplasm resources. In the present study, we genotyped six natural populations around the Shandong Peninsula in Northern China as well as populations of the artificially selected PR and ZY strains characterized by rapid growth and high survival. We identified 19,438 high-quality single-nucleotide polymorphisms (SNPs) that evenly distributed across the C. farreri genome. All populations exhibited high levels of genetic diversity, including high heterozygosity (Ho: 0.280–0.311) and low inbreeding (Fis: −0.087–0.019). They also displayed a high frequency of rare alleles (13.80%–23.63%). Hence, these germplasms could be applied toward the genetic improvement of this species. A population analysis revealed no obvious genetic differentiation among natural populations. In contrast, the artificially selected populations in general, and the ZY strain in particular, significantly differed from the natural ones (Fst > 0.05). A selective sweep analysis indicated that 95 regions containing 76 genes, and 118 regions containing 99 genes, showed significant selection signatures in PR and ZY, respectively. These candidate genes may regulate various biological processes including growth, reproduction, and stress resistance, and account for the rapid growth and stress-resistance characteristic ZY and PR. The results of this work provide valuable information for resource management and germplasm improvement in C. farreri, and contribute to understand the genetic basis of selective breeding in scallop.

CfIRF8-like interacts with the TBK1/IKKε family protein and regulates host antiviral innate immunity

The interferon regulatory factor (IRF) family, a class of transcription factors with key functions, are important in host innate immune defense and stress response. However, further research is required to determine the functions of IRFs in invertebrates. In this study, the coding sequence of an IRF gene was obtained from the Zhikong scallop (Chlamys farreri) and named CfIRF8-like. The open reading frame of CfIRF8-like was 1371 bp long and encoded 456 amino acids. Protein domain prediction revealed a typical IRF domain in the N-terminus of the CfIRF8-like protein and a typical IRF3 domain in the C-terminus. Multiple sequence alignment confirmed the conservation of the amino acid sequences of these two functional protein domains. Phylogenetic analysis showed that CfIRF8-like clustered with mollusk IRF8 proteins and then clustered with vertebrate IRF3, IRF4, and IRF5 subfamily proteins. Quantitative real-time PCR detected CfIRF8-like mRNA in all tested scallop tissues, with the highest expression in the gills. Simultaneously, the expression of CfIRF8-like transcripts in gills was significantly induced by polyinosinic–polycytidylic acid challenge. The results of protein interaction experiments showed that CfIRF8-like could directly bind the TBK1/IKKε family protein of scallop (CfIKK2) via its N-terminal IRF domain, revealing the presence of an ancient functional TBK1/IKKε-IRF signaling axis in scallops. Finally, dual-luciferase reporter assay results showed that the overexpression of CfIRF8-like in human embryonic kidney 293T cells could specifically activate the interferon β promoter of mammals and the interferon-stimulated response element promoter in dose-dependent manners. The findings of this preliminary analysis of the signal transduction and immune functions of scallop CfIRF8-like protein lay a foundation for an in-depth understanding of the innate immune function of invertebrate IRFs and the development of comparative immunology. The experimental results also provide theoretical support for the breeding of scallop disease-resistant strains.

Scallop interferon regulatory factor 1 interacts with myeloid differentiation primary response protein 88 and is crucial for antiviral innate immunity

The interferon regulatory factor (IRF) family comprises transcription factors that are crucial in immune defense, stress response, reproduction, and development. However, the function of IRFs in invertebrates is unclear. Here, the full-length cDNA of an IRF-encoding gene (CfIRF1) in the Zhikong scallop (Chlamys farreri) comprising 2007 bp with an open reading frame of 1053 bp that encoded 350 amino acids was characterized, and its immune function was studied. The CfIRF1 protein contained a typical IRF domain at its N-terminus. CfIRF1 was clustered with other proteins of the IRF1 subfamily, implying that they were closely related. CfIRF1 mRNA transcripts could be detected in all tested scallop tissues, with the highest expression observed in the gills and hepatopancreas. CfIRF1 expression was significantly induced by the polyinosinic–polycytidylic acid and acute viral necrosis virus challenge. CfIRF1 could directly interact with myeloid differentiation primary response protein 88 (MyD88), the key adaptor molecule of the toll-like receptor signaling pathway. CfIRF1 did not interact with scallop IKK1 (IKKα/β family protein), IKK2, IKK3 (IKKε/TBK1 family protein), or with other IRF family proteins (IRF2 or IRF3). However, CfIRF1 interacted with itself to form a homodimer. CfIRF1 could specifically activate the interferon β promoter of mammals and the promoter containing the interferon-stimulated response element (ISRE) in a dose-dependent manner. The truncated form of CfIRF1 had a significantly reduced ISRE activation ability, indicating that structural integrity was crucial for CfIRF1 to function as a transcription factor. Our findings provide insights into the functions of mollusk IRFs in innate immunity. The research results also provide valuable information that enriches the theory of comparative immunology and that can help prevent diseases in scallop farming.

Transcriptome analysis provides insight into adaptive mechanisms of scallops under environmental stress

High temperature and hypoxia greatly threaten marine life and aquaculture. Scallops, a diverse and ecologically important group of high economic value, mostly thrive in fluctuating environments, and are vulnerable to environmental stress. In the present study, the molecular response mechanism of scallops to a combination of environmental stressors was investigated via transcriptome analysis of the gill tissues in three scallop species, the Yesso scallop (Patinopecten yessoensis), Zhikong scallop (Chlamys farreri) and bay scallop (Argopecten irradians) that were exposed to transient heat, hypoxia and a combination thereof. The Yesso scallop had the most differentially expressed genes (DEGs) compared with the other two scallop species, indicating the highest sensitivity of the Yesso scallop to environmental stress. With increased temperature and decreased dissolved oxygen, the number of DEGs was greatly increased in the three scallop species, indicative of the enhancement in gene expression regulation in scallops in response to severe environmental changes. Heat and hypoxia had a synergistic effect on scallops. GO and KEGG enrichment analysis of DEGs under different stressors revealed overlapping molecular mechanisms of response in scallops following exposure to heat and hypoxia. Several immune and apoptosis-related pathways were highly enriched in the upregulated DEGs of the three scallops, suggesting that immune system activation and apoptosis promotion occurred in scallops in response to environmental stress. Heat shock proteins (HSPs) were significantly upregulated under heat and hypoxia, which likely assisted in correct protein folding to facilitate the adaption of the scallops to the altered environment. Additionally, the HIF-1 signaling pathway—the key pathway associated with hypoxia response—was triggered by extremely acute environmental changes. Comparative transcriptome analysis revealed 239 positively selected genes among the different scallops, including those involved in immune system and environmental adaptation, suggesting a long-term mechanism of environmental adaptation. The present study provides new insights into the molecular response mechanism in scallops to multiple environmental stressors and improves our understanding of the adaptive mechanisms of marine organisms under changing global climate conditions.

Transcriptome and Network Analyses Reveal the Gene Set Involved in PST Accumulation and Responses to Toxic Alexandrium minutum Exposure in the Gills of Chlamys farreri.

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 (Chlamys farreri) after 1, 3, 5, 10, and 15 day(s) exposure of PST-producing dinoflagellate Alexandrium minutum. Higher numbers of differentially expressed genes (DEGs) were detected at day 1 (1538) and day 15 (989) than that at day 3 (77), day 5 (82), and day 10 (80) after exposure, and most of the DEGs were only regulated at day 1 or day 15, highlighting different response mechanisms of scallop to PST-producing dinoflagellate at different stages of exposure. Functional enrichment results suggested that PST exposure induced the alterations of nervous system development processes and the activation of xenobiotic metabolism and substance transport processes at the acute and chronic stages of exposure, respectively, while the immune functions were inhibited by PST and might ultimately cause the activation of apoptosis. Furthermore, a weighted gene co-expression network was constructed, and ten responsive modules for toxic algae exposure were identified, among which the yellow module was found to be significantly correlated with PST content. Most of the hub genes in the yellow module were annotated as solute carriers (SLCs) with eight being OCTN1s, implying their dominant roles in regulating PST accumulation in scallop gills. Overall, our results reveal the gene set responding to and involved in PST accumulation in scallop gills, which will deepen our understanding of the molecular mechanism of bivalve resistance to PST.

Metabonomic Analysis Provides New Insights into the Response of Zhikong Scallop (Chlamys farreri) to Heat Stress by Improving Energy Metabolism and Antioxidant Capacity.

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.

Interferon Regulatory Factors Functioned as Activators of the Interferon Pathway in the Scallop Chlamys farreri

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.

Genome-Wide Identification and Expression Profiling of the COMMD Gene Family in Four Bivalve Molluscs

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.

Molecular allocation of PC4s provides implications for deciphering thermal response in Zhikong scallop (Chlamys farreri).

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 (3h, 6h, 12h, 24h, 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.

Scallop IKK1 Responds to Bacterial and Virus-Related Pathogen Stimulation and Interacts With MyD88 Adaptor of Toll-Like Receptor Pathway Signaling.

IKK proteins are key signaling molecules in the innate immune system of animals, and act downstream of pattern recognition receptors. However, research on IKKs in invertebrates, especially marine mollusks, remains scarce. In this study, we cloned CfIKK1 gene from the Zhikong scallop (Chlamys farreri) and studied its function and the signaling it mediates. The open reading frame of CfIKK1 was 2190 bp and encoded 729 amino acids. Phylogenetic analysis showed that CfIKK1 belonged to the invertebrate IKKα/IKKβ family. Quantitative real-time PCR analysis revealed the ubiquitous expression of CfIKK1 mRNA in all scallop tissues and challenge with lipopolysaccharide, peptidoglycan, or poly(I:C) significantly upregulated the expression of CfIKK1. Co-immunoprecipitation assays confirmed the interaction of CfIKK1 with scallop MyD88 (Myeloid differentiation actor 88, the key adaptor of the TLR signaling pathway) via its N-terminal kinase domain. Additionally, CfIKK1 protein could form homodimers and even oligomers, with N-terminal kinase domain and C-terminal scaffold dimerization domain playing key roles in this process. Finally, the results of RNAi experiments showed that when the scallop IKK1 gene was suppressed, the expression of IRF genes also decreased significantly. In conclusion, CfIKK1 could respond to PAMPs challenge and interact with MyD88 protein of scallop TLR signaling, with the formation of CfIKK1 dimers or oligomers. At the same time, the results of RNAi experiments revealed the close regulatory relationship between IKK1 and IRF genes of scallop. Therefore, as a key signal transduction molecule and immune activity regulator, CfIKK1 plays important roles in the innate immune system of scallops.

The Caspase Homologues in Scallop Chlamys farreri and Their Expression Responses to Toxic Dinoflagellates Exposure.

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 (Chlamys farreri) is a bivalve mollusc that is well adapted to complex marine environments, yet the diversity of caspase homologues and their expression patterns in the Zhikong scallop remain largely unknown. Here, we identified 30 caspase homologues in the genome of the Zhikong scallop and analysed their expression dynamics during all developmental stages and following exposure to paralytic shellfish toxins (PSTs). The 30 caspase homologues were classified as initiators (caspases-2/9 and caspases-8/10) or executioners (caspases-3/6/7 and caspases-3/6/7-like) and displayed increased copy numbers compared to those in vertebrates. Almost all of the caspase-2/9 genes were highly expressed throughout all developmental stages from zygote to juvenile, and their expression in the digestive gland and kidney was slightly influenced by PSTs. The caspase-8/10 genes were highly expressed in the digestive gland and kidney, while PSTs inhibited their expression in these two organs. After exposure to different Alexandrium PST-producing algae (AM-1 and ACDH), the number of significantly up-regulated caspase homologues in the digestive gland increased with the toxicity level of PST derivatives, which might be due to the higher toxicity of GTXs produced by AM-1 compared to the N-sulphocarbamoyl analogues produced by ACDH. However, the effect of these two PST-producing algae strains on caspase expression in the kidney seemed to be stronger, possibly because the PST derivatives were transformed into highly toxic compounds in scallop kidney, and suggested an organ-dependent response to PSTs. These results indicate the dedicated control of caspase gene expression and highlight their contribution to PSTs in C. farreri. This work provides a further understanding of the role of caspase homologues in the Zhikong scallop and can guide future studies focussing on the role of caspases and their interactions with PSTs.

Estimating genetic parameters of muscle imaging trait with 2b-RAD SNP markers in Zhikong scallop (Chlamys farreri)

Genetic improvement is a crucial tool to deal with the increasing demand for high-quality, sustainably produced meat in the scallop industry. Breeding programs are based on genetic parameters, such as heritability, genetic correlations, and breeding values (BVs). By X-ray technology, we used adductor muscle area percentage (AMAP) obtained by dividing adductor muscle area (AMA) by shell area (SA) in an X-ray scallop image as a novel muscle imaging trait in a non-invasive manner. The goals of this study are 1) to estimate the genetic parameters of the muscle imaging trait AMAP and the adductor muscle weight percentage (AMWP), which is traditionally determined by weighing after dissection causing low efficiency in measurement and failure to collect data in vivo, and 2) to compare estimation accuracies of breeding values of AMAP and AMWP in a Zhikong scallop (Chlamys farreri) population. 2b-RAD SNPs were applied in 449 two-year-old healthy and active Zhikong scallops randomly sampled from an artificially bred population in the same culture sea area. The estimates of SNP-based heritability conducted using all SNPs (17,495 SNPs with MAF > 2%) were 0.551 (S.E. 0.07) for AMAP and 0.515 (S.E. 0.08) for AMWP, respectively. Chromosome-wise heritabilities for AMAP and AMWP were regressed against the chromosome lengths with significant linear trends (P = 0.02969 and P = 0.03043), implying that the genetic contribution to muscle trait is scattered across the genome and driven by many loci with small effect rather than a few causal loci with a large effect. The phenotypic and genetic correlations between AMAP and AMWP were 0.769 (S.E. 0.002) and 0.917 (S.E 0.043), suggesting that we can use the image-based trait AMAP in substitution for the muscle growth trait AMWP for genetic improvement of meat in live scallops. The impact of population genome structure (e.g., the effective population size Ne), number of independent chromosome segments (Me), SNP marker densities, and training population sizes on genomic prediction accuracy of AMAP and AMWP was investigated as well. These results will provide important reference information and methodology basis for breeding through image-based traits in scallops.

A Systematical Survey on the TRP Channels Provides New Insight into Its Functional Diversity in Zhikong Scallop (Chlamys farreri).

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.

Chromosome Identification and Cytogenetic Map Construction of Zhikong Scallop (Chlamys farreri) Based on Fluorescence in situ Hybridization

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.

Bioaccumulation and biotransformation of inorganic arsenic in zhikong scallop (Chlamys farreri) after waterborne exposure

Arsenic (As) and As speciation in marine bivalves have been widely investigated. However, little is known about the bioaccumulation and biotransformation of inorganic As in different tissues of scallops. Therefore, the tissue-specific accumulation, biotransformation and subcellular partitioning of As were investigated in Chlamys farreri following 12 d inorganic As [arsenite (AsⅢ) and arsenate (AsⅤ)] exposure and 30 d depuration. Total As levels were highest in the kidneys and lowest in the adductor muscle after 12 d exposure for both As (Ⅲ) and As (Ⅴ) treatment groups, and the bioavailability of As (Ⅲ) was significantly higher than that of As (Ⅴ) for C. farreri. After 30 d elimination, total As levels were significantly decreased to the control levels. The subcellular fate of As in five different tissues was similar for different inorganic As treatment groups. The greatest proportion of As was found in the metallothionein-like protein fraction (MTLP) and the second was the cellular debris (CD). A little part of As (Ⅲ) could be oxidized to As (Ⅴ) in the gill and digestive gland for As (Ⅲ) treatment groups, and the reduction of As (Ⅴ) to As (Ⅲ) happened in the gill and kidney under As (Ⅴ) exposure. Although a high methylation activity was found in C. farreri, it varied in different tissues with different inorganic As species exposure. The present results indicated that exposure to As (Ⅲ) and As (Ⅴ) could induce different responses in bioaccumulation and biotransformation in five tissues of C. farreri.