iTRAQ-Based Quantitative Proteomic Analysis Reveals Toxicity Mechanisms in Chlamys farreri Exposed to Okadaic Acid

Okadaic acid (OA), produced by dinoflagellates during harmful algal blooms, is a principal diarrhetic shellfish poisoning toxin. This toxin poses a potential threat to bivalves with economic values. To better understand the toxicity mechanism of OA to bivalves, in this study, oxidative stress biomarkers (superoxide dismutase, SOD; catalase, CAT; glutathione S-transferase, GST; malondialdehyde, MDA) and the expression of detoxification genes (heat shock protein 70, HSP70; heat shock protein 90, HSP90; cytochrome P450, CYP450) were assessed in the gills of scallops Chlamys farreri after 24 h, 48 h and 96 h exposure to OA. In addition, the digestive glands of scallops exposed to OA for 96 h were dissected for an iTRAQ based quantitative proteomic analysis. The results of OA exposure experiments showed that OA induces oxidative stress and significant enhancement of the expression of detoxification genes in scallops. The proteomics analysis revealed that 159 proteins altered remarkably in OA-treated scallops, and these proteins were involved in phagosomes, regulation of actin cytoskeleton, adherens junction, tight junction, and focal adhesion. Amino acid biosynthesis, carbon metabolism, pentose phosphate pathway, fructose and mannose metabolism in the digestive glands were also significantly impacted. Our data shed new insights on the molecular responses and toxicity mechanisms of C. farreri to OA.

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-holomog 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.

Identification and Characterization of HSP90 Gene Family Reveals Involvement of HSP90, GRP94, and Not TRAP1 in Heat Stress Response in Chlamys farreri

Heat shock proteins 90 (HSP90s) are a class of ubiquitous, highly conserved, and multi-functional molecular chaperones present in all living organisms. They assist protein folding processes to form functional proteins. In the present study, three HSP90 genes, CfHSP90, CfGRP94 and CfTRAP1, were successfully identified in the genome of Chlamys farreri. The length of CfHSP90, CfGRP94 and CfTRAP1 were 7211 bp, 26457 bp, and 28699 bp, each containing an open reading frame (ORF) of 2181 bp, 2397 bp, and 2181bp, and encoding proteins of 726, 798, and 726 amino acids, respectively. A transcriptomic database demonstrated that CfHSP90 and CfGRP94 were the primary functional executors with high expression during larval development and in adult tissues, while CfTRAP1 expression was low. Furthermore, all of the three CfHSP90s showed higher expression in gonads and ganglia as compared with other tissues, which indicated their probable involvement in gametogenesis and nerve signal transmission in C. farreri. In addition, under heat stress, the expressions of CfHSP90 and CfGRP94 were significantly up-regulated in the mantle, gill, and blood, but not in the heart. Nevertheless, the expression of CfTRAP1 did not change significantly in the four tested tissues. Taken together, in coping with heat stress, CfHSP90 and CfGRP94 could help correct protein folding or salvage damaged proteins for cell homeostasis in C. farreri. Collectively, a comprehensive analysis of CfHSP90s in C. farreri was conducted. The study indicates the functional diversity of CfHSP90s in growth, development, and environmental response, and our findings may have implications for the subsequent in-depth exploration of HSP90s in invertebrates.

Transcriptional Responses of Flavin-Containing Monooxygenase Genes in Scallops Exposed to PST-Producing Dinoflagellates Implying Their Involvements in Detoxification

Flavin-containing monooxygenase (FMO) is one of the most prominent xenobiotic metabolic enzymes. It can catalyze the conversion of heteroatom-containing chemicals to polar, readily excretable metabolites and is considered an efficient detoxification system for xenobiotics. Bivalves can accumulate paralytic shellfish toxins (PSTs) produced by dinoflagellates, especially during outbreaks of harmful algal blooms. Exploring FMO genes in bivalves may contribute to a better understanding of the adaptation of these species and the mechanisms of PSTs bioavailability. Therefore, through genome screening, we examined the expansion of FMO genes in two scallops (Patinopecten yessoensis and Chlamys farreri) and found a new subfamily (FMO_like). Our expression analyses revealed that, in both scallops, members of the FMO_N-oxide and FMO_like subfamilies were mainly expressed from the D-stage larvae to juveniles, whereas the FMO_GS-OX subfamily genes were mainly expressed at and prior to the trochophore stage. In adult organs, higher expressions of FMOs were observed in the kidney and hepatopancreas than in other organs. After exposure to PST-producing algae, expression changes in FMOs occurred in hepatopancreas and kidney of both scallops, with more members being up-regulated in hepatopancreas than in kidney for Alexandrium catenella exposure, while more up-regulated FMOs were found in kidney than in hepatopancreas of C. farreri exposed to A. minutum. Our findings suggest the adaptive functional diversity of scallop FMO genes in coping with the toxicity of PST-producing algae.

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.

Identification, Characterization, and Expression Analysis Reveal Diverse Regulated Roles of Three MAPK Genes in Chlamys farreri Under Heat Stress

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.

Reproductive Toxicity Induced By Benzo[a]pyrene Exposure: First Exploration Highlighting The Multi-stage Molecular Mechanism In Female Scallop Chlamys Farreri

Reproductive toxicity induced by Benzo[a]pyrene (B[a]P) exposure have received great ecotoxicological concerns. However, huge gaps on molecular mechanism still exist in bivalves. In this study, reproduction-related indicators during reproductive periods (proliferative, growth, mature, and spawn stage) were investigated in female scallops Chlamys farreri, which under gradient concentration of B[a]P at 0, 0.04, 0.4 and 4 μg/L. To elucidate the potential molecular mechanisms of reproductive toxicology, a multi-stage ovarian transcriptome analysis under 4 μg/L B[a]P exposure was also conducted. The results indicated that life-cycle exposure to 0.4 and 4 μg/L B[a]P had significantly decreased GSI and sex steroid levels. Even 0.04 μg/L B[a]P could play the wicked role on DNA integrity at mature and spawn stages. Ovarian histological sections showed the inhibitions on oocyte maturation and ovulation of B[a]P with dose-dependent effects. Through the functional enrichment analysis of DEGs from transcriptome data, 18 genes involved in endocrine disruption effects, DNA damage and repair, and oogenesis damage were selected and further determined by qRT-PCR. The down-regulate of steroidogenic and estrogen signaling pathways genes indicated the endocrine disruption mechanisms by B[a]P, which emphasized the functions of receptor independent and dependent pathways under B[a]P exposure. The variation of DNA single strand break and repair gene expressions implied there might exist the similar toxic mechanism with that in vertebrates. Gene expression data involved in cell cycle, apoptosis and cell adhesion exhibited the possibly toxic mechanisms of oogenesis caused by B[a]P. Taken together, this study is a pioneer to take advantage of genome-wide transcriptomic analysis and its corresponding reproductive indicators to explore the toxic mechanism under B[a]P exposure in bivalves. Meanwhile, some selected genes were firstly identified in bivalves, and the expression data might be useful in establishing new hypotheses and discovering new biomarkers for marine biomonitoring.