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.

Identification and functional characterization of two Bcl-2 family protein genes in Zhikong scallop Chlamys farreri.

Apoptosis plays significant roles in maintenance of homeostasis, immune defense and development. The Bcl-2 family proteins are important regulators of the intrinsic apoptosis. In the study, we have characterized a Bcl-2-like gene (named CfBcl-2) and a Bax-like gene (named CfBax) from the Zhikong scallop Chlamys farreri. The full-length of the CfBcl-2 cDNA is 944 nucleotides (nt) encoding a putative protein of 225 amino acid residues (aa) that contains four Bcl-2 homology (BH) domains, and the CfBax cDNA is 505 nt encoding a putative protein of 115 aa that contains three Bcl-2 BH domains. Sequence and phylogenetic analysis demonstrate that CfBcl-2 and CfBax present typical domain organization of the corresponding Bcl-2 related proteins and are more similar and clustered with their homologues of other molluscs. The two genes are ubiquitously expressed in six tissues of C. farreri, with the highest expression level of CfBcl-2 in adductor muscle and highest expression level of CfBax in gill. The expressions of CfBcl-2 and CfBax in hemocytes were both significantly up-regulated after an in vivo exposure of scallops to air, injection with lipopolysaccharide and infection with acute viral necrobiotic disease virus, and the expression patterns of the two genes after the three treatments vary in different change magnitude and up-regulation timespan. Yeast two-hybrid assay reveals a direct interaction between the CfBcl-2 and CfBax proteins. These results indicate that the CfBcl-2 and CfBax may participate in the apoptosis-based stress and immune responses against noxious stimulation.

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.

Characterization and functional analysis of two inhibitor of apoptosis genes in Zhikong scallop Chlamys farreri.

The proteins of inhibitor of apoptosis (IAP) family play important roles in regulation of apoptosis, immunological response and cell proliferation. Here we reported two IAP genes (named CfIAP1 and CfIAP2) in Zhikong scallop Chlamys farreri. The full-length CfIAP1 cDNA contained 1552 nucleotides, encoding a predicted protein of 251 amino acids with two BIR domains. The full-length CfIAP2 cDNA contained 1243 nt, encoding a 356-aa protein with one BIR domain and one RING domain. The two genes are ubiquitously expressed in six types of tissue of C. farreri. The expression levels of CfIAP1 and CfIAP2 were significantly up-regulated after challenged with acute viral necrobiotic disease virus, lipopolysaccharide and exposure to air. Subcellular localization assay showed that CfIAP1 was mainly distributed in cytoplasm and CfIAP2 was in cytoplasm and nucleus. As assessed using a kit designed to test Caspase3 function in mammalian cells, the activity of CfCaspase3 was enhanced as a result of the down-regulation of CfIAP2 expression by dsRNA-mediated gene silencing. Our study indicated that CfIAP1 and CfIAP2 may participate in the innate immunity and stress responses and that CfIAP2 might block apoptosis via inhibiting CfCaspase3 indirectly through an unexplored mechanism in C. farreri.

Transcriptomic response to estrogen exposure in the male Zhikong scallop, Chlamys farreri.

The transcriptomes of Zhikong scallop exposed to 17ß-estradiol were determined using the Roche/454. A total of 51,997 unigenes, representing 45,030 contigs and 6967 singlets were obtained. And 14,028, 19,798 and 14,981 of these unigenes were annotated from the non-redundant nucleic acid database, non-redundant protein database and Swiss protein database, respectively. A total of 10,699 unigenes were further annotated to biological processes (9080), molecular functions (8692) and cellular components (7829) using the GO, and 8945 unigenes were mapped to biological pathways including the metabolism (2862) and genetic information processing (2263). Most importantly, 16,692 unigenes and 18,686 unigenes in testis, and 10,492 unigenes and 13,186 unigenes in digestive gland were up-regulated significantly after exposure to 50 and 500 ng E2/L; while 10,212 unigenes and 9409 unigenes in testis and 10,629 unigenes and 9463 unigenes in digestive gland were down-regulated. These valuable information provides insights into the mechanisms in invertebrate exposure to EDCs.

Evidence for the presence of sex steroid hormones in Zhikong scallop, Chlamys farreri.

To obtain evidence of the presence of sex steroid hormones in mollusks, hormone variation in the gonads of the Zhikong scallop Chlamys farreri was analyzed using UPLC-MS/MS. These were found, as expected, with concentrations of estrone (E1), 17beta-estradiol (E2), and testosterone (T) in the testes ranging from not detected (ND) to 0.07 ± 0.10, ND to 3.10 ± 2.00, and ND to 2.67 ± 1.55 ng/g wet weight, respectively. In the ovaries, these hormones ranged from ND to 2.45 ± 1.22, ND to 27.90 ± 4.23, and ND to 2.38 ± 1.56 ng/g ww, respectively. The levels of T in males and E2 in females followed a trend similar to the gonadal-somatic index over the course of the reproductive period. In addition, the gene expression of vitellogenin and calmodulin-2 showed similar patterns to T and E2, while the estrogen receptors and calmodulin-1 did not. These results indicate that sex steroids are present in the scallop and that they may regulate endocrine functions during the reproductive process.