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.

Toxin- and species-dependent regulation of ATP-binding cassette (ABC) transporters in scallops after exposure to paralytic shellfish toxin-producing dinoflagellates.

ATP-binding cassette (ABC) transporters are membrane-bound proteins involved in exporting various xenobiotic compounds from living cells. Bivalve mollusks can accumulate large amounts of paralytic shellfish toxins (PSTs) from marine dinoflagellates. For aquatic invertebrates, the importance of ABC proteins in multi-xenobiotic resistance has been demonstrated, however, the systematic identification of ABC transporters is very limited. In this study, 64 and 67 ABC genes containing all eight described subfamilies (A to H) were identified in Yesso scallop (Patinopecten yessoensis) and Zhikong scallop (Chlamys farreri), respectively, with massive gene expansion being observed in the ABCC and ABCG subfamilies. The kidney harbored more specifically expressed ABC genes than other organs/tissues, most of which belonged to ABCB, ABCC, and ABCG subfamilies. After feeding the scallops with PST-producing dinoflagellates, the expression of scallop ABC genes in the kidney was regulated in toxin- and species-dependent manners. In total, 20 and 24 ABC genes in Zhikong scallop (CfABCs) were induced after exposure to Alexandrium minutum and A. catenella, with the up-regulated members from both ABCC and ABCG subfamilies mainly showing acute and chronic induction by A. minutum and A. catenella, respectively, while the up-regulated CfABCBs mainly showing chronic induction by both dinoflagellates. In Yesso scallop, only eight ABC genes (PyABCs) were regulated after A. catenella exposure, and all the five up-regulated PyABCs were acutely induced. Our findings imply the functional diversity of scallop ABC genes in coping with PST accumulation, which may contribute to the lineage-specific adaptation of scallops for dealing with algal toxins challenge.

Correction to: Transcriptomic Profiling Provides Insights into Inbreeding Depression in Yesso Scallop Patinopecten yessoensis.

The original version of this article unfortunately contained a mistake in the authorgroup section. Author Zhenmin Bao's given name was incorrectly spelled as "Zhemin Bao".

Tissue-Biased and Species-Specific Regulation of Glutathione Peroxidase (GPx) Genes in Scallops Exposed to Toxic Dinoflagellates.

Marine bivalves could accumulate paralytic shellfish toxins (PSTs) produced by toxic microalgae, which might induce oxidative stress. Glutathione peroxidases (GPxs) are key enzymes functioning in the antioxidant defense, whereas our understanding of their roles in PST challenge in bivalves is limited. Herein, through genome-wide screening, we identified nine (CfGPx) and eight (PyGPx) GPx genes in Zhikong scallop (Chlamys farreri) and Yesso scallop (Patinopecten yessoensis), respectively, and revealed the expansion of GPx3 sub-family in both species. RNA-Seq analysis revealed high expression of scallop GPx3s after D stage larva during early development, and in adult hepatopancreas. However, in scallops exposed to PST-producing dinoflagellates, no GPx was significantly induced in the hepatopancreas. In scallop kidneys where PSTs were transformed to higher toxic analogs, most CfGPxs were up-regulated, with CfGPx3s being acutely and chronically induced by Alexandrium minutum and A. catenella exposure, respectively, but only one PyGPx from GPx3 subfamily was up-regulated by A. catenella exposure. Our results suggest the function of scallop GPxs in protecting kidneys against the oxidative stresses by PST accumulation or transformation. The tissue-, species-, and toxin-dependent expression pattern of scallop GPxs also implied their functional diversity in response to toxin exposure.

Solute carriers in scallop genome: Gene expansion and expression regulation after exposure to toxic dinoflagellate.

The solute carriers (SLCs) are membrane proteins that transport many endogenous and exogenous substances such as xenobiotic toxins. Bivalve mollusks, mainly feeding on microalgae, show marked capacity to accumulate paralytic shellfish toxins (PSTs), the most common and hazardous marine biotoxins produced by dinoflagellates. Exploring the SLCs related to PST accumulation in bivalve could benefit our understanding about the mechanisms of PST bioavailability in bivalve and the adaptations of these species. Herein, we provided the first systematic analysis of SLC genes in mollusks, which identified 673 SLCs (PySLCs, 48 subfamilies) in Yesso scallop (Patinopecten yessoensis), 510 (48 subfamilies) in Pacific oyster (Crassostrea gigas), and 350 (47 subfamilies) in gastropod owl limpet (Lottia gigantea). Significant expansion of subfamilies SLC5, SLC6, SLC16, and SLC23 in scallop, and SLC46 subfamily in both scallop and oyster were revealed. Different PySLC members were highly expressed in the developmental stages and adult tissues, and hepatopancreas harboured more specifically expressed PySLCs than other tissues/organs. After feeding the scallops with PST-producing dinoflagellate, 131 PySLCs were regulated and more than half of them were from the expanded subfamilies. The trend of expression fold change in regulated PySLCs was consistent with that of PST changes in hepatopancreas, implying the possible involvement of these PySLCs in PST transport and homeostasis. In addition, the PySLCs from the expanded subfamily were revealed to be under positive selection, which might be related to lineage-specific adaptation to the marine environments with algae derived biotoxins.

Genome-Wide Identification and Characterization of SODs in Zhikong Scallop Reveals Gene Expansion and Regulation Divergence after Toxic Dinoflagellate Exposure.

As filter-feeding animals mainly ingesting microalgae, bivalves could accumulate paralytic shellfish toxins (PSTs) produced by harmful algae through diet. To protect themselves from the toxic effects of PSTs, especially the concomitant oxidative damage, the production of superoxide dismutase (SOD), which is the only eukaryotic metalloenzyme capable of detoxifying superoxide, may assist with toxin tolerance in bivalves. To better understand this process, in the present study, we performed the first systematic analysis of SOD genes in bivalve Chlamys farreri, an important aquaculture species in China. A total of six Cu/Zn-SODs (SOD1-6) and two Mn-SODs (SOD7, SOD8) were identified in C. farreri, with gene expansion being revealed in Cu/Zn-SODs. In scallops exposed to two different PSTs-producing dinoflagellates, Alexandrium minutum and A. catenella, expression regulation of SOD genes was analyzed in the top ranked toxin-rich organs, the hepatopancreas and the kidney. In hepatopancreas, which mainly accumulates the incoming PSTs, all of the six Cu/Zn-SODs showed significant alterations after A. minutum exposure, with SOD1, 2, 3, 5, and 6 being up-regulated, and SOD4 being down-regulated, while no significant change was detected in Mn-SODs. After A. catenella exposure, up-regulation was observed in SOD2, 4, 6, and 8, and SOD7 was down-regulated. In the kidney, where PSTs transformation occurs, SOD4, 5, 6, and 8 were up-regulated, and SOD7 was down-regulated in response to A. minutum feeding. After A. catenella exposure, all the Cu/Zn-SODs except SOD1 were up-regulated, and SOD7 was down-regulated in kidney. Overall, in scallops after ingesting different toxic algae, SOD up-regulation mainly occurred in the expanded Cu/Zn-SOD group, and SOD6 was the only member being up-regulated in both toxic organs, which also showed the highest fold change among all the SODs, implying the importance of SOD6 in protecting scallops from the stress of PSTs. Our results suggest the diverse function of scallop SODs in response to the PST-producing algae challenge, and the expansion of Cu/Zn-SODs might be implicated in the adaptive evolution of scallops or bivalves with respect to antioxidant defense against the ingested toxic algae.

Transcriptomic Profiling Provides Insights into Inbreeding Depression in Yesso Scallop Patinopecten yessoensis.

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

Diverse expression regulation of Hsp70 genes in scallops after exposure to toxic Alexandrium dinoflagellates.

Heat shock proteins 70KD (Hsp70s) are highly conserved molecular chaperones with essential roles against biotic and abiotic stressors. Marine bivalves inhabit highly complex environments and could accumulate paralytic shellfish toxins (PSTs), the well-noted neurotoxins generated during harmful algal blooms. Here, we systematically analyzed Hsp70 genes (CfHsp70s) in Zhikong scallop (Chlamys farreri), an important aquaculture mollusk in China. Sixty-five CfHsp70s from eight sub-families were identified, and 47 of these genes showed expansion in the Hspa12 sub-family. After exposure to different PST-producing dinoflagellates, Alexandrium minutum and Alexandrium catenella, diverse CfHsp70s regulation presented in scallop hepatopancreas, mainly accumulating incoming PSTs, and kidneys, transforming PSTs into higher toxic analogs. All the up-regulated CfHsp70s were from CfHsp70B2, CfHspa12, and CfHspa5 sub-families. CfHsp70B2 sub-family was mainly induced in the hepatopancreas, and CfHspa12 sub-family was highly induced in the kidneys. CfHsp70s up-regulation under two dinoflagellates exposure was stronger in the kidneys (log2FC: 19.5 and 18.6) than that in hepatopancreas (log2FC: 4.3 and 6.1). Exposure to different Alexandrium species had varying effects, that in hepatopancreas, CfHsp70B2s were chronically induced only after A. catenella exposure, whereas in kidney, CfHspa12s were more acutely induced after exposure of A. minutum than A. caenella. Moreover, in Yesso scallops (Patinopecten yessoensis), only Hspa12s were up-regulated in hepatopancreas after A. catenella exposure, and all the Hsp70B2s were down-regulated. These organ-, toxin-, and species-dependent Hsp70 regulation suggested the functional diversity of duplicated Hsp70s in response to the stress by PST-producing algae. Our findings provide insights into the evolution and functional characteristics of Hsp70s in scallops.

The GRP94 gene of Yesso scallop (Patinopecten yessoensis): Characterization and expression regulation in response to thermal and bacterial stresses.

The 94-kDa glucose-regulated protein (GRP94) belonging to the HSP90 family is an endoplasmic reticulum (ER) chaperone. It plays critical roles in ER quality control, and has been implicated as a specialized immune chaperone to regulate both innate and adaptive immunity. In this study, we identified and characterized a GRP94 gene (PyGRP94) from Yesso scallop (Patinopecten yessoensis). The protein sequence of PyGRP94 is highly conserved with its homologs in vertebrates, with a signal sequence in N-terminal, an ER retrieval signal sequence in C-terminal and a HATPase_c domain. Expression analysis suggests that PyGRP94 transcripts in early embryos are maternally derived and the zygotic expression is started from D-shaped larvae. This gene is also expressed in almost all the adult tissues examined except smooth muscle, with the highest expression level in hemocytes. Besides, PyGRP94 was demonstrated to be induced by heat shock and both Gram-positive (Micrococcus luteus) and Gram-negative (Vibrio anguillarum) bacterial infection, with much more dramatic changes being observed after V. anguillarum challenge. Our results suggest the involvement of PyGRP94 in response to thermal stress, and that it might play an important role in the innate immune defense of scallop.

Expression of Cathepsin F in response to bacterial challenges in Yesso scallop Patinopecten yessoensis.

Cathepsin F is a unique papain cysteine proteinase with highly conserved structures: catalytic triad and a cystatin domain contained in the elongated N-terminal pro-region. It has been reported that cathepsin F is associated with the establishment of innate immune in several vertebrate including fish in aquaculture, but not known in bivalves. In this study, we firstly identified and characterized cathepsin F in the Yesso scallop (Patinopecten yessoensis). The protein structural and phylogenetic analyses were then conducted to determine its identity and evolutionary position. We've also investigated the expression levels of cathepsin F gene at different embryonic developmental stages, in healthy adult tissues and especially in the hemocytes and hepatopancreas after Gram-positive (Micrococcus luteus) and negative (Vibrio anguillarum) challenges using quantitative real-time PCR (qPCR). Cathepsin F was significantly up-regulated 3 h after infection of V. anguillarum in hemocytes, suggesting its participation in immune response. Our findings have provided strong evidence that cathepsin F may be a good target for enhancing the immune activity in Yesso scallop.