Physiological and transcriptomic analysis provides new insights into osmoregulation mechanism of Ruditapes philippinarum under low and high salinity stress.

The Manila clam (Ruditapes philippinarum) is a commercially important marine bivalve, which inhabits the estuarine and mudflat areas. The osmoregulation is of great significance for molluscs adaptation to salinity fluctuations. In this study, we investigated the effects of low salinity (10 psu) and high salinity (40 psu) stress on survival and osmoregulation of the R. philippinarum. The results of physiological parameters showed that the ion (Na+, K+, Cl-) concentrations and Na+/K+-ATPase (NKA) activity of R. philippinarum decreased significantly under low salinity stress, but increased significantly under high salinity stress, indicating that there are differences in physiological adaptation of osmoregulation of R. philippinarum. In addition, we conducted the transcriptome analysis in the gills of R. philippinarum exposed to low (10 psu) and high (40 psu) salinity challenge for 48 h using RNA-seq technology. A total of 153 and 640 differentially expressed genes (DEGs) were identified in the low salinity (LS) group and high salinity (HS) group, respectively. The immune (IAP, TLR6, C1QL4, Ank3), ion transport (Slc34a2, SLC39A14), energy metabolism (PCK1, LDLRA, ACOX1) and DNA damage repair-related genes (Gadd45g, HSP70B2, GATA4) as well as FoxO, protein processing in endoplasmic reticulum and endocytosis pathways were involved in osmoregulation under low salinity stress of R. philippinarum. Conversely, the ion transport (SLC6A7, SLC6A9, SLC6A14, TRPM2), amino acid metabolism (GS, TauD, ABAT, ALDH4A1) and immune-related genes (MAP2K6, BIRC7A, CTSK, GVIN1), and amino acid metabolism pathways (beta-Alanine, Alanine, aspartate and glutamate, Glutathione) were involved in the process of osmoregulation under high salinity stress. The results obtained here revealed the difference of osmoregulation mechanism of R. philippinarum under low and high salinity stress through physiological and molecular levels. This study contributes to the assessment of salinity adaptation of bivalves in the context of climate change and provides useful information for marine resource conservation and aquaculture.

The energy metabolism and transcriptomic responses of the Manila clam (Ruditapes philippinarum) under the low-temperature stress.

Low temperature in winter poses a threat to the Manila clam Ruditapes philippinarum in North China. However, a number of low-temperature-tolerant clams could survive such condition. It is therefore of interest to explore the survival mechanisms underlying the cold tolerance of R. philippinarum. The Zebra II population of R. philippinarum (Zebra II) from North China and the native Putian population from South China were used as experimental materials. Both populations were stressed with low-temperature and the differences in their survival rates, energy metabolism and transcriptional responses were compared. The results shown that after cold treatment at -1.9 °C, survival rate of Zebra II was higher than that of the Putian group. For both groups, the respiration, ammonia excretion, and ingestion rates continuously decreased till 0 with reductions temperature. In addition, RNA-seq revealed that as compared with the Putian group, there were 3682 up-regulated differentially expressed genes (DEGs) and 3361 down-regulated DEGs in Zebra II group. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that these DEGs were mostly enriched in the purine, pyrimidine, and pyruvate metabolism pathways in Zebra II under low-temperature stress. Furthermore, qRT-PCR analysis further confirmed that Zebra II responded to low-temperature stress through upregulating genes involved in purine, pyrimidine, and pyruvate metabolism pathways. Taken together, all these results indicated that Zebra II has higher cold tolerance than the Putian group. Therefore, Zebra II is capable for overwintering in the intertidal zone of North China.

The lgi-miR-2d is Potentially Involved in Shell Melanin Synthesis by Targeting mitf in Manila Clam Ruditapes philippinarum.

Shell color as an important economic trait is also the crucial target trait for breeding and production. MicroRNA (miRNA) is an endogenous small non-coding RNA that can post-transcriptionally regulate the expression of target genes, it plays important roles in many life activities and physiological processes, such as shell color, stress response, and disease traits. In this study, we investigated the function of lgi-miR-2d in shell melanin formation and the expression patterns of lgi-miR-2d and target gene Rpmitf in Manila clam Ruditapes philippinarum. We further explored and verified the relationship between Rpmitf and lgi-miR-2d and identified the expression level of shell color-related gene changes by RNAi and injecting the antagomir of lgi-miR-2d, respectively. Our results indicated that lgi-miR-2d antagomir affected the expression of its target gene Rpmitf. In addition, the dual-luciferase reporter assay was conducted to confirm the direct interaction between lgi-miR-2d and Rpmitf. The results showed that the expression levels of melanin-related genes such as Rpmitf and tyr were significantly decreased in the positive treatment group compared with the blank control group after the Rpmitf dsRNA injection, indicating Rpmitf plays a crucial role in the melanin synthesis pathway. Taken together, we speculated that lgi-miR-2d might be negatively modulating Rpmitf, which might regulate other shell color-related genes, thereby affecting melanin synthesis in R. philippinarum.

Transcriptome analysis provides new insights into the immune response of Ruditapes philippinarum infected with Vibrio alginolyticus.

Manila clam (Ruditapes philippinarum) is a bivalve species with commercial value, but it is easily infected by pathogenic microorganisms in aquaculture, which restricts the shellfish industry. Notably, the impact of Vibrio alginolyticus on clam culture is obvious. In this study, RNA-seq was performed to analyze clam hepatopancreas tissue in 48 h (challenge group, G48h) and 96 h (challenge group, G96h) after infection with V. alginolyticus and 0 h after injection of PBS (control group, C). The results showed that a total of 1670 differentially expressed genes were detected in the G48h vs C group, and 1427 differentially expressed genes were detected in the G96h vs C group. In addition, KEGG analysis showed that DEGs were significantly enriched in pathways such as Lysosome and Mitophagy. Moreover, 15 immune related DEGs were selected for qRT-PCR analysis to verify the accuracy of RNA-seq, and the results showed that the expression level of DEGs was consistent with that of RNA-seq. Therefore, the results obtained in this study provides a preliminary understanding of the immune defense of R. philippinarum and molecular insights for genetic breeding of V. alginolyticus resistance in Manila clam.

Comprehensive analysis of differentially expressed mRNA, lncRNA and miRNA, and their ceRNA networks in the regulation of shell color in the Manila clam (Ruditapes philippinarum).

The regulatory mechanism of ceRNA network plays an important role in molecular function and biological processes, however, the molecular mechanism in the shell color of Ruditapes philippinarum has not yet been reported. In this study, we performed transcriptome sequencing on the mantle of R. philippinarum with different shell colors, and screened for mRNA, miRNA, and lncRNA. A total of 61 mRNAs, 3725 lncRNAs and 90 miRNAs were obtained from all the shell color comparison groups (all mRNAs, lncRNAs and miRNAs P < 0.05), and 7 mRNAs, 8 lncRNAs, and 4 miRNAs of the porphyrin pathway and melanin pathway were screened for competitive endogenous RNA (ceRNA) network construction. The results indicate that the ceRNA network composed of mRNA and lncRNA, centered around efu-miR-101, mle-bantam-3p, egr-miR-9-5p, and sma-miR-75p, may play a crucial regulatory role in shell color formation. This study reveals for the first time the mechanism of ceRNA regulatory networks in the shell color of R. philippinarum and providing important reference data for molecular breeding of shell color in R. philippinarum.

Genome-Wide Identification and Characterization of MITF Genes in Ruditapes philippinarum and Their Involvement in the Immune Response to Vibrio anguillarum Infection.

Studies have shown that the shellfish have innate immune system, which is a very important immune form of shellfish, and they rely on the innate immune system to resist diseases. As a transcription factor, Microphthalmia-associated transcription factor (MITF) plays a regulatory role in immune response and the shell color is also an important index for the breeding of excellent varieties of R. philippinarum. The research on immune response mechanism of RPMITFs can provide important reference data for the breeding of excellent clam varieties. In the genome of R. philippinarum, the RPMITF genes family of shell color-related gene family was selected as the target gene of this experiment. There are 12 RpMITF genes named RpMITF1, RpMITF2, RpMITF3, RpMITF4, RpMITF5, RpMITF6, RpMITF7, RpMITF8, RpMITF9, RpMITF10, RpMITF11, and RpMITF12. The open reading frame length is 639, 1233, 996, 1239, 675, 624, 816, 1365, 612, 1614, 1122, and 486 bp, encoding 212, 410, 331, 412, 224, 207, 271, 454, 203, 537, 373, and 161 aa, respectively. The predicted molecular weight range of amino acids is 18.85-62.61 kda, and the isoelectric point range is 5.26-9.44. Real-time quantitative PCR was used to detect the gene expression of RpMITF gene family in hepatopancreas tissues of two populations of Manila clam at 6 time points (0, 3, 6, 12, 24, and 48 h) after Vibrio anguillarum stress. The results show that RpMITF gene family was significantly expressed in hepatopancreas of two clam populations after V. anguillarum stress (P < 0.05).

Neuroprotection of Emodin by Inhibition of Microglial NLRP3 Inflammasome-Mediated Pyroptosis.

BACKGROUND: Neuroinflammation triggered by chronic cerebral ischemia-induced microglial pyroptosis is a significant contributor to vascular cognitive impairment. It has been shown that emodin possesses anti-inflammatory and neuroprotective properties, however, it's potential molecular and signaling transduction pathway remains to be illuminated. This study researched the neuroprotective mechanisms of emodin focussing on emodin effects on lipopolysaccharide/adenosine triphosphate (LPS/ATP)-caused pyroptosis in BV2 cells and HT-22 hippocampal neurons. METHODS: To explore the neuroprotective effect of emodin, Emodin was applied to BV2 cells, HT-22 hippocampal neurons, and BV2/HT-22 co-cultures stimulated with LPS/ATP to evaluate the cell morphology, levels of inflammatory factors, NLRP3 inflammatory inflammasome activity and focal pyroptosis-related protein expression, as same as neuronal apoptosis. RESULTS: Emodin alleviated LPS/ATP-induced pyroptosis of BV2 cells by preventing the activity of the NLRP3 inflammasome and the cleavage of pyroptosis executive protein Gasdermin D (GSDMD). Furthermore, levels of interleukin (IL)-18, IL-1ß and tumor necrosis factor (TNF)-α were reduced, the apoptosis of HT-22 hippocampal neurons was attenuated, and cell viability was restored. CONCLUSIONS: Emodin can antagonize microglial neurotoxicity by inhibiting microglial pyroptosis, thereby exerting anti-inflammatory and neuroprotective effects.

Revealing the potential regulatory relationship between HSP70, HSP90 and HSF genes under temperature stress.

Heat Shock Protein (HSPs) gene family members play fundamental roles in different environmental stress tolerances, protect the structure and function of cells, and perform a significant task in cellular homeostasis. In this study, we conducted a genome-wide identification, evolutionary relationship analysis and gene expression analysis of the HSP70, HSP90, and HSF gene families in Ruditapes philippinarum. We identified 83 RpHSP70, 6 RpHSP90, and 3 RpHSF genes in R. philippinarum. The structural characteristics, chromosomal localization, and the gene structure map were constructed to reveal the characteristics of protein structures. Furthermore, the expression profiling of transcriptome data showed the expression pattern of HSP70, HSP90 and HSF genes in Manila clam from different populations, and under high and low temperature stress. In addition, we performed protein-protein interaction network analysis between HSP70, HSP90, and HSF gene family which enabled us to recognize the regulatory relationship between the two HSP gene families and the HSF gene family. Furthermore, the predicted sub-cellular location revealed a diversified subcellular distribution of HSP70, HSP90, and HSF proteins, which may be directly or indirectly associated with functional diversification under heat stress condition.

iTRAQ-based proteomic analysis reveals proteins associated with cold stress response in two different populations of Manila clam (Ruditapes philippinarum).

Water temperature is one of the key environmental factors for marine ectotherms and a change in temperature beyond and organism's capacity limits can cause a series of changes to physiological state and damage to the organism. Understanding how organisms adapt to complex environments is a central goal of evolutionary biology and ecology. Ruditapes philippinarum is an ecologically and scientifically important marine bivalve species. To uncover the molecular mechanisms of acclimation of R. philippinarum to low-temperature stress, iTRAQ-based quantitative proteomics was conducted to compare the proteomes of the north and south populations of R. philippinarum under low-temperature stress. The results showed a total of 6355 and 6352 proteins were identified in two populations, respectively. Among these, 94 and 83 were differentially abundant proteins (DAPs), and most of DAPs were related to oxidation-process, protein binding, or an integral component of membrane. According to the results of KEGG pathway enrichment analysis, most of DAPs in both populations are involved in immune-related pathways, while other population-specific significant abundance proteins of south population and north population were enriched in biosynthesis of amino acids (Enolase, Glutamine synthetase) and unsaturated fatty acids pathways (3-ketoacyl-CoA thiolase, Stearoyl-CoA desaturase), respectively, indicating that two population of clams may have different cold-stress regulation mechanisms. Our study provides new insights into different cold stress tolerance mechanisms in northern and southern populations of R. philippinarum using iTRAQ-based proteomics. This work contributes to a better understanding of molecular basis on cold stress response and adaptations, which shed lights on evolutionary biology and general ecophysiology of R. philippinarum.

Transient receptor potential (TRP) channels in the Manila clam (Ruditapes philippinarum): Characterization and expression patterns of the TRP gene family under heat stress in Manila clams based on genome-wide identification.

In this study, we identified a total of 40 transient receptor potential genes (RpTRP) in Manila clam by genome-wide identification and classified them into four categories (TRPV, TRPA, TRPM, TRPC) based on gene structure and subfamily relationships. The protein length of RpTRP genes ranges from 281 amino acids to 1601 amino acids. Molecular weight and theoretical PI values range from 182.82 kDa to 32.43 kDa, respectively, with PI values between 5.17 and 9.25. By comparing the expression profiles of TRP genes during heat stress in Manila clams at different latitudes, we found that most genes in the TRP gene family were up-regulated in expression during heat challenge. Therefore, we determined that TRP genes have an important role in the heat stress of Manila clams. This work provides a basis for further studies on the molecular mechanisms of TRP-mediated heat tolerance in Manila clam and for explaining differences in heat tolerance in Manila clam at different latitudes through key differential TRP genes at the molecular level.

Metabolomic analysis provides new insights into the heat-hardening response of Manila clam (Ruditapes philippinarum) to high temperature stress.

The temperature has always been a key environmental factor in Manila clam (Ruditapes philippinarum) culture. In this study, the Manila clam was treated to different temperature pre-heat (28 °C, 30 °C) and gained heat tolerance after recover of 12 h, and a survival rate (14.7 %-49.1 %) advantage after high temperature challenge (30 and 32 °C). To further investigate the physiological and metabolism changes in Manila clam that had experienced a heat stress, non-targeted metabolomics (LC-MS/MS) was used to analyze the metabolic responses of gills in three group Manila clams during the heat challenge. Metabolic profiles revealed that high temperature caused changes in fatty acid composition, energy metabolism, antioxidant metabolites, hydroxyl compounds, and amino acids in heat-hardened clams compared to non-hardened clams. We found a number of significantly enriched pathways, including cAMP signaling pathway, serotonergic synapse, and biosynthesis of unsaturated fatty acids in heat-hardened Manila clam compared with non-hardened and untreated Manila clam. After a brief high temperature treatment, the physiological maintenance ability of Manila clam was improved. Combined with metabolomics analysis, heat hardening treatment may improve the energy metabolism and antioxidant ability of Manila clam. These results provide new insights into the cellular and metabolic responses of Manila clams following high temperature stress.

Diffusion Tensor Imaging as a Tool to Evaluate the Cognitive Function of Patients With Vascular Dementia: A Meta-Analysis.

BACKGROUND: Vascular dementia (VaD) is the most common type of dementia secondary to Alzheimer's disease. The pathologic mechanism of VaD is complex, and VaD still lacks a more objective diagnosis and evaluation method. Diffusion tensor imaging (DTI) can better detect the organizational structure and functional characteristics compared with any other diagnosis methods. Therefore, DTI has broad application in evaluating the severity and prognosis of VaD. This study aimed to assess the value of DTI in evaluating the cognitive function of patients with VaD. METHODS: Authors searched Pubmed, Embase, and Cochrane Library, using the search terms, such as "diffusion tensor imaging," "DTI," "Vascular Dementia," "Arteriosclerotic Dementia," "Cognition," and "Cognitive." A voxel-based meta-analysis combined with quality statistics was performed, using the anisotropic effect-size version of the signed differential mapping method. RESULTS: A total of 8 case-control studies were included in this meta-analysis. The sample size of patients ranged from 35 to 60, including 166 patients in the VaD group and 177 healthy individuals. The DTI imaging of the brain tissue of VaD patients was significantly different from that of healthy individuals. CONCLUSIONS: DTI imaging of the brain tissue of VaD patients was clearly different from that of healthy controls. Therefore it may be feasible to use DTI imaging as a diagnostic method for VaD.

Metabolic adaptation of the clam Ruditapes philippinarum during air exposure and the positive effects of sodium nitroprusside pretreatment.

The Manila clam (Ruditapes philippinarum), as one of the shellfish living in the intertidal zone, is known for its strong ability to withstand air exposure. Sodium nitroprusside (SNP), a donor of nitric oxide (NO), has been shown to be useful for antioxidant and immune regulation in aquatic animals. In this study, an untargeted metabolomics (LC-MS/MS) technique was employed for the first time in Manila clam to analyze the metabolic and histological impacts after air exposure and the positive effects of SNP pretreatment. During air exposure, a significant increase in taurine, L-glutamate, and several polyunsaturated fatty acids in clams was detected, which indicates that clams may experience inflammatory reactions, oxidative stress, and an increase in blood ammonia content. When clams were exposed to SNP for 6 h, arginine, spermine, L-glutamic acid, and glutathione content were all upregulated, indicating that the SNP exposure induced NO production and improved antioxidant capacity in clams. When the clams were exposed to air after SNP pretreatment, there were no significant differences in the levels of taurine, L-glutamate, or aliphatic acids between the experimental and control groups. Gill tissue was more severely damaged in clams directly exposed to air than in those that experienced air exposure after SNP pretreatment, especially in clams exposed to air for a long time (72 h). Both metabolomics and tissue section structure indicated that SNP pretreatment decreased the stress responses caused by air exposure in R. philippinarum. These findings provided fresh insights and a theoretical foundation for understanding the tolerance to air exposure and physiological functions of SNP (or NO) in R. philippinarum.

Genome-wide investigation and expression analysis of TLR gene family reveals its immune role in Vibrio tolerance challenge of Manila clam.

Toll-like receptors (TLRs) play key roles in activating immune responses during infection. In this study, we identified TLR genes in Manila clam at the genome-wide level and characterized it into 9 types according to the Ruditapes philippinarum genome annotation, including TLR1 (1-10), TLR2 (1-10), TLR2-2 (1-5), TLR3 (1-3), TLR4 (1-9), TLR5, TLR6 (1-5), TLR7 (1-2), and TLR13 (1-4). The length of TLR proteins varied from 128 to 1257 amino acids. The molecular weights and theoretical isoelectric point (pI) values ranged from 14.63 to 143.32 kDa and 4.47 to 9.45, respectively. TLR genes showed universal expression levels in adductor muscle (AM), mantle (M), foot (F), gill (GI), pipe (P), digestive gland (DG), gonad (GO) and labial palp (L). Transcriptome analysis revealed that the expression level of TLR4, TLR5, TLR7 and TLR13 genes are significantly highly expressed in resistant individuals of Manila clam under Vibrio anguillarum challenge, indicating these TLR genes may play significant roles in response to invading pathogens. The results obtained in this work will provide valuable insights into the immune function of TLR gene in R. philippinarum.

Full Mitochondrial Genomes Reveal Species Differences between the Venerid Clams Ruditapes philippinarum and R. variegatus.

In natural sea areas along the coast of China, venerid clams Ruditapes philippinarum and R. variegatus exhibit similar adult shell forms and are especially difficult to distinguish as spat and juveniles. This study used comparative mitochondrial genome analysis to reveal differences between these species. The results showed that: (1) the mitochondrial genomes of R. philippinarum and R. variegatus share a large number of similar gene clusters arranged in consistent order, yet they also display noncommon genes, with both gene rearrangements and random losses found; (2) the 13 protein-coding genes in R. philippinarum as well as two-fold and four-fold degenerate sites in R. variegatus have an evident AT bias; (3) the Ka/Ks ratio of the mitochondrial ATP8 gene was significantly higher in R. philippinarum than in R. variegatus, and an analysis of selection pressure revealed that the mitochondrial NADH dehydrogenase subunit 2 gene and NADH dehydrogenase subunit 6 gene of R. variegatus were under great selective pressure during its evolution; and finally, (4) the two species clustered into one branch on a phylogenetic tree, further affirming their phylogenetic closeness. Based on these results, we speculate that the species differences between R. variegatus and R. philippinarum are largely attributable to adaptive evolution to the environment. The present findings provide a reference for the development of germplasm identification.

Comparative transcriptomic analysis revealed dynamic changes of distinct classes of genes during development of the Manila clam (Ruditapes philippinarum).

BACKGROUND: The Manila clam Ruditapesphilippinarum is one of the most economically important marine shellfish. However, the molecular mechanisms of early development in Manila clams are largely unknown. In this study, we collected samples from 13 stages of early development in Manila clam and compared the mRNA expression pattern between samples by RNA-seq techniques. RESULTS: We applied RNA-seq technology to 13 embryonic and larval stages of the Manila clam to identify critical genes and pathways involved in their development and biological characteristics. Important genes associated with different morphologies during the early fertilized egg, cell division, cell differentiation, hatching, and metamorphosis stages were identified. We detected the highest number of differentially expressed genes in the comparison of the pediveliger and single pipe juvenile stages, which is a time when biological characteristics greatly change during metamorphosis. Gene Ontology (GO) enrichment analysis showed that expression levels of microtubule protein-related molecules and Rho genes were upregulated and that GO terms such as ribosome, translation, and organelle were enriched in the early development stages of the Manila clam. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the foxo, wnt, and transforming growth factor-beta pathways were significantly enriched during early development. These results provide insights into the molecular mechanisms at work during different periods of early development of Manila clams. CONCLUSION: These transcriptomic data provide clues to the molecular mechanisms underlying the development of Manila clam larvae. These results will help to improve Manila clam reproduction and development.

Effects of high water temperature on physiology, survival, and resistance to high temperature air-exposure in the Manila clam Ruditapes philippinarum.

Ruditapes philippinarum is a typical burrowing shellfish, living in the intertidal zone. In natural conditions, the mortality of R. philippinarum is most affected by high water temperatures, high temperature air-exposure, and other environmental stresses. In this study, the mortality rates of the two populations of R. philippinarum under high water temperature stress were recorded, and catalase (CAT), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) antioxidant enzyme activities in the hepatopancreas were analyzed. The results showed that the survival times of cultured clams were longer than those of wild clams after acute high temperature stress. CAT, SOD, and T-AOC activities increased after acute high water temperature and high temperature air-exposure stress. These antioxidant enzyme activities gradually decreased to their initial levels after 2 days of recovery from these high temperature stresses. Based on these experimental results, we found that the cultured clam population had better heat and high temperature air-exposure resistances than the wild clams. CAT, SOD, and T-AOC enzymes play an important role in the antioxidant processes of R. philippinarum in response to high water temperature and high temperature air-exposure. This study provided a theoretical basis for the development of healthy aquacultural practices for these shellfish.

Examination of the potential roles of insulin-like peptide receptor in regulating the growth of Manila clam Ruditapes philippinarum.

The role of insulin/insulin-like growth factor (IGF) signaling pathway in the growth regulation of marine invertebrates has not been fully studied. In this study, the economically important species Ruditapes philippinarum was sacrificed to clarify the role of IGF system in the growth regulation of R. philippinarum by real-time quantitative PCR. Systematic bioinformatics analysis can identify the major genes of IGF signaling pathway and insulin-like peptide receptor (ILPR) - mediated signaling pathway in R. philippinarum. The expression levels of IGF and its downstream signaling pathway genes in larger clams were significantly higher than those in small clams, indicating that they were involved in the growth regulation of R. philippinarum. These results suggest that IGF signaling pathway and ILPR mediated signaling pathway to regulate the growth of R. philippinarum.

Transcriptome sequencing reveals improved ammonia nitrogen tolerance in Zebra II strain of the Manila clam Ruditapes philippinarum.

In this research, we identified genes associated with ammonia nitrogen (TAN) stress response and resistance in juveniles of the Zebra II strain and a wild population of the Manila clam Ruditapes philippinarum. Both groups were subjected to a 96 h acute toxicity test using TAN concentrations of 17.617 ± 0.634 and 16.670 ± 0.7 mg/l, respectively. We then collected samples, conducted transcriptome sequencing and screened the sequences for differentially expressed genes (DEGs) related to TAN stress response. We identified 2908 and 2861 DEGs in the Zebra II and wild clam groups, respectively, and the two groups had 626 DEGs in common. The verified DEGs had less of a detoxification effect in the wild population than that in the Zebra II group. Gene Ontology database analysis showed that Zebra II juveniles were mainly enriched in protein phosphorylation, purine nucleoside binding, and kinase activity, whereas the wild population juveniles were primarily enriched in oxidases activity, organic acid metabolic processes, and extracellular regions. Kyoto Encyclopedia of Genes and Genomes pathway analysis mainly highlighted aminoacyl tRNA biosynthesis in Zebra II juveniles and sphingolipid metabolism, FOXO signaling, biosynthesis of aminoacyl tRNA, and other pathways in the wild population. These results show that the toxic effect of TAN on the Manila clam is related to a variety of pathways, which are mainly related to immune response, inflammatory response, metabolic pathways, and nerve conduction. This study provides basic data and theoretical reference for revealing the molecular regulation mechanism of the improved TAN tolerance of Zebra II strain as compared with the wild population of Ruditapes philippinarum.

Genome-wide identification and analysis of HECT E3 ubiquitin ligase gene family in Ruditapes philippinarum and their involvement in the response to heat stress and Vibrio anguillarum infection.

E3 ubiquitin ligase (E3s) plays an important role in ubiquitin proteasome pathway, proteins containing homologous E6-AP carboxyl terminus (HECT) domains. However, the role of HECT E3 ubiquitin ligase in mollusk was rarely explored. In this study, we performed a genome-wide analysis of the HECT domain-containing gene in Ruditapes philippinarum to identify and predict the structural and functional characterization of HECT genes in response to abiotic and biotic stress. A total of sixteen members of HECT gene family were identified and analyzed for the gene structure, phylogenetic relation, three-dimensional structure, protein interaction network, and expression patterns. Experimental results demonstrated that Rph.HUWE1, Rph.HECTD1, Rph.Ubr5 were significantly up-regulated in response to heat stress and bacterial challenge. Taken together, our data provide insights into the potential function of HECT E3 ligase in heat stress and Vibrio anguillarum infection.