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.

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.

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.

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.

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.

lncRNA XIST induces Aß accumulation and neuroinflammation by the epigenetic repression of NEP in Alzheimer's disease.

Alzheimer's disease (AD) is the leading cause of dementia globally, but effective treatment is lacking. We aimed to explore lncRNA XIST role in AD and the mechanisms involved in the effect of changes in lncRNA XIST on the expression of Aß-degrading enzymes. The mouse model of AD and the cell model induced by Aß were established. LncRNA XIST, IDE, NEP, Plasmin, ACE, EZH2 expressions and distribution of XIST in the nucleus and cytoplasm were detected by qRT-PCR. Inflammatory cytokines IL-6, IL-1ß, TNFα, IL-8, and Aß42 levels were detected by ELISA. TUNEL was used to measure brain tissue damage. Cell proliferation was detected by CCK-8 assay. Flow cytometry detected cell apoptosis. RIP validated the combination of XIST and EZH2. ChIP verified that XIST recruits EZH2 to mediate enrichment of HEK27me3 in the NEP promoter region. The protein expression in brain tissues and cells was detected by Western blot. The expression of lncRNA XIST was increased in AD mice and cell models. Inflammation and injury of nerve cells occurred in AD mice and cell models. The knockdown of lncRNA XIST alleviated Aß-induced neuronal inflammation and damage. LncRNA XIST affected the expression of Aß-degrading enzyme NEP, and lncRNA XIST was negatively correlated with NEP expression in AD mice. LncRNA XIST regulated NEP expression partly through epigenetic regulation by binding with EZH2. LncRNA XIST mediated neuronal inflammation and injury through epigenetic regulation of NEP. Overall, our study found that lncRNA XIST induced Aß accumulation and neuroinflammation by the epigenetic repression of NEP in AD.

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.

Genome-wide investigation and expression analysis of MACPF gene family reveals its immune role in response to bacterial challenge of Manila clam.

In this study, 18 MACPF genes (RpMACPF) were identified and classed into three types (Macrophage-expressed gene 1, Apextrin, and MACPF domain contain protein) based on gene structure and phylogenetic relationship in R. philippinarum. The length of RpMACPF proteins varied from 287 to 785 amino acids. The molecular weights and Theoretical PI values ranged from 3.2 kDa to 8.7 kDa and 4.7 to 8.6, respectively. RNA-seq data analysis revealed that 14 of 18 RpMACPF genes were highly expressed at the pediveliger larvae stage indicate RpMACPF might contribute to the early development and metamorphosis processes of the R. philippinarum. Besides, we found RpMACPF genes were significantly regulated by pathogen-associated molecular patterns (PAMPs) and Vibrio parahemolyticus, which indicates RpMACPF genes may play significant roles in response to invading pathogens. The results obtained in this work will provide valuable insight into the immune function of MACPF gene in R. philippinarum.

Molecular mechanisms of wound healing and regeneration of siphon in the Manila clam Ruditapes philippinarum revealed by transcriptomic analysis.

Ruditapes philippinarum is an economically important marine shellfish aquaculture species, and it has the ability to regenerate its siphons. To gain a greater understanding of the molecular mechanisms at work during siphon regeneration and to provide evidence for morphological regeneration, we examined transcriptome responses of siphon tissue of R. philippinarum during regeneration and observed regenerative siphons under the stereomicroscope. The overall process of siphon regeneration was dissected based on the morphological changes of siphon and the identification of up-regulated key differentially expressed genes (DEGs). The protein biosynthesis and metabolism played important roles in wound healing and siphon regeneration of R. philippinarum. Transcriptomic analysis identified the Wnt and TGF-ß signaling pathways by focusing on the function and expression pattern of genes in these pathways during siphon regeneration. In addition, we carried out a genome-wide identification and phylogenetic analysis of TGF-ß superfamily in R. philippinarum. The expression profiles of the TGF-ß superfamily genes were analyzed in eight adult tissues (adductor muscle, mantle, foot, gill, siphon, digestive gland, gonad, and labial palp) and regenerative siphon. This study shed new light on the process of morphological regeneration and regenerative mechanism of siphon of R. philippinarum.

Transcriptomic analysis provides insights into candidate genes and molecular pathways involved in growth of Manila clam Ruditapes philippinarum.

Growth is one of the most important traits of aquaculture breeding programs. Understanding the mechanisms underlying growth differences between individuals can contribute to improving growth rates through more efficient breeding schemes. Ruditapes philippinarum is an economically important marine bivalve. In order to gain insights into the molecular mechanisms to growth variability in marine shellfish, we conducted the transcriptome sequencing and examined the expression differences in growth-related gene and molecular pathways involved in growth trait of R. philippinarum. In this study, we investigated the molecular and gene expression differences in fast-growing and slow-growing Manila clam and focused on the analysis of the differential expression patterns of specific genes associated with growth by RNA-seq and qPCR analysis. A total of 61 differentially expressed genes (DEGs) were captured significantly differentially expressed, and were categorized into Ras signaling pathway, hedgehog signaling pathway, AMPK signaling pathway, p53 signaling pathway, regulation of actin cytoskeleton, focal adhesion, mTOR signaling pathway, VEGF signaling pathway, and TGF-beta signaling pathway. A total of 34 growth-related genes were validated significantly and up/downregulated at fast growing and slow growing of R. philippinarum. Functional enrichment analysis revealed the insulin signaling pathway, PI3K-Akt signaling pathway, and mTOR signaling pathway play pivotal roles in molecular function and regulation of growth trait in R. philippinarum. The growth-related genes and pathways obtained here provide important insights into the molecular basis of physiological acclimation, metabolic activity, and growth variability in marine bivalves.

Analysis of differential gene expression by SRAP-cDNA in mantle tissue of Meretrix petechialis with different external shell color.

Gene expression of two shell colors in Meretrix petechialis were analyzed by sequence related amplified polymorphism-cDNA to screen the associated molecular markers. The two shell color genomes of M. petechialis were amplified using combinations of 30 primers; 11 pairs of primers showed differential fragments, and by recovery, cloning and sequencing, 18 different differential sequences were obtained. The sequencing results were analyzed by BlastX. Only one fragment shared high homology with memory-related protein-2 and TonB-dependent receptor was found that related to shell color. Sequence characterized amplified region primers were designed according to the difference sequences, and PCR amplification was performed in both 'yellow' and 'red' M. petechialis. Four pairs of differential primers were obtained. Using the population to verify the four markers (Me1-Em2, Me2-Em3, Me4-The Em11 and Me4-Em12), it was found that Me1-Em2 and Me2-Em3 were positive in the 'yellow' and Me4-The Em11 and Me4-Em12 were positive in the 'red' M. petechialis populations. All four markers can, therefore, be used as M. petechialis shell color related markers. This provides a theoretical basis for studying shell color regulation in M. petechialis, which may help to reveal the underlying molecular mechanisms more comprehensively.

Transcriptome analysis reveals the pigmentation-related genes in two shell color strains of the Manila clam Ruditapes philippinarum.

The Manila clam, Ruditapes philippinarum, is an ecologically and economically important marine bivalve species. In this study, we conducted transcriptomic sequencing of two different shell color strains (O and Z) before color appearance (uncolored juvenile clam) and pigmented shell color (colored juvenile clam) and investigated the analysis of the differential expression patterns of specific genes associated with pigmentation by RNA-seq and time course qPCR analysis. The transcription level of 16 differentially expressed genes (DEGs) related with shell color was analyzed by qRT-PCR to validate the performance of RNA-seq from Illumina sequence data where most of them were up-regulated. Two genes were down-regulated after the occurrence of zebra clam stripes compared with uncolored zebra clam. The trend of gene expression obtained by qPCR was basically consistent with that of RNA-seq. The synthesis of melanin in bivalves plays potential roles in the pigmentation of the shell and is closely related to the formation of the surface pattern. The porphyrin metabolism combined with tyrosinase and melanogenesis signaling pathway is a novel finding in shell color determination of R. philippinarum. This study sheds light on the pigmentation and coloration mechanism of the Manila clam.

Eutrophication likely prompts metal bioaccumulation in edible clams.

Coastal eutrophication is an indisputable reality and becoming a worldwide concern. However, whether and how eutrophication affects metal bioaccumulation in marine bivalves have not yet been elucidated. Here, we present the potential influence of coastal eutrophication on metal bioaccumulation in the Manila clam Ruditapes philippinarum. The degree of coastal eutrophication was examined monthly over a 1-year period at three sampling sites. The bioconcentration factor (BCF), biosediment accumulation factor (BSAF) and metal pollution index (MPI) were applied to evaluate the efficiency of metal bioaccumulation in R. philippinarum. BCF and BSAF indicated that eutrophication did not significantly affect the bioaccumulation of Cr, Cu, Zn, Cd, Pb, Hg, and As in R. philippinarum. However, up to 56% of MPI variation can be related to the level of eutrophication. Therefore, further research should address the synergistic effects of eutrophication and metal pollution on coastal ecosystems.

Transcriptome analysis reveals the pigmentation related genes in four different shell color strains of the Manila clam Ruditapes philippinarum.

Ruditapes philippinarum is an important marine bivalve species. In this study, we conducted the RNA-seq of four different shell color strains of the R. philippinarum and investigated the analysis of the differential expression patterns of specific genes associated with pigmentation. The maximum different genes was 13 between WZ vs O, WZ vs W and WZ vs O have same numbers of different genes, was 5, Z vs W has 4 genes of 18 DEGs, W vs O just have two DEGs, while there is no DEGs between WZ vs Z. The synthesis of melanin plays important roles in the pigmentation of the shell and is closely related to the formation of the surface pattern. We speculate the possible involvement of porphyrin and chlorophyll metabolism combined with calcium signaling pathway in shell color determination. This study sheds light on the pigmentation and coloration mechanism of the Manila clam.

New insights into the Manila clam and PAMPs interaction based on RNA-seq analysis of clam through in vitro challenges with LPS, PGN, and poly(I:C).

BACKGROUND: Manila clam (Ruditapes philippinarum) is a worldwide commercially important marine bivalve species. In recent years, however, microbial diseases caused high economic losses and have received increasing attention. To understand the molecular basis of the immune response to pathogen-associated molecular patterns (PAMPs) in R. philippinarum, transcriptome libraries of clam hepatopancreas were constructed at 24 h post-injection with Lipopolysaccharide (LPS), peptidoglycan (PGN), and polyinosinic-polycytidylic acid (poly(I:C)) and phosphate-buffered saline (PBS) control by using RNA sequencing technology (RNA-seq). RESULTS: A total of 832, 839, and 188 differentially expressed genes (DEGs) were found in LPS, PGN, and poly(I:C) challenge group compared with PBS control, respectively. Several immune-related genes and pathways were activated in response to the different PAMPs, suggesting these genes and pathways might specifically participate in the immune response to pathogens. Besides, the analyses provided useful complementary data to compare different PAMPs challenges in vivo. Functional enrichment analysis of DEGs demonstrated that PAMPs responsive signal pathways were related to apoptosis, signal transduction, immune system, and signaling molecules and interaction. Several shared or specific DEGs response to different PAMPs were revealed in R. philippinarum, including pattern recognition receptors (PRRs), antimicrobial peptides (AMPs), interferon-induced proteins (IFI), and some other immune-related genes were found in the present work. CONCLUSIONS: This is the first study employing high throughput transcriptomic sequencing to provide valuable genomic resources and investigate Manila clam response to different PAMPs through in vivo challenges with LPS, PGN, and poly(I:C). The results obtained here provide new insights to understanding the immune characteristics of R. philippinarum response to different PAMPs. This information is critical to elucidate the molecular basis of R. philippinarum response to different pathogens invasion, which potentially can be used to develop effective control strategies for different pathogens.

Transcriptome analysis reveals differential immune related genes expression in Ruditapes philippinarum under hypoxia stress: potential HIF and NF-κB crosstalk in immune responses in clam.

BACKGROUND: Hypoxia is an important environmental stressor in aquatic ecosystems, with increasingly impacts on global biodiversity. Benthic communities are the most sensitive parts of the coastal ecosystem to eutrophication and resulting hypoxia. As a filter-feeding organism living in the seafloor sediment, Ruditapes philippinarum represents an excellent "sentinel" species to assess the quality of marine environment. In order to gain insight into the molecular response and acclimatization mechanisms to hypoxia stress in marine invertebrates, we examined hypoxia-induced changes in immune-related gene expression and gene pathways involved in hypoxia regulation of R. philippinarum. RESULTS: We investigated the response of the Manila clam R. philippinarum to hypoxia under experimental conditions and focused on the analysis of the differential expression patterns of specific genes associated with hypoxia response by RNA-seq and time course qPCR analysis. A total of 75 genes were captured significantly differentially expressed, and were categorized into antioxidant/oxidative stress response, chaperones/heat shock proteins, immune alteration, and cell proliferation/apoptosis. Fourteen hypoxia responsive genes were validated significantly up/down regulated at different time 0, 2, 5, and 8 d in gills of R. philippinarum in hypoxia challenged group. Functional enrichment analysis revealed the HIF signaling pathway and NF-κB signaling pathway play pivotal roles in hypoxia tolerance and resistance in R. philippinarum. CONCLUSION: The HIF signaling pathway and NF-κB signaling pathway play a critical role in hypoxia tolerance and resistance in Manila clam. The immune and defense related genes and pathways obtained here gain a fundamental understanding of the hypoxia stress in marine bivalves and provide important insights into the physiological acclimation, immune response and defense activity under hypoxia challenge. The reduced metabolism is a consequence of counterbalancing investments in immune defense against other physiological processes.

Transcriptomic analysis of Manila clam Ruditapes philippinarum under lipopolysaccharide challenge provides molecular insights into immune response.

The Manila clam, Ruditapes philippinarum, is an economically important shellfish in marine aquaculture. A better understanding of the immune system in R. philippinarum will provide the basis for the development of strategies to mitigate the impact of infectious diseases affecting this species but can also be of relevance for other bivalves of commercial interest. In this study, the transcriptional response of the Manila clam under lipopolysaccharide (LPS) challenge was characterized using RNA sequencing. The transcriptomes of LPS challenged group of clams (LH1, LH2 and LH3), and the PBS control group (CH1, CH2 and CH3), were sequenced with the Illumina HiSeq platform. Compared with the unigene expression profile of the control group, 223 unigenes were up-regulated and 389 unigenes were down-regulated in the LPS challenged group. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that signal transduction, defense response, and immune-related pathways such as Chemokine signaling pathway, Complement and coagulation cascades, NOD-like receptor signaling pathway, and Inflammatory mediator regulation of TRP channels in sensory system were the most highly enriched pathways among the genes that were differentially expressed under LPS challenge. This study present understanding of the molecular basis underpinning response to LPS challenge and provides useful information for future work on the molecular mechanism of pathogen resistance and immunity in Manila clam.

Molecular cloning and expression analysis of mc5r like genes (mc5rl) in Ruditapes philippinarum (Manila clam) after aerial exposure and low-temperature stress.

The melanocortin-5 receptor (mc5r) plays an important role in exocrine function, lipid metabolism, obesity, and stress response in the vertebrate. However, the functions of the mc5r in mollusks have been rarely investigated. We cloned the full length of Ruditapes philippinarum mc5r like gene (mc5rl) and the sequence structure and phylogenetic relationship of mc5rl were analyzed. Besides, we detected the tissue distribution and the expression pattern of R. philippinarum mc5r like (mc5rl) genes after aerial exposure and low-temperature stress. The full-length cDNA of the mc5rl-1 was 2143 bp, consisting of a 1224 bp open reading frame encoding (ORF) 408 amino acids. Sequence and phylogenetic analyses revealed that the nucleotide and amino acid sequences of Manila clam mc5rl were highly homologous with mc5r of Crassostrea virginica, Crassostrea gigas, Mizuhopecten yessoensis, and Pecten maximus (32%-36%) and low homologous with vertebrates. The results of the distribution of mc5rl genes showed that mc5rl genes were dominant in the mantle, gonad, and hepatopancreas in R. philippinarum. The expression of mc5rl genes was significantly increased after aerial exposure and low-temperature stress in R. philippinarum in hepatopancreas. Aerial exposure and low-temperature stress could induce mc5rl expressed. Mc5rl might serve as a sensor and promote stress response in R. philippinarum. The cloning and expression characteristics of mc5rl will facilitate the investigation of its function in stress response and other physiological processes in R. philippinarum.

Molecular cloning and expression analysis of C-type lectin (RpCTL) in Manila clam Ruditapes philippinarum after lipopolysaccharide challenge.

The Manila clam, Ruditapes philippinarum, is one of the most commercially important marine bivalves. C-type lectins (CTLs) are pattern recognition receptors (PRRs) that play important roles in the identification and elimination of pathogens by the innate immune system. In this study, a new CTL (RpCTL) was identified in the Manila clam, R. philippinarum. The full-length RpCTL cDNA is 802 bp, with an open reading frame of 591 bp, encoding 196 amino acids, including an N-terminal signal peptide and a carbohydrate recognition domain (CRD). RpCTL contains conserved CRD disulfide bonds involving four cysteine residues (Cys30-Cys104, Cys124, and Cys132), and the EPN (Glu94-Pro95-Asn96) and WND (Trp119-Asn120-Asp121) motifs. Quantitative reverse transcription (RT)-PCR detected RpCTL transcripts mainly in the gill, siphon, and hepatopancreas in three shell-color strains (zebra, white, and white-zebra strains) and two unselected populations of R. philippinarum, and the gene was highly expressed in the hepatopancreas after lipopolysaccharide treatment. Antimicrobial activity assays of recombinant RpCTL against both Gram-positive and Gram-negative bacteria showed that RpCTL inhibits microorganismal growth. In a survival test, RpCTL inhibited and killed Vibrio anguillarum in R. philippinarum. These results suggest that RpCTL participates in the pathogen identification process of R. philippinarum as a PRR and in its immune defense system.