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).

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.

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.

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.

MiRNA-mRNA Integration Analysis Reveals the Regulatory Roles of MiRNAs in Shell Pigmentation of the Manila clam (Ruditapes philippinarum).

The shell color of the Manila clam (Ruditapes philippinarum) is an economically important trait. We used high-throughput sequencing and transcriptome analysis to study the molecular mechanisms that underlie shell color formation and regulation in this species. We constructed small RNA libraries from mantle tissues from four shell color strains of Manila clam, subjected them to high-throughput sequencing. Notably, the results suggested that a number of pigment-associated genes including Mitf, HERC2, were negatively regulated by nvi-miR-2a, tgu-miR-133-3p, respectively. They might be involved in melanin formation via the activation of the melanogenesis pathway. And aae-miR-71-5p and dme-miR-7-5p linked to shell formation-related genes such as Calmodulin and IMSP3 were considered to participate in the calcium signaling pathway. We then used quantitative PCR to verify the candidate miRNAs and target genes in different shell color groups. Our results indicated that miR-7, miR-71, and miR-133 may regulate target mRNAs to participate in shell color pigmentation. These results provide the foundation to further characterize miRNA effects on the regulation of shell color and have significant implications for the breeding of new varieties of clams.

Identification of shell-color-related microRNAs in the Manila clam Ruditapes philippinarum using high-throughput sequencing of small RNA transcriptomes.

Shell-color polymorphism is a common phenomenon in several mollusk species and has been associated with thermal capacity, developmental stability, shell strength, and immunity. Shell-color polymorphism has been related to the differential expression of genes in several signal transduction pathways; however, the functions of micro-RNAs (miRNAs) in shell-color formation remain unclear. In the present study, we compared high-quality, small-RNA transcriptomes in three strains of the Manila clam Ruditapes philippinarum with specific shell-color patterns, artificially selected for six generations. Totals of 114 known and 208 novel miRNAs were identified by high-throughput sequencing, of which nine known and one novel miRNA were verified by stem-loop quantitative real time-polymerase chain reaction. Predicted miRNA targets were subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. miR-137 and miR-216b and the Hedgehog signaling pathway and Wnt signaling pathway were identified as being potentially involved in pigment formation and regulation in R. philippinarum. These results may help to clarify the role of miRNAs in shell coloration and shed light on the mechanisms regulating color formation in bivalve shells.

Genome-wide identification and expression profiling of TYR gene family in Ruditapes philippinarum under the challenge of Vibrio anguillarum.

Tyrosinase (EC1.14.18.1, TYR) is also called phenol oxidase, is not only involved in pigmentation but also plays an important role in modulating innate immunity in invertebrates. Tyrosinase is a copper containing metalloenzyme. The tyrosinase protein has two copper binding sites and three conserved histidines. In this study, 21 tyrosinase genes (RpTYR) were obtained from the whole genome of Ruditapes philippinarum. Their open reading frames were from 951 to 5424 aa, the range of predicted relative molecular weight from 36.72 to 203.81 kDa, and the range of isoelectric point from 4.72 to 9.88. Transcriptome analysis showed that RpTYR gene was expressed specifically in different developmental stages, adult tissues, four strains and two groups with different shell colors. Besides, the expression profiles of 21 RpTYRs were investigated against the immune response of R. philippinarum to a Vibrio challenge. The qPCR results showed that RpTYRs were involved in the immune response of R. philippinarum after Vibrio anguillarum challenge. This study provides preliminary evidence that the tyrosinases genes are involved in the immune defense and the potential immune function of R. philippinarum. Overall, these findings suggested that the expansion of TYR genes may play vital roles in larval development, the formation of shell color pattern, and immune response in R. philippinarum.

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.

Population genetics of the Manila clam (Ruditapes philippinarum) in East Asia.

The Manila clam Ruditapes philippinarum is the world's second most important bivalve mollusk commercially farmed, whose indigenous populations are mainly distributed in the coastal areas of East Asia. However, with the development of commercialization, mixture of populations and loss of local germplasm have become prominent problems. Here, genetic differentiation of seven Manila clam populations from East Asia was investigated through analyzing the polymorphism of the mitochondrial cytochrome C oxidase subunit I (COI) gene as well as 20 simple sequence repeat (SSR) molecular loci. In total, 40 haplotypes were identified, among which 31 were unique. Moreover, two main haplotypes were detected with several radiating derived haplotypes. Populations in Japan-North Korea shared haplotype Hap_31, and populations in China shared haplotype Hap_7, suggesting that the natural geographical isolation of the Yangtze River and the Yalu River might have divided the East Asian indigenous populations into three groups, which were located in South China, North China, and Japan-North Korea, respectively. The Aquaculture breeding activities from South to North in China might have promoted gene exchange among Manila clam populations. Population in Laizhou had the highest genetic diversity and therefore could be an excellent germplasm source.

Physiological and gene expression analysis of the Manila clam Ruditapes philippinarum in response to cold acclimation.

Overwinter mortality of the Manila clam (Ruditapes philippinarum) is a major impediment to the aquaculture industry in China. Cold tolerance ability has a tremendous impact on the survivability of R. philippinarum during the overwintering season. In this study, we evaluated the effects of acute and chronic cold stress on the expression of Cold Shock Domain-containing E1 (CSDE1) and Antifreeze protein type II (AFPII) genes and the activities of lysozyme (LZM), catalase (CAT), and superoxide dismutase (SOD) in three cultivated strains (zebra, white, and white zebra) and two wild populations (northern and southern) of R. philippinarum. Under acute and chronic cold stress, the expression levels of CSDE1 and AFPII mRNA in the gills and hepatopancreas were significantly increased in all populations, but the increase varied among different strains and populations. Under acute cold stress, SOD activity significantly decreased in the two wild populations and the white zebra strain. LZM activity significantly decreased but CAT activity significantly increased in selected strains and populations after acute low temperature stress (P < 0.05). Under chronic cold stress, SOD activity significantly increased in the northern population and white zebra strain, while CAT activity significantly increased in the southern population and the white and zebra strains. These results provide useful information about the Manila clam response to cold stress that may be applied to improve the low temperature resistance of Manila clams in aquaculture environments.

Molecular cloning and expression analysis of tyrosinases (tyr) in four shell-color strains of Manila clam Ruditapes philippinarum.

The Manila clam (Ruditapes philippinarum) is an economically important molluscan bivalve with variation in pigmentation frequently observed in the shell. In nature, tyrosinase is widely distributed in invertebrates and vertebrates, and plays a crucial role in a variety of physiological activities. In this study, a tyrosinase gene (tyr 9) was cloned and the expression level of tyr genes (tyr 6, tyr 9, tyr 10, and tyr 11) were investigated in different shell colors. Quantitative real-time PCR showed that tyr genes were significantly expressed in the mantle, a shell formation and pigmentation-related tissue. Moreover, the expression pattern of the tyr genes in the mantle of different shell-color strains was different, suggesting that tyrosinases might be involved in different shell-color formation. In addition, the expression profile of tyr 6, tyr 9, tyr 10, and tyr 11 genes were detected at different early developmental stages and the expression level varied with embryonic and larval growth. RNA interference (RNAi) results showed that the expression level of tyr 9 in the RNAi group was significantly down-regulated compared to control and negative control groups, indicating that Rptyr 9 might participate in shell-color formation. Our results indicated that tyr genes were likely to play vital roles in the formation of shell and shell-color in R. philippinarum.

Genome-wide identification and characterization of the AMPK genes and their distinct expression patterns in response to air exposure in the Manila clam (Ruditapes philippinarum).

INTRODUCTION: AMP-activated protein kinases (AMPK) are heterotrimeric complexes. The main upstream phosphorylase has AMP-dependent LKB1 and Ca2+-dependent CaMKK beta. AMPK also includes an auto-inhibitory domain and a region associated with beta and gamma subunits, which regulate a variety of cellular activities and energy metabolism. The increase in the ratio of AMP/ATP can stimulate the activation of AMPK. Once AMPK is activated, pathways to ATP consumption (e.g., fat, cholesterol, and protein synthesis) will be shut down. The pathway to ATP generation (e.g., oxidation of fat and glycolysis pathway) will be activated. AMPK genes have not been systematically characterized in marine invertebrates. METHODS: In this study, we identified and characterized three AMPK genes, AMPK-α, AMPK-ß, and AMPK-γ, in the Manila clam (Ruditapes philippinarum). To gain insight into the role of AMPK genes during clam energy metabolism, quantitative real-time PCR was used to investigate the expression profiles in the different stages of clam development, in healthy adult tissues, and after air exposure at two different temperatures. RESULTS: Phylogenetic and protein structural analyses were conducted to determine the identity and evolutionary relationships of these genes. The structural features of the genes were relatively well-conserved, relative to the AMPK genes of other vertebrates. The expression of genes was significantly induced 3-48 h after air exposure. CONCLUSINON: AMPK-α, AMPK-ß and AMPK-γ are involved in clam energy metabolism. Increased expression levels of AMPK genes in the gill and intestine of Manila clam in response to air exposure implied a strong adaptability to the coastal environment.

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.

Molecular characterization and expression analysis of fibrinogen related protein (FREP) genes of Manila clam (Ruditapes philippinarum) after lipopolysaccharides challenge.

Ruditapes philippinarum has high economic value and is distributed all over the world. Fibrinogen associated protein (FREP) is a type of pattern recognition receptor, participates in the innate immune response to eliminate pathogens after the invasion of pathogenic microorganisms. In this study, three FREP genes (FREP-1, FREP-2, and FREP-3) were identified and characterized from R. philippinarum. The protein sequence of FREPs were highly conserved with those homologous in vertebrates, and FBG domain possessed significantly high structural conservation in polypeptide binding site and Ca2+ binding site. The tissues expression analysis of FREPs in three shell color strains and two population of R. philippinarum were examined, with the highest expression level in gill and hepatopancreas. Besides, FREP genes were demonstrated to be induced by lipopolysaccharides injection, the significantly changes were observed after LPS injected. Our results suggest the involvement of FREPs in response to LPS injection, and it might exert a significant function on the innate immune defense of the Manila clam.

Clam Genome Sequence Clarifies the Molecular Basis of Its Benthic Adaptation and Extraordinary Shell Color Diversity.

Ruditapes philippinarum is an economically important bivalve with remarkable diversity in its shell coloration patterns. In this study, we sequenced the whole genome of the Manila clam and investigated the molecular basis of its adaptation to hypoxia, acidification, and parasite stress with transcriptome sequencing and an RNA sequence analysis of different tissues and developmental stages to clarify these major issues. A number of immune-related gene families are expanded in the R. philippinarum genome, such as TEP, C3, C1qDC, Hsp70, SABL, and lysozyme, which are potentially important for its stress resistance and adaptation to a coastal benthic life. The transcriptome analyses demonstrated the dynamic and orchestrated specific expression of numerous innate immune-related genes in response to experimental challenge with pathogens. These findings suggest that the expansion of immune- and stress-related genes may play vital roles in resistance to adverse environments and has a profound effect on the clam's adaptation to benthic life.

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.

Publisher Correction: Construction of a High-Density Genetic Map and Quantitative Trait Locus Mapping in the Manila clam Ruditapes philippinarum.

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