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.

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.

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.

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.

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.

Genetic diversity and differentiation of nine populations of the hard clam (Meretrix petechialis) assessed by EST-derived microsatellites

BACKGROUND: Meretrix petechialis is one of the commercially important marine bivalves. In this study, we selected six highly polymorphic EST-derived microsatellite markers to assess the genetic diversity and population differentiation on nine wild populations of Meretrix petechialis. RESULTS: The number of alleles detected per-locus ranged from 4 to 30 (mean NA = 27.5) with a total of 165 alleles. The mean value of observed and expected heterozygosities varied from 0.717 to 0.861 and from 0.797 to 0.856, respectively. Meanwhile, the result of Neighbor-joining and overall FST = 0.214 (P < 0.01) reveled that M. petechialis populations from GX are the farthest populations, a certain degree of genetic variation among individuals in each population and the genetic differentiation is significant. CONCLUSIONS: GX population has high genetic diversity among individual, and there are certain differences in genetic characteristics among different populations. This study will provide a basis for the domestication and cultivation of genetic diversity of M. petechialis population and the protection of clam germplasm resources.

Genetic diversity and population structure of Meretrix petechialis in China revealed by sequence-related amplified polymorphism markers.

Genetic variation in nine stocks of Meretrix petechialis collected from China was analyzed using sequence-related amplified polymorphism (SRAP) markers. Eight primer pairs produced 132 polymorphic loci with an average of 16.5 loci per primer pair. A population from Jiangsu had the highest percentage of polymorphic loci at 27.27%, suggesting that these resources had a rich genetic diversity. The Nei's gene diversity of the nine populations ranged from 0.0647 to 0.0793; a population from Shandong was the lowest and a population from North Korea the highest. The Shannon's information index was between 0.1023 and 0.1202, with the lowest in the Shandong population and the highest in the Jiangsu population. The Nei's unbiased genetic distance between the nine populations was 0.0243-0.0570 and the genetic similarity was 0.9446-0.9760; the genetic distance between Guangxi and Shandong populations was the furthest (0.0570) and the genetic distance between Shandong and Jiangsu populations was the closest (0.0243). Nei's gene diversity analysis indicated that the genetic variance was mainly found within individual geographical populations, and the analysis of molecular variance revealed low but significant genetic differentiation among local and regional populations. The limited gene flow (Nm = 0.555) was inferred as a major reason for the extent of genetic differentiation in M. petechialis. The results obtained here indicated that M. petechialis have high degree of genetic diversity and the potential of further breeding with excellent germplasm resources. This study provides a scientific basis for the protection of germplasm resources and the breeding of M. petechialis.