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

ABSTRACT: 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.

SUBMITTER: Ding J

PROVIDER: S-EPMC8044141 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

Project description: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.

| S-EPMC7032058 | biostudies-literature

Transcriptomics of in vitro immune-stimulated hemocytes from the Manila clam Ruditapes philippinarum using high-throughput sequencing.

Project description:BackgroundThe Manila clam (Ruditapes philippinarum) is a worldwide cultured bivalve species with important commercial value. Diseases affecting this species can result in large economic losses. Because knowledge of the molecular mechanisms of the immune response in bivalves, especially clams, is scarce and fragmentary, we sequenced RNA from immune-stimulated R. philippinarum hemocytes by 454-pyrosequencing to identify genes involved in their immune defense against infectious diseases.Methodology and principal findingsHigh-throughput deep sequencing of R. philippinarum using 454 pyrosequencing technology yielded 974,976 high-quality reads with an average read length of 250 bp. The reads were assembled into 51,265 contigs and the 44.7% of the translated nucleotide sequences into protein were annotated successfully. The 35 most frequently found contigs included a large number of immune-related genes, and a more detailed analysis showed the presence of putative members of several immune pathways and processes like the apoptosis, the toll like signaling pathway and the complement cascade. We have found sequences from molecules never described in bivalves before, especially in the complement pathway where almost all the components are present.ConclusionsThis study represents the first transcriptome analysis using 454-pyrosequencing conducted on R. philippinarum focused on its immune system. Our results will provide a rich source of data to discover and identify new genes, which will serve as a basis for microarray construction and the study of gene expression as well as for the identification of genetic markers. The discovery of new immune sequences was very productive and resulted in a large variety of contigs that may play a role in the defense mechanisms of Ruditapes philippinarum.

| S-EPMC3334963 | biostudies-literature

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

Project description: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.

| S-EPMC7736867 | biostudies-literature

The Whole-Genome and Transcriptome of the Manila Clam (Ruditapes philippinarum).

Project description:The manila clam, Ruditapes philippinarum, is an important bivalve species in worldwide aquaculture including Korea. The aquaculture production of R. philippinarum is under threat from diverse environmental factors including viruses, microorganisms, parasites, and water conditions with subsequently declining production. In spite of its importance as a marine resource, the reference genome of R. philippinarum for comprehensive genetic studies is largely unexplored. Here, we report the de novo whole-genome and transcriptome assembly of R. philippinarum across three different tissues (foot, gill, and adductor muscle), and provide the basic data for advanced studies in selective breeding and disease control in order to obtain successful aquaculture systems. An approximately 2.56 Gb high quality whole-genome was assembled with various library construction methods. A total of 108,034 protein coding gene models were predicted and repetitive elements including simple sequence repeats and noncoding RNAs were identified to further understanding of the genetic background of R. philippinarum for genomics-assisted breeding. Comparative analysis with the bivalve marine invertebrates uncover that the gene family related to complement C1q was enriched. Furthermore, we performed transcriptome analysis with three different tissues in order to support genome annotation and then identified 41,275 transcripts which were annotated. The R. philippinarum genome resource will markedly advance a wide range of potential genetic studies, a reference genome for comparative analysis of bivalve species and unraveling mechanisms of biological processes in molluscs. We believe that the R. philippinarum genome will serve as an initial platform for breeding better-quality clams using a genomic approach.

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Transcriptome sequencing and microarray development for the Manila clam, Ruditapes philippinarum: genomic tools for environmental monitoring.

Project description:BackgroundThe Manila clam, Ruditapes philippinarum, is one of the major aquaculture species in the world and a potential sentinel organism for monitoring the status of marine ecosystems. However, genomic resources for R. philippinarum are still extremely limited. Global analysis of gene expression profiles is increasingly used to evaluate the biological effects of various environmental stressors on aquatic animals under either artificial conditions or in the wild. Here, we report on the development of a transcriptomic platform for global gene expression profiling in the Manila clam.ResultsA normalized cDNA library representing a mixture of adult tissues was sequenced using a ultra high-throughput sequencing technology (Roche 454). A database consisting of 32,606 unique transcripts was constructed, 9,747 (30%) of which could be annotated by similarity. An oligo-DNA microarray platform was designed and applied to profile gene expression of digestive gland and gills. Functional annotation of differentially expressed genes between different tissues was performed by enrichment analysis. Expression of Natural Antisense Transcripts (NAT) analysis was also performed and bi-directional transcription appears a common phenomenon in the R. philippinarum transcriptome. A preliminary study on clam samples collected in a highly polluted area of the Venice Lagoon demonstrated the applicability of genomic tools to environmental monitoring.ConclusionsThe transcriptomic platform developed for the Manila clam confirmed the high level of reproducibility of current microarray technology. Next-generation sequencing provided a good representation of the clam transcriptome. Despite the known limitations in transcript annotation and sequence coverage for non model species, sufficient information was obtained to identify a large set of genes potentially involved in cellular response to environmental stress.

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Population genetics of the Manila clam (Ruditapes philippinarum) introduced in North America and Europe.

Project description:Globally, the Manila clam (Ruditapes philippinarum) stands as the second most important bivalve species in fisheries and aquaculture. Native to the Pacific coast of Asia, it is now well-established in North America and Europe, where its on-going management reflects local economic interests. The historic record of transfers spans the 20th century and suggests sequential movement from Japan to North America, as a hitch-hiker on oysters, and then intentional introduction in Europe, but global genetic data are missing. We have studied mitochondrial DNA and microsatellite markers in nine populations from Asia, North America and Europe. The results from the two types of markers indicated a good concordance of present-day genetic structure with the reported history of clam transfers across continents, and no evidence of relevant concealed introductions from continental Asia in Europe and North America. However, European populations showed a loss of genetic variability and significant genetic differentiation as compared to their American counterparts. Our study shows that in spite of the increasing ease for species to spread out of their native range, in the case of the Manila clam this has not resulted in new invasion waves in the two studied continents.

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Filtration rates of the manila clam, Ruditapes philippinarum, in tidal flats with different hydrographic regimes.

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Effects of hypoxia on survival, behavior, metabolism and cellular damage of Manila clam (Ruditapes philippinarum).

Project description:The Manila clam Ruditapes philippinarum has become a common and dominant macrobenthic species in coastal areas of the northwestern Pacific and temperate waters of Europe; it is also a major cultured shellfish, with annual worldwide production exceeding 3.3 million tonnes. This species faces greater risk of exposure to hypoxia as eutrophication worsens throughout its coastal habitats; however, its tolerance to hypoxia remains unclear, and the toxicological indicators including LC50 and LT50 have not yet been assessed. Previous studies on the effects of hypoxia on marine benthos have focused largely on functional responses, such as metabolism and gene expression, leaving potential structural damage to the mitochondria or the cells unknown. In this study we assessed the effects of hypoxia on Manila clam in terms of survival, behavior, metabolism and cellular damage, using a newly designed automated hypoxia simulation device that features exceptional accuracy and good stability. The clams exhibited strong tolerance to hypoxia as the 20-day LC50 for dissolved oxygen (DO) was estimated to be 0.57 mg L-1, and the LT50 at 0.5 mg L-1 DO was 422 hours. Adaptations included fewer buried clams and a depressed metabolism, while the unexpected rise in the activities of key enzymes involved in glycolysis may indicate a diverse strategy of shellfish under hypoxia. Cellular damage was observed as collapse of the mitochondrial cristae and both cellular and mitochondrial vacuolization. This multi-level study complements and updates our knowledge of the effects of hypoxia on marine benthos, by improving our understanding of the potential for marine ecological transformation under hypoxic conditions and providing useful information for Manila clam farming.

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Population Genetics of Manila Clam (<i>Ruditapes philippinarum</i>) in China Inferred from Microsatellite Markers.

Project description:The Manila clam (Ruditapes philippinarum) is one of the most commercially important bivalves along the coast of China. With the continuous expansion of clam farming scale, it may lead to some serious problems, including loss of genetic variation, inbreeding depression, and reduced effective population size (Ne). In the present study, eleven microsatellite markers were used to investigate the genetic diversity and differentiation among 13 clam populations along the coast of China. As a result, 150 alleles were detected according to the genotyping results of eleven microsatellite loci. The observed heterozygosity (Ho) was estimated to be ranging from 0.437 to 0.678, while the expected heterozygosity (He) was calculated to be varying from 0.587 to 0.700. Fst values between populations ranged from 0.0046-0.1983. In particular, the Laizhou population had the highest genetic variability, which was significantly different from the others (all Fst values > 0.1). For all the clam populations, there was no significant linear regression between genetic and geographic distance, indicating that these populations do not follow a pattern of isolation by distance (IBD). Genetic structure was estimated according to NJ, principal coordinates (PCoA), and structure-based clustering. Estimates of effective population size range from dozens to thousands among different populations, based on linkage-disequilibrium and molecular coancestry methods. The results reveal the genetic diversity of clams and verify the hypothesis that clam population differentiation may be influenced by the mode of southern breeding and northern culture, providing guiding information for natural resource conservation and genetic breeding of clams.

| S-EPMC10135866 | biostudies-literature