The clam Ruditapes philippinarum is an important species in the marine aquaculture industry in China. However, in recent years, the aquaculture of R. philippinarum has been negatively impacted by various bacterial pathogens. In this study, the transcriptome libraries of R. philippinarum showing different levels of resistance to challenge with Vibrio anguillarum were constructed and RNA-seq was performed using the Illumina sequencing platform. Host immune factors were identified that responded to V. anguillarum infection, including C-type lectin domain, glutathione S-transferase 9, lysozyme, methyltransferase FkbM domain, heat shock 70 kDa protein, Ras-like GTP-binding protein RHO, C1q, F-box and BTB/POZ domain protein zf-C2H2. Ten genes were selected and verified by RT-qPCR, and nine of the gene expression results were consistent with those of RNA-seq. The lectin gene in the phagosome pathway was expressed at a significantly higher level after V. anguillarum infection, which might indicate the role of lectin in the immune response to V. anguillarum . Comparing the results from R. philippinarum resistant and nonresistant to V. anguillarum increases our understanding of the resistant genes and key pathways related to Vibrio challenge in this species. The results obtained here provide a reference for future immunological research focusing on the response of R. philippinarum to V. anguillarum infection.
Abstract 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.
Abstract 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.
Additional file 4. 75 differentially expressed unigenes, including 43 upregulated and 32 downregulated unigenes indentified between hypoxia stress group and normoxia control group (qvalue 1).
Microsatellite loci often used as a genetic tool for estimating genetic diversity population variation in a wide variety of different species. The application of microsatellite markers in genetics and breeding includes investigating the genetic differentiation of wild and cultured populations, assessing and determining the genetic relationship of different populations. The aim of this work is to develop several microsatellite markers via high-throughput sequencing and characterize these markers in commercially important bivalve Ruditapes philippinarum. Among the two populations of R. philippinarum studied, 110 alleles were detected. The number of alleles at the cultured population ranged from 3 to 17 (mean NA = 6.897) and wild population ranged from 2 to 15 (mean NA = 6.793). The observed and expected heterozygosities of cultured population ranged from 0.182 to 0.964, and from 0.286 to 0.900, with an average of 0.647 and 0.692, respectively. The observed and expected heterozygosities of wild population ranged from 0.138 to 1.000, and from 0.439 to 0.906, with an average of 0.674 and 0.693, respectively. The polymorphism information content ranged from 0.341 to 0.910 with an average of 0.687. Sixteen and thirteen microsatellite loci deviated significantly from Hardy–Weinberg equilibrium after correction for multiple tests in cultured and wild population, respectively. Twenty-nine novel microsatellite loci were developed using Illumina paired-end shotgun sequencing and characterized in two population of R. philippinarum. How to cite: Jiang L, Nie H, Li C, et al. The genetic diversity of wild and cultivated Manila clam (Ruditapes philippinarum) revealed by 29 novel microsatellite markers. Electron J Biotechnol 2018;34. https://doi.org/10.1016/j.ejbt.2018.05.003.
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.
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