Project description:Purpose:The mud crab Scylla paramamosain is an economically important marine crab in China suffering from severe outbreaks of infectious disease caused by marine bacteria such as Vibrio Parahaemolyticus, resulting in great economic losses. However, the mechanisms involved in the immune response of this crab to bacterial infection are not fully understood. To understand the molecular mechanisms underlying the immune response to such pathogenic bacteria, we used high-throughput deep sequencing technology to investigate the transcriptome and comparative expression profiles of the mud crab S.paramamosain infected with V.parahaemolyticus. Methods: The hemocytes sampled at 0-24h after infection with V.parahaemolyticus were used for transcriptome analysis. The hemocytes sampled at 24 h after injections with V.parahaemolyticus and no injected 0h(as control) were used for gene expression profiling analysis. Results: A total of 52,934,042 reads were obtained and assembled into 186,193 contigs in transcriptional responses of the V.parahaemolyticus-infected mud crab. Via annotation to the NCBI database and the Swissprot database, we obtained 48,934 identified unigenes. In total, 10,139(20.7%) unigenes were classified into Gene Ontology, and 25,349 unigenes were found in 20 KEGG categories. These genes included representatives from almost all functional categories. By using Solexa/Illumina's DeepSAGE, 1213 differentially expressed genes (P value < 0.05) were detected in comparative analysis of the expression profiles between V.parahaemolyticus-infected crabs and control crabs, including 538 remarkably upregulated genes and 675 remarkably downregulated genes. Conclusions: Based on our results, we conclude that the inflammatory response may play an important role in the early stages of infection. The signaling cascades such as the chemokine, JAK-STAT, and MAPK pathways are regulated by V.parahaemolyticus infection. These results revealed changes of multiple signaling pathways involved in immunity during V.parahaemolyticus infection, which will facilitate our comprehensive understanding of the mechanisms involved in the immune response to bacterial infection in the mud crab.

Project description:Intertidal zone organisms can experience transient freezing temperatures during winter low tides, but their extreme cold tolerance mechanisms are not known. Petrolisthes cinctipes is a temperate mid-high intertidal zone crab species that can experience wintertime habitat temperatures below the freezing point of seawater. We examined how cold tolerance changed during the initial phase of thermal acclimation to cold and warm temperatures, as well as the persistence of cold tolerance during long-term thermal acclimation. Thermal acclimation for as little as 6 hours at 8˚C enhanced crab tolerance during a 1h exposure to -2°C relative to crabs acclimated to 18˚C. Potential mechanisms for this enhanced tolerance were elucidated using cDNA microarrays to probe for differences in gene expression in cardiac tissue of warm and cold acclimated crabs during the first day of thermal acclimation. No changes in gene expression were detected until 12h of thermal acclimation. Genes strongly upregulated in warm acclimated crabs represented immune response and extracellular / intercellular processes, suggesting that warm acclimated crabs had a generalized stress response and may have been remodelling tissues or altering intercellular processes. Genes strongly upregulated in cold acclimated crabs included many that are involved in glucose production suggesting that cold acclimation involves increasing intracellular glucose as a cryoprotectant. Structural cytoskeletal proteins were also strongly represented among the genes upregulated in only cold acclimated crabs. There were no consistent changes in composition or the level of unsaturation of membrane phospholipid fatty acids with cold acclimation, which suggests that neither short- nor long-term changes in cold tolerance are mediated by changes in membrane fatty acid composition. Overall, our study demonstrates that initial changes in cold tolerance are likely not regulated by transcriptomic responses, but that gene expression-related changes in homeostasis begin within 12 hours – the length of a tidal cycle. all array data and raw images archived at the Porcelain Crab Array Database (http://array.sfsu.edu)

Project description:Intertidal zone organisms can experience transient freezing temperatures during winter low tides, but their extreme cold tolerance mechanisms are not known. Petrolisthes cinctipes is a temperate mid-high intertidal zone crab species that can experience wintertime habitat temperatures below the freezing point of seawater. We examined how cold tolerance changed during the initial phase of thermal acclimation to cold and warm temperatures, as well as the persistence of cold tolerance during long-term thermal acclimation. Thermal acclimation for as little as 6 hours at 8˚C enhanced crab tolerance during a 1h exposure to -2°C relative to crabs acclimated to 18˚C. Potential mechanisms for this enhanced tolerance were elucidated using cDNA microarrays to probe for differences in gene expression in cardiac tissue of warm and cold acclimated crabs during the first day of thermal acclimation. No changes in gene expression were detected until 12h of thermal acclimation. Genes strongly upregulated in warm acclimated crabs represented immune response and extracellular / intercellular processes, suggesting that warm acclimated crabs had a generalized stress response and may have been remodelling tissues or altering intercellular processes. Genes strongly upregulated in cold acclimated crabs included many that are involved in glucose production suggesting that cold acclimation involves increasing intracellular glucose as a cryoprotectant. Structural cytoskeletal proteins were also strongly represented among the genes upregulated in only cold acclimated crabs. There were no consistent changes in composition or the level of unsaturation of membrane phospholipid fatty acids with cold acclimation, which suggests that neither short- nor long-term changes in cold tolerance are mediated by changes in membrane fatty acid composition. Overall, our study demonstrates that initial changes in cold tolerance are likely not regulated by transcriptomic responses, but that gene expression-related changes in homeostasis begin within 12 hours – the length of a tidal cycle. all array data and raw images archived at the Porcelain Crab Array Database (http://array.sfsu.edu) n=264 specimens were divided into warm (18°C, n=96), cold (8°C, n=96), and control (13°C, n=72) acclimation groups. Crabs were sampled from the 13°C group at 0 h (the start of the experiment) and 24 h, the termination of the experiment. Crabs were sampled from the warm and cold acclimation groups at 6, 12, 18, and 24 hours following the start of thermal acclimation. At each time point, heart tissue from n=16 crabs from each group was dissected, flash frozen and stored at −80°C. A pooled total aRNA sample was prepared for each group by mixing equal quantities of total RNA from n=5 individuals in each group in order to have the same amount of biological diversity within each pooled RNA sample. For microarray hybridizations we used n=25 slides in an incomplete loop design where each sample was hybridized n=5 times, 2-3 times labelled with each Cy dye

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