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

Project description:Southern California (USA) populations of the intertidal marine snail Chlorostoma (formerly Tegula) funebralis are generally exposed to higher air and water temperatures than northern California populations. Previous studies have shown that southern populations are more tolerant of heat stress than northern populations. To assess the potential role of gene regulation in these regional differences, we examined transcriptome responses to thermal stress in two southern and two northern populations of C. funebralis. Snails from the four populations were acclimated to a common lab environment, exposed to a heat stress representative of natural low tide conditions, and then analyzed using RNA-Seq to characterize changes in gene expression associated with stress and differences in expression across geographic regions. Changes in expression following stress were dominated by genes involved in apoptosis, the inflammatory response, response to mis and unfolded proteins, and ubiquitination of proteins. Heat shock proteins (Hsps) were up-regulated in both northern and southern populations. However, while the magnitude of the response was significantly greater in northern populations for the majority of Hsp70s, the southern populations showed a greater up-regulation for roughly half of the Hsp40s, which are co-chaperones for Hsp70s. Differential expression analysis of the control versus treatment genes in the northern and southern populations respectively revealed that 56 genes, many involved in the inflammation and immune response, responded to heat stress only in the northern populations. Moreover, several of the molecular chaperones and antioxidant genes that were not differentially expressed in the southern populations instead showed higher constitutive expression under control conditions compared to the northern populations. The expression levels of some of these constitutive genes such as superoxide dismutase were also found to positively correlate with survival following heat stress. This suggests that expression of these genes has evolved a degree of M-bM-^@M-^\frontloadingM-bM-^@M-^] that may contribute to the higher thermal tolerance of southern populations. mRNA profiles of northern and southern California heat-stressed and control C. funebralis were generated by 100bp paired end sequencing, in duplicate, using Illumina HiSeq2000.

Project description:Southern California (USA) populations of the intertidal marine snail Chlorostoma (formerly Tegula) funebralis are generally exposed to higher air and water temperatures than northern California populations. Previous studies have shown that southern populations are more tolerant of heat stress than northern populations. To assess the potential role of gene regulation in these regional differences, we examined transcriptome responses to thermal stress in two southern and two northern populations of C. funebralis. Snails from the four populations were acclimated to a common lab environment, exposed to a heat stress representative of natural low tide conditions, and then analyzed using RNA-Seq to characterize changes in gene expression associated with stress and differences in expression across geographic regions. Changes in expression following stress were dominated by genes involved in apoptosis, the inflammatory response, response to mis and unfolded proteins, and ubiquitination of proteins. Heat shock proteins (Hsps) were up-regulated in both northern and southern populations. However, while the magnitude of the response was significantly greater in northern populations for the majority of Hsp70s, the southern populations showed a greater up-regulation for roughly half of the Hsp40s, which are co-chaperones for Hsp70s. Differential expression analysis of the control versus treatment genes in the northern and southern populations respectively revealed that 56 genes, many involved in the inflammation and immune response, responded to heat stress only in the northern populations. Moreover, several of the molecular chaperones and antioxidant genes that were not differentially expressed in the southern populations instead showed higher constitutive expression under control conditions compared to the northern populations. The expression levels of some of these constitutive genes such as superoxide dismutase were also found to positively correlate with survival following heat stress. This suggests that expression of these genes has evolved a degree of “frontloading” that may contribute to the higher thermal tolerance of southern populations.

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Expression data from porcelain crabs following heat stress

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