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: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:Background: The in vivo gene response associated with hyperthermia and subsequent return to homeostasis or development of heat illness is poorly understood. Early activation of gene networks in the heat stress response is likely to lead to the systemic inflammation, multi-organ functional impairment, and other pathophysiological states characteristic of heat illness. Here, we perform an unbiased global characterization of the multi-organ gene response using an in vivo model of heat stress in the conscious rat. Results: Rats were subjected to elevated temperatures until implanted thermal probes indicated a maximal core temperature of 41.8 M-BM-0C (Tc,Max). Liver, lung, kidney, and heart were harvested at Tc,Max, 24 hours, and 48 hours after heat stress in groups of experimental animals and time-matched controls kept at ambient temperature. Clinical chemistries suggested abnormal function in liver, kidney, and lung at Tc,Max, and cardiac histopathology at 48 hours supported persistent cardiac damage in 3 out of 6 animals. Microarray analysis identified 78 differentially expressed genes common to all 4 organs at Tc,Max (i.e., the consensus heat stress response). Gene set enrichment analysis of gene ontology terms identified 25 biological processes in 4 general gene ontology categories: protein folding, regulation of apoptosis, response to cytokines, and transcriptional responses. Functional analysis clustering of the 78 differentially expressed genes in the consensus heat stress response also identified functional categories of protein folding and regulation of apoptosis. Self-organizing maps identified gene-specific signatures corresponding to protein folding disorders specific to only heat-stressed rats with histopathologic evidence of cardiac injury at 48 hours. Enrichment analysis of differentially expressed proteins in heat-injured hearts at 48 hours corroborated gene enrichment analysis results. Quantitative proteomics analysis by iTRAQ demonstrated that differential protein expression was not comparable to transcript expression at Tc,Max and 24 hours. However, the profile of differentially expressed proteins closely matched the transcriptomic profile in heat-injured animals at 48 hours. Pathway analysis at both the transcript and protein levels supported catastrophic deficits in energetics and cellular metabolism, chronic proteotoxic response, and activation of the unfolded protein response. Calculation of protein super-saturation scores demonstrated an increased propensity of proteins to aggregate in the hearts of heat-injured animals at 48 hours, consistent with accumulation of misfolded proteins. Conclusions: Global transcriptomic and proteomic analysis identified networks of genes and proteins initiating an unfolded protein response, metabolic dysfunction, and mitochondrial energy crisis in animals with histopathologic evidence of persistent heat injury, providing the basis for a systems-level physiological model of heat illness and recovery. To induce the pathophysiological effects of heat stress, conscious rats were placed in an incubator at 37M-BM-:C and their core temperature was monitored. Animals were sacrificed when their core temperature reached 41.8M-BM-:C (Tc,Max), or they were returned to their normal housing environment and were allowed to recover for up to 24 or 48 hours prior to sacrifice. Time-matched controls were euthanized at times corresponding to Tc,Max, 24 hours, and 48 hours. For each condition n=6 for a total of 36 animals. Four tissues (liver, lung, kidney and heart) were analyzed from each animal for a total of 144 arrays; however a few arrays did not pass QC therefore only 140 are reported here: 34 liver, 35 lung, 36 kidney, or 35 heart.

Dataset Information

Expression data from porcelain crabs following heat or cold stress

Publications

Seasonal and latitudinal acclimatization of cardiac transcriptome responses to thermal stress in porcelain crabs, Petrolisthes cinctipes.

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