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The suborder Notothenioidae is comprised of Antarctic fishes, several of which have lost their ability to rapidly upregulate heat shock proteins in response to thermal stress, instead adopting a pattern of expression resembling constitutive genes. Given the cold-denaturing effect that sub-zero waters have on proteins, evolution in the Southern Ocean has likely selected for increased expression of molecular chaperones. These selective pressures may have also enabled retention of gene duplicates, bolstering quantitative output of cytosolic heat shock proteins (HSPs). Given that newly duplicated genes are under more relaxed selection, it is plausible that gene duplication enabled altered regulation of such highly conserved genes. To test for evidence of gene duplication, copy number of various isoforms within major heat shock gene families were characterized via qPCR and compared between the Antarctic notothen, Trematomus bernacchii, which lost the inducible heat shock response, and the non-Antarctic notothen, Notothenia angustata, which maintains an inducible heat shock response. The results indicate duplication of isoforms within the hsp70 and hsp40 super families have occurred in the genome of T. bernacchii. The findings suggest gene duplications may have been critical in maintaining protein folding efficiency in the sub-zero waters and provided an evolutionary mechanism of alternative regulation of these conserved gene families.

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BACKGROUND: Antarctic fishes of the Notothenioidei suborder constitutively upregulate multiple inducible chaperones, a highly derived adaptation that preserves proteostasis in extreme cold, and represent a system for studying the evolution of gene frontloading. We screened for Hsf1-binding sites, as Hsf1 is a master transcription factor of the heat shock response, and highly-conserved non-coding elements within proximal promoters of chaperone genes across 10 Antarctic notothens, 2 subpolar notothens, and 17 perciform fishes. We employed phylogenetic models of molecular evolution to determine whether (i) changes in motifs associated with Hsf1-binding and/or (ii) relaxed purifying selection or exaptation at ancestral cis-regulatory elements coincided with the evolution of chaperone frontloading in Antarctic notothens. RESULTS: Antarctic notothens exhibited significantly fewer Hsf1-binding sites per bp at chaperone promoters than subpolar notothens and Serranoidei, the most closely-related suborder to Notothenioidei included in this study. 90% of chaperone promoters exhibited accelerated substitution rates among Antarctic notothens relative to other perciformes. The proportion of bases undergoing accelerated evolution (i) was significantly greater in Antarctic notothens than in subpolar notothens and Perciformes in 70% of chaperone genes and (ii) increased among bases that were more conserved among perciformes. Lastly, we detected evidence of relaxed purifying selection and exaptation acting on ancestrally conserved cis-regulatory elements in the Antarctic notothen lineage and its major branches. CONCLUSION: A large degree of turnover has occurred in Notothenioidei at chaperone promoter regions that are conserved among perciform fishes following adaptation to the cooling of the Southern Ocean. Additionally, derived reductions in Hsf1-binding site frequency suggest cis-regulatory modifications to the classical heat shock response. Of note, turnover events within chaperone promoters were less frequent in the ancestral node of Antarctic notothens relative to younger Antarctic lineages. This suggests that cis-regulatory divergence at chaperone promoters may be greater between Antarctic notothen lineages than between subpolar and Antarctic clades. These findings demonstrate that strong selective forces have acted upon cis-regulatory elements of chaperone genes among Antarctic notothens.

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Despite the lack of an inducible heat shock response (HSR), the Antarctic notothenioid fish, Trematomus bernacchii, has retained a level of physiological plasticity that can at least partially compensate for the effects of acute heat stress. Over the last decade, both physiological and transcriptomic studies have signaled these fish can mitigate the effects of acute heat stress by employing other aspects of the cellular stress response (CSR) that help confer thermotolerance as well as drive homeostatic mechanisms during long-term thermal acclimations. However, the regulatory mechanisms that determine temperature-induced changes in gene expression remain largely unexplored in this species. Therefore, this study utilized next generation sequencing coupled with an in silico approach to explore the regulatory role of microRNAs in governing the transcriptomic level response observed in this Antarctic notothenioid with respect to the CSR. Using RNAseq, we characterized the expression of 125 distinct miRNA orthologues in T. bernacchii gill tissue. Additionally, we identified 12 miRNAs that appear to be thermally responsive based on differential expression (DE) analyses performed between fish acclimated to control (-1.5 °C) and an acute heat stress (+4 °C). We further characterized the functional role of these DE miRNAs using bioinformatics pipelines to identify putative gene targets of the DE miRNAs and subsequent gene set enrichment analyses, which together suggest these miRNAs are involved in regulating diverse aspects of the CSR in T. bernacchii.

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The adaptations used by notothenioid fish to combat extreme cold may have left these fish poorly poised to deal with a changing environment. As such, the expected environmental perturbations brought on by global climate change have the potential to significantly affect the energetic demands and subsequent cellular processes necessary for survival. Despite recent lines of evidence demonstrating that notothenioid fish retain the ability to acclimate to elevated temperatures, the underlying mechanisms responsible for temperature acclimation in these fish remain largely unknown. Furthermore, little information exists on the capacity of Antarctic fish to respond to changes in multiple environmental variables. We have examined the effects of increased temperature and pCO2 on the rate of oxygen consumption in three notothenioid species, Trematomus bernacchii, Pagothenia borchgrevinki, and Trematomus newnesi. We combined these measurements with analysis of changes in aerobic and anaerobic capacity, lipid reserves, fish condition, and growth rates to gain insight into the metabolic cost associated with acclimation to this dual stress. Our findings indicated that temperature is the major driver of the metabolic responses observed in these fish and that increased pCO2 plays a small, contributing role to the energetic costs of the acclimation response. All three species displayed varying levels of energetic compensation in response to the combination of elevated temperature and pCO2. While P. borchgrevinki showed nearly complete compensation of whole animal oxygen consumption rates and aerobic capacity, T. newnesi and T. bernacchii displayed only partial compensation in these metrics, suggesting that at least some notothenioids may require physiological trade-offs to fully offset the energetic costs of long-term acclimation to climate change related stressors.

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As we move into the Anthropocene, organisms inhabiting marine environments will continue to face growing challenges associated with changes in ocean pH (ocean acidification), dissolved oxygen (dead zones) and temperature. These factors, in combination with naturally variable environments such as the rocky intertidal zone, may create extreme physiological challenges for organisms that are already performing near their biological limits. Although numerous studies have examined the impacts of climate-related stressors on intertidal animals, little is known about the underlying physiological mechanisms driving adaptation to ocean acidification and how this may alter organism interactions, particularly in marine vertebrates. Therefore, we have investigated the effects of decreased ocean pH on the hypoxia response of an intertidal sculpin, Clinocottus analis. We used both whole-animal and biochemistry-based analyses to examine how the energetic demands associated with acclimation to low-pH environments may impact the fish's reliance on facultative air breathing in low-oxygen environments. Our study demonstrated that acclimation to ocean acidification resulted in elevated routine metabolic rates and acid-base regulatory capacity (Na+,K+-ATPase activity). These, in turn, had downstream effects that resulted in decreased hypoxia tolerance (i.e. elevated critical oxygen tension). Furthermore, we present evidence that these fish may be living near their physiological capacity when challenged by ocean acidification. This serves as a reminder that the susceptibility of teleost fish to changes in ocean pH may be underestimated, particularly when considering the multiple stressors that many experience in their natural environments.

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PURPOSE: The IPCC has reasserted the strong influence of anthropogenic CO2 contributions on global climate change and highlighted the polar-regions as highly vulnerable. With these predictions the cold adapted fauna endemic to the Southern Ocean, which is dominated by fishes of the sub-order Notothenioidei, will face considerable challenges in the near future. Recent physiological studies have demonstrated that the synergistic stressors of elevated temperature and ocean acidification have a considerable, although variable, impact on notothenioid fishes. The present study explored the transcriptomic response of Pagothenia borchgrevinki to increased temperatures and pCO2 after 7, 28 and 56days of acclimation. We compared this response to short term studies assessing heat stress alone and foretell the potential impacts of these stressors on P. borchgrevinki's ability to survive a changing Southern Ocean. RESULTS: P. borchgrevinki did demonstrate a coordinated stress response to the dual-stressor condition, and even indicated that some level of inducible heat shock response may be conserved in this notothenioid species. However, the stress response of P. borchgrevinki was considerably less robust than that observed previously in the closely related notothenioid, Trematomus bernacchii, and varied considerably when compared across different acclimation time-points. Furthermore, the molecular response of these fish under multiple stressors displayed distinct differences compared to their response to short term heat stress alone. CONCLUSIONS: When exposed to increased sea surface temperatures, combined with ocean acidification, P. borchgrevinki demonstrated a coordinated stress response that has already peaked by 7days of acclimation and quickly diminished over time. However, this response is less dramatic than other closely related notothenioids under identical conditions, supporting previous research suggesting that this notothenioid species is less sensitive to environmental variation.

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BACKGROUND: As global climate change progresses, the Southern Ocean surrounding Antarctica is poised to undergo potentially rapid and substantial changes in temperature and pCO2. To survive in this challenging environment, the highly cold adapted endemic fauna of these waters must demonstrate sufficient plasticity to accommodate these changing conditions or face inexorable decline. Previous studies of notothenioids have focused upon the short-term response to heat stress; and more recently the longer-term physiological response to the combined stress of increasing temperatures and pCO2. This inquiry explores the transcriptomic response of Trematomus bernacchii to increased temperatures and pCO2 at 7, 28 and 56 days, in an attempt to discern the innate plasticity of T. bernacchii available to cope with a changing Southern Ocean. RESULTS: Differential gene expression analysis supported previous research in that T. bernacchii exhibits no inducible heat shock response to stress conditions. However, T. bernacchii did demonstrate a strong stress response to the multi-stressor condition in the form of metabolic shifts, DNA damage repair, immune system processes, and activation of apoptotic pathways combined with negative regulation of cell proliferation. This response declined in magnitude over time, but aspects of this response remained detectable throughout the acclimation period. CONCLUSIONS: When exposed to the multi-stressor condition, T. bernacchii demonstrates a cellular stress response that persists for a minimum of 7 days before returning to near basal levels of expression at longer acclimation times. However, subtle changes in expression persist in fish acclimated for 56 days that may significantly affect the fitness T. bernacchii over time.

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Antarctic fish of the suborder Notothenioidei have evolved several unique adaptations to deal with subzero temperatures. However, these adaptations may come with physiological trade-offs, such as an increased susceptibility to oxidative damage. As such, the expected environmental perturbations brought on by global climate change have the potential to significantly increase the level of oxidative stress and cellular damage in these endemic fish. Previous single stressor studies of the notothenioids have shown they possess the capacity to acclimate to increased temperatures, but the cellular-level effects remain largely unknown. Additionally, there is little information on the ability of Antarctic fish to respond to ecologically relevant environmental changes where multiple variables change concomitantly. We have examined the potential synergistic effects that increased temperature and P(CO2) have on the level of protein damage in Trematomus bernacchii, Pagothenia borchgrevinki and Trematomus newnesi, and combined these measurements with changes in total enzymatic activity of catalase (CAT) and superoxide dismutase (SOD) in order to gauge tissue-specific changes in antioxidant capacity. Our findings indicate that total SOD and CAT activity levels displayed only small changes across treatments and tissues. Short-term acclimation to decreased seawater pH and increased temperature resulted in significant increases in oxidative damage. Surprisingly, despite no significant change in antioxidant capacity, cellular damage returned to near-basal levels, and significantly decreased in T. bernacchii, after long-term acclimation. Overall, these data suggest that notothenioid fish currently maintain the antioxidant capacity necessary to offset predicted future ocean conditions, but it remains unclear whether this capacity comes with physiological trade-offs.

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BACKGROUND: The notothenioids comprise a diverse group of fishes that rapidly radiated after isolation by the Antarctic Circumpolar Current approximately 14-25 million years ago. Given that evolutionary adaptation has led to finely tuned traits with narrow physiological limits in these organisms, this system provides a unique opportunity to examine physiological trade-offs and limits of adaptive responses to environmental perturbation. As such, notothenioids have a rich history with respect to studies attempting to understand the vulnerability of polar ecosystems to the negative impacts associated with global climate change. Unfortunately, despite being a model system for understanding physiological adaptations to extreme environments, we still lack fundamental molecular tools for much of the Nototheniidae family. RESULTS: Specimens of the emerald notothen, Trematomus bernacchii, were acclimated for 28 days in flow-through seawater tanks maintained near ambient seawater temperatures (-1.5°C) or at +4°C. Following acclimation, tissue specific cDNA libraries for liver, gill and brain were created by pooling RNA from n = 5 individuals per temperature treatment. The tissue specific libraries were bar-coded and used for 454 pyrosequencing, which yielded over 700 thousand sequencing reads. A de novo assembly and annotation of these reads produced a functional transcriptome library of T. bernacchii containing 30,107 unigenes, 13,003 of which possessed significant homology to a known protein product. Digital gene expression analysis of these extremely cold adapted fish reinforced the loss of an inducible heat shock response and allowed the preliminary exploration into other elements of the cellular stress response. CONCLUSIONS: Preliminary exploration of the transcriptome of T. bernacchii under elevated temperatures enabled a semi-quantitative comparison to prior studies aimed at characterizing the thermal response of this endemic fish whose size, abundance and distribution has established it as a pivotal species in polar research spanning several decades. The comparison of these findings to previous studies demonstrates the efficacy of transcriptomics and digital gene expression analysis as tools in future studies of polar organisms and has greatly increased the available genomic resources for the suborder Notothenioidei, particularly in the Trematominae subfamily.

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Although the physiological response of teleost fishes to increased temperature has been well documented, there is only a small body of literature that examines the effects of ocean acidification on fish under ecologically relevant scenarios. Furthermore, little data exists which examines the possible synergistic effects of increased sea surface temperatures and pCO(2) levels, although it is well established that both will co-committedly change in the coming centuries. In this study we examined the effects of increased temperature, increased pCO(2), and a combination of these treatments on the resting metabolic rate (RMR) of four species of notothenioid fish, Trematomus bernacchii, T. hansoni, T. newnesi, and Pagothenia borchgrevinki, acclimated to treatment conditions for 7, 14 or 28days. While most species appear capable of rapidly acclimating to increased pCO(2), temperature continues to impact RMRs for up to 28days. One species in particular, T. newnesi, displayed no acclimatory response to any of the treatments regardless of acclimation time and may have a reduced capacity to respond to environmental change. Furthermore, we present evidence that temperature and pCO(2) act synergistically to further elevate the RMR and slow acclimation when compared to temperature or pCO(2) increases alone.

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SUMMARY: The marine intertidal zone is characterized by large variation in temperature, pH, dissolved oxygen and the supply of nutrients and food on seasonal and daily time scales. These oceanic fluctuations drive of ecological processes such as recruitment, competition and consumer-prey interactions largely via physiological mehcanisms. Thus, to understand coastal ecosystem dynamics and responses to climate change, it is crucial to understand these mechanisms.Here we utilize transcriptome analysis of the physiological response of the mussel Mytilus californianus at different spatial scales to gain insight into these mechanisms. We used mussels inhabiting different vertical locations within Strawberry Hill on Cape Perpetua, OR and Boiler Bay on Cape Foulweather, OR to study inter- and intra-site variation of gene expression.The results highlight two distinct gene expression signatures related to the cycling of metabolic activity and perturbations to cellular homeostasis. Intermediate spatial scales show a strong influence of oceanographic differences in food and stress environments between sites separated by ~65 km.Together, these new insights into environmental control of gene expression may allow understanding of important physiological drivers within and across populations.

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Previous studies have shown fertilization and development of marine species can be significantly inhibited when the pH of sea water is artificially lowered. Little mechanistic understanding of these effects exists to date, but previous work has linked developmental inhibition to reduced cleavage rates in embryos. To explore this further, we tested whether common cell cycle checkpoints were involved using three cellular biomarkers of cell cycle progression: (1) the onset of DNA synthesis, (2) production of a mitotic regulator, cyclin B, and (3) formation of the mitotic spindle. We grew embryos of the purple sea urchin, Strongylocentrotus purpuratus, in seawater artifically buffered to a pH of ∼7.0, 7.5, and 8.0 by CO(2) infusion. Our results suggest the reduced rates of mitotic cleavage are likely unrelated to common cell cycle checkpoints. We found no significant differences in the three biomarkers assessed between pH treatments, indicating the embryos progress through the G(1)/S, G(2)/M and metaphase/anaphase transitions at relatively similar rates. These data suggest low pH environments may not impact developmental programs directly, but may act through secondary mechanisms such as cellular energetics.

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The Hsp70-interacting protein Hip has been identified as a transient participant in the assembly of both glucocorticoid (GR) and progesterone receptor complexes. Although it has been difficult to identify a physiological role for Hip, it is believed to have intrinsic chaperoning properties and has been identified as a potential anti-apoptotic target of Granzyme B. In vitro assays have provided evidence that Hip may interact with GR complexes in an Hsp70 independent manner and can enhance the function of GR in hormone based reporter assays. In this study, a cDNA for human Hip was used in mutational analysis to map Hip function to critical structural elements. A single amino acid substitution (L211S) resulted in a loss of Hip function. This mutation also appears to disrupt the interaction of Hip with Hsp70 in vitro. Failure to recover Hip-L211S constructs in co-immunoprecipitation assays with an Hsp70 monoclonal antibody suggests that the mutation is unlikely to result in a misfolded substrate.

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To test the temperature sensitivity of molecular chaperones in poikilothermic animals, we purified the molecular chaperone Hsc70 from 2 closely related notothenioid fishes--the Antarctic species Trematomus bernacchii and the temperate New Zealand species Notothenia angustata--and characterized the effect of temperature on Hsc70 adenosine triphosphatase (ATPase) activity. Hsc70 ATPase activity was measured using [alpha-32P]-adenosine triphosphate (ATP)-based in vitro assays followed by separation of adenylates by thin-layer chromatography. For both species, a significant increase in Hsc70 ATPase activity was observed across a range of temperatures that was ecologically relevant for each respective species. Hsc70 from T bernacchii hydrolyzed 2-fold more ATP than did N angustata Hsc70 at 0 degrees C, suggesting that the Antarctic molecular chaperone may be adapted to function more efficiently at extreme cold temperatures. In addition, Q10 measurements indicate differential temperature sensitivity of the ATPase activity of Hsc70 from these differentially adapted fish that correlates with the temperature niche inhabited by each species. Hsc70 from T bernacchii was relatively temperature insensitive, as indicated by Q10 values calculated near 1.0 across each temperature range measured. In the case of Hsc70 purified from N angustata, Q10 values indicated thermal sensitivity across the temperature range of 0 degrees C to 10 degrees C, with a Q10 of 2.714. However, Hsc70 from both T bernacchii and N angustata exhibited unusually high thermal stabilities with ATPase activity at temperatures that far exceeded temperatures encountered by these fish in nature. Overall, as evidenced by in vitro ATP hydrolysis, Hsc70 from T bernacchii and N angustata displayed biochemical characteristics that were supportive of molecular chaperone function at ecologically relevant temperatures.

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Although a great deal is known about the cellular function of molecular chaperones in general, very little is known about the effect of temperature selection on the function of molecular chaperones in nonmodel organisms. One major unanswered question is whether orthologous variants of a molecular chaperone from differential thermally adapted species vary in their thermal responses. To address this issue, we utilized a comparative approach to examine the temperature interactions of a major cytosolic molecular chaperone, Hsc70, from differently thermally adapted notothenioids. Using in vitro assays, we measured the ability of Hsc70 to prevent thermal aggregation of lactate dehydrogenase (LDH). We further compared the capacity of Hsc70 to refold chemically denatured LDH over the temperature range of -2 to +45 degrees C. Hsc70 purified from the temperate species exhibited greater ability to prevent the thermal denaturation of LDH at 55 degrees C compared with Hsc70 from the cold-adapted species. Furthermore, Hsc70 from the Antarctic species lost the ability to competently refold chemically denatured LDH at a lower temperature compared with Hsc70 from the temperate species. These data indicate the function of Hsc70 in notothenioid fishes maps onto their thermal history and that temperature selection has acted on these molecular chaperones.

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The Antarctic fishes, isolated over evolutionary history in the sub-zero waters of the Southern Ocean, are an ideal group for studying the processes of cold adaptation. One species of Antarctic notothenioid fish, Trematomus bernacchii, has lost the ability to induce heat shock proteins (Hsps) in response to exposure to acute thermal stress or to the toxic heavy metal cadmium, an important part of the cellular defense response to such stressors. To elucidate the mechanism responsible for the lack of Hsp induction, we examined several stages of the hsp gene expression pathway, including transcription factor activity, Hsp70 mRNA production and protein synthesis patterns, in hepatocytes from T. bernacchii. Hsp70 mRNA was detected, as was heat shock factor 1 (HSF1) with DNA-binding activity. However, exposure to elevated temperature and to chemical inducers of the heat shock response failed to increase Hsp70 mRNA levels, HSF1 activity or the concentration of any size class of Hsps. These results suggest that Hsps, inducible in nearly every other species, are expressed constitutively in the cold-adapted T. bernacchii.

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The role and function of molecular chaperones has been widely studied in model systems (e.g. yeast, Escherichia coli and cultured mammalian cells), however, comparatively little is known about the function of molecular chaperones in eurythermal ectotherms. To investigate the thermal sensitivity of molecular chaperone function in non-model ectotherms, we examined the in vitro activity of Hsc70, a constitutively expressed member of the 70-kDa heat-shock protein gene family, purified from white muscle of the eurythermal marine goby Gillichthys mirabilis. The activity of G. mirabilis Hsc70 was assessed with an in vitro refolding assay where the percent refolding of thermally denatured luciferase was monitored using a luminometer. Assays were conducted from 10-40 degrees C, a range of temperatures that is ecologically relevant for this estuarine species. The results showed that isolated Hsc70 displayed chaperone characteristics in vitro, and was relatively thermally insensitive across the range of experimental temperatures. In addition, the thermal stability of the luciferase refolding capacity of Hsc70 was relatively stable, with refolding activity occurring as high as 50 degrees C. Overall, Hsc70 from G. mirabilis displayed thermal properties in vitro that suggest that the molecular chaperone is capable of binding and chaperoning proteins at temperatures that the goby encounters in nature.

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Previous research on the Antarctic notothenioid fish Trematomus bernacchii demonstrated the loss of the heat shock response (HSR), a classical cellular defense mechanism against thermal stress, characterized by the rapid synthesis of heat shock proteins (Hsps). In the current study, we examined potential mechanisms for the apparent loss of the HSR in Antarctic notothenioids and, in addition, compared expression patterns of two genes from the 70-kDa Hsp family (hsc71 and hsp70) in tissues from T. bernacchii to expression patterns in tissues of two closely related temperate notothenioid fishes from New Zealand, Bovichtus variegatus and Notothenia angustata. The results showed that transcript for both the constitutive and inducible genes in the Hsp70 gene family were expressed in detectable levels in all three species. However, only the cold-temperate New Zealand fishes displayed the ability to upregulate the inducible transcript, hsp70. Although hsp70 was present in detectable levels in several tissues of the Antarctic notothen T. bernacchii, in vitro thermal stresses failed to produce a significant increase in mRNA levels. In all species, the expression of the constitutive transcript hsc71 was variable and nonresponsive to temperature increases, even at temperatures as high as 10 degrees C above the ecologically relevant range for the species under study. Field-collected tissues from T. bernacchii (sampled immediately after capture) indicated that hsp70 mRNA was expressed at high levels in field-acclimatized fishes. Thus upregulation of molecular chaperones suggested that low-temperature stress may be significantly denaturing to cellular proteins in Antarctic fish, an observation that was supported by elevated levels of ubiquitin-conjugated protein.

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The intertidal zone has historically functioned as an important natural laboratory for testing ideas about how physical factors such as temperature influence organismal physiology and in turn influence the distribution patterns of organisms. Key to our understanding of how the physical environment helps structure organismal distribution is the identification of physiological processes that have ecological relevance. We have focused on biochemical- and molecular-level physiology that would contribute to thermal tolerance and maintenance of a functional intracellular protein pool in the face of extreme and fluctuating environmental temperatures. Past research has addressed processes central to protein homeostasis (e.g., protein ubiquitination) and the molecular ecology of molecular chaperones, a.k.a. heat shock proteins (Hsps), in ectothermic animals. In this presentation, we focus on two new developments regarding the biology of heat shock proteins as molecular chaperones in intertidal organisms. First, we present data on the functional characteristics of the transcriptional factor, HSF1 and discuss how these data relate to the plasticity of Hsp gene expression observed in intertidal organisms in nature. Second, we present data on the biochemical function of heat shock proteins purified from our non-model study organisms and discuss the temperature relationships of these molecules as they assist in protein folding in situ.