Bradley A. Buckley

The cellular response to heat stress in the goby Gillichthys mirabilis: a cDNA microarray and protein-level analysis.

SUMMARY The cellular response to stress relies on the rapid induction of genes encoding proteins involved in preventing and repairing macromolecular damage incurred as a consequence of environmental insult. To increase our understanding of the scope of this response, a cDNA microarray, consisting of 9207 cDNA clones, was used to monitor gene expression changes in the gill and white muscle tissues of a eurythermic fish, Gillichthys mirabilis (Gobiidae) exposed to ecologically relevant heat stress. In each tissue, the induction or repression of over 200 genes was observed. These genes are associated with numerous biological processes, including the maintenance of protein homeostasis, cell cycle control, cytoskeletal reorganization, metabolic regulation and signal transduction, among many others. In both tissues, the molecular chaperones, certain transcription factors and a set of additional genes with various functions were induced in a similar manner; however, the majority of genes displayed tissue-specific responses. In gill, thermal stress induced the expression of the major structural components of the cytoskeleton, whereas these same genes did not respond to heat in muscle. In muscle, many genes involved in promoting cell growth and proliferation were repressed, perhaps to conserve energy for repair and replacement of damaged macromolecules, but a similar repression was not observed in the gill. Many of the observed changes in gene expression were similar to those described in model species whereas many others were unexpected. Measurements of the concentrations of the protein products of selected genes revealed that in each case an induction in mRNA synthesis correlated with an increase in protein production, though the timing and magnitude of the increase in protein was not consistently predicted by mRNA concentration, an important consideration in assessing the condition of the stressed cell using transcriptomic analysis.

Regulation of heat shock genes in isolated hepatocytes from an Antarctic fish, Trematomus bernacchii.

SUMMARY 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.

cDNA microarray analysis reveals the capacity of the cold-adapted Antarctic fish Trematomus bernacchii to alter gene expression in response to heat stress

During their many millions of years of evolution in the extreme and stable cold, Antarctic notothenioid fishes have acquired profoundly cold-adapted physiologies. Gene expression profiling via cDNA microarray was used to determine the extent to which one species of notothenioid, Trematomus bernacchii, has retained the ability to alter gene expression in response to heat stress. While an inability to up-regulate the expression of any size class of heat shock proteins (except for a 1.1-fold induction of the co-chaperone Hsp40) was observed, hundreds of additional genes, associated with a broad range of cellular processes, were responsive to heat. Many of these genes are associated with central aspects of the evolutionarily conserved cellular stress response (CSR), which plays a pivotal role in responding to physical and chemical stresses. The inability of T. bernacchii to mount a heat shock response underscores the potential susceptibility of this species to the effects of global warming.

Molecular Chaperones in Ectothermic Marine Animals: Biochemical Function and Gene Expression

Abstract 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.

Heterologous hybridization to a complementary DNA microarray reveals the effect of thermal acclimation in the endothermic bluefin tuna (Thunnus orientalis)

The temperature stress that pelagic fishes experience can induce physiological and behavioural changes that leave a signature in gene expression profiles. We used a functional genomics approach to identify genes that were up‐ or down‐regulated following thermal stress in the Pacific bluefin tuna. Following the acclimation period, 113, 81 and 196 genes were found to be differentially expressed between the control (20 °C) and cold (15°) treatment groups, in ventricle, red muscle and white muscle, respectively. The genes whose expression levels were responsive to thermal acclimation varied according to muscle fibre type, perhaps reflecting the tissue‐specific degrees of endothermy characteristic of this species.

Transcriptomic responses to environmental temperature in eurythermal and stenothermal fishes

ABSTRACT Ectothermic species like fishes differ greatly in the thermal ranges they tolerate; some eurythermal species may encounter temperature ranges in excess of 25°C, whereas stenothermal species in polar and tropical waters live at essentially constant temperatures. Thermal specialization comes with fitness trade-offs and as temperature increases due to global warming, the physiological basis of specialization and thermal plasticity has become of great interest. Over the past 50 years, comparative physiologists have studied the physiological and molecular differences between stenothermal and eurythermal fishes. It is now well known that many stenothermal fishes have lost an inducible heat shock response (HSR). Recent advances in transcriptomics have now made it possible to examine genome-wide changes in gene expression (GE) in non-model ecologically important fish, broadening our view beyond the HSR to regulation of genes involved in hundreds of other cellular processes. Here, we review the major findings from transcriptomic studies of extreme eurythermal and stenothermal fishes in response to acute and long-term exposure to temperature, both time scales being critically important for predicting climate change responses. We consider possible molecular adaptations that underlie eurythermy and stenothermy in teleosts. Furthermore, we highlight the challenges that still face the field of comparative environmental genomics and suggest fruitful paths of future investigation. Summary: Comparative transcriptomics is rapidly accelerating our understanding of how fish respond to thermal stress; this review summarizes and synthesizes gene expression patterns from stenothermal and eurythermal species responding to acute and long-term exposure to temperature.

Sub-lethal heat stress causes apoptosis in an Antarctic fish that lacks an inducible heat shock response.

The endemic fish fauna of the Southern Ocean are cold-adapted stenotherms and are acutely sensitive to elevated temperature. Many of these species lack a heat shock response and cannot increase the production of heat shock proteins in their tissues. However, some species retain the ability to induce other stress-responsive genes, some of which are involved in cell cycle arrest and apoptosis. Here, the effect of heat on cell cycle stage and its ability to induce apoptosis were tested in thermally stressed hepatocytes from a common Antarctic fish species from McMurdo Sound in the Ross Sea. Levels of proliferating cell nuclear antigen were also measured as a marker of progression through the cell cycle. The results of these studies demonstrate that even sub-lethal heat stress can have deleterious impacts at the cellular level on these environmentally sensitive species.

Cold tolerance of the invasive Carcinus maenas in the east Pacific: molecular mechanisms and implications for range expansion in a changing climate

Physiological studies have long been utilized to understand the role of environmental temperature in the distribution of native organisms within marine communities. For the invasive crab Carcinus maenas, temperature has been implicated as the main predictor of establishment success across temperate regions. Therefore, we determined whether the lower temperature tolerances of this non-native crab would restrict it from spreading farther poleward from a relatively new recipient environment. Cold tolerance capacity was determined in the laboratory by holding crabs sampled from Vancouver Island, British Columbia (BC)—near the present northern limit for the northeast Pacific metapopulation to an overwintering thermal profile generated from Sitka, Alaska, USA. These crabs were physiologically capable of overwintering north of their present range boundary. The cellular response to cold stress was investigated using two functional categories of the cellular stress response. We measured cyclin D1, a cell-cycle regulator, and Hsp70, a protein chaperone, after laboratory acclimation and acute cold stress on two populations of C. maenas from the west coast of North America that have disparate thermal histories (crabs sampled from CA or BC). We found site-specific differential expression of cyclin D1 after cold acclimation and cold shock, perhaps affecting invasion capacity in this species. Determining what physiological mechanisms are in place with respect to thermal tolerance and preference can give insight into what makes an invasive organism successful and aid in predicting probable distribution of such species within a new environment.

Acute heat stress and thermal acclimation induce CCAAT/enhancer-binding protein delta in the goby Gillichthysmirabilis

Members of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors have regulatory control over numerous processes related to cell fate determination, including differentiation, proliferation, cell cycle arrest and apoptosis. In mammals, abnormalities in the expression of some isoforms of C/EBPs are pathogenic and are implicated as being involved in myeloid leukemia and breast cancers. Next to nothing is known about their regulation, function or stress-responsiveness in poikilotherms. Here, both acute heat stress and thermal acclimation were demonstrated to induce the expression of one isoform, C/EBP-δ, in the liver, white muscle and gill of the eurythermal estuarine goby, Gillichthys mirabilis. The established role of C/EBP-δ in causing cell cycle arrest and/or promoting apoptosis in other vertebrates suggests that the heat-inducibility of this protein in poikilotherms may be part of the conserved cellular stress response with the hypothesized role of causing temporary cessation of cell growth and/or programmed cell death during bouts of environmental stress. The observed regulation of c/ebp-δ during hyperthermia represents a novel, heat-inducible signaling pathway in fishes.

Rapid Change in Shallow Water Fish Species Composition in an Historically Stable Antarctic Environment

Abstract McMurdo Sound, Antarctica, is home to a unique marine biota with an ecology that has evolved in this frigid environment over millions of years. The region is one of the least disturbed, and possibly the last pristine, marine ecosystem on Earth. Here, the results of three seasons of fishing in the shallow nearshore waters of McMurdo Sound are reported. A shift in the composition of small fish species at one site, Inaccessible Island, has been observed in just five years. The shift in shallow water species composition occurred during a period that followed the maturation of a commercial fishery for the Antarctic toothfish, Dissostichus mawsoni Norman, a predator of smaller fish, and the presence of a large iceberg, termed B-15, at the mouth of McMurdo Sound during the early 2000s that trapped the annual sea ice in the area leading to the unusual accrual of multi-year sea ice. The data presented here provide a current record of species composition and physiological condition of small, shallow water fishes at three sites in McMurdo Sound, providing a current baseline for the assessment of future changes wrought by environmental changes and unprecedented fishery pressures in the Ross Sea.