Tissue-specific effects of temperature and salinity on the cell cycle and apoptosis in the Nile Tilapia (Oreochromis niloticus).

The Nile Tilapia (Oreochromis niloticus) evolved in warm, freshwater rivers, but possesses a broad physiological tolerance to a range of environmental conditions. Due to this hardiness and resilience, this species has been successfully introduced to regions widely outside of its native range. Here, we examine the impact of temperature and salinity variation on this species at the sub-lethal level. Specifically, Nile Tilapia were exposed to two temperatures (21 °C or 14 °C) and three salinities (0, 16, 34 ppt) for 1-h. Given their native habitat, the 21 °C / 0 ppt exposure was considered the control condition. Both cell cycle arrest and apoptosis represent sub-lethal but deleterious responses to environmental stress. Flow cytometry was used to assess the percentage of cells in a given stage of the cell cycle as a metric of cell cycle arrest in spleen and liver. Percentage of apoptotic cells were also quantified. Spleen was more sensitive to cold stress, demonstrating an increase in cells in the G2/M phase after experimental treatment. Liver, however, was more sensitive to salinity stress, with a significant increase in cells stalled in G2/M phase at higher salinities, which is in keeping with the freshwater evolutionary history of the species. A modest apoptotic signal was observed in liver but not in spleen. Together, these findings demonstrate that even short, acute exposures to cold temperatures and elevated salinity can cause sub-lethal damage in a species that is otherwise tolerant of environmental stress at the whole organism level.

Life on the Edge-the Biology of Organisms Inhabiting Extreme Environments: An Introduction to the Symposium.

Life persists, even under extremely harsh conditions. While the existence of extremophiles is well known, the mechanisms by which these organisms evolve, perform basic metabolic functions, reproduce, and survive under extreme physical stress are often entirely unknown. Recent technological advances in terms of both sampling and studying extremophiles have yielded new insight into their evolution, physiology and behavior, from microbes and viruses to plants to eukaryotes. The goal of the "Life on the Edge-the Biology of Organisms Inhabiting Extreme Environments" symposium was to unite researchers from taxonomically and methodologically diverse backgrounds to highlight new advances in extremophile biology. Common themes and new insight that emerged from the symposium included the important role of symbiotic associations, the continued challenges associated with sampling and studying extremophiles and the important role these organisms play in terms of studying climate change. As we continue to explore our planet, especially in difficult to reach areas from the poles to the deep sea, we expect to continue to discover new and extreme circumstances under which life can persist.

Total Mercury in Six Antarctic Notothenioid Fishes.

We analyzed white muscle samples from six species of Antarctic fish (suborder Notothenioidei) collected in 2011 from McMurdo Sound, Ross Sea, Antarctica, to assess levels of total mercury (THg). Gymnodraco acuticeps and Trematomus bernacchii exhibited the highest concentrations of THg followed by Trematomus pennellii, Trematomus nicolai, Trematomus newnesi and Pagothenia borchgrevinki, (71.3, 53.9±32.1, 45.8±27.3, 37.2±18.6, 35.7±23.6, and 21.9±2.8 ng/g wet weight, respectively). The results from this study suggest that THg has the potential to bioaccumulate from various marine Antarctic ecosystems into biota.

Transcriptomic responses to environmental temperature in eurythermal and stenothermal fishes.

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.

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.

Cardiovascular oxygen transport limitations to thermal niche expansion and the role of environmental Po2 in Antarctic notothenioid fishes.

The notothenioid fishes of the Southern Ocean possess some of the lowest upper thermal thresholds of any species and display a range of cardiovascular features that distinguish them from other fishes. Some species lack hemoglobin, and it has been posited that the inability to deliver sufficient oxygen at elevated temperature may in part determine upper thermal thresholds. Here, we provide an analysis of systemic O2 transport based on circulatory resistance, cardiac outputs, and cardiac power for three species of Antarctic fishes, including species that possess hemoglobin (Trematomus bernacchii, Pagothenia borchgrevinki) and a species lacking hemoglobin (Chaenocephalus aceratus) and that differ in their cardiovascular characteristics. This analysis supports the hypothesis that the mutation resulting in the lack of hemoglobin would be metabolically prohibitive at elevated temperatures. The analysis also suggests that such a mutation would be least detrimental to species with greater cardiac power outputs and lower total peripheral resistance. Decreased environmental Po2 has the greatest detrimental effect on the metabolic capacity in the species without hemoglobin. These data indicate that differences in cardiovascular characteristics of the notothenioid fishes place varying limits on thermal niche expansion in these species, but any significant increase in environmental temperature or decrease in environmental Po2 will prohibit maintenance of cardiovascular systemic O2 transport in all species. These data also suggest an evolutionary sequence of events such that a reduction in hematocrit, to reduce blood viscosity and resistance, was a first step in the invasion of low-temperature habitats and loss of hemoglobin was followed by increased cardiac power output to achieve sustainable metabolic rates.

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

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.

Expression of HSP70 in Mytilus californianus following exposure to caffeine.

Caffeine, a biologically active drug with many known molecular targets, is recognized as a contaminant of marine systems. Although the concentrations of caffeine reported from aquatic systems are low (ng/l-µg/l), harmful ecological effects not detected by traditional toxicity tests could occur as a result of caffeine contamination. We used Hsp70, a molecular biomarker of cellular stress, to investigate the sub-lethal cellular toxicity of environmentally relevant concentrations of caffeine on the mussel Mytilus californianus, a dominant species in the rocky intertidal zone along the Oregon Coast. Hsp70 concentrations in the gill and mantle tissue of mussels exposed to 0.05, 0.2, and 0.5 µg/l of caffeine for 10, 20, and 30 days were compared to basal levels in control mussels. Hsp70 in the gill tissue of M. californianus had an initial attenuation of the stress protein followed by a significant up-regulation relative to controls in all but the 0.5 µg/l treatment. Hsp70 in the mantle tissue of mussels exposed to caffeine did not differ from control mussels. This study provides laboratory evidence that environmentally relevant concentrations of caffeine can exert an effect on M. californianus gill tissue at the molecular-level.

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 degrees C) and cold (15 degrees) 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.

Comparative environmental genomics in non-model species: using heterologous hybridization to DNA-based microarrays.

The emerging field of comparative environmental genomics involves the cross-species comparison of broad-scale patterns of gene expression. Often, the goal is to elucidate the evolutionary basis or ecological implications of genomic responses to environmental stimuli. DNA-based microarrays represent powerful means with which to investigate gene expression, and the application of genomic tools to studies on non-model species is becoming increasingly feasible. The use of a microarray generated from one species to probe gene expression in another, a method termed 'heterologous hybridization', eliminates the need to fabricate novel microarray platforms for every new species of interest. In this review, recent advances in heterologous hybridization are reviewed, and the technical caveats of this approach are discussed.

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

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.

Magnitude and duration of thermal stress determine kinetics of hsp gene regulation in the goby Gillichthys mirabilis.

The stress-induced transcription of heat shock genes is controlled by heat shock transcription factor 1 (HSF1), which becomes activated in response to heat and other protein denaturants. In previous research on the eurythermal goby Gillichthys mirabilis, thermal activation of HSF1 was shown to vary as a function of acclimation temperature, suggesting the mechanistic importance of HSF1 activation to the plasticity of heat shock protein (Hsp) induction temperature. We examined the effect of season on the thermal activation of HSF1 in G. mirabilis, as well as the relative kinetics of HSF1 activation and Hsp70 mRNA production at ecologically relevant temperatures. There was no predictable seasonality in the thermal activation of HSF1, perhaps due to the existence of stressors, in addition to heat, acting in the field. Concentrations of Hsp70, a negative regulator of HSF1, as well as those of HSF1, varied with collection date. The rapidity of HSF1 activation and of Hsp70 mRNA synthesis increased with laboratory exposure temperature. Furthermore, Hsp70 mRNA production was more sustained at 35 degrees C than at 30 degrees C. Therefore, both the magnitude and the duration of a heat shock are important in determining the intensity of heat shock gene induction.

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

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.

Thermal acclimation changes DNA-binding activity of heat shock factor 1 (HSF1) in the goby Gillichthys mirabilis: implications for plasticity in the heat-shock response in natural populations.

The intracellular build-up of thermally damaged proteins following exposure to heat stress results in the synthesis of a family of evolutionarily conserved proteins called heat shock proteins (Hsps) that act as molecular chaperones, protecting the cell against the aggregation of denatured proteins. The transcriptional regulation of heat shock genes by heat shock factor 1 (HSF1) has been extensively studied in model systems, but little research has focused on the role HSF1 plays in Hsp gene expression in eurythermal organisms from broadly fluctuating thermal environments. The threshold temperature for Hsp induction in these organisms shifts with the recent thermal history of the individual but the mechanism by which this plasticity in Hsp induction temperature is achieved is unknown. We examined the effect of thermal acclimation on the heat-activation of HSF1 in the eurythermal teleost Gillichthys mirabilis. After a 5-week acclimation period (at 13, 21 or 28 degrees C) the temperature of HSF1 activation was positively correlated with acclimation temperature. HSF1 activation peaked at 27 degrees C in fish acclimated to 13 degrees C, at 33 degrees C in the 21 degrees C group, and at 36 degrees C in the 28 degrees C group. Concentrations of both HSF1 and Hsp70 in the 28 degrees C group were significantly higher than in the colder acclimated fish. Plasticity in HSF1 activation may be important to the adjustable nature of the heat shock response in eurythermal organisms and the environmental control of Hsp gene expression.

Molecular chaperones in ectothermic marine animals: biochemical function and gene expression.

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.