Anne E. Todgham

Heat shock protein genes and their functional significance in fish

Despite decades of intensive investigation, important questions remain regarding the functional, ecological, and evolutionary roles of heat shock proteins. In this paper, we discuss the utility of fish as a model system to address these questions, and review the relevant studies of heat shock protein genes and the regulation of their expression in fish. Although molecular studies of the heat shock proteins in fish are still in their early descriptive phase, data are rapidly being collected. More is known about the biotic and abiotic factors regulating heat shock proteins. We briefly review these studies and focus on the role of heat shock proteins in development, their regulation by the endocrine system, and their importance in fish in nature. Functional genomics approaches will provide the tools necessary to gain a comprehensive understanding of the significance of heat shock proteins in the cellular stress response, in the physiological processes at higher levels of organization, and in the whole animal in its natural environment.

Are hsps suitable for indicating stressed states in fish

SUMMARY In response to most stressors, fish will elicit a generalized physiological stress response, which involves the activation of the hypothalamic-pituitary-interrenal axis (HPI). As in other vertebrates, this generalized stress response comprises physiological responses that are common to a wide range of environmental, physical and biological stressors. Recently, several families of heat shock proteins (hsps) have been proposed as indicators of a generalized stress response at the cellular level. Recent findings that hsp levels, in various fish tissues, respond to a wide range of stressors have supported the use of these proteins as indicators of stressed states in fish. However, the cellular stress response can vary, for example, according to tissue, hsp family and type of stressor. This brief overview of these responses in fish asks the question of whether changes in levels and families of hsps can be used as a suitable indicator of stressed states in fish. By casting this question in the context of the well-established generalized physiological stress response in fish, we argue that the use of hsps as indicators of stressed states in fish in general is premature.

Is cold the new hot? Elevated ubiquitin-conjugated protein levels in tissues of Antarctic fish as evidence for cold-denaturation of proteins in vivo

Levels of ubiquitin (Ub)-conjugated proteins, as an index of misfolded or damaged proteins, were measured in notothenioid fishes, with both Antarctic (Trematomus bernacchii, T. pennellii, Pagothenia borchgrevinki) and non-Antarctic (Notothenia angustata, Bovichtus variegatus) distributions, as well as non-notothenioid fish from the Antarctic (Lycodichthys dearborni, Family Zoarcidae) and New Zealand (Bellapiscis medius, Family Tripterygiidae), in an effort to better understand the effect that inhabiting a sub-zero environment has on maintaining the integrity of the cellular protein pool. Overall, levels of Ub-conjugated proteins in cold-adapted Antarctic fishes were significantly higher than New Zealand fishes in gill, liver, heart and spleen tissues suggesting that life at sub-zero temperatures impacts protein homeostasis. The highest tissue levels of ubiquitinated proteins were found in the spleen of all fish. Ub conjugate levels in the New Zealand N. angustata, more closely resembled levels measured in other Antarctic fishes than levels measured in other New Zealand species, likely reflecting their recent shared ancestry with Antarctic notothenioids.

The response of the tidepool sculpin, Oligocottus maculosus, to hypoxia in laboratory, mesocosm and field environments

Animals living in the intertidal zone experience regular, predictable fluctuations in physical parameters including temperature, oxygen and salinity and rely on behavioural, physiological and biochemical mechanisms to cope with environmental variation. In the present study, behavioural strategies induced by aquatic hypoxia (e.g. emergence) were performed at similar oxygen tensions across laboratory, mesocosm and field environments; the number of individuals performing these behaviours at any one time was similar in mesocosms and the field. The use of aquatic surface respiration (ASR) was more plastic than emergence behaviour, occurring at a lower oxygen tension in juveniles than adults and being influenced by the addition of alarm substance. Oxygen uptake was lower in air than in water in adults but, in contrast, oxygen uptake was not influenced by the respiratory medium in juveniles. In the laboratory, 72 h of forced emergence did not affect whole body concentrations of lactate but when ASR and emergence were prevented within mesocosm environments there was a significant elevation of lactate. The present study highlights the benefits of transcending traditional laboratory/field boundaries allowing the responses of laboratory-held animals to environmental fluctuation to be integrated with how these animals perform in their natural environment.

Testing local and global stressor impacts on a coastal foundation species using an ecologically realistic framework

Despite the abundance of literature on organismal responses to multiple environmental stressors, most studies have not matched the timing of experimental manipulations with the temporal pattern of stressors in nature. We test the interactive effects of diel-cycling hypoxia with both warming and decreased salinities using ecologically realistic exposures. Surprisingly, we found no evidence of negative synergistic effects on Olympia oyster growth; rather, we found only additive and opposing effects of hypoxia (detrimental) and warming (beneficial). We suspect that diel-cycling provided a temporal refuge that allowed physiological compensation. We also tested for latent effects of warming and hypoxia to low-salinity tolerance using a seasonal delay between stressor events. However, we did not find a latent effect, rather a threshold survival response to low salinity that was independent of early life-history exposure to warming or hypoxia. The absence of synergism is likely the result of stressor treatments that mirror the natural timing of environmental stressors. We provide environmental context for laboratory experimental data by examining field time series environmental data from four North American west coast estuaries and find heterogeneous environmental signals that characterize each estuary, suggesting that the potential stressor exposure to oysters will drastically differ over moderate spatial scales. This heterogeneity implies that efforts to conserve and restore oysters will require an adaptive approach that incorporates knowledge of local conditions. We conclude that studies of multiple environmental stressors can be greatly improved by integrating ecologically realistic exposure and timing of stressors found in nature with organismal life-history traits.

Effects of the Natural Tidal Cycle and Artificial Temperature Cycling on Hsp Levels in the Tidepool Sculpin Oligocottus maculosus

The rocky intertidal zone is characterized by a predictable cycle of environmental change cued by the ebb and flow of the tides. Tidepools are thus an excellent environment in which to determine whether predictability of environmental change can entrain an endogenous rhythmicity in heat shock protein (Hsp) levels. In this study, we monitored changes in Hsp mRNA and protein levels that occurred over the tidal cycle in tidepool sculpins and investigated whether there was an endogenous tidal rhythm in Hsp expression that persisted once the sculpins were transferred to a stable environment. Fluctuations in the tidepool environment increased hsc70, hsp70, and hsp90 mRNA levels, which translated into increased Hsc/Hsp70 and Hsp90 protein levels; however, this was not due to an endogenous tidal rhythm in Hsp levels because sculpins held under constant conditions did not show any rhythmicity in the expression of these genes. By exposing sculpins to an artificial temperature cycling regime that mimicked the temperature changes of a mid‐intertidal pool, we were able to account for the direct role of temperature in regulating Hsp expression. However, there are additional extrinsic factors that likely integrate with temperature and result in differences between the hsp induction profiles that were observed in sculpins inhabiting their natural environment and those in cycling conditions in the laboratory.

The Onset Temperature of the Heat-Shock Response and Whole-Organism Thermal Tolerance Are Tightly Correlated in both Laboratory-Acclimated and Field-Acclimatized Tidepool Sculpins (Oligocottus maculosus)

We examined the relationship between thermal tolerance, measured as critical thermal maximum (CTmax), and aspects of the heat-shock response in tidepool sculpins (Oligocottus maculosus) acclimated to constant laboratory temperatures or acclimatized to field conditions. The CTmax of fish laboratory acclimated to 6°, 13°, and 20°C were , , and , respectively, increasing linearly by 0.2°C for each 1°C increase in acclimation temperature. The CTmax of field-acclimatized fish from the low intertidal () was significantly lower than that of fish from the mid- () and high () intertidal. CTmax and the onset temperature of hsp70 induction in gill (Ton) were highly correlated in both laboratory-acclimated and field-acclimatized sculpins, with Ton occurring at 2°C below CTmax in all cases. However, there was no consistent relationship between CTmax and the maximum levels of gill hsp70 mRNA. Predicted “acclimation” temperature () and mean habitat temperature () were similar for sculpins from low intertidal pools, but this relationship was not apparent in mid- and high intertidal fish. Mark-recapture experiments indicated that approximately 80% of fish from low intertidal pools were residents of that pool, but residency rates were less than 50% in mid- and high intertidal pools, which may explain the lack of correlation between CTmax and habitat variables in these groups. These data indicate that gill hsp70 Ton and CTmax are highly correlated indicators of the thermal performance of tidepool sculpins in both laboratory and field settings.

Ocean acidification exerts negative effects during warming conditions in a developing Antarctic fish

Polar areas are experiencing some of the most rapid impacts of climate change, yet we have a limited understanding of biological vulnerability to multiple stressors. Increased temperature broadly affected early embryo physiology in the naked dragonfish, while ocean acidification interacted synergistically with temperature to decrease survival and alter developmental rate.

Effects of exercise on nitrogen excretion, carbamoyl phosphate synthetase III activity and related urea cycle enzymes in muscle and liver tissues of juvenile rainbow trout (Oncorhynchus mykiss)

The purpose of this study was to determine if carbamoyl phosphate synthetase III (CPSase III) and related urea cycle enzyme activities in skeletal muscle tissue of juvenile rainbow trout (Oncorhynchus mykiss) increase during short- or long-term exercise, in parallel with changes in whole-body urea excretion rates. Urea excretion was elevated by 65% in fish that swam at high-speed (50 cm/s) vs. low-speed (20 cm/s) over a 2-h period, with no significant changes in CPSase III, ornithine transcarbamoylase or glutamine synthetase activities in muscle tissue. Fish that swam for 4 days at high-speed had higher rates of ammonia excretion and GSase activity in muscle and liver tissue relative to low-speed swimmers. Calculations showed that 47-53% of excreted urea, theoretically could be accounted for by total muscle CPSase III activity in juvenile and adult trout. The data indicate that increases in the rate of urea excretion during short-term high intensity exercise are not linked to higher activities of urea cycle enzymes in muscle tissue, but this does not rule out the possibility of increased flux through muscle CPSase III and related enzymes. Furthermore, these results indicate that urea cycle enzyme activities in skeletal muscle tissue can account for a significant portion of total urea excretion in juvenile and adult trout.

Thermal physiology of the fingered limpet Lottia digitalis under emersion and immersion.

SUMMARY Marine animals living high in the rocky intertidal zone experience long durations of aerial emersion, sometimes enduring rapid increases in temperature. To date, much of our understanding of the thermal physiology of intertidal organisms comes from studies in which organisms are exposed to increasing temperatures when immersed, with the added effect of aerial emersion rarely considered. In this study, we examined the physiological response of the finger limpet, Lottia digitalis, to increases in temperature under both immersed and emersed conditions. We investigated the thermal sensitivity and upper temperature tolerance of limpets through assessment of cardiac performance, metabolic rate, glycogen depletion and maintenance of protein integrity. Cardiac performance in response to ecologically relevant increases in temperature was similar in emersed and immersed limpets from 15 to 35°C and showed multiple break patterns in heart rate as temperature was increased. Overall, emersed limpets had a greater upper thermal limit on cardiac performance, with the ability to maintain heart rate at a temperature 3–5°C higher than that for immersed limpets. Metabolism in limpets also differed significantly between emersion and immersion, where a significant depression in aerobic metabolic rate was observed under immersion with increasing temperature. Greater levels of ubiquitin-conjugated proteins were found under emersed conditions compared with immersed limpets. Maintaining cardiac performance and aerobic metabolism to higher temperatures under emersed conditions is likely reflective of physiological adaptations to live in an aerially exposed environment. Measured field temperatures where fingered limpets were collected demonstrated that limpets have a narrow thermal safety margin for aerobic performance, and currently experience multiple days where summer temperatures might exceed their threshold limits.