Capacity-limited Thermal Tolerance Research Articles

Relatedness of hypoxia and hyperthermia tolerances in the Nile tilapia (Oreochromis niloticus) and their relationships with cardiac and gill traits

In fish, thermal and hypoxia tolerances may be functionally related, as suggested by the oxygen- and capacity-limited thermal tolerance (OCLTT) concept, which explains performance failure at high temperatures due to limitations in oxygen delivery. In this study the interrelatedness of hyperthermia and hypoxia tolerances in the Nile tilapia (Oreochromis niloticus), and their links to cardiorespiratory traits were examined. Different groups of O. niloticus (n = 51) were subjected to hypoxia and hyperthermia challenges and the O2 tension for aquatic surface respiration (ASR pO2) and critical thermal maximum (CTmax) were assessed as measurement endpoints. Gill filament length, total filament number, ventricle mass, length and width were also measured. Tolerance to hypoxia, as evidenced by ASR pO2 thresholds of the individual fish, was highly variable and varied between 0.26 and 3.39 kPa. ASR events increased more profoundly as O2 tensions decreased below 2 kPa. The CTmax values recorded for the O. niloticus individuals ranged from 43.1 to 44.8 °C (Mean: 44.2 ± 0.4 °C). Remarkably, there was a highly significant correlation between ASR pO2 and CTmax in O. niloticus (r = −0.76, p < 0.0001) with ASR pO2 increasing linearly with decreasing CTmax. There were, however, no discernible relationships between the measured cardiorespiratory properties and hypoxia or hyperthermia tolerances. The strong relationship between hypoxia and hyperthermia tolerances in this study may be related to the ability of the cardiorespiratory system to provide oxygen to respiring tissues under thermal stress, and thus provides some support for the OCLTT concept in this species, at least at the level of the entire organism.

Thermal preference does not align with optimal temperature for aerobic scope in zebrafish (Danio rerio).

Warming is predicted to have negative consequences for fishes by causing a mismatch between oxygen demand and supply, and a consequent reduction in aerobic scope (AS) and performance. This oxygen and capacity limited thermal tolerance (OCLTT) hypothesis features prominently in the literature but remains controversial. Within the OCLTT framework, we hypothesised that fish would select temperatures that maximise their AS, and thus their performance. We tested this hypothesis using intermittent flow respirometry to measure AS at, above (+2.5°C) and below (-2.5°C) the self-selected, preferred temperature (Tpref) of individual zebrafish (Danio rerio). AS was greatest 2.5°C above Tpref, which was driven by an increase in maximal metabolic rate. This mismatch between Tpref and the optimal temperature for AS suggests that factor(s) aside from AS maximisation influence the thermal preference of zebrafish.

Controversial Roles of Oxygen in Organismal Responses to Climate Warming.

AbstractDespite the global ecological importance of climate change, controversy surrounds how oxygen affects the fate of aquatic ectotherms under warming. Disagreements extend to the nature of oxygen bioavailability and whether oxygen usually limits growth under warming, explaining smaller adult size. These controversies affect two influential hypotheses: gill oxygen limitation and oxygen- and capacity-limited thermal tolerance. Here, we promote deeper integration of physiological and evolutionary mechanisms. We first clarify the nature of oxygen bioavailability in water, developing a new mass-transfer model that can be adapted to compare warming impacts on organisms with different respiratory systems and flow regimes. By distinguishing aerobic energy costs of moving oxygen from environment to tissues from costs of all other functions, we predict a decline in energy-dependent fitness during hypoxia despite approximately constant total metabolic rate before reaching critically low environmental oxygen. A new measure of oxygen bioavailability that keeps costs of generating water convection constant predicts a higher thermal sensitivity of oxygen uptake in an amphipod model than do previous oxygen supply indices. More importantly, by incorporating size- and temperature-dependent costs of generating water flow, we propose that oxygen limitation at different body sizes and temperatures can be modeled mechanistically. We then report little evidence for oxygen limitation of growth and adult size under benign warming. Yet occasional oxygen limitation, we argue, may, along with other selective pressures, help maintain adaptive plastic responses to warming. Finally, we discuss how to overcome flaws in a commonly used growth model that undermine predictions of warming impacts.

Warmer temperature decreases the maximum length of six species of marine fishes, crustacean, and squid in New Zealand

As global oceans continue to warm and deoxygenate, it is expected that marine ectotherms will reduce in body size resulting from the interactive effects of temperature and dissolved oxygen availability. A temperature-size response describes how wild populations of ectothermic species grow faster and reach a smaller size within warmer temperatures. While temperature-size responses are well observed in marine ectotherms, the mechanisms underpinning such a reduction in body size remain debated. Here, we analyse the relative influence of temperature, dissolved oxygen concentration, and geographic location (which encompasses multiple latent variables), on the maximum body length of four fish, one crustacean, and one squid species, which inhabit shallow to deep sea (1000 m) New Zealand waters across a temperature gradient of 1.5 to 18 °C. We found that all study species displayed a temperature-size response, with the strongest response exhibited by the largest species, hoki (Macruronus novaezelandiae). We also found that temperature was more important than dissolved oxygen concentration in determining maximum body length, as dissolved oxygen levels were at or near saturation in the study area. Our results suggest that larger-bodied species may experience the strongest temperature-size responses, and support expectations from the gill-oxygen limitation theory (GOLT) and the oxygen and capacity limited thermal tolerance (OCLTT) concept that increases in oxygen demand may be size- and temperature-dependent, thus driving a reduction in maximum body length of marine ectotherms with warming.

The role of mechanistic physiology in investigating impacts of global warming on fishes.

Warming of aquatic environments as a result of climate change is already having measurable impacts on fishes, manifested as changes in phenology, range shifts and reductions in body size. Understanding the physiological mechanisms underlying these seemingly universal patterns is crucial if we are to reliably predict the fate of fish populations with future warming. This includes an understanding of mechanisms for acute thermal tolerance, as extreme heatwaves may be a major driver of observed effects. The hypothesis of gill oxygen limitation (GOL) is claimed to explain asymptotic fish growth, and why some fish species are decreasing in size with warming; but its underlying assumptions conflict with established knowledge and direct mechanistic evidence is lacking. The hypothesis of oxygen- and capacity-limited thermal tolerance (OCLTT) has stimulated a wave of research into the role of oxygen supply capacity and thermal performance curves for aerobic scope, but results vary greatly between species, indicating that it is unlikely to be a universal mechanism. As thermal performance curves remain important for incorporating physiological tolerance into models, we discuss potentially fruitful alternatives to aerobic scope, notably specific dynamic action and growth rate. We consider the limitations of estimating acute thermal tolerance by a single rapid measure whose mechanism of action is not known. We emphasise the continued importance of experimental physiology, particularly in advancing our understanding of underlying mechanisms, but also the challenge of making this knowledge relevant to the more complex reality.

Advances in understanding the impacts of global warming on marine fishes farmed offshore: Sparus aurata as a case study.

Monitoring variations in proteins involved in metabolic processes, oxidative stress responses, cell signalling and protein homeostasis is a powerful tool for developing hypotheses of how environmental variations affect marine organisms' physiology and biology. According to the oxygen- and capacity-limited thermal tolerance hypothesis, thermal acclimation mechanisms such as adjusting the activities of enzymes of intermediary metabolism and of antioxidant defence mechanisms, inducing heat shock proteins (Hsps) or activating mitogen-activated protein kinases may all shift tolerance windows. Few studies have, however, investigated the molecular, biochemical and organismal responses by fishes to seasonal temperature variations in the field to link these to laboratory findings. Investigation of the impacts of global warming on fishes farmed offsore, in the open sea, can provide a stepping stone towards understanding effects on wild populations because they experience similar environmental fluctuations. Over the last 30 years, farming of the gilthead sea bream Sparus aurata (Linnaeus 1758) has become widespread along the Mediterranean coastline, rendering this species a useful case study. Based on available information, the prevailing seasonal temperature variations expose the species to the upper and lower limits of its thermal range. Evidence for this includes oxygen restriction, reduced feeding, reduced responsiveness to environmental stimuli, plus a range of molecular and biochemical indicators that change across the thermal range. Additionally, close relationships between biochemical pathways and seasonal patterns of metabolism indicate a connection between energy demand and metabolic processes on the one hand, and cellular stress responses such as oxidative stress, inflammation and autophagy on the other. Understanding physiological responses to temperature fluctuations in fishes farmed offshore can provide crucial background information for the conservation and successful management of aquaculture resources in the face of global change.

Growth and energy storage responses vary seasonally in the Australasian snapper Chrysophrys auratus with only modest changes in aerobic scope

Many temperate marine species cope with profound seasonal changes in temperature. One way in which these species have adapted to these conditions is by adopting life-history traits that employ seasonally dependent growth, maximising growth in the warmer summer period before experiencing a period of negligible growth and maintenance in the cooler winter period. This strategy is considered to ensure that temperate species survive the unproductive winter period. However, in the field of eco-physiology, the inability to grow in low temperature environments is considered to result from physiological limitations on growth and digestion imposed by low aerobic scope (AS) at low temperatures. In this study, we investigate the seasonal growth, bioenergetic changes and metabolic oxygen requirements (including AS) of the Australasian snapper (Sparidae: Chrysophrys auratus) over natural seasonal cycles. We demonstrate that snapper undergo marked growth over a 7 mo period spanning spring, summer and autumn, then negligible (or even negative) growth in the winter. These growth responses coincide with marked changes in physiological character, including changes in energy storage, body composition, gonadal development and haematological variation. The biological changes observed occur in combination with a broad AS curve that was relatively insensitive to the seasonal temperatures experienced in their natural range. Within this broad AS curve, variations in growth rate could not be explained by changes in AS, and vastly different rates of biological activity were observed despite only modest change in AS availability. The relevance of the oxygen capacity-limited thermal tolerance framework to the seasonal responses of snapper is discussed.

Heat tolerance and thermal preference of the copepod Tigriopus californicus are insensitive to ecologically relevant dissolved oxygen levels

Shifting climate patterns may impose novel combinations of abiotic conditions on animals, yet understanding of the present-day interactive effects of multiple stressors remains under-developed. We tested the oxygen and capacity limited thermal tolerance (OCLTT) hypothesis and quantified environmental preference of the copepod Tigriopus californicus, which inhabits rocky-shore splashpools where diel fluctuations of temperature and dissolved oxygen (DO) are substantial. Egg-mass bearing females were exposed to a 5 h heat ramp to peak temperatures of 34.1–38.0 °C crossed with each of four oxygen levels: 22, 30, 100 and 250% saturation (4.7–5.3, 5.3–6.4, 21.2–21.3, and 50.7–53.3 kPa). Survival decreased at higher temperatures but was independent of DO. The behavioral preference of females was quantified in seven combinations of gradients of both temperature (11–37 °C) and oxygen saturation (17–206% or 3.6–43.6 kPa). Females avoided high temperatures regardless of DO levels. This pattern was more pronounced when low DO coincided with high temperature. In uniform temperature treatments, the distribution shifted toward high DO levels, especially in uniform high temperature, confirming that Tigriopus can sense environmental pO2. These results question the ecological relevance of OCLTT for Tigriopus and raise the possibility of microhabitat selection being used within splashpool environments to avoid physiologically stressful combinations of conditions.

Oxygen supply limits the chronic heat tolerance of locusts during the first instar only

Although scientists know that overheating kills many organisms, they do not agree on the mechanism. According to one theory, referred to as oxygen- and capacity-limitation of thermal tolerance, overheating occurs when a warming organism’s demand for oxygen exceeds its supply, reducing the organism’s supply of ATP. This model predicts that an organism’s heat tolerance should decrease under hypoxia, yet most terrestrial organisms tolerate the same amount of warming across a wide range of oxygen concentrations. This point is especially true for adult insects, who deliver oxygen through highly efficient respiratory systems. However, oxygen limitation at high temperatures may be more common during immature life stages, which have less developed respiratory systems. To test this hypothesis, we measured the effects of heat and hypoxia on the survival of South American locusts (Schistocerca cancellata) throughout development and during specific instars. We demonstrate that the heat tolerance of locusts depends on oxygen supply during the first instar but not during later instars. This finding provides further support for the idea that oxygen limitation of thermal tolerance depends on respiratory performance, especially during immature life stages.

Seasonal cellular stress responses of commercially important invertebrates at different habitats of the North Aegean Sea

In many aquatic species, the negative effect of temperature variations has a significant impact on physiological performance since beyond Tp (upper pejus) and Tc (critical temperatures), according to the oxygen- and capacity-limited thermal tolerance (OCLTT), transition to hypoxemia and mitochondrial metabolism triggers the increase in reactive oxygen species (ROS) production. However, climate change may have different spatial impact, and as a result, areas with more favorable climatic conditions (refugia) can be identified. The aim of the present study, based on cellular stress responses, is the demarcation of these areas and the preservation of commercially important marine species. Under this prism, individuals of the species Callinectes sapidus (blue crab), Sepia officinalis (common cuttlefish), Holothuria tubulosa (sea cucumber) and Venus verrucosa (clam) from Thermaikos, Pagasitikos and Vistonikos gulf were collected seasonally. The results showed an increase in the levels of several stress indicators exhibiting the triggering of Heat Shock Response, MAPK activation, apoptotic phenomena and increased ubiquitilination during the summer sampling in relation to the spring and autumn samplings concerning blue crab and clam, while no changes were observed for common cuttlefish and sea cucumber. It seems that these cellular responses consist a cytoprotective mechanism against environmental thermal stress. Regarding collection sites, for all examined species, higher cellular stress levels were observed in Pagasitikos, and lower in Vistonikos gulf. This analysis of biochemical and molecular markers is expected to provide a clearer picture for the definition of “refugia” for the above species.

Using aerobic exercise to evaluate sub-lethal tolerance of acute warming in fishes

ABSTRACTWe investigated whether fatigue from sustained aerobic swimming provides a sub-lethal endpoint to define tolerance of acute warming in fishes, as an alternative to loss of equilibrium (LOE) during a critical thermal maximum (CTmax) protocol. Two species were studied, Nile tilapia (Oreochromis niloticus) and pacu (Piaractus mesopotamicus). Each fish underwent an incremental swim test to determine gait transition speed (UGT), where it first engaged the unsteady anaerobic swimming mode that preceded fatigue. After suitable recovery, each fish was exercised at 85% of their own UGT and warmed 1°C every 30 min, to identify the temperature at which they fatigued, denoted as CTswim. Fish were also submitted to a standard CTmax, warming at the same rate as CTswim, under static conditions until LOE. All individuals fatigued in CTswim, at a mean temperature approximately 2°C lower than their CTmax. Therefore, if exposed to acute warming in the wild, the ability to perform aerobic metabolic work would be constrained at temperatures significantly below those that directly threatened survival. The collapse in performance at CTswim was preceded by a gait transition qualitatively indistinguishable from that during the incremental swim test. This suggests that fatigue in CTswim was linked to an inability to meet the tissue oxygen demands of exercise plus warming. This is consistent with the oxygen and capacity limited thermal tolerance (OCLTT) hypothesis, regarding the mechanism underlying tolerance of warming in fishes. Overall, fatigue at CTswim provides an ecologically relevant sub-lethal threshold that is more sensitive to extreme events than LOE at CTmax.

Thermal performance of the European flat oyster, Ostrea edulis (Linnaeus, 1758)—explaining ecological findings under climate change

Climate change challenges marine organisms by constraining their temperature-dependent scope for performance, fitness, and survival. According to the concept of Oxygen and Capacity Limited Thermal Tolerance (OCLTT), the overall thermal performance curve relates to an organism’s aerobic power budget, its overall aerobic scope for growth, exercise, reproduction, and other performances. We hypothesize that physiological principles shaping tolerance in extant ecosystems have also been operative during climatic changes in the distant past. To compare response patterns in extant fauna and their palaeo-relatives, we started here by studying the metabolic background of performance in the European flat oyster Ostrea edulis at organismic and cellular levels, focusing on the acute thermal window and the metabolic changes towards upper lethal temperatures. We investigated the response of the oysters (pre-acclimated at 12 °C) to a short-term warming protocol (by 2 °C every 48 h) from 14 to 36 °C which we identified as the lethal temperature. At the organismic level, heart and filtration rates were recorded. Gill metabolites were studied by 1H NMR spectroscopy to address thermal responses at the cellular level. Feeding activity by O. edulis (assessed by the filtration rates) was highest between 18 and 24 °C when overall energy expenditure (indicated by heart rate as a proxy for routine metabolic rate) was moderate. We conclude that this range reflects the thermal optimum of this species. Beyond 26 °C, the gill tissue of O. edulis became partly anaerobic, and cardiac dysfunction (arrhythmia) developed at 28 °C followed by an Arrhenius break point (30 °C). This mirrors performance constraints and indicates a wide temperature range of passive tolerance which may be a long-standing characteristic of ostreids supporting survival in extreme environments as well as during past and present climate oscillations.

The effect of ambient oxygen on the thermal performance of a cockroach, Nauphoeta cinerea.

The oxygen and capacity-limited thermal tolerance (OCLTT) hypothesis proposes that the thermal tolerance of an animal is shaped by its capacity to deliver oxygen in relation to oxygen demand. Studies testing this hypothesis have largely focused on measuring short-term performance responses in animals under acute exposure to critical thermal maximums. The OCLTT hypothesis, however, emphasises the importance of sustained animal performance over acute tolerance. The present study tested the effect of chronic hypoxia and hyperoxia during development on moderate to long-term performance indicators at temperatures spanning the optimal temperature for growth in the speckled cockroach, Nauphoeta cinerea In contrast to the predictions of the OCLTT hypothesis, development under hypoxia did not significantly reduce growth rate or running performance, and development under hyperoxia did not significantly increase growth rate or running performance. The effects of developmental temperature and oxygen on tracheal morphology and metabolic rate were also not consistent with OCLTT predictions, suggesting that oxygen delivery capacity is not the primary driver shaping thermal tolerance in this species. Collectively, these findings suggest that the OCLTT hypothesis does not explain moderate to long-term thermal performance in N.cinerea, which raises further questions about the generality of the hypothesis.

Temperature-related heart rate in water and air and a comparison to other temperature-related measures of performance in the fiddler crab Leptuca pugilator (Bosc 1802).

Performance in poikilotherms is known to be sensitive to temperature, often with a low-sloping increase with temperature to a peak, and a steep decline with increasing temperature past the peak. We complemented past measures of performance by measuring heartbeat rates of the fiddler crab Leptuca pugilator in water and in air as a function of a range of temperatures previously shown to affect other measures of performance. In water over a range of 20-50°C, heartbeat increased steadily to a peak at 40°C and then steeply declined to near zero at 50°C. In air, heartbeat also increased, but to a peak at 35°C and then with a gentler decline than was found in water. Part of this different response may be due to evaporative water loss, which reduced body temperature in air, and therefore thermal stress, relative to body temperature when crabs were immersed in water. Increased availability of oxygen from air, according to the oxygen and capacity-limited thermal tolerance hypothesis, likely increased aerobic scope past the thermal peak, relative to within water, where oxygen delivery at higher temperatures may have been curtailed. We compared the heart rate performance relations to two previous measures of performance - endurance on a treadmill and sprint speed, both done in air. The peak performance temperature increased in the order: treadmill endurance time, sprint speed, heart rate in air, and heart rate in water, which demonstrates that different performance measures give different perspectives on the relation of thermal tolerance and fitness to temperature. Endurance may therefore be the limiting upper thermal stress factor in male fiddler crabs, when on hot sand flats. Temperature preference, found to be for temperatures <30°C in air, could be a bet-hedging evolutionary strategy to avoid aerobic scope affecting endurance.

Oxygen Limitation Does Not Drive the Decreasing Heat Tolerance of Grasshoppers during Development.

Thermal physiology changes as organisms grow and develop, but we do not understand what causes these ontogenetic shifts. According to the theory of oxygen- and capacity-limited thermal tolerance, an organism's heat tolerance should change throughout ontogeny as its ability to deliver oxygen varies. As insects grow during an instar, their metabolic demand increases without a proportional increase in the size of tracheae that supply oxygen to the tissues. If oxygen delivery limits heat tolerance, the mismatch between supply and demand should make insects more susceptible to heat and hypoxia as they progress through an instar. We tested this hypothesis by measuring the heat tolerance of grasshoppers (Schistocerca americana) on the second and seventh days of the sixth instar, in either a normoxic or a hypoxic atmosphere (21% or 10% O2, respectively). As expected, heat tolerance decreased as grasshoppers grew larger. Yet contrary to expectation, hypoxia had no effect on heat tolerance across all stages and sizes. Although heat tolerance declines as grasshoppers grow, this pattern must stem from a mechanism other than oxygen limitation.

Hypoxia but not shy-bold phenotype mediates thermal preferences in a threatened freshwater fish, Notropis percobromus.

For ectothermic animals, ambient temperature strongly influences developmental growth rate and individual fitness. While many ectotherms live in environments that are spatially hetero-thermal, the coupling between behavioural phenotypes (e.g., shy or bold behaviour) and thermal preferences remains uncertain. Relative to shy counterparts, bolder phenotypes may exert higher preference for ambient temperatures that are closer to their thermal optimum, thereby accelerating development. In addition, ectotherms should select colder temperatures in low oxygen conditions (hypoxia) according to the oxygen- and capacity-limited thermal tolerance (OCLTT) hypothesis. Using wild caught carmine shiner (Notropis percobromus), this study examined thermoregulatory behaviour in individuals exhibiting consistent behavioural phenotypes along the shy-bold continuum and between ecologically relevant normal oxygen concentration (normoxic) and hypoxic treatments. Furthermore, the behaviour observed in the laboratory was compared to environmental data from the natal stream. Results demonstrated that individual shy-bold behavioural phenotype was consistent before and after a simulated aerial predator attack, indicating consistency of behaviour across situations. Individual preferred and avoidance temperatures varied substantially, but were unrelated to shy-bold behavioural phenotypes. In contrast, individual preferred and maximum avoidance temperatures were significantly reduced in hypoxia, consistent with the OCLTT hypothesis. These findings might indicate suppressed development rates in hypoxia, not only by the limited oxygen for aerobic metabolism, but also by the preference for colder water in hypoxia. Furthermore, the tolerated thermal ranges were reduced in hypoxia. Using test conditions confirmed by field data, our study demonstrates the strong influence of oxygen availability on thermoregulatory behaviours and preferences in aquatic environments.

Oxygen limitation is not the cause of death during lethal heat exposure in an insect.

Oxygen- and capacity-limited thermal tolerance (OCLTT) is a controversial hypothesis claiming to explain variation in, and mechanistically determine, animal thermal limits. The lack of support from Insecta is typically argued to be a consequence of their high-performance respiratory systems. However, no studies have reported internal body oxygen levels during thermal ramping so it is unclear if changes in ambient gas are partially or fully offset by a compensatory respiratory system. Here we provide such an assessment by simultaneously recording haemolymph oxygen (pO2) levels-as an approximation of tissue oxygenation-while experimentally manipulating ambient oxygen and subjecting organisms to thermal extremes in a series of thermolimit respirometry experiments using pupae of the butterfly Pieris napi. The main results are that while P. napi undergo large changes in haemolymph pO2 that are positively correlated with experimental oxygen levels, haemolymph pO2 is similar pre- and post-death during thermal assays. OCLTT predicts that reduction in body oxygen level should lead to a reduction in CTmax. Despite finding the former, there was no change in CTmax across a wide range of body oxygen levels. Thus, we argue that oxygen availability is not a functional determinant of the upper thermal limits in pupae of P. napi.

Approaches to Reconsider Literature on Physiological Effects of Environmental Change: Examples From Ocean Acidification Research

Understanding links between the abiotic environment and organism fitness and function is a central challenge of biology, and an issue of growing relevance due to anthropogenic environmental changes. To date, our understanding of these links has largely been based on the findings of isolated experimental studies. This command may, however, be enhanced where currently disparate data are synthesized. By outlining a range of approaches appropriate in bringing together the findings of studies considering ocean acidification effects, we hope to provide insight as to how they may be used in in the future. Specifically, approaches discussed in this narrative literature review include established literature review methods, as well as emerging schemes structured around biological theories (i.e. dynamic energy budget, oxygen- and capacity-limited thermal tolerance, multiple performance-multiple optima), and strategies developed in other disciplines (i.e. adverse outcome pathways). In the future approaches to use such frameworks in creative combinations may be developed. Regardless of the approach taken, synthesizing scientific literature represents a potentially powerful method to enhance understanding of the influence of the abiotic environment on whole organism fitness.

Physiological and regulatory underpinnings of geographic variation in reptilian cold tolerance across a latitudinal cline.

Understanding the mechanisms that produce variation in thermal performance is a key component to investigating climatic effects on evolution and adaptation. However, disentangling the effects of local adaptation and phenotypic plasticity in shaping patterns of geographic variation in natural populations can prove challenging. Additionally, the physiological mechanisms that cause organismal dysfunction at extreme temperatures are still largely under debate. Using the green anole, Anolis carolinensis, we integrate measures of cold tolerance (CTmin ), standard metabolic rate, heart size, blood lactate concentration and RNAseq data from liver tissue to investigate geographic variation in cold tolerance and its underlying mechanisms along a latitudinal cline. We found significant effects of thermal acclimation and latitude of origin on variation in cold tolerance. Increased cold tolerance correlates with decreased rates of oxygen consumption and blood lactate concentration (a proxy for oxygen limitation), suggesting elevated performance is associated with improved oxygen economy during cold exposure. Consistent with these results, co-expression modules associated with blood lactate concentration are enriched for functions associated with blood circulation, coagulation and clotting. Expression of these modules correlates with thermal acclimation and latitude of origin. Our findings support the oxygen and capacity-limited thermal tolerance hypothesis as a potential contributor to variation in reptilian cold tolerance. Moreover, differences in gene expression suggest regulation of the blood coagulation cascade may play an important role in reptilian cold tolerance and may be the target of natural selection in populations inhabiting colder environments.

AMPK affects thermal tolerance in decapod crustaceans

AMP‐activated protein kinase (AMPK) is a cellular master regulator of energy metabolism, that is activated during stress to ensure a stable ATP pool in the cell. Additional to substantial work on the role of AMPK in mammals, especially humans, it becomes increasingly clear that AMPK is also involved in multiple processes in marine invertebrates, like crustaceans. Following the Oxygen and Capacity‐Limited Thermal Tolerance Hypothesis (OCLTT) marine invertebrates' thermal tolerance is dependent on sufficient oxygen delivery to the tissues and subsequent cellular ATP supply. Recent work has shown AMPK activation during heat stress in the rock crab, Cancer irroratus and the lobster, Homarus americanus. However, heat stress did not activate AMPK in the green crab Carcinus maenas. In the current project we tested whether pharmaceutically activating AMPK prior to a heat stress affects subsequent thermal tolerance. We injected Metformin in C. irroratus and activated AMPK about 3 fold above control levels, without affecting HSP70 protein levels or glucose levels in the hemolymph, indicating that this treatment did not trigger a broader stress response. A subsequent progressive temperature challenge between 11 and 32°C led in the Metformin treated animals to a drop in the maximum heart rate from 150 to 100 BPM at 29 and 23°C (control vs. Metformin treated, respectively), and a drop of the critical temperature, indicated by a switch to anaerobic metabolism and lactate accumulation, from 30 to 26°C. Therefore, AMPK activation diminished thermal tolerance in C. irroratus. Injecting Metformin into C. maenas showed a 30% increase in AMPK activity in the gills, with no substantial increase in AMPK activity in the hepatopancreas or heart. First results on the effect on injecting another AMPK activator, AICAR, don't lead to a substantial increase in AMPK activity yet, and require further adjustments of the treatment protocol.Pharmaceutically activating AMPK and investigating the subsequent effect on stress tolerance will contribute to a mechanistic understanding of energy allocations during stress in the context of the dynamic energy budget framework.Support or Funding InformationSupported by a summer research fellowship from the American Physiological Society to P.L., and NSF grant DUE 1431955 to M.F.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.