SUMMARYExposure of marine invertebrates to high temperatures leads to a switch from aerobic to anaerobic metabolism, a drop in the cellular ATP concentration ([ATP]), and subsequent death. In mammals, AMP-activated protein kinase (AMPK) is a major regulator of cellular [ATP] and activates ATP-producing pathways, while inhibiting ATP-consuming pathways. We hypothesized that temperature stress in marine invertebrates activates AMPK to provide adequate concentrations of ATP at increased but sublethal temperatures and that AMPK consequently can serve as a stress indicator (similar to heat shock proteins, HSPs). We tested these hypotheses through two experiments with the rock crab, Cancer irroratus. First, crabs were exposed to a progressive temperature increase (6°C h -1 ) from 12 to 30°C. AMPK activity, total AMPK protein and HSP70 levels, reaction time, heart rate and lactate accumulation were measured in hearts at 2°C increments. AMPK activity remained constant between 12 and 18°C, but increased up to 9.1(±1.5)-fold between 18 and 30°C. The crabsʼ reaction time also decreased above 18°C. By contrast, HSP70 (total and inducible) and total AMPK protein expression levels did not vary significantly over this temperature range. Second, crabs were exposed for up to 6 h to the sublethal temperature of 26°C. This prolonged exposure led to a constant elevation of AMPK activity and levels of HSP70 mRNA. AMPK mRNA continuously increased, indicating an additional response in gene expression. We conclude that AMPK is an earlier indicator of temperature stress in rock crabs than HSP70, especially during the initial response to high temperatures. We discuss the temperature-dependent increase in AMPK activity in the context of Shelfordʼs law of tolerance. Specifically, we describe AMPK activity as a cellular marker that indicates a thermal threshold, called the pejus temperature, T p . At T p the animals leave their optimum range and enter a temperature range with a limited aerobic scope for exercise. This T p is reached periodically during annual temperature fluctuations and has higher biological significance than earlier described critical temperatures, at which the animals switch to anaerobic metabolism and HSP expression is induced.

Marine invertebrates in the intertidal and subtidal zones are often exposed to highly variable environmental conditions, especially rapid changes in temperature. The ability to survive at different temperatures has previously been described using an extended version of Shelford's law of tolerance, with optimum, pejus (Latin: 'turning worse'), and pessimum ranges, and the respective thresholds, critical (T c ) and pejus (T p ) temperatures, that mark the transition from one range into the next. The width of the pejus range, in which the scope for activity gradually declines, varies among species. We tested the hypothesis that the width of the pejus range is correlated to the temperature stability of the species' respective habitats. We used locomotor activity, heart rate, lactate accumulation, heat shock protein 70 (HSP70) levels, and the activation of AMPactivated protein kinase (AMPK) to identify T c and T p in 3 decapod crustaceans: green crab Carcinus maenas, rock crab Cancer irroratus, and lobster Homarus americanus. We found speciesspecific patterns of temperature-induced changes in all parameters, especially in HSP70 protein and AMPK activity. The width of the pejus range (between T p and T c ) was 8 to 12°C for rock crabs and 12 to 16°C for lobsters. Most importantly, green crab, the most temperature-tolerant of our 3 species and which lives in a highly variable habitat, switched directly from optimum to pessimum range, meaning that the pejus range was eliminated completely. Additionally, even lethal temperatures did not activate AMPK in green crabs, pointing to a different cellular tolerance strategy than in rock crabs and lobsters. This modified tolerance pattern might represent a broader strategy to enhance physiological tolerance in a highly variable habitat. KEY WORDS: AMP-activated protein kinase · AMPK · Heat shock protein 70 · HSP70 · Temperature stress · Critical temperatures Resale or republication not permitted without written consent of the publisherMar Ecol Prog Ser 444: 263-274, 2012 264 ranges at both extremes. T c characterizes the transition into the pessimum range. However, in its natural environment an animal is frequently exposed to suboptimal conditions, often well before T c is attained. Consequently, Frederich & Pörtner (2000) added 'pejus temperature' (T p ) and 'pejus range' (from the Latin for 'turning worse') to this concept. In the pejus range, which is between the optimum and pessimum ranges, animals can still survive, but with a reduced scope for aerobic activity. Subsequent studies have identified T p and pejus ranges in various species of marine invertebrates and fish (reviewed by Pört-ner 2010). T p and T c differ by several degrees, thus in dicating a well-defined and broad pejus range. However, the width of the pejus range appears to vary among species, and the outstanding question of whether this is associated with habitat stability needs to be addressed.In a previous study on the rock crab Cancer irroratus, we showed that T p can be identified on the cell...

Measurements of body temperatures in the field have shown that spatial and temporal patterns are often far more complex than previously anticipated, particularly in intertidal regions, where temperatures are driven by both marine and terrestrial climates. We examined the effects of body size, body position within the sediment, and microhabitat (presence or absence of Spartina alterniflora) on the body temperature of the mussel Geukensia demissa. We then used these data to develop a laboratory study exposing mussels to an artificial "stressful" day, mimicking field conditions as closely as possible. Results suggested that G. demissa mortality increases greatly at average daily peak temperatures of 45 degrees C and higher. When these temperatures were compared to field data collected in South Carolina in the summer of 2004, our data indicated that mussels likely experienced mortality due to high-temperature stress at this site during this period. Our results also showed that body position in the mud is the most important environmental modifier of body temperature. This experiment suggested that the presence of marsh grass leads to increases in body temperature by reducing convection, overwhelming the effects of shading. These data add to a growing body of evidence showing that small-scale thermal variability can surpass large-scale gradients.

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Sessile aquatic invertebrates are at great risk for temperature stress. Changes in ambient temperature affect metabolic demands, thus altering energy budgets, and often reducing performance or survival of these species. Zebra mussels are highly invasive, yet little is known about their physiology under biologically relevant conditions, especially with regard to cellular parameters. This study examined the effect of temperature on zebra mussel physiology and investigated whether the levels of two cellular markers, HSP70 and AMPK activity, could serve as indicators of chronic thermal stress. Mussels were collected from a site in central Illinois, slowly acclimated to either 10, 20, or 30°C, and held at these temperatures for four weeks. Size, mortality, and the cellular markers were measured. Size and mortality data indicate heat stress at 30°C. Elevation in HSP70 levels confirmed this temperature elicits a stress response. Elevation in AMPK activity was not detected at 30°C, most likely indicating this temperature is beyond the scope for this marker, and therefore at or near the lethal limit. These data suggest this zebra mussel population experiences reduced performance and potential mortality in the field during summer months. Interestingly, cold acclimation resulted in a temporary elevation in AMPK activity, a result that has not been reported previously in ectotherms and is likely attributable to the metabolic demands of thermal acclimation.