Arrhenius Break Research Articles

Effect of temperature and thermal stress on the hemodynamics of the scallop Chlamys farreri, as indicated by Doppler ultrasonography

The effect of temperature and thermal stress on cardiac activity and hemodynamics of a temperate scallop species Chlamys farreri was revealed using Doppler echocardiography. The variation scope of eight hemodynamic parameters within the optimal temperature range (8–26 °C) was determined, including heart beat rate (HR), acceleration of blood flow (ABF), peak systolic velocity (PS), end diastolic velocity (ED), maximum & minimum instantaneous blood flow (Max & Min-IBF), resistive index (RI) and PS/ED.The effects of two thermal stress factors were examined, prolonging high water temperature (28–30 °C) and fast heating (2 °C h−1), on hemodynamics of gill blood vessels. The HR-based Arrhenius break temperatures (ABTs) of the scallop under two stress factors were 29.32 °C and 32.37 °C respectively. Results showed that the scallop has a short-term tolerance to acute thermal stress compared with prolonging high temperature. When temperature rose above 28 °C, the PS/ED ratio and RI exceeded the thresholds of 3.0 and 0.63, respectively, indicating potential damage and malfunction of the blood vessels. Low water temperature (6 °C) may also have negative impacts on blood circulation. When faced with a rapidly rising temperature, the scallop cannot adjust its heartbeat in time, but in compensation, the scallop still supplied sufficient hemolymphs to the gill by expanding the diameter of the arteries. The cause of the scallop's death may be different under two thermal stresses: prolonging high temperature caused cardiac failure and physical damage of the artery, whereas rapid heating caused serious tachycardia accompanied by cardiac failure.

Thermal tolerance traits of the undulated surf clam Paphia undulata based on heart rate and physiological energetics

Thermal tolerance traits are important factors that affect the ability of an aquaculture species to survive, grow, and reproduce in new habitats. Paphia undulata, a species of saltwater clam, has been farmed in China for over thirty years. Despite this, the ecophysiology of P. undulata and its effects on the aquaculture of this species are poorly understood. To investigate the thermal tolerance of P. undulata, the semi-lethal temperature, cardiac activity, and scope for growth (SFG) of the organisms were measured under different thermal stresses in a laboratory-simulated muddy habitat condition. Our results showed that the cardiac activity, feeding behaviors, and respiration rates of P. undulata were positively correlated with temperature changes from 10 °C to 32 °C. The upper incipient lethal temperature (192-h UILT50) and Arrhenius break temperature (ABT) of this species were 33.49 ± 0.02 °C and 35.64 ± 0.77 °C, respectively. SFG trend had negative values at temperatures between 10 °C and 16 °C and positive values at temperatures between 18 °C to 32 °C. These data suggest that the suitable thermal window that provided the energy required for the efficient growth and reproduction of P. undulata in the Dongshan Bay was approximately nine months out of the year. Taken together, these results indicate that P. undulata is proficient at adapting to thermal stress in the subtidal zone. Moreover, our results may also provide information that assists in both the management of wild populations of P. undulata and the improvement of P. undulata aquaculture.

Anaerobic metabolism and thermal tolerance: The importance of opine pathways on survival of a gastropod after cardiac dysfunction.

Organisms on rocky shores are frequently exposed to high temperatures, which cause impairment of cardiac function and retard cellular oxygen delivery. However, some gastropods can survive at several degrees Celsius higher than their Arrhenius break temperature of cardiac function (ABT), indicating the importance of anaerobic metabolism for their thermal tolerance. We measured the global molecular responses to heat stress in limpet Cellana toreuma using 454 GS-FLX to investigate the variations of genes involved in anaerobic metabolism at high temperatures. Next, the gene expression levels of 4 anaerobic enzymes and activity of alanopine dehydrogenase (AlDH), which is involved in opine pathway, were measured in response to elevated temperature. A total of 19 heat shock proteins (HSPs) were determined using real-time PCR at different temperatures. At high temperatures, the extensive upregulation of HSP genes was an effective but energetically expensive form of protection to prevent thermal damage. The upregulation of hypoxia-inducible factor 1 alpha mRNA indicated the condition of cellular hypoxia and the high gene expression and enzyme activity of AlDH suggested that opine pathway was the main anaerobic pathway. These results implied that anaerobic metabolism was enhanced to provide energy in the face of thermal stress. Our findings highlight the ecological significance of the anaerobic metabolism of gastropods to thermal adaptation. For predicting the ecological impact of global warming on the distribution of gastropods, the role of anaerobic pathways should be evaluated.

Influence of crude oil exposure on cardiac function and thermal tolerance of juvenile rainbow trout and European sea bass.

Oil spills pose a threat to aquatic organisms. However, the physiological effects of crude oil on cardiac function and on thermal tolerance of juvenile fish are still poorly understood. Consequently, in this paper, we will present results of two separate experiments where we exposed juvenile rainbow trout and European sea bass to crude oil and made cardiac thermal tolerances and maximum heart rate (f Hmax) measurements after 1week (rainbow trout) and 6-month recovery (sea bass). In both species, the f Hmax was lower in crude oil-exposed fish than in the control ones at temperatures below the optimum but this difference disappeared at higher temperatures. More importantly, the oil-exposed fish had significantly higher Arrhenius break point temperature for f Hmax, which gave an estimate for optimum temperature, than the control fish in both species even though the exposure conditions and recovery times differed between species. The results indicated that exposure of juvenile fish to crude oil did not have a significant negative impact upon their cardiac performance in high temperatures and upper thermal tolerance increased when the fish were tested 1week or 6months after the exposure. Our findings suggest that the cardiac function and thermal tolerance of juvenile fish are relatively resistant to a crude oil exposure.

Divergent thermal sensitivities among different life stages of the pulmonate limpet Siphonaria japonica

The different life-history stages of organisms may have divergent thermal limits and thus respond differently to global warming. For comprehensive understanding of the ecological impacts of global warming, it is crucial to determine thermal limits among different life stages and to understand how close an organism lives to its thermal limits at different life stages. Here, we measured the effect of temperature on hatching and the lethal temperature (LT50, lethal temperature of 50%) of larvae to explore effects of high temperature on the encapsulated and planktonic stages of the pulmonate limpet Siphonaria japonica. We measured cardiac performance to determine adult limpets’ thermal limit. At the encapsulated stage, heat shock at 34.5 °C affected larval hatching significantly. Pelagic larvae showed a high LT50 (45.03 °C), indicating a high thermal tolerance for the pelagic larval stage. For the adults, the Arrhenius break temperature and flatline temperature were 38.49 ± 1.27 and 44.27 ± 1.08 °C, respectively. The integrated study of environmental temperatures and thermal sensitivities at different life stages of S. japonica indicates that the egg ribbon and adult stages are potentially most sensitive to the future temperature increases on subtropical shores.

Cardiac performance: a thermal tolerance indicator in scallops

Thermal tolerance has become an active research area in marine poikilotherms due to the influence of increased sea temperature caused by global warming. Previous indicators of thermal tolerance in bivalves are generally laborious and time-consuming and even require killing the specimens. In this study, we demonstrated that heart rate (HR) was a stable and reliable indicator for scallop physiological status by applying an infrared-based cardiac performance monitoring system. The feasibility of HR-based Arrhenius break temperatures (ABTs) as a scallop thermal tolerance indicator was evaluated by investigating ABTs of four species with different thermal limits, including the Yesso scallop (Patinopecten yessoensis), the Zhikong scallop (Chlamys farreri), the bay scallop (Argopecten irradians), and the Catarina scallop (Argopecten ventricosus). In accordance with the thermal limits, ABTs of the Yesso scallop, Zhikong scallop, bay scallop, and Catarina scallop were 22.03 ± 0.19, 29.10 ± 0.25, 32.20 ± 0.25, and 34.09 ± 0.19 °C, respectively, suggesting that the ABT could indicate thermal limits in interspecific scallops. Variations in the ABTs were observed among intraspecific scallops with different sizes, weights, and ages, suggesting that smaller and younger scallops tend to have higher thermal limits. Significant differences in ABT were also observed between pre- and post-spawning individuals, implying that spawning behavior could decrease scallop thermal limits. The above results suggest that HR-based ABT can detect not only interspecific but also intraspecific thermal tolerance in scallops. This study reports the feasibility of infrared-based cardiac performance as a rapid, efficient, and noninvasive indicator for bivalve thermal tolerance.

Assessment of the thermal tolerance of abalone based on cardiac performance in Haliotis discus hannai, H. gigantea and their interspecific hybrid

Temperature stress is one of the major threats to the economic and ecological sustainability of marine fisheries and aquaculture. The abalone aquaculture in China has been in development for more than twenty years, while summer mortality for Pacific abalone (Haliotis discus hannai) is still the most serious issue for its aquaculture in southern China. Therefore, an effective and accurate measurement for evaluating the abalone's thermal tolerance is crucial for both scientific researchers and farmers. In this study, we evaluate the thermal tolerance of two abalone species: H. discus hannai (DD), H. gigantea (GG) and their offspring H. gigantea ♀×H. discus hannai ♂ (GD) using Arrhenius break temperatures (ABT) of cardiac performance. These three groups possessed different thermal limits, as indicated by ABT, GD (32.50±0.71°C)>GG (31.30±1.52°C)>DD (30.00±1.23°C). The results were similar to further assessments of lethal temperature, Kaplan–Meier cumulative survival curves and critical thermal maximum. These results indicate that cardiac performance is an effective method to assess heat tolerance and can represent the different physiological statuses of abalones. Moreover, the results also suggested that global warming may have a profound influence on abalone aquaculture in China in the near future. Statement of relevanceThe results of the present study may facilitate in better understanding the difference on the thermal tolerance ability of H. discus hannai, H. gigantea and their offspring H. gigantea ♀×H. discus hannai ♂ using Arrhenius break temperatures (ABT) of cardiac performance. The results were similar to further assessments of lethal temperature, Kaplan–Meier cumulative survival curves and critical thermal maximum. These findings indicate that cardiac performance is an effective method to assess heat tolerance and can represent the different physiological statuses of abalone. The ABT of cardiac performance could afford an efficient method to predict abalone thermal performance, which would be greatly beneficial in aquaculture and breeding projects.Moreover, it may have potentially broad application in the study of thermal tolerance of abalone and other commercially aquaculture species.

Exposure to solar radiation drives organismal vulnerability to climate: Evidence from an intertidal limpet

Understanding the physiological abilities of organisms to cope with heat stress is critical for predictions of species’ distributions in response to climate change. We investigated physiological responses (respiration and heart beat rate) of the ectotherm limpet Patella vulgata to heat stress events during emersion and the role of seasonal and microclimatic acclimatization for individual thermal tolerance limits. Individuals were collected from 5 microhabitats characterized by different exposure to solar radiation in the high intertidal zone of a semi-exposed rocky shore in winter and summer of 2014. Upper thermal tolerance limits (heat coma temperatures – HCTs, and heart rate Arrhenius break temperatures - ABTs) were determined for individuals from each microhabitat in both seasons under laboratory conditions. While we found a clear seasonal acclimatization, i.e., higher HCTs and ABTs in summer than in winter, we did not find evidence for microhabitat-specific responses that would suggest microclimatic acclimatization. However, operative limpet temperatures derived from in-situ temperature measurements suggest that individuals from sun exposed microhabitats have a much narrower thermal safety margins than those from less exposed surfaces or within crevices. Microhabitat specific thermal safety margins caused by high thermal heterogeneity at small spatial scales and the lack of short term acclimatization will likely shape small scale distribution patterns of intertidal species in response to the predicted increase in the frequency and intensity of heat waves.

Temperate Drosophila preserve cardiac function at low temperature

Most insects are chill susceptible and will enter a coma if exposed to sufficiently low temperature. This chill coma has been associated with a failure of the neuromuscular system. Insect heart rate (HR) is determined by intrinsic regulation (muscle pacemaker) with extrinsic (nervous and humoral) input. By examining the continually active heart of five Drosophila species with markedly different cold tolerance, we investigated whether cardiac performance is related to the whole animal critical thermal minimum (CTmin). Further, to separate the effects of cold on extrinsic and intrinsic regulators of HR, we measured HR under similar conditions in decapitated flies as well as amputated abdomens of Drosophila montana. Cardiac performance was assessed from break points in HR–temperature relationship (Arrhenius break point, ABP) and from the HR cessation temperature. Among the five species, we found strong relationships for both the HR-ABP and HR cessation temperatures to whole animal CTmin, such that temperate Drosophila species maintained cardiac function at considerably lower temperatures than their tropical congeners. Hearts of amputated abdomens, with reduced extrinsic input, had a higher thermal sensitivity and a significantly lower break point temperature, suggesting that central neuronal input is important for stimulating HR at low temperatures.

Modulation of Active Site Picosecond Dynamics in Mutant Forms of a Thermophilic Alcohol Dehydrogenase (HT-ADH)

Previous kinetic and solvent-exchange data have suggested that an Arrhenius break observed at 30oC is attributed to a shift in the equilibrium populations of inactive and active microstates accompanied with a change in protein flexibility [1,2]. We employed time-correlated single photon counting to investigate the statistical nature of the fluorescence dynamics as a function of temperature in ht-ADH. Two single-tryptophan variants were used as reporter probes; Trp87 is fortuitously located at the active site and Trp167 is a surface tryptophan >25A from the catalytic center. The temperature dependence of the time-resolved Stokes shifts reveal that the active site Trp contains highly temperature-dependent relaxation dynamics while the surface Trp is virtually temperature-independent. Though no break is observed that correlates to catalysis, such results are discussed in the context of slower, more global dynamics as a major contributor to catalytic behavior. Using both single-Trp variants, Ala was then introduced to Tyr25 at the dimer interface and Val260 within the cofactor binding site. Such alterations were previously shown to either eliminate or exacerbate the magnitude of the temperature break, respectively [3,4]. The fluorescence dynamics reveal that the temperature-dependent dynamics at Trp87 are drastically altered relative to its wild-type counterpart, while Trp167 shows minor differences. Such results are discussed in the context of an altered conformational landscape that further corroborates an equilibration process occurring more slowly than the ps-timescale.[1] Kohen, A., et al (1999) Nature, 399, 496-499.[2] Liang, Z. X., et al (2004) PNAS, 101, 9556-9561.[3] Nagel, Z. D., et al (2011) PNAS, 108, 10520-10525.[4] Nagel, Z. D., et al (2013) JBC, 288, 14807-14097.

Identification of a Long-range Protein Network That Modulates Active Site Dynamics in Extremophilic Alcohol Dehydrogenases

A tetrameric thermophilic alcohol dehydrogenase from Bacillus stearothermophilus (ht-ADH) has been mutated at an aromatic side chain in the active site (Trp-87). The ht-W87A mutation results in a loss of the Arrhenius break seen at 30 °C for the wild-type enzyme and an increase in cold lability that is attributed to destabilization of the active tetrameric form. Kinetic isotope effects (KIEs) are nearly temperature-independent over the experimental temperature range, and similar in magnitude to those measured above 30 °C for the wild-type enzyme. This suggests that the rigidification in the wild-type enzyme below 30 °C does not occur for ht-W87A. A mutation at the dimer-dimer interface in a thermolabile psychrophilic homologue of ht-ADH, ps-A25Y, leads to a more thermostable enzyme and a change in the rate-determining step at low temperature. The reciprocal mutation in ht-ADH, ht-Y25A, results in kinetic behavior similar to that of W87A. Collectively, the results indicate that flexibility at the active site is intimately connected to a subunit interaction 20 Å away. The convex Arrhenius curves previously reported for ht-ADH (Kohen, A., Cannio, R., Bartolucci, S., and Klinman, J. P. (1999) Nature 399, 496-499) are proposed to arise, at least in part, from a change in subunit interactions that rigidifies the substrate-binding domain below 30 °C, and impedes the ability of the enzyme to sample the catalytically relevant conformational landscape. These results implicate an evolutionarily conserved, long-range network of dynamical communication that controls C-H activation in the prokaryotic alcohol dehydrogenases.

Metabolic energy sensors (AMPK and SIRT1), protein carbonylation, and cardiac failure as biomarkers of thermal stress in an intertidal limpet: linking energetic allocation with environmental temperature during aerial emersion

The effects of heat stress on organisms are manifested at the levels of organ function, metabolic activity, protein stability and gene expression. Here, we examined effects of high temperature on the intertidal limpet Cellana toreuma to determine how the temperatures at which (1) organ failure (cardiac function), (2) irreversible protein damage (carbonylation) and (3) expression of genes encoding proteins involved in molecular chaperoning (hsp70 and hsp90) and metabolic regulation (ampk and sirt1) occur compare with field temperatures, which commonly exceed 30°C and can reach 46°C. Heart failure, indexed by the Arrhenius break temperature, occurred at 34.3°C. Protein carbonylation rose significantly at 38°C. Genes for heat shock proteins HSP70 (hsp70) and HSP90 (hsp90), for two subunits of AMP-activated protein kinase (AMPK) (ampkα and ampkβ) and for histone/protein deacetylase SIRT1 (sirt1) all showed increased expression at 30°C. Temperatures of maximal expression differed among genes, as did temperatures at which upregulation ceased. Expression patterns for ampk and sirt1 indicate that heat stress influenced cellular energy homeostasis; above ~30°C, upregulation of ATP-generating pathways is suggested by elevated expression of genes for ampk; an altered balance between reliance on carbohydrate and lipid fuels is indicated by changes in expression of sirt1. These results show that C. toreuma commonly experiences temperatures that induce expression of genes associated with the stress response (hsp70 and hsp90) and regulation of energy metabolism (ampk and sirt1). At high temperatures, there is likely to be a shift away from anabolic processes such as growth to catabolic processes, to provide energy for coping with stress-induced damage, notably to proteins.

Using maximum heart rate as a rapid screening tool to determine optimum temperature for aerobic scope in Pacific salmon Oncorhynchus spp.

The mean ±s.e. optimum temperature (T(opt)) for aerobic scope in juvenile coho salmon Oncorhynchus kisutch was determined to be 17·0 ± 0·7° C. The repeated measures protocol took 3 weeks to complete the T(opt) determination using 12 fish tested at five temperatures separated by 2° C increments. This experiment also demonstrated that the T(opt) was associated with maximum heart rate (f(H)) failing to maintain a Q(10) -related increase with temperature. When maximum f(H) was produced in anaesthetized fish with pharmacological stimulation and f(H) measured from electrocardiogram recordings during acute warming, the Arrhenius break temperature (ABT) for Q(10) discontinuities in maximum f(H) (mean ±s.e. = 17·1 ± 0·5° C for 15 ppm clove oil and 16·5 ± 0·2° C for 50 ppm MS-222) was statistically indistinguishable from the T(opt) measured using aerobic scope. Such a determination took only 3 days rather than 3 weeks. Therefore, it is proposed that determining ABT for discontinuities in maximum f(H) in anaesthetized fish presents itself as a valuable, high-throughput screening tool to assess T(opt) in fishes, a metric that has become recognized as being extremely valuable in fish biology and fisheries management.

Neural Thermal Performance in Porcelain Crabs, GenusPetrolisthes

Neurons are highly temperature sensitive. Temperature-induced nerve failure may play an important role in determining organismal thermal tolerance limits and distribution patterns. To expand our understanding of the role of neuronal thermal performance in setting thermal limits, we compared the thermal performance of neurons from five porcelain crab (genus Petrolisthes) congeners that differ in thermal habitat. In experiment 1, neuronal performance of sensory neurons was determined by extracellular recording of spontaneous action potentials during thermal ramps. Arrhenius break temperatures of action potential generation were used to calculate maximum critical temperature (CT(max)) and minimum critical temperature (CT(min)) for neuronal performance. CT(max) and CT(min) were related to habitat temperature across the five species and were found to respond to acclimation temperature. In experiment 2, we assessed the performance of neurons from Petrolisthes cinctipes acclimated at 8°, 18°, and 25°C when placed at 30°C (near the whole-organism CT(max) of this species) and demonstrated that neural performance near whole-organism CT(max) increases with increasing acclimation temperature. In experiment 3, we compared the thermal limits of sensory afferents and pacemaker efferents and found that they were correlated, although pacemaker efferents tended to have a higher CT(max) and reduced plasticity. Our final analysis, which was of transcriptomic data in cardiac tissue, leads us to hypothesize that nerve membrane K(+) conductance may underlie variation in nerve thermal tolerance.

Mitochondrial function in Antarctic notothenioid fishes that differ in the expression of oxygen-binding proteins

State III respiration rates were measured in mitochondria isolated from hearts of Antarctic notothenioid fishes that differ in the expression of hemoglobin (Hb) and myoglobin (Mb). Respiration rates were measured at temperatures between 2 and 40°C in Gobionotothen gibberifrons (+Hb/+Mb), Chaenocephalus aceratus (–Hb/–Mb) and Chionodraco rastrospinosus (–Hb/+Mb). Blood osmolarity was measured in all three species and physiological buffers prepared for isolating mitochondria and measuring respiration rates. Respiration rates were higher in mitochondria from G. gibberifrons compared to those from C. aceratus at 2°C, but were similar among all species at temperatures between 10 and 26°C. Respiration rates were significantly lower in icefishes at 35 and 40°C compared to G. gibberifrons. The respiratory control ratio of isolated mitochondria was lower in C. aceratus compared to G. gibberifrons at all temperatures below 35°C. At 35 and 40°C, mitochondria were uncoupled in all species. The Arrhenius break temperature of state III respiration was similar among all three species (30.5 ± 0.9°C) and higher than values previously reported for Antarctic notothenioids, likely due to the higher osmolarity of buffers used in this study. These results suggest that differences in mitochondrial structure, correlated with the expression of oxygen-binding proteins, minimally impact mitochondrial function.

Pea seed mitochondria are endowed with a remarkable tolerance to extreme physiological temperatures.

Most seeds are anhydrobiotes, relying on an array of protective and repair mechanisms, and seed mitochondria have previously been shown to harbor stress proteins probably involved in desiccation tolerance. Since temperature stress is a major issue for germinating seeds, the temperature response of pea (Pisum sativum) seed mitochondria was examined in comparison with that of mitochondria from etiolated epicotyl, a desiccation-sensitive tissue. The functional analysis illustrated the remarkable temperature tolerance of seed mitochondria in response to both cold and heat stress. The mitochondria maintained a well-coupled respiration between -3.5 degrees C and 40 degrees C, while epicotyl mitochondria were not efficient below 0 degrees C and collapsed above 30 degrees C. Both mitochondria exhibited a similar Arrhenius break temperature at 7 degrees C, although they differed in phospholipid composition. Seed mitochondria had a lower phosphatidylethanolamine-to-phosphatidylcholine ratio, fewer unsaturated fatty acids, and appeared less susceptible to lipid peroxidation. They also accumulated large amounts of heat shock protein HSP22 and late-embryogenesis abundant protein PsLEAm. The combination of membrane composition and stress protein accumulation required for desiccation tolerance is expected to lead to an unusually wide temperature tolerance, contributing to the fitness of germinating seeds in adverse conditions. The unique oxidation of external NADH at low temperatures found with several types of mitochondria may play a central role in maintaining energy homeostasis during cold shock, a situation often encountered by sessile and ectothermic higher plants.

Evolutionary and Acclimation-Induced Variation in the Thermal Limits of Heart Function in Congeneric Marine Snails (GenusTegula): Implications for Vertical Zonation

We analyzed the thermal limits of heart function for congeneric species of the marine snail Tegula that have different patterns of vertical zonation. T. funebralis is found in the low to mid-intertidal zone, and T. brunnea and T. montereyi live in the low-intertidal or subtidally. As indices of thermal limits of heart function, we used the temperature at which heart rate initially decreased rapidly during heating (the Arrhenius break temperature, or ABT) and the temperature at which heart ceased to beat with either heating or cooling (the flatline temperature, or FLT(hot) or FLT(cold), respectively). These three indices provide an estimate of the thermal range within which Tegula heart function is maintained. For field-acclimatized specimens, the thermal range of the high-intertidal T. funebralis was greater than those of its two lower-occurring congeners (higher ABT, higher FLT(hot), lower FLT(cold)). We also demonstrated the effects of constant thermal acclimation on the heart rate response to heat stress. Acclimation to 14 degrees C and 22 degrees C resulted in increases in ABT and FLT(hot), with the largest changes in T. brunnea and T. montereyi. Although T. funebralis is more heat tolerant and eurythermal than its two lower-occurring congeners, it can encounter field body temperatures that exceed ABT, indicating that T. funebralis faces a larger threat from heat stress, in situ. These findings are consistent with recent studies on other taxa of marine invertebrates that have shown, somewhat paradoxically, that warm-adapted, eurythermal intertidal species may be more impacted by global warming than congeneric subtidal species that are less heat tolerant.

Mitochondrial function and critical temperature in the Antarctic bivalve, Laternula elliptica

Thermal sensitivities of maximum respiration and proton leakage were compared in gill mitochondria of the Antarctic bivalve Laternula elliptica for an assessment of the contribution of mitochondrial mechanisms to limiting temperature tolerance. Proton leakage was measured as the oxygen consumption rate during blockage of oxidative phosphorylation (state IV respiration+oligomycin). The maximum capacity of NADP dependent mitochondrial isocitrate dehydrogenase (IDH) was investigated as part of a proposed mitochondrial substrate cycle provoking proton leakage by the action of transhydrogenase. State III and IV+respiration rose exponentially with temperature. Thermal sensitivities of proton leakage and IDH were unusually high, in accordance with the hypothesis that H + leakage is an enzyme catalysed process with IDH being involved. In contrast to proton leakage, state III respiration exhibited an Arrhenius break temperature at 9°C, visible as a drop in thermal sensitivity close to, but still above the critical temperature of the species (3–6°C). Progressive uncoupling of mitochondria led to a drop in RCR values and P/O ratios at high temperature. The same discontinuity as for state III respiration was found for the activity of mitochondrial IDH suggesting that this enzyme may influence the thermal control of mitochondrial respiration. In general, the high thermal sensitivity of proton leakage may cause an excessive rise in mitochondrial oxygen demand and a decreased efficiency of oxidative phosphorylation. This may exceed the whole animal capacity of oxygen uptake and distribution by ventilation and circulation and set a thermal limit, characterized by the transition to anaerobic metabolism.

The SR Ca 2+ ATPase of the Antarctic scallop Adamussium colbecki : cold adaptation and heavy metal effects

Cell calcium is accumulated in intracellular stores by sarco-endoplasmic reticulum Ca2+ ATPases functionally interacting with the membrane lipid environment. Cold adaptations of membrane lipids in Antarctic Sea organisms suggest possible adaptive effects also on sarco-endoplasmic reticulum Ca2+ ATPases. We investigated the SR Ca2+ ATPase of an Antarctic scallop, Adamussium colbecki, by characterising the enzyme activity and studying temperature effects. Ca2+ ATPase, assayed by following ATP hydrolysis, was thapsigargin- and vanadate-sensitive, showed maximum activity under 2 μM Ca2+, 200 mM KCl and pH 7.2, and had a KM for ATP of 22 ± 7 μM. Temperature effects showed an Arrhenius inversion between −1.8 and 0°C, indicating cold adaptation, an Arrhenius break at 10°C, and a collapse above 20°C. A. colbecki accumulates high amounts of cadmium in the digestive gland; heavy metal effects on sarco-endoplasmic reticulum Ca2+ ATPases were therefore tested, finding an IC50 = 0.9 μM for Hg2+ and 3 μM for Cd2+. Finally, SDS-PAGE analysis showed a main band at about 100 kDa, which was identified as sarco-endoplasmic reticulum Ca2+ ATPase after trypsin digestion, and accounted for 60% total protein.

Effects of temperature on mitochondrial function in the Antarctic fish Trematomus bernacchii

Effects of temperature on O2 consumption by mitochondria of the Antarctic fish Trematomus bernacchii were compared with effects obtained with mitochondria from tropical (Sarotheridon mossambica) and temperate zone fishes (Sebastes carnatus and Sebastes mystinus). Arrhenius plots of O2 consumption versus temperature exhibited slope discontinuities (“breaks”) at temperatures (Arrhenius break temperatures: ABTs) reflective of the species' adaptation temperatures. The ABT for mitochondria of T. bernacchii is the lowest reported for any animal and is ∼12 °C below the value predicted by a regression equation based on ABT data for several invertebrates and fishes. The temperature at which the acceptor control ratio (ACR), an index of efficiency of coupling of electron transport to synthesis of ATP, began to decrease with rising temperature also reflected adaptation temperature. The decrease in ACR with rising temperature began at ∼18 °C for mitochondria of T. bernacchii, in contrast to ∼35 °C for mitochondria of Sarotheridon mossambica. Maintaining T. bernacchii at 4 °C for 2 weeks led to no changes in ABT or in the response of ACR to temperature. The thermal sensitivities of mitochondria of T. bernacchii reflect the high level of cold adaptation and stenothermy that is characteristic of Antarctic Notothenioid fishes.