Chilling-injury and disturbance of ion homeostasis in the coxal muscle of the tropical cockroach (Nauphoeta cinerea)

Adults of warm- and cold-acclimated tropical cockroaches, Nauphoeta cinerea were exposed to low temperatures of 0 or 5 degrees C for various time intervals (hours to days). Development of chilling-injury (defects in crawling and uncoordinated movements) and mortality during the exposure were assessed and correlated with the changes in concentrations of metal ions (Na(+), K(+) and Mg(2+)) in the haemolymph and coxal muscle tissue. Warm-acclimated insects entered chill-coma at both low temperatures. In their haemolymph, the [Na(+)] and [Mg(2+)] linearly decreased and [K(+)] increased with the increasing time of exposure. The rate of concentration changes was higher at 0 than at 5 degrees C. The concentration changes resulted in gradually dissipating equilibrium potentials across the muscle cell membranes. For instance, E(K) decreased from -49.8 to -20.7 mV during 7 days at 5 degrees C. Such a disturbance of ion homeostasis was paralleled by the gradual development of chilling-injury and mortality. Most of the cockroaches showed chilling-injury when the molar ratio of [Na(+)]/[K(+)] in their haemolymph decreased from an initial of 4.4 to 2.1-2.5. In contrast, the cold-acclimated cockroaches did not enter chill-coma. They maintained constant concentrations of ions in their haemolymph, constant equilibrium potentials across muscle cell membranes and the development of chilling-injury was significantly suppressed at 5 degrees C for 7 days.     

Temperature effects on the Na and Ca currents in rat and hedgehog ventricular muscle

Cardiac transmembrane potentials and Na and Ca currents were recorded at different temperatures in rat and hedgehog ventricular muscle. At 35 degrees C in both species resting potential was about -80 mV and upstroke velocity (Vmax) of the action potential above 100 V/s. The shape of the action potential in hedgehog ventricular cells at 35 degrees C was similar to that in the rat showing a fast repolarization phase. When temperature was decreased, the membrane resting potential depolarized and action potential amplitude and Vmax declined. In rat ventricular cells at 10 degrees C, the resting potential was about -40 to -50 mV and Vmax was reduced to about 5 V/s. In hedgehog ventricular cells, however, the transmembrane potentials and Vmax were better maintained at low temperature. Phase 3 of the action potential was markedly prolonged below 20 degrees C in hedgehog but not in rat ventricular cells. When temperature was decreased to 10 degrees C the availability curve of the Na current shifted toward more negative potentials and ICa.peak declined in rat ventricular cells. In hedgehog cardiac preparations, the Na current was less influenced by the cooling and ICa.peak did not change very much at low temperatures. A transient inward current usually considered to induce cardiac arrhythmias could be recorded in rat ventricular cells below 20 degrees C but not in hedgehog preparations. These features of hedgehog cardiac membranes may contribute to the cold tolerance and the resistance to ventricular fibrillation during the hypothermia in mammalian hibernators.     

Response to selection for rapid chill-coma recovery in Drosophila melanogaster: physiology and life-history traits

Resistance to low temperatures can vary markedly among invertebrate species and is directly related to their distribution. Despite the ecological importance of cold resistance this trait has rarely been studied genetically, mainly because low and variable fitness of offspring from cold-stressed mothers makes it difficult to undertake selection experiments and compare cold resistance of parents and offspring. One measure of cold resistance that varies geographically in Drosophila melanogaster and that is amenable to genetic analysis is chill-coma recovery. Three replicate lines of D. melanogaster were selected every second generation, for over 30 generations, for decreased recovery time following exposure to 0 degrees C. Correlated responses were scored to characterize underlying physiological traits and to investigate interactions with other traits. Lines responded rapidly to the intermittent selection regime with realized heritabilities varying from 33% to 46%. Selected lines showed decreased recovery time after exposure to a broad range of low temperatures and also had a lower mortality following a more severe cold shock, indicating that a general mechanism underlying cold resistance had been selected. The selection response was independent of plastic changes in cold resistance because the selected lines maintained their ability to harden (i.e. a short-term exposure to cool temperature resulted in decreased recovery time in subsequent chill-coma assays). Changes in cold resistance were not associated with changes in resistance to high temperature exposure, and selected lines showed no changes in wing size, development time or viability. However, there was a decrease in longevity in the selected lines due to an earlier onset of ageing. These results indicate that chill-coma recovery can be rapidly altered by selection, as long as selection is undertaken every second generation to avoid carry-over effects, and suggest that lower thermal limits can be shifted towards increased cold resistance independently of upper thermal limits and without tradeoffs in many life-history traits.     

Induction of rapid cold hardening by cooling at ecologically relevant rates in Drosophila melanogaster

Over a decade ago it was hypothesized that the rapid cold hardening process allows an organism's overall cold tolerance to track changes in environmental temperature, as would occur in nature during diurnal thermal cycles. Although a number of studies have since focused on characterizing the rapid cold hardening process and on elucidating the physiological mechanisms upon which it is based, the ecological relevance of this phenomenon has received little attention. We present evidence that in Drosophila melanogaster rapid cold hardening can be induced during cooling at rates which occur naturally, and that the protection afforded in such a manner benefits the organism at ecologically relevant temperatures. Drosophila melanogaster cooled at natural rates (0.05 and 0.1 degrees C min(-1)) exhibited significantly higher survival after one hour of exposure to -7 and -8 degrees C than did those directly transferred to these temperatures or those cooled at 0.5, or 1.0 degrees C min(-1). Protection accrued throughout the cooling process (e.g., flies cooled to 0 degrees C were more cold tolerant than those cooled to 11 degrees C). Whereas D. melanogaster cooled at 1.0 degrees C min(-1) had a critical thermal minimum (i.e., the temperature at which torpor occurred) of 6.5+/-0.6 degrees C, those cooled at an ecologically relevant rate of 0.1 degrees C min(-1) had a significantly lower value of 3.9+/-0.9 degrees C.     

A comparison of Hsp70 expression and thermotolerance in adults and larvae of three Drosophila species

Heat shock proteins (Hsps) and other molecular chaperones perform diverse physiological roles. One is to facilitate, in part, organismal thermotolerance, of which the functional consequences depend on Hsp70 concentration and developmental stage in Drosophila melanogaster. To test whether an Hsp70-thermotolerance relationship is a general phenomenon within Drosophila, I assayed Hsp70 concentration at a range of temperatures in intact larvae and adults of three species, D. melanogaster, D. simulans, and D. mojavensis, and compared those results to the increase in survival to heat shock that occurs after an Hsp70 inducing pretreatment. Larvae of D. melanogaster and D. simulans responded similarly to heat; they expressed Hsp70 maximally at 36-37 degrees C, and their tolerance of 1 h heat shocks increased by 1.5-2 degrees C. By contrast, D. mojavensis, which tolerates higher temperatures than do D. melanogaster and D. simulans, expressed Hsp70 only at higher temperatures, although the 36 degrees C pretreatment still increased thermotolerance. Critically, the temperature that maximally induced Hsp70 was a poor inducer of thermotolerance in D. mojavensis and may have harmed larvae. Results for Drosophila adults, which tolerated heat poorly compared to larvae, likewise suggest that a close link between peak Hsp70 expression and maximal induction of thermotolerance is a feature of D. melanogaster, and not of the other species. Neither D. simulans nor D. mojavensis adults increased tolerance after exposure to the temperatures that maximally induced Hsp70.     

Microtubule-associated proteins from Antarctic fishes

Microtubules and presumptive microtubule-associated proteins (MAPs) were isolated from the brain tissues of four Antarctic fishes (Notothenia gibberifrons, N. coriiceps neglecta, Chaenocephalus aceratus, and a Chionodraco sp.) by means of a taxol-dependent, microtubule-affinity procedure (cf. Vallee: Journal of Cell Biology 92:435-442, 1982). MAPs from these fishes were similar to each other in electrophoretic pattern. Prominent in each preparation were proteins in the molecular weight ranges 410,000-430,000, 220,000-280,000, 140,000-155,000, 85,000-95,000, 40,000-45,000, and 32,000-34,000. The surfaces of MAP-rich microtubules were decorated by numerous filamentous projections. Exposure to elevated ionic strength released the MAPs from the microtubules and also removed the filamentous projections. Addition of fish MAPs to subcritical concentrations of fish tubulins at 0-5 degrees C induced the assembly of microtubules. Both the rate and the extent of this assembly increased with increasing concentrations of the MAPs. Sedimentation revealed that approximately six proteins, with apparent molecular weights between 60,000 and 300,000, became incorporated into the microtubule polymer. Bovine MAPs promoted microtubule formation by fish tubulin at 2-5 degrees C, and proteins corresponding to MAPs 1 and 2 co-sedimented with the polymer. MAPs from C. aceratus also enhanced the polymerization of bovine tubulin at 33 degrees C, but the microtubules depolymerized at 0 degrees C. We conclude that MAPs are part of the microtubules of Antarctic fishes, that these proteins promote microtubule assembly in much the same way as mammalian MAPs, and that they do not possess special capacities to promote microtubule assembly at low temperatures or to prevent cold-induced microtubule depolymerization.     

Further study on selective transmission of mitochondrial DNA in heteroplasmic lines of Drosophila melanogaster.

The temperature-dependent transmission of mitochondrial DNA (mtDNA) was investigated in heteroplasmic lines of Drosophila melanogaster established by germ-plasm transplantation. Using D. melanogaster, D. simulans and D. mauritiana as germ-plasm donors, five recipient-donor combinations of heteroplasmy, differing from those previously examined (Matsuura et al., 1991), were constructed. For intraspecific reciprocal combinations, donor mtDNA in one combination was retained at 25 degrees C but was almost lost by the tenth generation at 19 degrees C. In the reciprocal, the proportion of the same type of recipient mtDNA decreased more quickly at 19 degrees C than 25 degrees C. Decreasing rates at 19 degrees C in the reciprocals differed from each other. For interspecific combinations, two species were used as germ-plasm donors. Donor mtDNA derived from D. simulans was lost at both temperatures and the rate of decrease was greater at 19 degrees C than 25 degrees C. The proportion of donor mtDNA derived from D. mauritiana increased at a greater rate at 25 degrees C than 19 degrees C when using two different strains of D. melanogaster as recipients. These results suggest that both the nuclear and two types of mitochondrial genomes are involved in the selective transmission of mtDNA.     

Deuterium oxide and temperature effects on the properties of endplate channels at the frog neuromuscular junction

The effects of deuterium oxide (D2O) and temperature on the properties of endplate channels were studied in voltage-clamped muscle fibers from the frog Rana pipiens. Studies were performed at temperatures of 8, 12, 16, and 20 degrees C. The single channel conductance (gamma) and mean channel lifetime (tau) were calculated from fluctuation analysis of the acetylcholine-induced end-plate currents. The reversal potential was determined by interpolation of the acetylcholine-induced current-voltage relation. The mean reversal potential was slightly more negative in D2O Ringer's (-7.9 +/- 0.1 mV [+/- SEM]) compared with H2O Ringer's (-5.2 +/- 0.6 mV, P less than 0.01). The single channel conductance was decreased in D2O. This decrease was greater than could be accounted for by the increased viscosity of D2O solutions, and the amount of the decrease was greater at higher temperatures. For example, gamma was 38.4 +/- 1.3 pS (+/- SEM) in H2O Ringer's and 25.7 +/- 1.0 pS in D2O Ringer's for a holding potential of -70 mV at 12 degrees C. The mean channel lifetime was significantly shorter in D2O, and the effect was greater at lower temperatures. There was not a strong effect of solvent on the temperature dependence of gamma. On the other hand, the temperature dependence of the reciprocal mean channel lifetime, alpha (where alpha = 1/tau), was strongly dependent upon the solvent. The single channel conductances showed no demonstrable voltage dependence over the range of -90 to -50 mV in both solvents. The reciprocal mean channel lifetime showed a voltage dependence, which could be described by the relation alpha = B exp(AV). The slope A was not strongly affected by either temperature or the solvent. On the other hand, the intercept B was a strong function of temperature and was weakly dependent upon the solvent, with most values greater in D2O. The D2O effects on alpha were what would be expected if they were due to the properties of D2O as a solvent (solvent isotope effects), while the D2O effects on gamma must also include the exchange of D for H in the vicinity of the selectivity filter (primary and/or secondary kinetic isotope effects).     

Stenotherms at sub-zero temperatures: thermal dependence of swimming performance in Antarctic fish

We examined the burst swimming performance of two Antarctic fishes, Trematomus bernacchii and T. centronotus, at five temperatures between -1 degrees C and 10 degrees C. As Antarctic fishes are considered one of the most cold specialised and stenothermal of all ectotherms, we predicted they would possess a narrow thermal performance breadth for burst swimming and a correlative decrease in performance at high temperatures. Burst swimming was assessed by videotaping swimming sequences with a 50-Hz video camera and analysing the sequences frame-by-frame to determine maximum velocity, the distance moved throughout the initial 200 ms, and the time taken to reach maximum velocity. In contrast to our prediction, we found both species possessed a wide thermal performance breadth for burst swimming. Although maximum swimming velocity for both T. bernacchii and T. centronotus was significantly highest at 6 degrees C, maximum velocity at all other test temperatures was less than 20% lower. Thus, it appears that specialisation to a highly stable and cold environment is not necessarily associated with a narrow thermal performance breadth for burst swimming in Antarctic fish. We also examined the ability of the Antarctic fish Pagothenia borchgrevinki to acclimate their burst-swimming performance to different temperatures. We exposed P. borchgrevinki to either -1 degrees C or 4 degrees C for 4 weeks and tested their burst-swimming performance at four temperatures between -1 degrees C and 10 degrees C. Burst-swimming performance of Pagothenia borchgrevinki was unaffected by exposure to either -1 degrees C or 4 degrees C for 4 weeks. Maximum swimming velocity of both acclimation groups was thermally independent over the total temperature range of 1 degrees C to 10 degrees C. Therefore, the loss of any capacity to restructure the phenotype and an inability to thermally acclimate swimming performance appears to be associated with inhabiting a highly stable thermal environment.     

Electrical and mechanical effects induced by cold temperatures in the ventricle of isolated Rana ridibunda hearts

A temperature decrease changes the contractility of the amphibian heart, but the underlying mechanisms are not totally understood. The objectives of the present work were to better understand the intrinsic mechanisms supporting contractility changes induced by a rapid temperature decrease in the ventricle of Rana ridibunda, and to investigate how fast they develop. Ventricular mechanical cycles (VMCs) and monophasic action potentials (MAPs) recorded from 15 isolated hearts were measured at 15, 30, 45, 60, 90, 120 and 150 s after the application of Ringer solutions of 20, 10 and 5 degrees C. Treatment with 10 and 5 degrees C Ringer solutions decreased the heart rate, and increased the magnitude of the ventricular contraction and the duration of the contraction and relaxation periods. The electrical changes included prolongation of the MAP depolarization plateau, which also decreased in amplitude as an effect of perfusion with 5 degrees C Ringer solution. In addition, treatment with 5 degrees C Ringer solution increased the latency of contraction. The block of L-type channels totally abolished the depolarization plateau at all perfusion temperatures, but failed to inhibit ventricular contraction. In conclusion, treatment with cold temperatures changes the electrical activity of the ventricular myocardium in R. ridibunda hearts, which results in modified ventricular contractility. Data suggest that in addition to L-type Ca2+ channels, other components that support calcium elevation are present R. ridibunda ventricular cells.     

Cryoanalgesia: electrophysiology at different temperatures

Somatosensory evoked potentials (SEP) and sensory conduction velocity (SCV) were measured in rabbit sciatic nerves following graded cold lesioning. The SEP disappeared when injury was induced at temperatures below -60 degrees C, but returned on day 41+/-4 (mean+/-SD). SEP returned on day 56+/-11 days when the lesion was induced at 100 to -180 degrees C. The SEP latency was prolonged after creating lesions at -100 to -180 degrees C, compared with both the sham operated and the -20 degrees C groups. These experiments suggest the cryolesions produced at temperatures between -60 and -100 degrees C are most suitable for altering the electrophysiological conduction of the nerve, and may result in suitable post-operative analgesia.     

Is glutamate the transmitter of crustacean motoneurons?

1. Bath-application of L-glutamate to crayfish opener muscle causes depolarization and resistance changes which both increase with falling temperature. At temperatures above 15 degrees C there is usually a resistance increase, at lower temperatures the resistance is decreased. 2. Meso-gamma . gamma'-diaminosuberic acid-dihydrochloride (meso-di-GABA) and dl-diamino-nonanedicarboxylic acid dihydrochloride (C-9) were newly synthesized as potential glutamate blockers. 3. Meso-di-GABA (10(-4) to 10(-3)M) usually caused a significant increase (15 degrees C) or decrease (7 degrees C) of membrane resistance and slight depolarization. Excitatory junction potentials (ejps) were reversibly depressed or blocked while the effects of glutamate were potentiated. The depression or block of neuromuscular transmission was not prevented by picrotoxin or by concanavaline A. 4. C-9 (3 x 10(-4) M) depressed or blocked the effect of applied glutamate with little or no effect on ejps. 5. The results are best explained by assuming that bath-applied glutamate acts mainly on extrasynaptic receptors. Meso-di-GABA is assumed to block synaptic receptors and to activate non-synaptic receptors while C-9 seems to act mainly as a blocker of glutamate action on non-synaptic receptors.     

Large changes in intracellular pH and calcium observed during heat shock are not responsible for the induction of heat shock proteins in Drosophila melanogaster.

Heat shock caused significant changes in intracellular pH (pHi) and intracellular free calcium concentration [( Ca2+]i) which occurred rapidly after temperature elevation. pHi fell from a resting level value at 25 degrees C of 7.38 +/- 0.02 (mean +/- standard error of the mean, n = 15) to 6.91 +/- 0.11 (n = 7) at 35 degrees C. The resting level value of [Ca2+]i in single Drosophila melanogaster larval salivary gland cells was 198 +/- 31 nM (n = 4). It increased approximately 10-fold, to 1,870 +/- 770 nM (n = 4), during a heat shock. When salivary glands were incubated in calcium-free, ethylene glycol-bis(beta-aminoethyl ether)-N,N',N'-tetraacetic acid (EGTA)-buffered medium, the resting level value of [Ca2+]i was reduced to 80 +/- 7 nM (n = 3), and heat shock resulted in a fourfold increase in [Ca2+]i to 353 +/- 90 nM (n = 3). The intracellular free-ion concentrations of Na+, K+, Cl-, and Mg2+ were 9.6 +/- 0.8, 101.9 +/- 1.7, 36 +/- 1.5, and 2.4 +/- 0.2 mM, respectively, and remained essentially unchanged during a heat shock. Procedures were devised to mimic or block the effects of heat shock on pHi and [Ca2+]i and to assess their role in the induction of heat shock proteins. We report here that the changes in [Ca2+]i and pHi which occur during heat shock are not sufficient, nor are they required, for a complete induction of the heat shock response.     

Complexity of the cold acclimation response in Drosophila melanogaster

Insects can increase their resistance to cold stress when they are exposed to non-lethal conditions prior to the stress; these plastic responses are normally described only in terms of immediate effects on mortality. Here we examine in Drosophila melanogaster the short- and longer-term effects of different conditions on several measures of cold resistance, but particularly chill coma recovery. Short-term exposure to sublethal temperature (cold hardening) did not decrease chill coma recovery times even though it decreased mortality. Exposure to 12 degrees C for 2 days (acclimation) decreased chill coma recovery times for a range of stressful temperatures when flies were cultured at 25 degrees C, but did not usually affect recovery times when flies were cultured at 19 degrees C. In contrast, 2-day exposure to 12 degrees C decreased mortality regardless of rearing temperature. Rearing at 19 degrees C decreased mortality and chill coma recovery time relative to rearing at 25 degrees C. Acclimation increased the eclosion rate of eggs from stressed females, but did not affect development time or size of the offspring. These results indicate that plastic responses to cold in D. melanogaster are complex when resistance is scored in different ways, and that effects can extend across generations.     

Metabolism and thermoregulation in the springhare (<Emphasis Type="Italic">Pedetes capensis</Emphasis>)

Springhares are large, nocturnally active, diurnally fossorial rodents that typically inhabit arid and semi-arid areas. This lifestyle means that they need to balance excessive heat loss when foraging at night against insufficient heat loss in a potentially warm, humid burrow and both of these against the need to minimize water turnover and energy requirements. In this study we investigated metabolism and thermoregulation in these animals. Basal metabolic rate averaged 8.62+/-1.37 J g(-1) h(-1) and minimum thermal conductance 0.386+/-0.062 J g(-1) h(-1) degrees C(-1). These were higher and lower than expected, respectively. This, along with a relatively low, lower critical temperature and broad thermal neutral zone indicate that springhares are physiologically well suited to the low night-time temperatures, which they typically encounter. Body temperatures were quite labile but springhares became hyperthermic at temperatures above 30 degrees C suggesting that they are poor thermoregulators at high temperatures. This is attributed to their seldom, if ever, encountering temperatures in this range. Insufficient heat loss under normal resting conditions does not appear to be a problem, as springhares inhabit deep burrows in which the temperature never exceeds the upper critical temperature. Excess heat generated during vigorous underground exercise is presumably stored and dissipated to the cool night air or the cooler soil when subsequently resting. Water turnover and energy expenditure are presumably adequately addressed by other physiological and behavioural characteristics.     

Body temperature-related structural transitions of monotremal and human hemoglobin

In this study, temperature-related structural changes were investigated in human, duck-billed platypus (Ornithorhynchus anatinus, body temperature T(b) = 31-33 degrees C), and echidna (Tachyglossus aculeatus, body temperature T(b) = 32-33 degrees C) hemoglobin using circular dichroism spectroscopy and dynamic light scattering. The average hydrodynamic radius (R(h)) and fractional (normalized) change in the ellipticity (F(obs)) at 222 +/- 2 nm of hemoglobin were measured. The temperature was varied stepwise from 25 degrees C to 45 degrees C. The existence of a structural transition of human hemoglobin at the critical temperature T(c) between 36-37 degrees C was previously shown by micropipette aspiration experiments, viscosimetry, and circular dichroism spectroscopy. Based on light-scattering measurements, this study proves the onset of molecular aggregation at T(c). In two different monotremal hemoglobins (echidna and platypus), the critical transition temperatures were found between 32-33 degrees C, which are close to the species' body temperature T(b). The data suggest that the correlation of the structural transition's critical temperature T(c) and the species' body temperature T(b) is not mere coincidence but, instead, is a more widespread structural phenomenon possibly including many other proteins.     

Cold tolerance in Drosophila: adaptive variations revealed by the analysis of starvation survival reaction norms

Three species of Drosophila were investigated for their capacity to survive without food (starvation tolerance) at seven different temperatures ranging from 0 to 25 degrees C. In all cases biphasic response curves (reaction norms) were observed, corresponding either to special deleterious effects of cold or to a progressive exhaustion of reserves proportional to metabolic rate. The temperature at which survival was longest was called the threshold temperature. The position of the threshold exhibited adaptive changes, either due to acclimation in the same species, or to genetic variations evidenced between species. In D. melanogaster, adults grown at lower temperature (12 degrees C) were more tolerant to cold than adults grown at higher temperatures (21, 25 or 30 degrees C). This acclimation process shifted, in an adaptive way, the position of the threshold temperature from 6.2 to 7.5 degrees C. A comparison of three different species grown at a single developmental temperature (21 degrees C) revealed similar but greater adaptive differences in their threshold temperature: 4.8 degrees C in the temperate D. subobscura, 7 degrees C in the cosmopolitan D. melanogaster and 14.6 degrees C in the tropical D. ananassae.     

Transmembrane electrical characteristics of cultured human skeletal muscle cells

Skeletal muscle explants from normal subjects were established from biopsy material on collagen. Cellular outgrowth appeared within 3-4 days, and fusion of myoblasts was observed in 5-10 days. Multinucleated myotubes were impaled under high optical magnification, at 37 degrees C, with conventional glass microelectrodes. The mean resting potential was -44.4 mV +/- 2.4 (n = 399); -33 +/- 2.3 mV at 9 days (n = 10) vs -48 +/- 2.5 mV (n = 15) at 27 days. The average input resistance (Rin) was 9.7 M omega (n = 83). Action potentials could be elicited by electrical stimulation and had a mean amplitude of 55.9 +/- 2.1 mV with a mean maximum rate of rise (Vmax) of 72.1 +/- 7.5 V/s. The mean overshoot was 13.9 +/- 2.3 mV, and the action potential duration determined at 50% of repolarization (APD50) was 8.0 msec (n = 7). The resting membrane potential showed a depolarization of 23 mV/decade for extracellular potassium ion concentration ([K]o) between 4.5-100 mM. Thus, we have established the normal resting potential and maximum rate of rise of the action potential for human myotubes in culture. We have shown that the values for these are less than those previously reported in cultured avian and rodent cells. In addition, we have shown that the response in our system of the resting potential to change in extracellular potassium concentration is blunted compared to studies using isolated muscle, suggesting an increase in ratio of sodium to potassium permeability. Cultured human muscle cells depolarized in the presence of ouabain.     

A comparative study of the short term cold resistance response in distantly related Drosophila species: the role of regucalcin and frost

The molecular basis of short term cold resistance (indexed as chill-coma recovery time) has been mostly addressed in D. melanogaster, where candidate genes (Dca (also known as smp-30) and Frost (Fst)) have been identified. Nevertheless, in Drosophila, the ability to tolerate short term exposure to low temperatures evolved several times independently. Therefore, it is unclear whether variation in the same candidate genes is also responsible for short term cold resistance in distantly related Drosophila species. It should be noted that Dca is a candidate gene for cold resistance in the Sophophora subgenus only, since there is no orthologous gene copy in the Drosophila subgenus. Here we show that, in D. americana (Drosophila subgenus), there is a north-south gradient for a variant at the 5' non-coding region of regucalcin (a Dca-like gene; in D. melanogaster the proteins encoded by the two genes share 71.9% amino acid identities) but in our D. americana F2 association experiment there is no association between this polymorphism and chill-coma recovery times. Moreover, we found no convincing evidence that this gene is up-regulated after cold shock in both D. americana and D. melanogaster. Size variation in the Fst PEST domain (putatively involved in rapid protein degradation) is observed when comparing distantly related Drosophila species, and is associated with short term cold resistance differences in D. americana. Nevertheless, this effect is likely through body size variation. Moreover, we show that, even at two hours after cold shock, when up-regulation of this gene is maximal in D. melanogaster (about 48 fold expression change), in D. americana this gene is only moderately up-regulated (about 3 fold expression change). Our work thus shows that there are important differences regarding the molecular basis of cold resistance in distantly related Drosophila species.     

Viability and rate of development at different temperatures in Drosophila: a comparison of constant and alternating thermal regimes

Populations belonging to the sibling species Drosophila melanogaster and D. simulans were collected in Southwestern France and Southern Spain, and investigated under constant (CT) and alternating (AT) temperature regimes. Development under CT was possible between 11 and 32 degrees C and egg-to-adult viability curves were almost 'rectangular', with a sharp decrease below 14 and above 29 degrees C. Rate of development followed a complex non-linear curve. A model described the curve as an exponential below a critical temperature (T(C)), and above T(C) as the difference between this function and another exponential which is assumed to show deleterious effects of heat. Developmental rates under two daily 12-h phases with various mid-temperatures and thermal amplitudes were compared to expected rates calculated from the above model. Acceleration effects were observed at four AT (in increasing order: 12-30, 9-21, 11-21, 16-26 degrees C); retardation occurred at three other ones (in increasing order, 7-21, 5-15, 7-29 degrees C). When expressed by the ratio observed/expected, the effects could be predicted using a multiple regression, as a positive function of the thermal amplitude and a negative one of the mid-temperature. Viability under AT was analysed considering an equivalent developmental temperature (EDT), that is the CT which would produce the same rate or development. Very low viabilities occurred under broad amplitude regimes, but the deleterious effects of some extreme temperatures, that would be lethal under CT, could be recovered by daily return to a moderate temperature. The two species exhibited slight but significant differences in their characteristic temperatures: developmental zero, critical temperature, temperature of maximum rate, upper developmental limit. All data may be interpreted by considering that D. simulans compared to D. melanogaster is more tolerant to cold but less tolerant to heat.