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.     

Temperature-related kinetic differentiation of glucosephosphate isomerase alleloenzymes isolated from the blue mussel,Mytilus edulis

Two glucosephosphate isomerase (GPI; D-glucose-6-phosphate ketolisomerase; EC 5.3.1.9) alleloenzymes from the blue mussel, Mytilus edulis, were purified to homogeneity. The steady-state kinetic properties of GPI1.00 and GPI.96, which exhibit latitudinal clines in frequency along the Atlantic coast of North America, were determined in both the glycolytic and the gluconeogenic reaction directions at physiological temperatures and pH levels. The two alleloenzymes are catalytically similar at low temperatures (5-10 degrees C), while GPI1.00 diverges to become more efficient at higher physiological temperatures (15-25 degrees C). This pattern of differentiation is consistent with the latitudinal distributions of the alleloenzymes and is due to the greater temperature sensitivities of GP1.00 Vmax/Km values; the Vmax values of the two alleloenzymes are virtually the same over the physiological range of temperatures. The observed pattern of catalytic differentiation is similar to that seen for interspecific GPI variants.     

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 genetic variant of beta-glucuronidase in Drosophila melanogaster

The beta-glucuronidase activity of Drosophila melanogaster exists as two chromatographically separable forms, both of which are glycoproteins. Form I is membrane-bound in vivo, has a pI of 8.0-8.5, and can be irreversibly inactivated either by incubation at 55 degrees C for 20 min or by incubation at 37 degrees C in the presence of 6 M urea. Form II exists both membrane-bound as well as membrane-free, has a pI of 4.5, and is resistant to the conditions which inactivate form I. The two forms are similar in Km and Vmax for the artificial substrate 4-methylumbelliferyl-beta-D-glucuronide and both forms are precipitated by antibody to form II. A natural genetic variant, beta-GluL1, completely lacks from I beta-glucuronidase. This variant behaves in a co-dominant fashion for the determination of the presence of form I and has been localized to the extreme distal portion of chromosome 3R. Other data indicate that at least one genetic determinant for the amount of form II is also localized to this portion of chromosome 3R.     

Cell fusion between temperature-sensitive mutants of a Drosophila melanogaster cell line.

Temperature-sensitive (ts) mutants were isolated in a cell line of Drosophila melanogaster, GM1, by ethyl methanesulfate treatment. Two of them, ts15 and ts58, formed colonies at 23 degrees C but not at 30 degrees when inoculated at densities of/or less than 10(5) cells per 60 X 15-mm dish. By using these ts mutants, cell fusion was attempted with polyethylene glycol (PEG) 6000. Several colonies per dish developed at 30 degrees C when different ts mutants were mixed, treated with PEG, and inoculated at a density of 10(4) cells per dish. Cells in some of the colonies thus developed were propagated and their temperature-sensitive character and karyotypes were studied. The results indicated that cell fusion could be induced with PEG and that the cells which formed colonies at 30 degrees C after PEG treatment were the hybrids in which the temperature-sensitive lesions in the mutants were complemented.     

Modulation of the catalytic activity of cruzipain, the major cysteine proteinase from Trypanosoma cruzi, by temperature and pH.

Cysteine proteinases are relevant to several aspects of the parasite life cycle and of parasite-host relationships. Here, a quantitative investigation of the effect of temperature and pH on the total substrate inhibition of cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi, is reported. Values of the apparent catalytic and inhibition parameters Km, Vmax, Vmax/Km, and K(i) for the cruzipain-catalysed hydrolysis of N-alpha-benzyloxycarbonyl-L-phenylalanyl-L-arginine-(7-amino-4-methylcoumarin) (Z-Phe-Arg-AMC) and azocasein were determined between 10.0 degrees C and 40.0 degrees C and between pH 4.5 and 8.5. Values of Km were independent of temperature and pH, whereas values of Vmax, Vmax/Km, and K(i) were temperature-dependent and pH-dependent. Over the whole pH range explored, values of logVmax, log(Vmax/Km), and logK(i) increased linearly with respect to T(-1). Values of Vmax and Vmax/Km were affected by the acid-base equilibrium of one temperature-independent ionizing group (i.e. pK(unl)' = pK(lig)' = 5.7 +/- 0.1, at 25.0 degrees C). Moreover, values of K(i) were affected by the alkaline pK shift of one ionizing group of active cruzipain (from pK(unl)" = 5.7 +/- 0.1 to pK(lig)" = 6.1 +/- 0.1, at 25.0 degrees C) upon Z-Phe-Arg-AMC binding. Values of logK(unl)', logK(lig)', and logK(lig)" were temperature-independent. Conversely, values of logK(unl)" were linearly dependent on T(-1). As a whole, total substrate inhibition of cruzipain decreased with increasing temperature and pH. These data suggest that both synthetic and protein substrates can bind to the unique active centre of cruzipain either productively or following a binding mode which results in enzyme inhibition. However, allosteric effect(s) cannot be excluded.     

Temperature-dependency of electron-transport activity in mitochondria with exogenous mitochondrial DNA in Drosophila.

In mitochondrial DNA (mtDNA) heteroplasmy induced artificially in Drosophila melanogaster (Matsuura et al., 1989), foreign mtDNA derived from D. mauritiana was selectively transmitted at 25 degrees C but was lost at 19 degrees C (Niki et al., 1989; Matsuura et al., 1990, 1991). To investigate temperature-dependent factors in the selective transmission of mtDNA, the temperature-dependency of electron-transport activity of mitochondria from D. melanogaster in which endogenous mtDNA was completely replaced by the foreign mtDNA was compared with that of D. melanogaster and D. mauritiana. For NADH-oxidase activity, the optimum temperature of D. mauritiana mitochondria was 35 degrees C while for two types of mitochondria from D. melanogaster each possessing either endogenous or exogenous mtDNA, maximum activity was noted at 32 degrees C. This observation suggests that the temperature-dependency of mitochondrial electron-transport activity is mainly determined by a nuclear genome. NADH-cytochrome c reductase and cytochrome c oxidase activities were not significantly different among the three types of mitochondria. The temperature-dependency of mitochondrial function apparently is not involved in the temperature-dependent selective transmission of mtDNA in the heteroplasmic state.     

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.     

Effects of temperature on allosteric and catalytic properties of the cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver

We have investigated effects of temperature on the catalytic and allosteric properties of the cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver. Vmax for cAMP and cGMP increased as assay temperature increased from 5 to 45 degrees C. At substrate concentrations below Kmapp, however, hydrolysis increased as temperature decreased from 45 to 5 degrees C and was much greater at 5 degrees C than at 45 degrees C. As assay temperature decreased, Kmapp for cAMP and cGMP decreased. Hill coefficients for cAMP and cGMP were approximately 1.9 at 45 degrees C and 1.2-1.0 at 5 degrees C. cGMP stimulated hydrolysis of 0.5 microM [3H]cAMP at all assay temperatures. Although maximal activity stimulated by cGMP, like Vmax, was lowest at 5 degrees C, presumably because of the effect of temperature on catalytic activity, the apparent activation constant (K alpha app) for cGMP stimulation was lower at 5 degrees C than at 45 degrees C. Thus, affinity for both substrate and effector was increased at 5 degrees C, suggesting that low temperature promotes transitions of the cGMP-stimulated phosphodiesterase to a "high affinity" state. That cGMP stimulated cAMP hydrolysis at 5 degrees C suggests that temperature-induced transitions are incomplete and/or readily reversible. In assays at 30 degrees C competitive inhibitors, like substrates, induce allosteric transitions which result in enhanced hydrolysis of low substrate (1.0 microM [3H] cAMP) concentrations. At higher substrate concentrations (50 microM [3H]cAMP), with the enzyme in the "activated" state, inhibitors compete with substrate at catalytic sites and reduce hydrolysis. At 45 degrees C, as at 30 degrees C, 1-methyl-3-isobutylxanthine (IBMX) and papaverine increased hydrolysis of 1.0 microM [3H]cAMP and reduced hydrolysis of 50 microM [3H]cAMP. At 5 degrees C, however, IBMX and papaverine inhibited hydrolysis of both 1.0 and 50 microM [3H]cAMP. Enzyme activity was relatively more sensitive to inhibition by IBMX at 5 degrees C than at 45 degrees C. Taken together, these observations support the notion that low temperature induces incomplete or readily reversible transitions to the high affinity state for substrates, effectors, and inhibitors. These observed effects of temperature also point out that enzyme determinants and topographical features responsible for transitions to the high affinity state and expression of catalytic activity can be regulated independently.     

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.     

Selective transmission of mitochondrial DNA in heteroplasmic lines for intra- and interspecific combinations in Drosophila melanogaster

The transmission of mitochondrial DNA (mtDNA) was investigated in the heteroplasmic lines of Drosophila melanogaster at 19 degrees C and at 25 degrees C. The selective transmission of one type of mtDNA was dependent on the temperature at which the lines were maintained. In heteroplasmic lines for an intraspecific combination induced by germ-plasm transplantation using D. melanogaster as a germ-plasm donor, the proportion of donor mtDNA decreased in four out of five lines examined, the decreasing rate of which being greater at 25 degrees C than at 19 degrees C. Donor mtDNA was lost by the 20th generation at 25 degrees C. For an interspecific combination using D. mauritiana as a germ-plasm donor, the proportion of donor mtDNA increased and endogenous mtDNA was replaced with donor mtDNA at 25 degrees C. But donor mtDNA was almost lost at 19 degrees C by the 14th generation in all four lines examined. Possible mechanisms involved in the temperature-dependent modes of mtDNA transmission are discussed.     

Kinetic characteristics of extracellular catalase from Penicillium piceum F-648 and variants of fungi,adapted to hydrogen peroxide

A comparative kinetic study of extracellular catalases produced by Penicillium piceum F-648 and their variants adapted to H2O2 was performed in culture liquid filtrates. The specific activity of catalase, the maximum rate of catalase-induced H2O2 degradation (Vmax),Vmax/KM ratio, and the catalase inactivation rate constant in the enzymatic reaction (kin, s-1) were estimated in phosphate buffer (pH 7.4) at 30 degrees C. The effective constant representing the rate of catalase thermal inactivation (kin*, s-1) was determined at 45 degrees C. In all samples, the specific activity and KM for catalase were maximum at a protein concentration in culture liquid filtrates of 2.5-3.5 x 10(-4) mg/ml. The effective constants describing the rate of H2O2 degradation (k, s-1) were similar to that observed in the initial culture. These values reflected a twofold decrease in catalase activity in culture liquid filtrates. We hypothesized that culture liquid filtrates contain two isoforms of extracellular catalase characterized by different activities and affinities for H2O2. Catalases from variants 5 and 3 with high and low affinities for H2O2, respectively, had a greater operational stability than the enzyme from the initial culture. The method of adaptive selection for H2O2 can be used to obtain fungal variants producing extracellular catalases with improved properties.     

An investigation of the in vitro inhibition of acetylcholinesterase by the carbamate inhibitor eserine sulphate in eserine resistant strains of Drosophila melanogaster

1. The kinetics of inhibition of AChE by the carbamate inhibitor eserine sulphate were investigated in five resistant strains of Drosophila melanogaster. 2. The dissociation constant (Kd) was higher and the carbamylation constant was lower relative to the control in four of these. 3. No change was observed in the decarbamylation constants (k3) of the five strains. 4. The Vmax of AChE was higher in the five resistant stocks than in the Canton-S/TM2 controls but no change in the Km of AChE for acetylthiocholine was observed. No electrophoretic variants of AChE were detected. 5. No increase in the activity of nonspecific esterases was detected in the resistant lines. 6. These results indicate that resistance to eserine sulphate may occur in D. melanogaster by a reduction in affinity of AChE for the inhibitor.     

Genetic Variants Affecting Phenoloxidase Activity in Drosophila melanogaster

In Drosophila melanogaster, two new variants affecting the activity of phenoloxidase were found in natural populations at Gomel in Belorussia and at Krasnodar in Russia. Prophenoloxidases, A ₁ and A ₃ , in these variants had the same mobilities on native electrophoresis as the wild type. However, enzymatic activities in their activated states were much lower than in the wild type, whereas the existence of prophenoloxidase proteins was demonstrated. Egg-to-adult and relative viabilities in the variants did not decrease at temperatures between 18 and 29°C. Genetic analyses indicated that the genes showing the phenotype of variants are new alleles of Mox and Dox-3 on the second chromosome.     

Parental and developmental temperature effects on the thermal dependence of fitness in Drosophila melanogaster

Cross-generational effects refer to nongenetic influences of the parental phenotype or environment on offspring phenotypes. Such effects are commonly observed, but their adaptive significance is largely unresolved. We examined cross-generational effects of parental temperature on offspring fitness (estimated via a serial-transfer assay) at different temperatures in a laboratory population of Drosophila melanogaster. Parents were reared at 18 degrees C, 25 degrees C, or 29 degrees C (Tpar) and then their offspring were reared at 18 degrees C, 25 degrees C, or 29 degrees C (Toff) to evaluate several competing hypotheses (including an adaptive one) involving interaction effects of parental and offspring temperature on offspring fitness. The results clearly show that hotter parents are better; in other words, the higher the temperature of the parents, the higher the fitness of their offspring, independent of offspring thermal environment. These data contradict the adaptive cross-generational hypothesis, which proposes that offspring fitness is maximal when the offspring thermal regime matches the parental one. Flies with hot parents have high fitness seemingly because their own offspring develop relatively quickly, not because they have higher fecundity early in life.     

The pH and temperature dependence of the activity of the high Km cyclic nucleotide phosphodiesterase of bakers' yeast

The hydrolysis of adenosine 3':5'-monophosphate by the high Km cyclic nucleotide phosphodiesterase of bakers' yeast was studied over a range of temperature and pH at I = 0.17. The effects of ionic strength and MgCl2 concentration were studied at pH 7.7 and 30 degrees C. Km and Vmax were insensitive to changes in the MgCl2 concentration between 1 and 30 mM, implying that this enzyme (which does not require free divalent metal ions) does not discriminate between free cyclic AMP- and the Mg-cyclic AMP+ complex. Vmax decreased below pH 6.8 because of protonation of a group required in the basic form in the enzyme x substrate complex. On the basis of its pK (5.46 at 30 degrees C) and delta H (23 kJ/mol) this group was tentatively identified as imidazole. Vmax/Km decreased above pH 6.8 because of ionization of a group required in the acid form in the free enzyme, with a pK of 7.88 at 30 degrees C and a delta H of about 13 kJ/mol. Several possibilities exist for the identity of this group, the most likely being a second imidazole, sulfhydryl, or a water molecule bonded to tightly bound zinc. At pH 7.90, log Vmax and log Km both changed linearly with 1/T (between 12 degrees C and 37 degrees C) with enthalpies of 47 and 55 kJ/mol, respectively. Consequently, at low enough cyclic AMP concentration, the rate of reaction at pH 7.90 decreases slightly when the temperature is increased. This is also true at higher pH, but in the physiological pH range (6.4 to 7.5) Vmax/Km and, therefore, the rate of reaction at very low cyclic AMP concentration were nearly independent of temperature. Under physiological conditions, the Km approaches the upper limit of in vivo cyclic AMP concentrations in yeast, and at normal in vivo cyclic AMP concentrations the pH optimum is within or below the physiological range of pH in yeast.     

产气荚膜梭菌促进黑腹果蝇的生长和发育

【目的】黑腹果蝇Drosophila melanogaster肠道中栖生着众多微生物,通过分离和研究其内共生菌,以研究肠道菌群的多态性和作用。【方法】利用Hungate滚管技术从黑腹果蝇成虫肠道分离厌氧细菌;通过记录果蝇的发育历期和生长速率,检测该细菌对果蝇发育和生长的影响。【结果】首次从黑腹果蝇肠道内分离到一株产气荚膜梭菌Clostridium perfringens。该菌能够有效地定植到果蝇肠道内,是果蝇肠道共生菌。产气荚膜梭菌显著地缩短无菌果蝇的发育历期,将无菌果蝇成蛹天数由20 d缩短到8.1 d,羽化天数由30 d缩短到12.7 d。该菌还可以提高果蝇生长速率。【结论】本研究揭示了产气荚膜梭菌是果蝇的内共生菌,可以通过提高生长速率而有效地促进果蝇的生长和发育。     

Temperature dependence of the inhibitory effects of orthovanadate on shortening velocity in fast skeletal muscle.

We have investigated the effects of the orthophosphate (P(i)) analog orthovanadate (Vi) on maximum shortening velocity (Vmax) in activated, chemically skinned, vertebrate skeletal muscle fibers. Using new "temperature-jump" protocols, reproducible data can be obtained from activated fibers at high temperatures, and we have examined the effect of increased [Vi] on Vmax for temperatures in the range 5-30 degrees C. We find that for temperatures < or = 20 degrees C, increasing [Vi] inhibits Vmax; for temperatures > or = 25 degrees C, increasing [Vi] does not inhibit Vmax. Attached cross-bridges bound to Vi are thought to be an analog of the weakly bound actin-myosin.ADP-P(i) state. The data suggest that the weakly bound Vi state can inhibit velocity at low temperature, but not at high temperature, with the transition occurring over a narrow temperature range of < 5 degrees C. This suggests a highly cooperative interaction. The data also define a Q10 for Vmax of 2.1 for chemically skinned rabbit psoas fibers over the temperature range of 5-30 degrees C.     

Effects of acclimation temperature on thermal tolerance and membrane phospholipid composition in the fruit fly Drosophila melanogaster

Adaptative responses of ectothermic organisms to thermal variation typically involve the reorganization of membrane glycerophospholipids (GPLs) to maintain membrane function. We investigated how acclimation at 15, 20 and 25 degrees C during preimaginal development influences the thermal tolerance and the composition of membrane GPLs in adult Drosophila melanogaster. Long-term cold survival was significantly improved by low acclimation temperature. After 60 h at 0 degrees C, more than 80% of the 15 degrees C-acclimated flies survived while none of the 25 degrees C-acclimated flies survived. Cold shock tolerance (1h at subzero temperatures) was also slightly better in the cold acclimated flies. LT50 shifted down by ca 1.5 degrees C in 15 degrees C-acclimated flies in comparison to those acclimated at 25 degrees C. In contrast, heat tolerance was not influenced by acclimation temperature. Low temperature acclimation was associated with the increase in proportion of ethanolamine (from 52.7% to 58.5% in 25 degrees C-acclimated versus 15 degrees C-acclimated flies, respectively) at the expense of choline in GPLs. Relatively small, but statistically significant changes in lipid molecular composition were observed with decreasing acclimation temperature. In particular, the proportions of glycerophosphoethanolamines with linoleic acid (18:2) at the sn-2 position increased. No overall change in the degree of fatty acid unsaturation was observed. Thus, cold tolerance but not heat tolerance was influenced by preimaginal acclimation temperature and correlated with the changes in GPL composition in membranes of adult D. melanogaster.