Experimental manipulation of the cost of thermal acclimation in Drosophila melanogaster

Acclimation to environmental change can impose costs on organisms. One potential cost is the energy and nutrients consumed by a physiological response, e.g. the resources required for expression of heat-shock proteins (Hsps). We examined the significance of this cost by genetic manipulation. We isolated four isofemale lines from a Drosophila melanogaster population previously transformed with a hsp70-kcZ fusion. Lines were similar in Hsp70 expression but differed in P-galactosidase expression upon heat shock, and replicates of each line were reared on a high quantity and low quantity medium. Multiple heat shock reduced survival in all lines, but did not increase developmental time. Variation in expression of β-galactosidase among lines, which differed more than 4-fold in response to heat treatment, was unrelated to the decreased survival. Thus the predicted effects of β-galactosidase expression on components of fitness were not evident. The superimposition of costs upon those normal for acclimation had no effect on mortality or developmental time, even when resources were especially limiting.     

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.     

Investigating thermal acclimation effects before and after a cold shock in Drosophila melanogaster using behavioural assays

Acclimation to environmental change can impose both costs and benefits to organisms. In this study we explored to what extent locomotor behaviour of Drosophila melanogaster is influenced by developmental temperature and adult temperature in both the laboratory and the field. Following development at 15, 25, or 31 °C, adult flies were tested for locomotor activity at all developmental temperatures in the laboratory before and after exposure to a cold shock and in the field for their ability to locate resources after a cold shock. Both test (15, 25, and 31 °C) and developmental temperatures strongly affected locomoter activity, with flies developed at 25 °C having the highest activity at all three test temperatures before the cold shock. After the cold shock flies developed at 15 °C had higher activity compared with flies developed at 25 and 31 °C when tested at 15 and 25 °C, and flies developed at 25 °C had the highest activity when tested at 31 °C. Furthermore, flies developed at 31 °C showed longer recovery times following the cold shock at test temperatures of 15 and 25 °C. However, flies acclimated at 15 °C during development did not recover faster at 15 and 25 °C compared with flies developed at 25 °C. There were no significant correlations between recovery time and locomotor activity at any of the test temperatures. Flies developed at 15 °C and exposed to a cold shock before release in the field were much more successful in locating a resource at low field temperatures compared with flies developed at 25 and 31 °C. Our results provide support for both the beneficial acclimation hypothesis and the optimal developmental temperature hypothesis, but the results are highly context dependent and change with the temperature experienced by the individual during its lifetime.     

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.     

Hardening trumps acclimation in improving cold tolerance of Drosophila melanogaster larvae

Abstract Chill‐susceptible insects are able to improve their survival of acute cold exposure over both the short term (i.e. hardening at a relatively severe temperature) and longer term (i.e. acclimation responses at milder temperatures over a longer time frame). However, the mechanistic overlap of these responses is not clear. Four larval stages of four different strains of Drosophila melanogaster are used to test whether low temperature acclimation (10 °C for 48 h) improves the acute cold tolerance (LT₉₀, ～2 h) of larvae, and whether acclimated larvae still show hardening responses after brief exposures to nonlethal cold or heat, or a combination of the two. Acclimation results in increased cold tolerance in three of four strains, with variation among instars. However, if acclimation is followed by hardening pre‐treatments, there is no improvement in acute cold survival. It is concluded that short‐term thermal responses (e.g. hardening) may be of more ecological relevance to short‐lived life stages such as larvae, and that the mechanisms of low temperature hardening and acclimation in D. melanogaster may be antagonistic, rather than complementary.     

Wolbachia modifies thermal preference in Drosophila melanogaster

Environmental variation can have profound and direct effects on fitness, fecundity, and host–symbiont interactions. Replication rates of microbes within arthropod hosts, for example, are correlated with incubation temperature but less is known about the influence of host–symbiont dynamics on environmental preference. Hence, we conducted thermal preference (T_p) assays and tested if infection status and genetic variation in endosymbiont bacterium Wolbachia affected temperature choice of Drosophila melanogaster. We demonstrate that isogenic flies infected with Wolbachia preferred lower temperatures compared with uninfected Drosophila. Moreover, T_p varied with respect to three investigated Wolbachia variants (wMel, wMelCS, and wMelPop). While uninfected individuals preferred 24.4°C, we found significant shifts of −1.2°C in wMel- and −4°C in flies infected either with wMelCS or wMelPop. We, therefore, postulate that Wolbachia-associated T_p variation within a host species might represent a behavioural accommodation to host–symbiont interactions and trigger behavioural self-medication and bacterial titre regulation by the host.     

Proteomic analysis of the wing imaginal discs of Drosophila melanogaster

We have combined high-resolution two-dimensional (2-D) gel electrophoresis and mass spectrometry with the aim of identifying proteins represented in the 2-D gel database of the wing imaginal discs of Drosophila melanogaster. First, we obtained a high-resolution 2-D gel pattern of [35S]methionine + [35S]cysteine-labeled polypeptides of Schneider cells, a permanent cell line of Drosophila embryonic origin, and compared it with the standard pattern of polypeptides of the wing imaginal disc. These studies reveal qualitative and quantitative differences between the two samples, but have more than 600 polypeptides in common. Second, we carried out preparative 2-D polyacrylamide gel electrophoresis using Schneider cells mixed with radioactively labeled wing imaginal discs in order to isolate some of the shared polypeptides and characterize them by matrix-assisted laser desorption/ionization-time of flight MALDI-TOF analysis. Using this strategy we identified 100 shared proteins represented in the database, and in each case confirmed their identity by MALDI-TOF/TOF analysis.     

Developmental thermal plasticity among Drosophila melanogaster populations

Many biotic and abiotic variables influence the dispersal and distribution of organisms. Temperature has a major role in determining these patterns because it changes daily, seasonally and spatially, and these fluctuations have a significant impact on an organism's behaviour and fitness. Most ecologically relevant phenotypes that are adaptive are also complex and thus they are influenced by many underlying loci that interact with the environment. In this study, we quantified the degree of thermal phenotypic plasticity within and among populations by measuring chill‐coma recovery times of lines reared from egg to adult at two different environmental temperatures. We used sixty genotypes from six natural populations of Drosophila melanogaster sampled along a latitudinal gradient in South America. We found significant variation in thermal plasticity both within and among populations. All populations exhibit a cold acclimation response, with flies reared at lower temperatures having increased resistance to cold. We tested a series of environmental parameters against the variation in population mean thermal plasticity and discovered the mean thermal plasticity was significantly correlated with altitude of origin of the population. Pairing our data with previous experiments on viability fitness assays in the same populations in fixed and variable environments suggests an adaptive role of this thermal plasticity in variable laboratory environments. Altogether, these data demonstrate abundant variation in adaptive thermal plasticity within and among populations.     

Metabolomic profiling of the effects of allopurinol on Drosophila melanogaster

Metabolomic profiling using hydrophilic interaction chromatography in combination with Fourier transform mass spectrometry was used to study the effects of the xanthine oxidase inhibitor allopurinol on wild type Drosophila melanogaster. Allopurinol treatment phenocopied the rosy mutation causing an elevation in the levels of xanthine and hypoxanthine and a fall in the levels of uric acid and allantoin. However, in addition there were some unexpected metabolic changes after treatment. Ascorbic acid levels were undetectable, glutathione levels fell and glutathione disulphide levels rose, methionine S-oxide levels rose and riboflavin levels fell. The origin of this oxidative stress was not immediately apparent; however, there was a strong suggestion that it might be related to a fall in NADPH levels linked to a reduction in glucose-6-phosphate dehydrogenase activity, resulting in reduced levels of some metabolites in the pentose phosphate pathway. In addition to producing oxidative stress there were marked effects on tryptophan metabolism with most of the metabolites in the kynurenine pathway being lowered by allopurinol treatment. The effects on the kynurenine pathway could be related to the established use of allopurinol in treating schizophrenia.     

Molecular thermal telemetry of free-ranging adult Drosophila melanogaster

The expression of two temperature-sensitive reporter genes, hsp70 and an hsp70-LacZ fusion, in free-ranging adult Drosophila melanogaster indicates that natural thermal stress experienced by such small and mobile insects may be either infrequent or not severe. Levels of the heat-shock protein Hsp70, the major inducible Hsp of Drosophila, were similar in most wild Droso- phila captured after warm days to levels previously reported for unstressed flies in the laboratory. In a transgenic strain transformed with an hsp70-LacZ fusion (i.e., the structural gene encoding bacterial β-galactosidase under control of a heat shock promoter), exposure to temperatures ≥32°C in the laboratory typically resulted in β-galactosidase activities exceeding 140 mOD₄₅₀ h^–1μg^–1 soluble protein. Flies caged in sun frequently had β-galactosidase activities in excess of this level, whereas flies caged in shade and flies released and recaptured on cool days did not. Most flies (>80%) released on warm, sunny days had low β-galactosidase activities upon recapture. Although the balance of recaptured flies had elevated β-galactosidase activities on these days, their β-galactosidase activities were <50% of levels for flies caged in direct sunlight or exposed to laboratory heat shock. These data suggest that even on warm days most flies may avoid thermal stress, presumably through microhabitat selection, but that a minority of adult D. melanogaster undergo mild thermal stress in nature. Both temperature-sensitive reporter genes, however, are limited in their ability to infer thermal stress and demonstrate its absence. Received: 14 July 1999 / Accepted: 21 December 1999     

Indirect thermal selection in Drosophila melanogaster and adaptive consequences

Summary Short-term indirect selection in Drosophila melanogaster for heat-sensitivity and heat resistance resulted in two strains, one heat sensitive and another heat resistant, and correlated responses were found for the rate of heat shock protein synthesis, behavioral patterns (asymmetrical sexual isolation) and fitness components (fecundity, fertility, viability, developmental time), as well as for several enzyme activities (MDH, G-6-PDH, ADH, ACHE). These responses associated with temperature selection may reflect the effects of differential inbreeding depression caused by homozygosity of temperature sensitive mutations with different pleiotropic effects. Selection even of a very short duration can induce significant adaptive and evolutionary changes.     

Metabolomic profiling of rapid cold hardening and cold shock in Drosophila melanogaster

A short exposure to a mild cold stress is sufficient to increase cold tolerance in many insects. This phenomenon, termed rapid cold hardening (RCH) expands the thermal interval that can be exploited by the insect. To investigate the possible role of altered metabolite levels during RCH, the present study used untargeted (1)H NMR metabolomic profiling to examine the metabolomic response in Drosophila melanogaster during the 72 h following RCH and cold shock treatment. These findings are discussed in relation to the costs and benefits of RCH that are measured in terms of survival and reproductive output. Cold shock caused a persistent disturbance of the metabolite profile that correlated well with a delayed onset of cold shock mortality. The disruption of metabolite homeostasis was smaller following RCH, where control levels were fully recovered after 72 h. RCH improved both survival and reproductive output after a subsequent cold shock but the RCH treatment alone was associated with costs in terms of reduced survival and reproductive output. The most pronounced changes following the RCH treatment were elevated levels of glucose and trehalose. Although, it is difficult to discern if a change in a specific metabolite is linked to physiological processes of adaptive, neutral or detrimental nature we observed that the onset and magnitude of the increased sugar levels correlated tightly with the improved chill tolerance following RCH. These findings suggest a putative role of cryoprotectants during RCH which are discussed in the light of the existing literature on the mechanistic background of RCH.     

Brief carbon dioxide exposure blocks heat hardening but not cold acclimation in Drosophila melanogaster

Carbon dioxide is a commonly used anaesthetic in Drosophila research. While any detrimental effects of CO2 exposure on behaviour or traits are largely unknown, a recent study observed significant effects of CO2 exposure on rapid cold hardening and chill-coma recovery in Drosophila melanogaster. In this study we investigated the effect of a brief CO2 exposure on heat hardening and cold acclimation in D. melanogaster, measuring heat knockdown and chill-coma recovery times of flies exposed to CO2 for 1 min after hardening or acclimation. CO2 anaesthesia had a significant negative effect on heat hardening, with heat knockdown rates in hardened flies completely reduced to those of controls after CO2 exposure. Chill-coma recovery rates also significantly increased in acclimated flies that were exposed to CO2, although not to the same extent seen in the heat populations. CO2 exposure had no impact on heat knockdown rates of control flies, while there was a significant negative effect of the anaesthetic on chill-coma recovery rates of control flies. In light of these results, we suggest that CO2 should not be used after hardening in heat resistance assays due to the complete reversal of the heat hardening process upon exposure to CO2.     

Hesperidin modulates the rhythmic proteomic profiling in Drosophila melanogaster under oxidative stress

The circadian clock regulates vital aspects of physiology including protein synthesis and oxidative stress response. In this investigation, we performed a proteome-wide scrutiny of rhythmic protein accrual in Drosophila melanogaster on exposure to rotenone, rotenone + hesperidin and hesperidin in D. melanogaster. Total protein from fly samples collected at 6 h intervals over the 24 h period was subjected to two-dimensional gel electrophoresis and mass spectrometry. Bioinformatics tool, Protein ANalysis THrough Evolutionary Relationships classification system was used to the determine the biological processes of the proteins of altered abundance. Conspicuous variations in the proteome (151 proteins) of the flies exposed to oxidative stress (by rotenone treatment) and after alleviating oxidative stress (by hesperidin treatment) were observed during the 24 h cycle. Significantly altered levels of abundance of a wide variety of proteins under oxidative stress (rotenone treatment) and under alleviation of oxidative stress (rotenone + hesperidin treatment) and hesperidin (alone) treatment were observed. These proteins are involved in metabolism, muscle activity, heat shock response, redox homeostasis, protein synthesis/folding/degradation, development, ion-channel/cellular transport, and gustatory and olfactory function of the flies. Our data indicates that numerous cellular processes are involved in the temporal regulation of proteins and widespread modulations happen under rotenone treatment and, action of hesperidin could also be seen on these categories of proteins.     

Rapid thermal adaptation during field temperature variations in Drosophila melanogaster

Under natural conditions, the fruit fly (Drosophila melanogaster) is constantly exposed to variations in temperature and light. Laboratory investigations have demonstrated that D. melanogaster and other insects adapt quickly to temperature variations, but only few studies have investigated this ability under natural temperature variations. Here we placed laboratory raised female D. melanogaster in field cages and exposed them to natural variations in light and temperature over a 2 day period (temperature range: 12–25 °C). During this period we sampled flies every 6 h and measured their ability to survive heat and cold shock. There was a significant positive correlation between field temperature and heat shock survival and a significant negative correlation between field temperature and cold shock survival indicating that D. melanogaster are constantly adapting to their surrounding environment. The results also suggest that heat and cold resistance are obtained at a cost as these two traits were negatively correlated.     

Variation in resistance and acclimation to low-temperature stress among three geographical strains of Drosophila melanogaster

Variation in cold resistance was examined in cold acclimated and non-acclimated Drosophila melanogaster from three geographical strains representing Morocco, France and Finland. Resistance was estimated as survival of adults at 0°C; the acclimation treatment involved a long-term exposure to 11°C starting from the late pupal stage and continuing with adults. After the cold stress, two fitness traits, percentage of fertile individuals and the number of adult progeny, were scored in both acclimated and non-acclimated flies. Acclimation dramatically increased survival in all strains, but did not affect the pattern of geographic variation in cold resistance. The European flies tended to be more resistant than the African ones and the ranking from most to least resistant strain was France>Finland>Morocco. In the absence of acclimation, females showed a higher survival than males. Percentage of fertile males in all strains and the number of progeny in the Finnish and French strains were decreased after acclimation. Without cold acclimation, the number of progeny was higher in the European flies as compared with the African ones. The results suggest that populations of D. melanogaster from cold climates are better adapted to low stressful temperatures and among-population variation in cold resistance may be due to non-plastic rather than plastic genetic changes. The deleterious effects of cold pretreatment on the life-history parameters indicate a possibility for acclimation costs in reproduction.