Jesper Sørensen

Full genome gene expression analysis of the heat stress response in Drosophila melanogaster

Abstract The availability of full genome sequences has allowed the construction of microarrays, with which screening of the full genome for changes in gene expression is possible. This method can provide a wealth of information about biology at the level of gene expression and is a powerful method to identify genes and pathways involved in various processes. In this study, we report a detailed analysis of the full heat stress response in Drosophila melanogaster females, using whole genome gene expression arrays (Affymetrix Inc, Santa Clara, CA, USA). The study focuses on up- as well as downregulation of genes from just before and at 8 time points after an application of short heat hardening (36°C for 1 hour). The expression changes were followed up to 64 hours after the heat stress, using 4 biological replicates. This study describes in detail the dramatic change in gene expression over time induced by a short-term heat treatment. We found both known stress responding genes and new candidate genes, and processes to be involved in the stress response. We identified 3 main groups of stress responsive genes that were early–upregulated, early– downregulated, and late–upregulated, respectively, among 1222 differentially expressed genes in the data set. Comparisons with stress sensitive genes identified by studies of responses to other types of stress allow the discussion of heat-specific and general stress responses in Drosophila. Several unexpected features were revealed by this analysis, which suggests that novel pathways and mechanisms are involved in the responses to heat stress and to stress in general. The majority of stress responsive genes identified in this and other studies were downregulated, and the degree of overlap among downregulated genes was relatively high, whereas genes responding by upregulation to heat and other stress factors were more specific to the stress applied or to the conditions of the particular study. As an expected exception, heat shock genes were generally found to be upregulated by stress in general.

Costs and benefits of cold acclimation in field-released Drosophila

One way animals can counter the effects of climatic extremes is via physiological acclimation, but acclimating to one extreme might decrease performance under different conditions. Here, we use field releases of Drosophila melanogaster on two continents across a range of temperatures to test for costs and benefits of developmental or adult cold acclimation. Both types of cold acclimation had enormous benefits at low temperatures in the field; in the coldest releases only cold-acclimated flies were able to find a resource. However, this advantage came at a huge cost; flies that had not been cold-acclimated were up to 36 times more likely to find food than the cold-acclimated flies when temperatures were warm. Such costs and strong benefits were not evident in laboratory tests where we found no reduction in heat survival of the cold-acclimated flies. Field release studies, therefore, reveal costs of cold acclimation that standard laboratory assays do not detect. Thus, although physiological acclimation may dramatically improve fitness over a narrow set of thermal conditions, it may have the opposite effect once conditions extend outside this range, an increasingly likely scenario as temperature variability increases under global climate change.

Altitudinal variation for stress resistance traits and thermal adaptation in adult Drosophila buzzatii from the New World.

Multiple stress resistance traits were investigated in the cactophilic fly Drosophila buzzatii. Adults from seven populations derived from North‐Western Argentina were compared with respect to traits relevant for thermal stress resistance and for resistance to other forms of environmental stress. The populations were collected along an altitudinal gradient spanning more than 2000 m in height, showing large climatic differences. The results suggest that knock‐down resistance to heat stress, desiccation resistance and Hsp70 expression at a relatively severe stressful temperature best reflect thermal adaptation in this species. Furthermore, cold resistance seemed to be of less importance than heat resistance, at least for the adult life stage, in these populations. Clinal variation in thermal resistance traits over short geographical distances suggests relatively strong adaptive differentiation of the populations. This study provides the first evidence for altitudinal differentiation in stress‐related traits, and suggests that Hsp70 expression level can be related to altitudinal clines of heat‐stress resistance.

Water loss in insects: An environmental change perspective

In the context of global environmental change much of the focus has been on changing temperatures. However, patterns of rainfall and water availability have also been changing and are expected to continue doing so. In consequence, understanding the responses of insects to water availability is important, especially because it has a pronounced influence on insect activity, distribution patterns, and species richness. Here we therefore provide a critical review of key questions that either are being or need to be addressed in this field. First, an overview of insect behavioural responses to changing humidity conditions and the mechanisms underlying sensing of humidity variation is provided. The primary sensors in insects belong to the temperature receptor protein superfamily of cation channels. Temperature-activated transient receptor potential ion channels, or thermoTRPs, respond to a diverse range of stimuli and may be a primary integrator of sensory information, such as environmental temperature and moisture. Next we touch briefly on the components of water loss, drawing attention to a new, universal model of the water costs of gas exchange and its implications for responses to a warming, and in places drying, world. We also provide an overview of new understanding of the role of the sub-elytral chamber for water conservation, and developments in understanding of the role of cuticular hydrocarbons in preventing water loss. Because of an increasing focus on the molecular basis of responses to dehydration stress we touch briefly on this area, drawing attention to the role of sugars, heat shock proteins, aquaporins, and LEA proteins. Next we consider phenotypic plasticity or acclimation responses in insect water balance after initial exposures to altered humidity, temperature or nutrition. Although beneficial acclimation has been demonstrated in several instances, this is not always the case. Laboratory studies show that responses to selection for enhanced ability to survive water stress do evolve and that genetic variation for traits underlying such responses does exist in many species. However, in others, especially tropical, typically narrowly distributed species, this appears not to be the case. Using the above information we then demonstrate that habitat alteration, climate change, biological invasions, pollution and overexploitation are likely to be having considerable effects on insect populations mediated through physiological responses (or the lack thereof) to water stress, and that these effects may often be non-intuitive.

Larval crowding in Drosophila melanogaster induces Hsp70 expression, and leads to increased adult longevity and adult thermal stress resistance.

In this study we show for the first time that moderate high larval density induces Hsp70 expression in Drosophila melanogaster larvae. Larval crowding led to both increased mean and maximal longevity in adults of both sexes. Two different measures of heat-stress resistance increased in adult flies developed at high density compared to flies developed at low density. The hardening-like effect of high larval density carried over to the adult life stage. The hardening memory (the period of increased resistance after hardening) was long compared to hardening of adult flies, and possibly lasts throughout life. The increase in resistance in adults following development at high larval density seemed not to be connected to Hsp70 itself, since Hsp70 expression level in adult flies after hardening was independent of whether larvae developed at low or high densities. More likely, Hsp70 may be one of many components of the stress response resulting in hardening.

Gene expression profile analysis of Drosophila melanogaster selected for resistance to environmental stressors

Here, we report a detailed analysis of changes in gene expression in Drosophila melanogaster selected for ecologically relevant environmental stress resistance traits. We analysed females from seven replicated selection regimes and one control regime using whole genome gene expression arrays. When compared with gene expression profiles of control lines, we were able to detect consistent selection responses at the transcript level in each specific selection regime and also found a group of differentially expressed genes that were changed among all selected lines. Replicated selection lines showed similar changes in gene expression (compared with controls) and thus showed that 10 generations of artificial selection give a clear signal with respect to the resulting gene expression profile. The changes in gene expression in lines selected for increased longevity, desiccation and starvation resistance, respectively, showed high similarities. Cold resistance‐selected lines showed little differentiation from controls. Different methods of heat selection (heat survival, heat knock down and constant 30 °C) showed little similarity verifying that different mechanisms are involved in high temperature adaptation. For most individual selection regimes, and in the comparison of all selected lines and controls, the gene expression changes were exclusively in one direction, although the different selection regimes varied in the direction of response. The responses to selection restricted to individual selection regimes can be interpreted as stress specific, whereas the response shared among all selected lines can be considered as a general stress response. Here, we identified genes belonging to both types of responses to selection for stress resistance.

Effects of cold- and heat hardening on thermal resistance in Drosophila melanogaster

The effects of cold- and heat hardening on resistance to both low and high temperature stress was examined in Drosophila melanogaster lines selected for resistance to either cold or heat. The hardening effect was positive when the hardening was of the same type as the stress in all selection regimes. The effect of cold hardening on survival after heat stress was further examined in the lines selected for cold resistance and corresponding controls. A cross-protection effect (increased heat resistance after cold hardening) was present and this effect was lower in the lines selected for resistance to cold than in the controls. The level of Hsp70 expression induced by a non-lethal cold hardening was examined, showing that cold hardening induced Hsp70 expression. The results suggest that the cross-protection effect is at least partly due to Hsp70 expression induced by cold exposure.

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.

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 degrees C). During this period we sampled flies every 6h 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.

How to assess Drosophila cold tolerance: chill coma temperature and lower lethal temperature are the best predictors of cold distribution limits

Summary 1. Thermal tolerance may limit and therefore predict ectotherm geographic distributions. However, which of the many metrics of thermal tolerance best predict distribution is often unclear, even for drosophilids, which constitute a popular and well-described animal model. 2. Five metrics of cold tolerance were measured for 14 Drosophila species to determine which metrics most strongly correlate with geographic distribution. The species represent tropical to temperate regions but all were reared under similar (common garden) conditions (20 °C). The traits measured were: chill coma temperature (CTmin), lethal temperature (LTe50), lethal time at low temperature (LTi50), chill coma recovery time (CCRT) and supercooling point (SCP). 3. Measures of CTmin, LTe50 and LTi50 proved to be the best predictors to describe the variation in realized latitudinal distributions (R 2 =0 699, R 2 =0 741 and 0550, respectively) and estimated environmental cold exposure (R 2 =0 633, R 2 =0 641 and 0511, respectively). Measures of CCRT also correlated significantly with estimated minimum temperature (R 2 =0 373), while the SCP did not. These results remained consistent after phylogenetically independent analysis or when applying nonlinear regression. Moreover, our findings were supported by a similar analysis based on existing data compiled from the Drosophila cold tolerance literature. 4. Trait correlations were strong between LTe50, LTi50 and CTmin, respectively (083 > R 2 >0 55). However, surprisingly, there was only a weak correlation between the entrance into coma (CTmin) and the recovery from chill coma (CCRT) (R 2 =0 256). 5. Considering the findings of the present study, data from previous studies and the logistical constraints of each measure of cold tolerance, we conclude that CTmin and LTe50 are superior measures when estimating the ecologically relevant cold tolerance of drosophilids. Of these two traits, CTmin requires less equipment, time and animals and thereby presents a relatively fast, simple and dynamic measure of cold tolerance.