Reduced Enzyme Activity Following Hsp70 Overexpression in Drosophila melanogaster

Acclimation to environmental change can impose costs to organisms. One potential cost is the change in cell metabolism that follows a physiological response, e.g., high expression of heat shock proteins may alter specific activity of important enzymes. We examined the significance of this cost in a pair of Drosophila melanogaster lines transformed with additional copies of a gene that encodes the heat shock protein, Hsp70. Heat shock induces Hsp70 expression in all lines, but lines with extra copies produce much more Hsp70 than do excision control strains. The consequence of this supranormal Hsp70 expression is to reduce specific activity of both enzymes analyzed, adult alcohol dehydrogenase (ADH), which is heat sensitive, and lactate dehydrogenase, which is not. Strain differences were most pronounced under those conditions where Hsp70 expression was maximized, and not where the heat stress denatured proteins. That result supported the idea that Hsp70 expression is constrained evolutionarily by its tendency to bind nascent peptides when overabundant within the cell.     

Clinal variation in developmental time and viability,and the response to thermal treatments in two species of Drosophila

The present study first addressed the question of whether developmental time (DT) and viability (VT) vary clinally along latitudinal and altitudinal gradients in Drosophila buzzatii, an autochthonous specialist and the generalist invasive Drosophila melanogaster. Coincident and positive altitudinal clines across species and, direct and inverse latitudinal clines were observed for DT in D. melanogaster and D. buzzatii, respectively. Opposing latitudinal and altitudinal clines were detected for VT only in D. melanogaster. The patterns observed along altitudinal gradients prompted us to investigate whether flies living at lowland and highland environments may respond differentially to thermal treatments consisting of regimes of constant and alternating temperatures. Flies reared at higher mean temperature developed faster than at lower mean temperature in both species. By contrast, the response in VT differed greatly between species. Highland D. melanogaster were more viable than lowland regardless the treatment, whereas, in D. buzzatii, highland flies were more viable than lowland in alternating thermal regimes and the reverse was true in treatments of constant temperature. The results obtained suggest that thermal amplitude may be an important factor that should be considered in investigations of thermal adaptation. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 233–245.     

Long-term changes in the chromosomal inversion polymorphism of Drosophila subobscura. I. Mediterranean populations from southwestern Europe

The chromosomal polymorphism of seven Mediterranean populations of Drosophila subobscura has been compared with that of the same populations collected 26 to 35 years ago. Significant latitudinal clines for the frequencies of A(ST), E(ST), O(ST). and U(ST) chromosomal arrangements have been detected in the old and new samples. Standard gene arrangements are frequent in the north and decrease in frequency towards the south. Significant negative regression coefficients between latitude and transformed frequency have also been observed for the more frequent nonstandard gene arrangements. The pattern of the clines is practically the same in the old and new collections. Furthermore, the frequencies of gene arrangements of all chromosomes have changed significantly during this period in a systematic way: an increase in the frequency of those arrangements typical of southern latitudes and a decrease for those more common in northern latitudes is observed in all populations. These changes could be due to climatic factors that are correlated with latitude, making the chromosomal composition of this species more "southern.'     

Long‐term changes in the chromosomal inversion polymorphism of Drosophila subobscura. II. European populations*

The chromosomal polymorphism of 13 European populations of Drosophila subobscura has been compared with that of the same populations collected 15–35 years ago. The chromosomal polymorphism of the old populations differs significantly from that of the new populations, mainly for chromosomes U and O. There is a very good agreement between the geographical space and the genetic space as shown by a graphical representation of the 26 statistical populations (13 old and 13 new) obtained by a principal coordinate analysis. This reflects both the existence of significant latitudinal clines for the frequencies of some chromosomal arrangements in the old and new samples and systematic changes that have taken place in these populations during the period that elapsed between the two surveys. An increase in the frequency of those arrangements typical of southern latitudes and a decrease for those common in northern latitudes is observed in all populations – Mediterranean, Atlantic and Central European. Furthermore, the genetic distances of the new populations to a southern population of reference have decreased in comparison with those of the old populations. These changes could be the result of climatic factors that are correlated with latitude. In particular, the assumption that global warming is responsible for all the changes observed appears rather likely. Whether these systematic changes of the chromosomal polymorphism are a consequence of local adaptations or have been produced by migration from the south remains an open question.     

Chromosomal inversions promote genomic islands of concerted evolution of Hsp70 genes in the Drosophila subobscura species subgroup

Heat‐shock (HS) assays to understand the connection between standing inversion variation and evolutionary response to climate change in Drosophila subobscura found that “warm‐climate” inversion O₃₊₄ exhibits non‐HS levels of Hsp70 protein like those of “cold‐climate” O_ST after HS induction. This was unexpected, as overexpression of Hsp70 can incur multiple fitness costs. To understand the genetic basis of this finding, we have determined the genomic sequence organization of the Hsp70 family in four different inversions, including O_ST, O₃₊₄ , O₃₊₄₊₈ and O₃₊₄₊₁₆ , using as outgroups the remainder of the subobscura species subgroup, namely Drosophila madeirensis and Drosophila guanche. We found (i) in all the assayed lines, the Hsp70 family resides in cytological locus 94A and consists of only two genes, each with four HS elements (HSEs) and three GAGA sites on its promoter. Yet, in O_ST, the family is comparatively more compact; (ii) the two Hsp70 copies evolve in concert through gene conversion, except in D. guanche; (iii) within D. subobscura, the rate of concerted evolution is strongly structured by inversion, being higher in O_ST than in O₃₊₄ ; and (iv) in D. guanche, the two copies accumulated multiple differences, including a newly evolved “gap‐type” HSE2. The absence of concerted evolution in this species may be related to a long‐gone‐unnoticed observation that it lacks Hsp70 HS response, perhaps because it has evolved within a narrow thermal range in an oceanic island. Our results point to a previously unrealized link between inversions and concerted evolution, with potentially major implications for understanding genome evolution.     

Natural hyperthermia and expression of the heat shock protein Hsp70 affect developmental abnormalities in Drosophila melanogaster

We demonstrate that natural heat stress on wild larval Drosophila melanogaster results in severe developmental defects in >10% of eclosing adults, and that increased copy number of the gene encoding the major inducible heat shock protein of D. melanogaster, Hsp70, is sufficient to reduce the incidence of such abnormalities. Specifically, non-adult D. melanogaster inhabiting necrotic fruit experienced severe, often lethal heat stress in natural settings. Adult flies eclosing from wild larvae that had survived natural heat stress exhibited severe developmental anomalies of wing and abdominal morphology, which should dramatically affect fitness. The frequency of developmental abnormalities varied along two independent natural thermal gradients, exceeding 10% in adults eclosing from larvae developing in warm, sunlit fruit. When exposed to natural heat stress, D. melanogaster larvae with the wild-type number of hsp70 genes (n=10) developed abnormal wings significantly more frequently than a transgenic sister strain with 22 copies of the hsp70 gene. Received: 19 April 1999 / Accepted: 16 July 1999     

Knockdown resistance to heat stress and slow recovery from chill coma are genetically associated in a quantitative trait locus region of chromosome 2 in Drosophila melanogaster

In insects, two ecologically relevant traits of thermal adaptation are knockdown resistance to high temperature (KRHT) and chill-coma recovery (CCR). Chromosome 2 of Drosophila melanogaster was tested for quantitative trait loci (QTL) affecting both CCR and KRHT in backcrosses between homosequential lines that are fixed for the standard (noninverted) sequence of this autosome. These lines were obtained by artificial selection on KRHT and subsequent inbreeding from a stock that was derived from a single wild population. Heat-induced expression of the 70KD heat-shock protein (Hsp70) was also examined for variation between the lines. Composite interval mapping was performed for each trait on each reciprocal backcross, identifying one QTL region in the middle of chromosome 2 for both KRHT and CCR. The largest estimates of additive effects were found in pericentromeric regions of chromosome 2, accounting for 10–14% (CCR) and 10–17% (KRHT) of the phenotypic variance in BC populations. No QTL was found in the region of the heat-shock factor ( hsf ) gene. However, the two parental lines have diverged in the heat-induced Hsp70 expression. Distribution of KRHT QTL on chromosome 2 was similar between this study based on crosses between lines selected from a single wild population and previous work based on crosses between selection lines from different continents. Colocalized QTL showed a trade–off association between CCR and KRHT, which should be the result of either multiple, tightly linked trait-specific genes or a single gene with pleiotropic effects on the traits. We discuss candidate loci contained within the QTL regions.     

Changes in thermotolerance and Hsp70 expression with domestication in Drosophila melanogaster

To examine how the duration of laboratory domestication may affect Drosophila stocks used in studies of thermotolerance, we measured expression of the inducible heat‐shock protein Hsp70 and survival after heat shock in D. melanogaster strains recently collected from nature and maintained in laboratory culture for up to 50 or more generations. After an initial increase in both Hsp70 expression and thermotolerance immediately after transfer to laboratory medium, both traits remained fairly constant over time and variation among strains persisted through laboratory domestication. Furthermore, variation in heat tolerance and Hsp70 expression did not correlate with the length of time populations evolved in the laboratory. Therefore, while environmental variation likely contributed most to early shifts in strain tolerance and Hsp70 expression, other population parameters, for example genetic drift, inbreeding, and selection likely affected these traits little. As long as populations are maintained with large numbers of individuals, the culture of insects in the laboratory may have little effect on the tolerance of different strains to thermal stress.     

Effects of inbreeding and rate of inbreeding in Drosophila melanogaster- Hsp70 expression and fitness

Induction of heat shock proteins (Hsp) is a well-known mechanism through which cells cope with stressful conditions. Hsp are induced by a variety of extrinsic stressors. However, recently intrinsic stressors (aging and inbreeding) have been shown to affect expression of Hsp. Increased homozygosity due to inbreeding may disrupt cellular homeostasis by causing increased expression of recessive deleterious mutations and breakdown of epistatic interactions. We investigated the effect of inbreeding and the rate of inbreeding on the expression of Hsp70, larval heat resistance and fecundity. In Drosophila melanogaster we found that inbred lines (F approximately 0.67) had significantly up-regulated expression of Hsp70, and reduced heat resistance and fecundity as compared with outbred control lines. A significant negative correlation was observed between Hsp70 expression and resistance to an extreme heat stress in inbred lines. We interpreted this as an increased requirement for Hsp70 in the lines suffering most from inbreeding depression. Inbreeding depression for fecundity was reduced with a slower rate of inbreeding compared with a fast rate of inbreeding. Thus, the effectiveness of purging seems to be improved with a slower rate of inbreeding.     

Effects of exposure to a 1950 MHz radio frequency field on expression of Hsp70 and Hsp27 in human glioma cells

Human glioma MO54 cells were used to investigate whether radio frequency (RF) field exposure could activate stress response genes. Cells were exposed to continuous wave 1950 MHz or sham conditions for up to 2 h. Specific absorption rates (SARs) were 1, 2, and 10 W/kg. For the cell growth experiment, cell numbers were counted at 0-4 days after exposure. Expression of Hsp27 and Hsp70, as well as the level of phosphorylated Hsp27 (78Ser) protein, was determined by Western blotting. It was found that sham exposed and RF exposed cells demonstrated a similar growth pattern up to 4 days after RF field exposure. RF field exposure at both 2 and 10 W/kg did not affect the growth of MO54 cells. In addition, there were no significant differences in protein expression of Hsp27 and Hsp70 between sham exposed and RF exposed cells at a SAR of 1, 2, or 10 W/kg for 1 and 2 h. However, exposure to RF field at a SAR of 10 W/kg for 1 and 2 h decreased the protein level of phosphorylated Hsp27 (78Ser) significantly. Our results suggest that although exposure to a 1950 MHz RF field has no effect on cell proliferation and expression of Hsp 27 and Hsp70, it may inhibit the phosphorylation of Hsp27 at Serine 78 in MO54 cells.     

Evolution of total net fitness in thermal lines: Drosophila subobscura likes it 'warm'

Fisher's fundamental theorem states that heritable variation for net fitness sets a limit to the rate of response to natural selection. How will temperate (i.e. cold‐tolerant) species cope with contemporary rapid global warming? Using three‐fold replicated lines of Drosophila subobscura that had been allowed to evolve for 4 years (between 32 and 59 generations) at 13 °C (cold), 18 °C (the supposed optimum temperature), and 22 °C (warm) I assess here how net fitness changes according to thermal environments. Net fitness was estimated following the classical approach in population genetics of competing over a number of generation in outbred experimental populations multiple wild‐type O chromosomes (homologous to arm 3R in D. melanogaster) independently derived from each base thermal stock in an otherwise homogeneous genetic background against a balancer chromosome. Warm‐adapted populations (‘warm‐adapted O chromosomes’) performed comparatively well at all tested temperatures. However, net fitness was severely reduced in cold‐adapted populations when transferred to warmer conditions. It seems, therefore, that thermal fitness breath for D. subobscura flies is positively associated to temperature. These findings are discussed in relation to the fast world‐wide clinal shifts in the frequency of genetic markers correlated with current climate change.     

Evolutionary potential of thermal preference and heat tolerance in Drosophila subobscura

Evolutionary change of thermal traits (i.e., heat tolerance and behavioural thermoregulation) is one of the most important mechanisms exhibited by organisms to respond to global warming. However, the evolutionary potential of heat tolerance, estimated as narrow‐sense heritability, depends on the methodology employed. An alternative adaptive mechanism to buffer extreme temperatures is behavioural thermoregulation, although the association between heat tolerance and thermal preference is not clearly understood. We suspect that methodological effects associated with the duration of heat stress during thermal tolerance assays are responsible for missing this genetic association. To test this hypothesis, we estimated the heritabilities and genetic correlations for thermal traits in Drosophila subobscura, using high‐temperature static and slow ramping assays. We found that heritability for heat tolerance was higher in static assays (h² = 0.134) than in slow ramping assays (h² = 0.084), suggesting that fast assays may provide a more precise estimation of the genetic variation of heat tolerance. In addition, thermal preference exhibited a low heritability (h² = 0.066), suggesting a reduced evolutionary response for this trait. We also found that the different estimates of heat tolerance and thermal preference were not genetically correlated, regardless of how heat tolerance was estimated. In conclusion, our data suggest that these thermal traits can evolve independently in this species. In agreement with previous evidence, these results indicate that methodology may have an important impact on genetic estimates of heat tolerance and that fast assays are more likely to detect the genetic component of heat tolerance.     

Toxicity of cypermethrin: hsp70 as a biomarker of response in transgenic Drosophila

Heat shock protein induction is often associated with a cellular response to a harmful environment or to adverse life conditions. The main aims of our study were (1) to evaluate the cytotoxic potential of cypermethrin; and (2) to investigate the suitability of stress-induced heat shock protein Hsp70 as a biomarker for environmental pollutants in transgenic Drosophila melanogaster (Hsp70-lacZ)Bg⁹. Different concentrations of cypermethrin (0.002, 0.2, 0.5 and 50.0 p.p.m.) were mixed with food. Third instar larvae of transgenic Drosophila melanogaster were allowed to feed on these mixtures for different time intervals (2, 4, 6, 12, 24 and 48h). Following feeding, hsp70 induction and tissue damage were evaluated. In the highest concentration treatment group (50 p.p.m.), 100% larval mortality was recorded after 12 h exposure. Hsp70 was found to be induced even at the lowest concentration (0.002 p.p.m.) of the insecticide, while tissue damage was observed in the larvae exposed for 48 h. While an insignificant decline in hsp70 expression was observed in the larvae exposed to cypermethrin at a dietary concentration of 0.002 p.p.m. after 48 h compared with those exposed for 24 h, in the next two higher concentrations of the toxicant, a similar but significant decline in hsp70 expression was evident in the exposed larvae after 48 h. The present study reveals the cytotoxic potential of cypermethrin and further proposes that hsp70 induction in transgenic Drosophila melanogaster could be used as a sensitive biomarker in risk assessment.     

Toxicity of cypermethrin: hsp70 as a biomarker of response in transgenic Drosophila

Heat shock protein induction is often associated with a cellular response to a harmful environment or to adverse life conditions. The main aims of our study were (1) to evaluate the cytotoxic potential of cypermethrin; and (2) to investigate the suitability of stress-induced heat shock protein Hsp70 as a biomarker for environmental pollutants in transgenic Drosophila melanogaster (Hsp70-lacZ)Bg⁹. Different concentrations of cypermethrin (0.002, 0.2, 0.5 and 50.0 p.p.m.) were mixed with food. Third instar larvae of transgenic Drosophila melanogaster were allowed to feed on these mixtures for different time intervals (2, 4, 6, 12, 24 and 48h). Following feeding, hsp70 induction and tissue damage were evaluated. In the highest concentration treatment group (50 p.p.m.), 100% larval mortality was recorded after 12 h exposure. Hsp70 was found to be induced even at the lowest concentration (0.002 p.p.m.) of the insecticide, while tissue damage was observed in the larvae exposed for 48 h. While an insignificant decline in hsp70 expression was observed in the larvae exposed to cypermethrin at a dietary concentration of 0.002 p.p.m. after 48 h compared with those exposed for 24 h, in the next two higher concentrations of the toxicant, a similar but significant decline in hsp70 expression was evident in the exposed larvae after 48 h. The present study reveals the cytotoxic potential of cypermethrin and further proposes that hsp70 induction in transgenic Drosophila melanogaster could be used as a sensitive biomarker in risk assessment.