Heat-induced hormesis in longevity is linked to heat-stress sensitivity across laboratory populations from diverse altitude of origin in Drosophila buzzatii.

Heat-induced hormesis in longevity is the increase in life span resulting from the previous exposure to a mild heat stress early in life. Here we examined heat-induced hormesis of Drosophila buzzatii in five mass-mating populations, which were derived from five wild populations along an elevation gradient from 202 to 1855 m above sea level in North-Western Argentina. Five day old flies were exposed to 37.5 °C for 90 min to induce hormesis and its possible variation across altitudinal populations. This heat treatment strongly extended longevity in lowland-derived flies from the most heat-resistant population only. Both heat-induced effects on longevity and heat-knockdown time (heat-stress sensitivity) were negatively correlated to altitude of population of origin. Hormesis was positively correlated to heat-knockdown time across populations. These results indicate that variation in heat-induced hormesis can not be considered as independent of heat-stress sensitivity (or heat-knockdown time) in populations of insects.

Mating success at elevated temperature is associated to thermal adaptation in a set of recombinant inbred lines of Drosophila melanogaster.

In insects, mating ability at elevated temperature can be relevant for adaptation to heat-stressed environments and global warming. Here, we examined copulation latency (T1), copulation duration (T2), and mating frequency (T3, an index of mating success) in two related sets of recombinant inbred lines (RIL) in Drosophila melanogaster at both elevated (33 °C) and benign (25 °C) temperatures. One of these RIL sets (RIL-SH2) was shown to be consistently more resistant in both heat knockdown and heat-shock survival assays than its related set (RIL-D48) in previous studies. Negative correlations across RILs were found between T1 and T3 in this study. Flies from the heat-resistant set of RIL (RIL-SH2) were better able to mate at elevated temperature than flies from the heat-susceptible set (RIL-D48). Quantitative trait locus (QTL) mapping identified temperature-dependent QTLs for all traits (T1, T2 and T3) on all the three major chromosomes. Mating success at elevated temperature was found to be influenced by multiple QTLs. At elevated temperature, several QTLs for mating traits co-localized with QTLs that were previously associated with thermotolerance. The genetic basis for T1, T2 and T3 at the elevated temperature was found to be largely different from the genetic basis controlling the variation for mating success at benign temperature, as there was only a very low (or even null) number of QTLs overlapping across temperatures.

Genetic variation in the heat-stress survival of embryos is largely decoupled from adult thermotolerance in an intercontinental set of recombinant lines of Drosophila melanogaster.

In insects, thermal adaptation works on the genetic variation for thermotolerance of not only larvae and adults but also of the immobile stages of the life cycle including eggs. In contrast to adults and larvae, the genetic basis for thermal adaptation in embryos (eggs) remains to be tested in the model insect Drosophila melanogaster. Quantitative-trait loci (QTL) for heat-stress resistance in embryos could largely differ from previously identified QTL for larvae and adults. Here we used an intercontinental set of recombinant inbred lines (RIL), which were previously used to identify thermotolerance-QTLs in adults and larvae because of their high variation segregating for adult thermotolerance. Eggs appeared to be more heat resistant than larvae and adults from previous studies on these RIL, though different heat-shock assays were used in previous studies. We found that variation in thermotolerance in embryos can be, at least partially, genetically decoupled from thermotolerance in the adult insect. Some RIL that are heat resistant in the adult and larvae can be heat susceptible in embryos. Only one small-effect QTL out of five autosomal QTL co-localized between embryo and other ontogenetic stages. These results suggest that selection for thermal adaptation in adult flies and larvae is predicted to have only a small impact on embryo thermotolerance. In addition, heat-stress tolerance of insects can be measured across ontogenetic stages including embryos in order to better predict thermal adaptive limits of populations and species.

Cardiac performance in heat-stressed flies of heat-susceptible and heat-resistant Drosophila melanogaster.

Thermotolerance is a complex trait that can greatly differ between heat-susceptible (HS) and heat-adapted populations of small insects including Drosophila, with short-term effects after a sub-lethal level of heat stress on many physiological functions. Cardiac performance could accordingly be more robust in heat-resistant (HR) than in HS individuals under heat stress. Here, we tested heart performance under heat-stress effects in two recombinant inbred lines (RIL) of Drosophila melanogaster that dramatically differ in heat knockdown resistance. Heart rate did not strongly differ between heat-susceptible and heat-tolerant flies after a sub-lethal heat stress. Instead, heat-susceptible flies showed a much higher arrhythmia incidence, a longer duration of each heartbeat, and a larger amount of bradycardia than heat-tolerant flies. The highly conserved cardiac proteins SERCA, RyR and NCX that participate in the excitation/contraction coupling, did not differ in activity level between HR and HS flies. Available information for both RIL suggests that heart performance under heat stress may be linked, at least partially, to candidate genes of previously identified quantitative trait loci (QTL) for thermotolerance. This study indicates that HR flies can be genetically more robust in their heart performance than HS flies under even sub-lethal levels of heat stress.

Heat knockdown resistance and chill-coma recovery as correlated responses to selection on mating success at high temperature in Drosophila buzzatii.

Reproduction and related traits such as mating success are strongly affected by thermal stress. We tested direct and correlated responses to artificial selection in replicated lines of Drosophila buzzatii that were selected for mating success at high temperature. Knockdown resistance at high temperature (KRHT) and chill-coma recovery (CCR) were tested as correlated selection responses. Virgin flies were allowed to mate for four hours at 33°C in three replicated lines (S lines) to obtain the selected flies and then returned at 25°C to lay eggs. Other three replicated lines were maintained at 25°C without any selection as control (C lines). After 15 selection generations, KRHT and CCR were measured. Both traits were assessed in flies that did not receive any hardening pretreatments as well as in flies that were either heat or cold hardened. Thermotolerance traits showed significant correlated responses with higher KRHT in S than in C lines, both with a heat-hardening pretreatment and without a heat-hardening pretreatment. CCR time was longer in S than in C lines both with a cold-hardening pretreatment and without a cold-hardening pretreatment. Hardening treatments improved both KRHT and CCR in all cases excepting KRHT in C lines. Overall, KRHT and CCR showed an antagonistic pattern of correlated responses to our selection regime, suggesting either pleiotropy or tightly linked trait-specific genes partially affecting KRHT and CCR.

Negative genetic correlation between longevity and its hormetic extension by dietary restriction in Drosophila melanogaster.

Longevity is a highly malleable trait which is influenced by many genetic and environmental factors including nutrition. Mild stress of dietary restriction (DR) is often beneficial by extending longevity in many organisms. Here, DR-induced effects on longevity were tested for genetic variation in a set of recombinant inbred lines (RIL) in D. melanogaster. Genetic variability was significant in the longevity response following a DR-treatment across RIL, with detrimental effects in several RIL but beneficial effects in other RIL. One quantitative trait locus (QTL) was consistently significant in the middle of chromosome 2 for DR-induced changes in longevity, including hormesis (an increase in longevity by DR). Another QTL co-localized with a previously found QTL for starvation resistance in females. Several other QTL were also significant on most chromosomal arms. Longevity in controls was negatively correlated to DR effects across RIL for longevity in females, the sex showing higher DR-induced hormesis. This negative genetic correlation highlights the importance to further investigate the effects of genetic variation in the strength of DR-induced hormesis in longevity and its sex-specificity.

Quantitative Trait Loci and Antagonistic Associations for Two Developmentally Related Traits in the Drosophila Head.

In insects, some developmentally related traits are negatively correlated. Here, we mapped Quantitative Trait Loci (QTL) for traits of eye size and head capsule, in an intercontinental set of recombinant inbred lines (RILs) of Drosophila melanogaster Composite interval mapping identified QTL on all major chromosomes. Two negatively correlated traits (size of eyes and between-eyes distance) were influenced by one QTL that appeared to be antagonistic between the traits (QTL cytological range is 25F5-30A6), consistent with a negative genetic correlation between these traits of the head capsule. Comparisons of QTL across traits indicated a nonrandom distribution over the genome, with a considerable overlap between some QTL across traits. Developmentally-related traits were influenced by QTL in a pattern that is consistent both with 1) the sign of the genetic correlation between the traits and 2) a constraint in the micro-evolutionary differentiation in the traits.

Elevated extension of longevity by cyclically heat stressing a set of recombinant inbred lines of Drosophila melanogaster throughout their adult life.

An extremely high (about 100 %) increase in longevity is reported for a subset of recombinant inbred lines (RILs) of Drosophila melanogaster subjected to a cyclic heat stress throughout the adult life. Previous work showed that both longevity and heat sensitivity highly differed among RILs. The novel heat stress treatment used in this study consisted of 5 min at 38 °C applicated approximately every 125 min throughout the adult life starting at the age of 2 days. In spite of the exceptionally high increase in longevity in a set of RILs, the same heat stress treatment reduced rather than increased longevity in other RILs, suggesting that heat-induced hormesis is dependent on the genotype and/or the genetic background. Further, one quantitative trait locus (QTL) was identified for heat-induced hormesis on chromosome 2 (bands 28A1-34D2) in one RIL panel (RIL-D48) but it was not significant in its reciprocal panel (RIL-SH2). The level of heat-induced hormesis showed a sexual dimorphism, with a higher number of lines exhibiting higher hormesis effects in males than in females. The new heat stress treatment in this study suggests that longevity can be further extended than previously suggested by applying a cyclic and mild stress throughout the life, depending on the genotype.

Effects of dietary composition on life span of Drosophila buzzatii and its short-lived sibling species D. koepferae.

Two sibling Drosophila species dramatically divergent in longevity, Drosophila buzzatii and D. koepferae, were examined for possible effects of both developmental culture medium and dietary composition (DC) on longevity. Longevity was greatly increased in the longer lived D. buzzatii when flies were reared and fed on a rich-in-nutrient and cactus-based culture (R-CBC) as compared to longevity in a poor nutrient culture (PNC). In D. buzzatii, life span was further increased by exposing flies to short periods of a poor-in-nutrient and cactus-based culture (P-CBC). In contrast, variation in the here used nutrient composition did not change life span in the shorter lived D. koepferae, as longevity in this species did not differ among R-CBC, P-CBC and PNC cultures. Hormesis is a plausible explanation for the beneficial biological effects against aging arising from brief exposure to a lowed calorie food source in D. buzzatii. This study shows that genetic variation between closely related species is substantial for dietary effects on longevity.

QTL for survival to UV-C radiation in Drosophila melanogaster.

PURPOSE: The aim of this study was to investigate tolerance to UV-C (ultraviolet C, 280-100 nm) radiation in Drosophila melanogaster, implementing a quantitative trait locus (QTL) mapping approach. This is of interest to test for genetic variation in survival to UV (ultraviolet) radiation. MATERIALS AND METHODS: We performed a QTL scan in D. melanogaster recombinant inbred lines (RIL) constructed from parental stocks derived from a crossing between northern and southern hemisphere populations that segregated substantial genetic variation in thermal resistance in a previous study. Here, two experimental treatments were implemented: Continuous and cyclic UV-C radiation. RESULTS: Significant QTL were detected on all three major chromosomes. Among these, multiple trait composite interval mapping revealed a significant QTL in the pericentromeric region of chromosome 2, a genome region consistently implicated in thermotolerance in previous studies. CONCLUSIONS: This study shows substantial genetic variation for UV-C radiation resistance in D. melanogaster, with QTL for survival to UV-C radiation generally overlapping with major thermotolerance QTL. The genetic architecture of UV-C radiation resistance appears to be more complex in continuously irradiated individuals.

Survival of heat stress with and without heat hardening in Drosophila melanogaster: interactions with larval density.

Survival of a potentially lethal high temperature stress is a genetically variable thermal adaptation trait in many organisms. Organisms cope with heat stress by basal or induced thermoresistance. Here, we tested quantitative trait loci (QTL) for heat stress survival (HSS) in Drosophila melanogaster, with and without a cyclic heat-hardening pre-treatment, for flies that were reared at low (LD) or high (HD) density. Mapping populations were two panels of recombinant inbred lines (RIL), which were previously constructed from heat stress-selected stocks: RIL-D48 and RIL-SH2, derived from backcrosses to stocks of low and high heat resistance, respectively. HSS increased with heat hardening in both LD and HD flies. In addition, HSS increased consistently with density in non-hardened flies. There was a significant interaction between heat hardening and density effects in RIL-D48. Several QTL were significant for both density and hardening treatments. Many QTL overlapped with thermotolerance QTL identified for other traits in previous studies based on LD cultures only. However, three new QTL were found in HD only (cytological ranges: 12E-16F6; 30A3-34C2; 49C-50C). Previously found thermotolerance QTL were also significant for flies from HD cultures.

Quantitative trait loci for longevity in heat-stressed Drosophila melanogaster.

Longevity is a typical quantitative trait which is influenced by multiple genes. Here we explore the genetic variation in longevity of Drosophila melanogaster in both mildly heat-stressed and control flies. Quantitative trait loci (QTL) analysis for longevity was performed in a single-sex environment at 25°C with and without a mild heat-stress pre-treatment, using a previously reported set of recombinant inbred lines (RIL). QTL regions for longevity in heat-stressed flies overlapped with QTL for longevity in control flies. All longevity QTL co-localized with QTL for longevity identified in previous studies using very different sets of RIL in mixed sex environments, though the genome is nearly saturated with QTL for longevity when considering all previous studies. Heat stress decreased the number of significant QTL for longevity if compared to the control environment. Our mild heat-stress pre-treatment had a beneficial effect (hormesis) more often in shorter-lived than in longer-lived RIL.

Consistent effects of a major QTL for thermal resistance in field-released Drosophila melanogaster.

Molecular genetic markers can be used to identify quantitative trait loci (QTL) for thermal resistance and this has allowed characterization of a major QTL for knockdown resistance to high temperature in Drosophila melanogaster. The QTL showed trade-off associations with cold resistance under laboratory conditions. However, assays of thermal tolerance conducted in the laboratory may not necessarily reflect performance at varying temperatures in the field. Here we tested if lines with different genotypes in this QTL show different thermal performance under high and low temperatures in the field using a release recapture assay. We found that lines carrying the QTL genotype for high thermal tolerance were significantly better at locating resources in the field releases under hot temperatures while the QTL line carrying the contrasting genotype were superior at cold temperatures. Further, we studied copulatory success between the different QTL genotypes at different temperatures. We found higher copulatory success in males of the high tolerance QTL genotype under hot temperature conditions, while there was no difference in females at cold temperatures. The results allow relating components of field fitness at different environmental temperatures with genotypic variation in a QTL for thermal tolerance.

Dominance variation in the correlation between longevity and heat-stress resistance in Drosophila melanogaster.

The possible genetic correlation between longevity and knockdown resistance to high temperature (KRHT) was tested in reciprocal backcrosses between nearly-homozygous lines of D. melanogaster. These baselines differ dramatically for KRHT. Because nearly-homozygous lines were used, reciprocal backcrosses were informative to test for dominant versus additive genetic associations between the traits. The line of high KRHT was longer lived than the low KRHT line. The correlation between longevity and KRHT was dependent on the backcross (BC). When a BC was set up with males from the low KRHT line, the most KRHT resistant individuals were in turn the longest-lived flies. However, this was not found in the reciprocal backcross, indicating dominance. Alleles that confer increased KRHT are dominant in the correlation between longevity and KRHT. The between-line variation in the traits studied is consistent with the hypothesis that stress-resistant genotypes correspond to long-lived individuals.

Quantitative trait locus for starvation resistance in an intercontinental set of mapping populations of Drosophila melanogaster.

Starvation resistance (SR) is an important trait for survival of insects in the wild. We used recombinant inbred lines (RIL) to search for quantitative trait loci (QTL) in crosses between intercontinental inbred lines that were originally selected for heat-knockdown resistance. SR was measured as the time of survival under repeated events of starvation. SR was consistently higher in females than in males. Composite interval mapping identified one QTL region (cytological range 64D-66E2) on the left arm of chromosome 3 in males, and no QTL was found in females. Many candidate genes that were identified in previous studies of QTL for stress resistance are included within this QTL region. The QTL-allele that decreased SR was found in the line originating from the colder population (Denmark). We discuss our results with regard to multiple candidate genes, noncolocalization with thermotolerance QTL, and possible geographical variation.

Combined expression patterns of QTL-linked candidate genes best predict thermotolerance in Drosophila melanogaster.

Knockdown resistance to high temperature (KRHT) is a thermal adaptation trait in Drosophila melanogaster. Here we used quantitative real-time PCR (qRT-PCR) to test for possible associations between KRHT and the expression of candidate genes within quantitative trait loci (QTL) in eight recombinant inbred lines (RIL). hsp60 and hsc70-3 map within an X-linked QTL, while CG10383, catsup, ddc, trap1, and cyp6a13 are linked in a KRHT-QTL on chromosome 2. hsc70-3 expression increased by heat-hardening. Principal Components analysis revealed that catsup, ddc and trap1 were either co-expressed or combined in their expression levels. This composite expression variable (e-PC1) was positively associated to KRHT in non-hardened RIL. In heat-hardened flies, hsp60 was negatively related to hsc70-3 on e-PC2, with effects on KRHT. These results are consistent with the notion that QTL can be shaped by expression variation in combined candidate loci. We found composite variables of gene expression (e-PCs) that best correlated to KRHT. Network effects with other untested linked loci are apparent because, in spite of their associations with KRHT phenotypes, e-PCs were sometimes uncorrelated with their QTL genotype.

QTL for the thermotolerance effect of heat hardening, knockdown resistance to heat and chill-coma recovery in an intercontinental set of recombinant inbred lines of Drosophila melanogaster.

The thermotolerance effect of heat hardening (also called short-term acclimation), knockdown resistance to high temperature (KRHT) with and without heat hardening and chill-coma recovery (CCR) are important phenotypes of thermal adaptation in insects and other organisms. Drosophila melanogaster from Denmark and Australia were previously selected for low and high KRHT, respectively. These flies were crossed to construct recombinant inbred lines (RIL). KRHT was higher in heat-hardened than in nonhardened RIL. We quantify the heat-hardening effect (HHE) as the ratio in KRHT between heat-hardened and nonhardened RIL. Composite interval mapping revealed a more complex genetic architecture for KRHT without heat-hardening than for KRHT in heat-hardened insects. Five quantitative trait loci (QTL) were found for KRHT, but only two of them were significant after heat hardening. KRHT and CCR showed trade-off associations for QTL both in the middle of chromosome 2 and the right arm of chromosome 3, which should be the result of either pleiotropy or linkage. The major QTL on chromosome 2 explained 18% and 27-33% of the phenotypic variance in CCR and KRHT in nonhardened flies, respectively, but its KRHT effects decreased by heat hardening. We discuss candidate loci for each QTL. One HHE-QTL was found in the region of small heat-shock protein genes. However, HHE-QTL explained only a small fraction of the phenotypic variance. Most heat-resistance QTL did not colocalize with CCR-QTL. Large-effect QTL for CCR and KRHT without hardening (basal thermotolerance) were consistent across continents, with apparent transgressive segregation for CCR. HHE (inducible thermotolerance) was not regulated by large-effect QTL.

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.

Heat-induced hormesis in longevity of two sibling Drosophila species.

Previous work showed that mild-heat stress induces longevity hormesis in a model organism, D. melanogaster. Here we compared the possible heat-induced hormesis in longevity of other species of Drosophila, D. buzzatii and its sibling species D. koepferae, in a single-sex environment. Possible correlations between longevity and heat-stress resistance were also tested by measuring longevity, heat-knockdown resistance and the heat-induced Hsp70 expression for each species in a common environment. D. buzzatii was longer lived than D. koepferae at benign temperature. Knockdown resistance to heat stress was positively correlated to longevity within species. However, the shorter-lived species was more resistant to knockdown by heat stress than the longer-lived species. The heat-induced Hsp70 expression was similar between species. A heat-shock treatment (37 degrees C for 1 h at 4 days of age) extended mean longevity in the longer lived species but not in the shorter lived species. In D. koepferae, the demographic rate of senescence decreased but the baseline mortality rate increased by heat-shock, resulting in no extension of mean longevity. Sympatric populations of closely related species can be differentially sensitive to temperature and exhibit different patterns of 37 degrees C-induced hormesis in demographic senescence and longevity. The results also show that positive correlations between stress resistance and life span within species can shift in sign across closely related species. Finally, this study shows that heat-induced hormesis in longevity can be found across different Drosophila species, as hormetic effects are not limited to the previously studied D. melanogaster.