Temperature and nutrition do not interact to shape the evolution of metabolic rate.

Metabolic cold adaptation, or Krogh's rule, is the controversial hypothesis that predicts a monotonically negative relationship between metabolic rate and environmental temperature for ectotherms living along thermal clines measured at a common temperature. Macrophysiological patterns consistent with Krogh's rule are not always evident in nature, and experimentally evolved responses to temperature have failed to replicate such patterns. Hence, temperature may not be the sole driver of observed variation in metabolic rate. We tested the hypothesis that temperature, as a driver of energy demand, interacts with nutrition, a driver of energy supply, to shape the evolution of metabolic rate to produce a pattern resembling Krogh's rule. To do this, we evolved replicate lines of Drosophila melanogaster at 18, 25 or 28°C on control, low-calorie or low-protein diets. Contrary to our prediction, we observed no effect of nutrition, alone or interacting with temperature, on adult female and male metabolic rates. Moreover, support for Krogh's rule was only in females at lower temperatures. We, therefore, hypothesize that observed variation in metabolic rate along environmental clines arises from the metabolic consequences of environment-specific life-history optimization, rather than because of the direct effect of temperature on metabolic rate. This article is part of the theme issue 'The evolutionary significance of variation in metabolic rates'.

Phylogenomic analyses of the genus Drosophila reveals genomic signals of climate adaptation.

Many Drosophila species differ widely in their distributions and climate niches, making them excellent subjects for evolutionary genomic studies. Here, we have developed a database of high-quality assemblies for 46 Drosophila species and one closely related Zaprionus. Fifteen of the genomes were newly sequenced, and 20 were improved with additional sequencing. New or improved annotations were generated for all 47 species, assisted by new transcriptomes for 19. Phylogenomic analyses of these data resolved several previously ambiguous relationships, especially in the melanogaster species group. However, it also revealed significant phylogenetic incongruence among genes, mainly in the form of incomplete lineage sorting in the subgenus Sophophora but also including asymmetric introgression in the subgenus Drosophila. Using the phylogeny as a framework and taking into account these incongruences, we then screened the data for genome-wide signals of adaptation to different climatic niches. First, phylostratigraphy revealed relatively high rates of recent novel gene gain in three temperate pseudoobscura and five desert-adapted cactophilic mulleri subgroup species. Second, we found differing ratios of nonsynonymous to synonymous substitutions in several hundred orthologues between climate generalists and specialists, with trends for significantly higher ratios for those in tropical and lower ratios for those in temperate-continental specialists respectively than those in the climate generalists. Finally, resequencing natural populations of 13 species revealed tropics-restricted species generally had smaller population sizes, lower genome diversity and more deleterious mutations than the more widespread species. We conclude that adaptation to different climates in the genus Drosophila has been associated with large-scale and multifaceted genomic changes.

Latitudinal clines in heat tolerance, protein synthesis rate and transcript level of a candidate gene in Drosophila melanogaster.

The occurrence of climatic adaptation in Drosophila melanogaster is highlighted by the presence of latitudinal clines in several quantitative traits, particularly clines in adult heat knockdown tolerance that is higher in tropical populations. However the presence of latitudinal patterns in physiological characteristics that may underlie these traits have rarely been assessed. Protein synthesis has been implicated as an important physiological process that influences thermal tolerance, and this has not been examined in a clinal context. Here, we characterise latitudinal variation in D. melanogaster from eastern Australia in both adult heat knockdown tolerance and rates of protein synthesis following rearing at both 25 °C, approximating summer conditions, and 18 °C approximating winter development. We also examined clinal variation in the predominant nuclear transcript of the heat-inducible RNA gene hsr-omega, which has been implicated in regulating protein synthesis. We find significant clines in heat-hardened tolerance when cultured at both 18 and 25 °C - tolerance increased towards the low latitude tropics. Rates of protein synthesis measured in ovarian tissue also associated negatively with latitude, however the presence of the clines depended on rearing temperature and heat stress conditions. Finally, omega-n levels measured without heat stress showed a positive linear cline. When measured after a mild heat stress higher levels of omega-n were detected and the clinal pattern became parabolic - mid-latitude populations had lower levels of the transcript. While congruent latitudinal trends were detected for these three traits, only a low level of positive association was detected between protein synthesis and thermal tolerance providing little evidence that these traits are related at the level of cellular physiology. However the new clinal patterns of protein synthesis and hsr-omega variation suggest that these variables exert important influences on traits involved with latitudinal climatic adaptation.

Transcripts from the Drosophila heat-shock gene hsr-omega influence rates of protein synthesis but hardly affect resistance to heat knockdown.

While hundreds of genes have recently been implicated in an organism's response to thermal stress, our insight into the cellular and physiological mechanisms affected by these genes has advanced to a lesser extent. We focus on an enigmatic Drosophila heat stress RNA gene, hsr-omega, which encodes two RNA transcripts that are constitutively expressed in almost all developing and adult tissues, omega-n in the nucleus and omega-c in the cytoplasm; both being readily induced to high levels by mild heat stress. We derived three hsr-omega mutant lines via imprecise P-element excision and characterised them for changes in expression, in both the presence and absence of heat stress. Viability estimates indicate that a low level of omega-n is required for normal development. Consistent with the model of omega-n as a negative regulator of intron-processed mRNA levels the mutants displayed a 1.5-fold increase in rates of protein synthesis measured in ovarian tissue in the absence of heat stress, a result suggesting that an important function of hsr-omega is the modulation of general protein synthesis. The mutants had little effect on two measures commonly used to assess heat tolerance, heat-knockdown time and heat hardening ability, suggesting that more subtle heat-related fitness components need to be examined for effects of these mutations.

Adult heat tolerance variation in Drosophila melanogaster is not related to Hsp70 expression.

Expression of heat-inducible Hsp70 is considered closely linked to thermotolerance in Drosophila melanogaster and other ectotherms. However, intra-specific variation of Hsp70 expression levels and its relationship to heat resistance has only been investigated in a few studies. Although in Drosophila larvae Hsp70 expression may be a key determinant of heat tolerance, the evidence for this in adults is equivocal. We therefore examined heat-induced Hsp70 expression and several measurements of adult heat tolerance in three independent collections of D. melanogaster, measured in three laboratories and using slightly different protocols. Expression levels of Hsp70 were quantified using ELISA or Western blots on extracts from adult females. Both Hsp70 and heat tolerance exhibited substantial within-population variation as previously reported. However, in all experiments there were no significant correlation between Hsp70 expression and laboratory assays of adult heat tolerance commonly used in Drosophila. When combining data across three studies we had high power to detect associations but the results showed that variation in Hsp70 expression is only likely to explain a small proportion of variation in adult heat tolerance. Therefore, although Hsp70 expression is a major component of the cellular heat stress response, its influence on intra-specific heat tolerance variation may be life-stage specific.