How hot is too hot? Metabolic responses to temperature across life stages of a small ectotherm.

To understand how global warming will impact biodiversity, we need to pay attention to those species with higher vulnerability. However, to assess vulnerability we also need to consider the thermoregulatory mechanisms, body size and thermal tolerance of species. Studies addressing thermal tolerance on small ectotherms have mostly focused on insects, while other arthropods such as arachnids remain understudied. Here, we quantified the physiological thermal sensitivity of the pseudoscorpion Dactylochelifer silvestris using a respirometry setup with a ramping temperature increase. Overall, we found that D. silvestris has a much lower metabolic rate than other organisms of similar size. As expected, metabolic rate increased with body size, with adults having larger metabolic rates, but the overall metabolic scaling exponent was low. Both the temperature at which metabolism peaked and the critical thermal maxima were high (above 44°C) and comparable to those of other arachnids. The activation energy, which characterizes the rising portion of the thermal sensitivity curve, was 0.66 eV, consistent with predictions for insects and other taxa in general. Heat tolerances and activation energy did not differ across life stages. We conclude that D. silvestris has low metabolic rates and a high thermal tolerance, which would likely influence how all stages and sexes of this species could endure climate change.

Functional and evolutionary analysis of host Synaptogyrin-2 in porcine circovirus type 2 susceptibility.

Mammalian evolution has been influenced by viruses for millions of years, leaving signatures of adaptive evolution within genes encoding for viral interacting proteins. Synaptogyrin-2 (SYNGR2) is a transmembrane protein implicated in promoting bacterial and viral infections. A genome-wide association study of pigs experimentally infected with porcine circovirus type 2b (PCV2b) uncovered a missense mutation (SYNGR2 p.Arg63Cys) associated with viral load. In this study, CRISPR/Cas9-mediated gene editing of the porcine kidney 15 (PK15, wtSYNGR2+p.63Arg) cell line generated clones homozygous for the favorable SYNGR2 p.63Cys allele (emSYNGR2+p.63Cys). Infection of edited clones resulted in decreased PCV2 replication compared to wildtype PK15 (P<0.05), with consistent effects across genetically distinct PCV2b and PCV2d isolates. Sequence analyses of wild and domestic pigs (n>700) revealed the favorable SYNGR2 p.63Cys allele is unique to domestic pigs and more predominant in European than Asian breeds. A haplotype defined by the SYNGR2 p.63Cys allele was likely derived from an ancestral haplotype nearly fixed within European (0.977) but absent from Asian wild boar. We hypothesize that the SYNGR2 p.63Cys allele arose post-domestication in ancestral European swine. Decreased genetic diversity in homozygotes for the SYNGR2 p.63Cys allele compared to SYNGR2 p.63Arg, corroborates a rapid increase in frequency of SYGNR2 p.63Cys via positive selection. Signatures of adaptive evolution across mammalian species were also identified within SYNGR2 intraluminal loop domains, coinciding with the location of SYNGR2 p.Arg63Cys. Therefore, SYNGR2 may reflect a novel component of the host-virus evolutionary arms race across mammals with SYNGR2 p.Arg63Cys representing a species-specific example of putative adaptive evolution.

Seasonal plasticity in morphology and metabolism differs between migratory North American and resident Costa Rican monarch butterflies.

Environmental heterogeneity in temperate latitudes is expected to maintain seasonally plastic life-history strategies that include the tuning of morphologies and metabolism that support overwintering. For species that have expanded their ranges into tropical latitudes, it is unclear the extent to which the capacity for plasticity will be maintained or will erode with disuse. The migratory generations of the North American (NA) monarch butterfly Danaus plexippus lead distinctly different lives from their summer generation NA parents and their tropical descendants living in Costa Rica (CR). NA migratory monarchs postpone reproduction, travel thousands of kilometers south to overwinter in Mexico, and subsist on little food for months. Whether recently dispersed populations of monarchs such as those in Costa Rica, which are no longer subject to selection imposed by migration, retain ancestral seasonal plasticity is unclear. To investigate the differences in seasonal plasticity, we reared the NA and CR monarchs in summer and autumn in Illinois, USA, and measured the seasonal reaction norms for aspects of morphology and metabolism related to flight. NA monarchs were seasonally plastic in forewing and thorax size, increasing wing area and thorax to body mass ratio in autumn. While CR monarchs increased thorax mass in autumn, they did not increase the area of the forewing. NA monarchs maintained similar resting and maximal flight metabolic rates across seasons. However, CR monarchs had elevated metabolic rates in autumn. Our findings suggest that the recent expansion of monarchs into habitats that support year-round breeding may be accompanied by (1) the loss of some aspects of morphological plasticity as well as (2) the underlying physiological mechanisms that maintain metabolic homeostasis in the face of temperature heterogeneity.

Physical bioenergetics: Energy fluxes, budgets, and constraints in cells.

Cells are the basic units of all living matter which harness the flow of energy to drive the processes of life. While the biochemical networks involved in energy transduction are well-characterized, the energetic costs and constraints for specific cellular processes remain largely unknown. In particular, what are the energy budgets of cells? What are the constraints and limits energy flows impose on cellular processes? Do cells operate near these limits, and if so how do energetic constraints impact cellular functions? Physics has provided many tools to study nonequilibrium systems and to define physical limits, but applying these tools to cell biology remains a challenge. Physical bioenergetics, which resides at the interface of nonequilibrium physics, energy metabolism, and cell biology, seeks to understand how much energy cells are using, how they partition this energy between different cellular processes, and the associated energetic constraints. Here we review recent advances and discuss open questions and challenges in physical bioenergetics.

Evolution of genome structure in the Drosophila simulans species complex.

The rapid evolution of repetitive DNA sequences, including satellite DNA, tandem duplications, and transposable elements, underlies phenotypic evolution and contributes to hybrid incompatibilities between species. However, repetitive genomic regions are fragmented and misassembled in most contemporary genome assemblies. We generated highly contiguous de novo reference genomes for the Drosophila simulans species complex (D. simulans, D. mauritiana, and D. sechellia), which speciated ∼250,000 yr ago. Our assemblies are comparable in contiguity and accuracy to the current D. melanogaster genome, allowing us to directly compare repetitive sequences between these four species. We find that at least 15% of the D. simulans complex species genomes fail to align uniquely to D. melanogaster owing to structural divergence-twice the number of single-nucleotide substitutions. We also find rapid turnover of satellite DNA and extensive structural divergence in heterochromatic regions, whereas the euchromatic gene content is mostly conserved. Despite the overall preservation of gene synteny, euchromatin in each species has been shaped by clade- and species-specific inversions, transposable elements, expansions and contractions of satellite and tRNA tandem arrays, and gene duplications. We also find rapid divergence among Y-linked genes, including copy number variation and recent gene duplications from autosomes. Our assemblies provide a valuable resource for studying genome evolution and its consequences for phenotypic evolution in these genetic model species.

Phenotypic Variation in Mitochondria-Related Performance Traits Across New Zealand Snail Populations.

Mitochondrial function is critical for energy homeostasis and should shape how genetic variation in metabolism is transmitted through levels of biological organization to generate stability in organismal performance. Mitochondrial function is encoded by genes in two distinct and separately inherited genomes-the mitochondrial genome and the nuclear genome-and selection is expected to maintain functional mito-nuclear interactions. The documented high levels of polymorphism in genes involved in these mito-nuclear interactions and wide variation for mitochondrial function demands an explanation for how and why variability in such a fundamental trait is maintained. Potamopyrgus antipodarum is a New Zealand freshwater snail with coexisting sexual and asexual individuals and, accordingly, contrasting systems of separate vs. co-inheritance of nuclear and mitochondrial genomes. As such, this snail provides a powerful means to dissect the evolutionary and functional consequences of mito-nuclear variation. The lakes inhabited by P. antipodarum span wide environmental gradients, with substantial across-lake genetic structure and mito-nuclear discordance. This situation allows us to use comparisons across reproductive modes and lakes to partition variation in cellular respiration across genetic and environmental axes. Here, we integrated cellular, physiological, and behavioral approaches to quantify variation in mitochondrial function across a diverse set of wild P. antipodarum lineages. We found extensive across-lake variation in organismal oxygen consumption and behavioral response to heat stress and differences across sexes in mitochondrial membrane potential but few global effects of reproductive mode. Taken together, our data set the stage for applying this important model system for sexual reproduction and polyploidy to dissecting the complex relationships between mito-nuclear variation, performance, plasticity, and fitness in natural populations.

A role for triglyceride lipase brummer in the regulation of sex differences in Drosophila fat storage and breakdown.

Triglycerides are the major form of stored fat in all animals. One important determinant of whole-body fat storage is whether an animal is male or female. Here, we use Drosophila, an established model for studies on triglyceride metabolism, to gain insight into the genes and physiological mechanisms that contribute to sex differences in fat storage. Our analysis of triglyceride storage and breakdown in both sexes identified a role for triglyceride lipase brummer (bmm) in the regulation of sex differences in triglyceride homeostasis. Normally, male flies have higher levels of bmm mRNA both under normal culture conditions and in response to starvation, a lipolytic stimulus. We find that loss of bmm largely eliminates the sex difference in triglyceride storage and abolishes the sex difference in triglyceride breakdown via strongly male-biased effects. Although we show that bmm function in the fat body affects whole-body triglyceride levels in both sexes, in males, we identify an additional role for bmm function in the somatic cells of the gonad and in neurons in the regulation of whole-body triglyceride homeostasis. Furthermore, we demonstrate that lipid droplets are normally present in both the somatic cells of the male gonad and in neurons, revealing a previously unrecognized role for bmm function, and possibly lipid droplets, in these cell types in the regulation of whole-body triglyceride homeostasis. Taken together, our data reveal a role for bmm function in the somatic cells of the gonad and in neurons in the regulation of male-female differences in fat storage and breakdown and identify bmm as a link between the regulation of triglyceride homeostasis and biological sex.

Beyond the Powerhouse: Integrating Mitonuclear Evolution, Physiology, and Theory in Comparative Biology.

Eukaryotes are the outcome of an ancient symbiosis and as such, eukaryotic cells fundamentally possess two genomes. As a consequence, gene products encoded by both nuclear and mitochondrial genomes must interact in an intimate and precise fashion to enable aerobic respiration in eukaryotes. This genomic architecture of eukaryotes is proposed to necessitate perpetual coevolution between the nuclear and mitochondrial genomes to maintain coadaptation, but the presence of two genomes also creates the opportunity for intracellular conflict. In the collection of papers that constitute this symposium volume, scientists working in diverse organismal systems spanning vast biological scales address emerging topics in integrative, comparative biology in light of mitonuclear interactions.

Lactate dehydrogenase and glycerol-3-phosphate dehydrogenase cooperatively regulate growth and carbohydrate metabolism during Drosophila melanogaster larval development.

The dramatic growth that occurs during Drosophila larval development requires rapid conversion of nutrients into biomass. Many larval tissues respond to these biosynthetic demands by increasing carbohydrate metabolism and lactate dehydrogenase (LDH) activity. The resulting metabolic program is ideally suited for synthesis of macromolecules and mimics the manner by which cancer cells rely on aerobic glycolysis. To explore the potential role of Drosophila LDH in promoting biosynthesis, we examined how Ldh mutations influence larval development. Our studies unexpectedly found that Ldh mutants grow at a normal rate, indicating that LDH is dispensable for larval biomass production. However, subsequent metabolomic analyses suggested that Ldh mutants compensate for the inability to produce lactate by generating excess glycerol-3-phosphate (G3P), the production of which also influences larval redox balance. Consistent with this possibility, larvae lacking both LDH and G3P dehydrogenase (GPDH1) exhibit growth defects, synthetic lethality and decreased glycolytic flux. Considering that human cells also generate G3P upon inhibition of lactate dehydrogenase A (LDHA), our findings hint at a conserved mechanism in which the coordinate regulation of lactate and G3P synthesis imparts metabolic robustness to growing animal tissues.

Temperature-Sensitive Reproduction and the Physiological and Evolutionary Potential for Mother's Curse.

Strict maternal transmission of mitochondrial DNA (mtDNA) is hypothesized to permit the accumulation of mitochondrial variants that are deleterious to males but not females, a phenomenon called mother's curse. However, direct evidence that mtDNA mutations exhibit such sexually antagonistic fitness effects is sparse. Male-specific mutational effects can occur when the physiological requirements of the mitochondria differ between the sexes. Such male-specific effects could potentially occur if sex-specific cell types or tissues have energy requirements that are differentially impacted by mutations affecting energy metabolism. Here we summarize findings from a model mitochondrial-nuclear incompatibility in the fruit fly Drosophila that demonstrates sex-biased effects, but with deleterious effects that are generally larger in females. We present new results showing that the mitochondrial-nuclear incompatibility does negatively affect male fertility, but only when males are developed at high temperatures. The temperature-dependent male sterility can be partially rescued by diet, suggesting an energetic basis. Finally, we discuss fruitful paths forward in understanding the physiological scope for sex-specific effects of mitochondrial mutations in the context of the recent discovery that many aspects of metabolism are sexually dimorphic and downstream of sex-determination pathways in Drosophila. A key parameter of these models that remains to be quantified is the fraction of mitochondrial mutations with truly male-limited fitness effects across extrinsic and intrinsic environments. Given the energy demands of reproduction in females, only a small fraction of the mitochondrial mutational spectrum may have the potential to contribute to mother's curse in natural populations.

Genetic Variation for Ontogenetic Shifts in Metabolism Underlies Physiological Homeostasis in Drosophila.

Organismal physiology emerges from metabolic pathways and subcellular structures like the mitochondria that can vary across development and among individuals. Here, we tested whether genetic variation at one level of physiology can be buffered at higher levels of biological organization during development by the inherent capacity for homeostasis in physiological systems. We found that the fundamental scaling relationship between mass and metabolic rate, as well as the oxidative capacity per mitochondria, changed significantly across development in the fruit fly Drosophila However, mitochondrial respiration rate was maintained at similar levels across development. Furthermore, larvae clustered into two types-those that switched to aerobic, mitochondrial ATP production before the second instar, and those that relied on anaerobic, glycolytic production of ATP through the second instar. Despite genetic variation for the timing of this metabolic shift, metabolic rate in second-instar larvae was more robust to genetic variation than was the metabolic rate of other instars. We found that larvae with a mitochondrial-nuclear incompatibility that disrupts mitochondrial function had increased aerobic capacity and relied more on anaerobic ATP production throughout development relative to larvae from wild-type strains. By taking advantage of both ways of making ATP, larvae with this mitochondrial-nuclear incompatibility maintained mitochondrial respiratory capacity, but also had higher levels of whole-body reactive oxygen species and decreased mitochondrial membrane potential, potentially as a physiological defense mechanism. Thus, genetic defects in core physiology can be buffered at the organismal level via physiological plasticity, and natural populations may harbor genetic variation for distinct metabolic strategies in development that generate similar organismal outcomes.

Energy demand and the context-dependent effects of genetic interactions underlying metabolism.

Genetic effects are often context dependent, with the same genotype differentially affecting phenotypes across environments, life stages, and sexes. We used an environmental manipulation designed to increase energy demand during development to investigate energy demand as a general physiological explanation for context-dependent effects of mutations, particularly for those mutations that affect metabolism. We found that increasing the photoperiod during which Drosophila larvae are active during development phenocopies a temperature-dependent developmental delay in a mitochondrial-nuclear genotype with disrupted metabolism. This result indicates that the context-dependent fitness effects of this genotype are not specific to the effects of temperature and may generally result from variation in energy demand. The effects of this genotype also differ across life stages and between the sexes. The mitochondrial-nuclear genetic interaction disrupts metabolic rate in growing larvae, but not in adults, and compromises female, but not male, reproductive fitness. These patterns are consistent with a model where context-dependent genotype-phenotype relationships may generally arise from differences in energy demand experienced by individuals across environments, life stages, and sexes.

The Mitochondrial Contribution to Animal Performance, Adaptation, and Life-History Variation.

Animals display tremendous variation in their rates of growth, reproductive output, and longevity. While the physiological and molecular mechanisms that underlie this variation remain poorly understood, the performance of the mitochondrion has emerged as a key player. Mitochondria not only impact the performance of eukaryotes via their capacity to produce ATP, but they also play a role in producing heat and reactive oxygen species and function as a major signaling hub for the cell. The papers included in this special issue emerged from a symposium titled "Inside the Black Box: The Mitochondrial Basis of Life-history Variation and Animal Performance." Based on studies of diverse animal taxa, three distinct themes emerged from these papers. (1) When linking mitochondrial function to components of fitness, it is crucial that mitochondrial assays are performed in conditions as close as the intracellular conditions experienced by the mitochondria in vivo. (2) Functional plasticity allows mitochondria to retain their performance, as well as that of their host, over a range of exogenous conditions, and selection on mitochondrial and nuclear-derived proteins can optimize the match between the environment and the bioenergetic capacity of the mitochondrion. Finally, (3) studies of wild and wild-derived animals suggest that mitochondria play a central role in animal performance and life history strategy. Taken as a whole, we hope that these papers will foster discussion and inspire new hypotheses and innovations that will further our understanding of the mitochondrial processes that underlie variation in life history traits and animal performance.

Mitochondrial Dysfunction and Infection Generate Immunity-Fecundity Tradeoffs in Drosophila.

Physiological responses to short-term environmental stressors, such as infection, can have long-term consequences for fitness, particularly if the responses are inappropriate or nutrient resources are limited. Genetic variation affecting energy acquisition, storage, and usage can limit cellular energy availability and may influence resource-allocation tradeoffs even when environmental nutrients are plentiful. Here, we utilized Drosophila mitochondrial-nuclear genotypes to test whether disrupted mitochondrial function interferes with nutrient-sensing pathways, and whether this disruption has consequences for tradeoffs between immunity and fecundity. We found that an energetically-compromised genotype was relatively resistant to rapamycin-a drug that targets nutrient-sensing pathways and mimics resource limitation. Dietary resource limitation decreased survival of energetically-compromised flies. Furthermore, survival of infection with a natural pathogen was decreased in this genotype, and females of this genotype experienced immunity-fecundity tradeoffs that were not evident in genotypic controls with normal energy metabolism. Together, these results suggest that this genotype may have little excess energetic capacity and fewer cellular nutrients, even when environmental nutrients are not limiting. Genetic variation in energy metabolism may therefore act to limit the resources available for allocation to life-history traits in ways that generate tradeoffs even when environmental resources are not limiting.

Maternal loading of a small heat shock protein increases embryo thermal tolerance in Drosophila melanogaster.

Maternal investment is likely to have direct effects on offspring survival. In oviparous animals whose embryos are exposed to the external environment, maternal provisioning of molecular factors like mRNAs and proteins may help embryos cope with sudden changes in the environment. Here, we sought to modify the maternal mRNA contribution to offspring embryos and test for maternal effects on acute thermal tolerance in early embryos of Drosophila melanogaster We drove in vivo overexpression of a small heat shock protein gene (Hsp23) in female ovaries and measured the effects of acute thermal stress on offspring embryonic survival and larval development. We report that overexpression of the Hsp23 gene in female ovaries produced offspring embryos with increased thermal tolerance. We also found that brief heat stress in the early embryonic stage (0-1 h old) caused decreased larval performance later in life (5-10 days old), as indexed by pupation height. Maternal overexpression of Hsp23 protected embryos against this heat-induced defect in larval performance. Our data demonstrate that transient products of single genes have large and lasting effects on whole-organism environmental tolerance. Further, our results suggest that maternal effects have a profound impact on offspring survival in the context of thermal variability.

Experimental test and refutation of a classic case of molecular adaptation in Drosophila melanogaster.

Identifying the genetic basis for adaptive differences between species requires explicit tests of historical hypotheses concerning the effects of past changes in gene sequence on molecular function, organismal phenotype and fitness. We address this challenge by combining ancestral protein reconstruction with biochemical experiments and physiological analysis of transgenic animals that carry ancestral genes. We tested a widely held hypothesis of molecular adaptation-that changes in the alcohol dehydrogenase protein (ADH) along the lineage leading to Drosophila melanogaster increased the catalytic activity of the enzyme and thereby contributed to the ethanol tolerance and adaptation of the species to its ethanol-rich ecological niche. Our experiments strongly refute the predictions of the adaptive ADH hypothesis and caution against accepting intuitively appealing accounts of historical molecular adaptation that are based on correlative evidence. The experimental strategy we employed can be used to decisively test other adaptive hypotheses and the claims they entail about past biological causality.

Incompatibility between mitochondrial and nuclear genomes during oogenesis results in ovarian failure and embryonic lethality.

Mitochondrial dysfunction can cause female infertility. An important unresolved issue is the extent to which incompatibility between mitochondrial and nuclear genomes contributes to female infertility. It has previously been shown that a mitochondrial haplotype from D. simulans (simw501 ) is incompatible with a nuclear genome from the D. melanogaster strain Oregon-R (OreR), resulting in impaired development, which was enhanced at higher temperature. This mito-nuclear incompatibility is between alleles of the nuclear-encoded mitochondrial tyrosyl-tRNA synthetase (Aatm) and the mitochondrial-encoded tyrosyl-tRNA that it aminoacylates. Here, we show that this mito-nuclear incompatibility causes a severe temperature-sensitive female infertility. The OreR nuclear genome contributed to death of ovarian germline stem cells and reduced egg production, which was further enhanced by the incompatibility with simw501  mitochondria. Mito-nuclear incompatibility also resulted in aberrant egg morphology and a maternal-effect on embryonic chromosome segregation and survival, which was completely dependent on the temperature and mito-nuclear genotype of the mother. Our findings show that maternal mito-nuclear incompatibility during Drosophila oogenesis has severe consequences for egg production and embryonic survival, with important broader relevance to human female infertility and mitochondrial replacement therapy.

The Roles of Compensatory Evolution and Constraint in Aminoacyl tRNA Synthetase Evolution.

Mitochondrial protein translation requires interactions between transfer RNAs encoded by the mitochondrial genome (mt-tRNAs) and mitochondrial aminoacyl tRNA synthetase proteins (mt-aaRS) encoded by the nuclear genome. It has been argued that animal mt-tRNAs have higher deleterious substitution rates relative to their nuclear-encoded counterparts, the cytoplasmic tRNAs (cyt-tRNAs). This dynamic predicts elevated rates of compensatory evolution of mt-aaRS that interact with mt-tRNAs, relative to aaRS that interact with cyt-tRNAs (cyt-aaRS). We find that mt-aaRS do evolve at significantly higher rates (exemplified by higher dN and dN/dS) relative to cyt-aaRS, across mammals, birds, and Drosophila. While this pattern supports a model of compensatory evolution, the level at which a gene is expressed is a more general predictor of protein evolutionary rate. We find that gene expression level explains 10-56% of the variance in aaRS dN/dS, and that cyt-aaRS are more highly expressed in addition to having lower dN/dS values relative to mt-aaRS, consistent with more highly expressed genes being more evolutionarily constrained. Furthermore, we find no evidence of positive selection acting on either class of aaRS protein, as would be expected under a model of compensatory evolution. Nevertheless, the signature of faster mt-aaRS evolution persists in mammalian, but not bird or Drosophila, lineages after controlling for gene expression, suggesting some additional effect of compensatory evolution for mammalian mt-aaRS. We conclude that gene expression is the strongest factor governing differential amino acid substitution rates in proteins interacting with mitochondrial versus cytoplasmic factors, with important differences in mt-aaRS molecular evolution among taxonomic groups.

Similar Efficacies of Selection Shape Mitochondrial and Nuclear Genes in Both Drosophila melanogaster and Homo sapiens.

Deleterious mutations contribute to polymorphism even when selection effectively prevents their fixation. The efficacy of selection in removing deleterious mitochondrial mutations from populations depends on the effective population size (Ne) of the mitochondrial DNA and the degree to which a lack of recombination magnifies the effects of linked selection. Using complete mitochondrial genomes from Drosophila melanogaster and nuclear data available from the same samples, we reexamine the hypothesis that nonrecombining animal mitochondrial DNA harbor an excess of deleterious polymorphisms relative to the nuclear genome. We find no evidence of recombination in the mitochondrial genome, and the much-reduced level of mitochondrial synonymous polymorphism relative to nuclear genes is consistent with a reduction in Ne. Nevertheless, we find that the neutrality index, a measure of the excess of nonsynonymous polymorphism relative to the neutral expectation, is only weakly significantly different between mitochondrial and nuclear loci. This difference is likely the result of the larger proportion of beneficial mutations in X-linked relative to autosomal loci, and we find little to no difference between mitochondrial and autosomal neutrality indices. Reanalysis of published data from Homo sapiens reveals a similar lack of a difference between the two genomes, although previous studies have suggested a strong difference in both species. Thus, despite a smaller Ne, mitochondrial loci of both flies and humans appear to experience similar efficacies of purifying selection as do loci in the recombining nuclear genome.

Thermal adaptation of cellular membranes in natural populations of Drosophila melanogaster.

Changes in temperature disrupt the fluidity of cellular membranes, which can negatively impact membrane integrity and cellular processes. Many ectotherms, including Drosophila melanogaster (Meigen), adjust the glycerophospholipid composition of their membranes to restore optimal fluidity when temperatures change, a type of trait plasticity termed homeoviscous adaptation.Existing data suggest that plasticity in the relative abundances of the glycerophospholipids phosphatidylethanolamine (PE) and phosphatidylcholine (PC) underlies cellular adaptation to temporal variability in the thermal environment. For example, laboratory populations of D. melanogaster evolved in the presence of temporally variable temperatures have greater developmental plasticity of the ratio of PE to PC (PE/PC) and greater fecundity than do populations evolved at constant temperatures.Here, we extend this work to natural populations of D. melanogaster by evaluating thermal plasticity of glycerophospholipid composition at different life stages, in genotypes isolated from Vermont, Indiana and North Carolina, USA. We also quantify the covariance between developmental and adult (reversible) plasticity, and between adult responses of the membrane to cool and warm thermal shifts.As predicted by physiological models of homeoviscous adaptation, flies from all populations decrease PE/PC and the degree of lipid unsaturation in response to warm temperatures. Furthermore, these populations have diverged in their degree of membrane plasticity. Flies from the most variable thermal environment (Vermont, USA) decrease PE/PC to a greater extent than do other populations when developed at a warm temperature, a pattern that matches our previous observation in laboratory-evolved populations. We also find that developmental plasticity and adult plasticity of PE/PC covary across genotypes, but that adult responses to cool and warm thermal shifts do not.When combined with our previous observations of laboratory-evolved populations, our findings implicate developmental plasticity of PE/PC as a mechanism of thermal adaptation in temporally variable environments. While little is known about the genetic bases of plastic responses to temperature, our observations suggest that both environmentally sensitive and environmentally specific alleles contribute to thermal adaptation of membranes, and that costs of plasticity may arise when the adult environment differs from that experienced during development.