Extended lifespan and sex-specific fertility loss in cold-acclimated flies of the sibling species Drosophila buzzatii and Drosophila koepferae.

Survival and reproduction are the core elements of Darwinian fitness. In the context of a fixed energy budget, organisms tend to allocate resources in order to maximize one at the expense of the other, in what has been called the lifespan-reproduction trade-off. Reproductive arrest and extended lifespan are common responses to low temperatures in many insects including fruit flies. In this study, we aim to understand the overwintering strategy of two closely-related Drosophila species with contrasting distribution ranges. We compared survival, lifespan, ovarian maturation, and reproductive output (fecundity and fertility) of virgin and mated adults of both Drosophila buzzatii and Drosophila koepferae after long-term cold exposure at dormancy-inducing conditions (10 °C, 10:14 L:D) and controls (25 °C, 12:12 L:D). Virgin flies of D. buzzatii showed the longest lifespan (averaging 102 days) under dormancy-inducing conditions. Cold-induced reproductive arrest preserves reproductive capacity mainly in virgin females that mated after reproductive dormancy, indicating that males were much more susceptible to fertility loss than females, in both species. Notably, females of D. buzzatii were capable of protecting stored sperm from cold damage and produced viable progeny. Even if, in D. buzzatii, fertility of flies mated after the cold-exposure was extremely low, cold temperature likely sterilized D. koepferae males, indicating that cold carry-over effects are stronger for the species with the shorter lifespan. Such species-specific effects of low temperature over fitness likely contributed to the divergence of these closely-related species and to the spread of D. buzzatii into cooler environments.

Divergent metabolomic profiles of cold-exposed mature and immature females of tropical versus temperate Drosophila species.

Temperate species, contrary to their tropical counterparts, are exposed not only to thermally variable environments with low temperatures but also to long winters. Different selective pressures may have driven divergent physiological adaptations in closely related species with different biogeographic origins. To survive unfavourable winter conditions, Drosophila species in temperate areas generally undergo a period of reproductive dormancy, associated with a cold-induced cessation of oogenesis and metabolic reorganization. This work aims to compare cold tolerance and metabolic signatures of cold-exposed females exhibiting different reproductive maturity status (mature and immature females) of four Drosophila species from tropical vs. temperate origins. We expected that the capacity for delayed reproduction of immature females could result in the redirection of the energy-related metabolites to be utilized for surviving the cold season. To do so, we studied an array of 45 metabolites using quantitative target GC-MS profiling. Reproductively immature females of temperate species showed the lower CTmin and the faster chill coma recovery time (i.e. the most cold-tolerant group). Principal component analysis captured differences across species, but also between reproductive maturity states. Notably, temperate species exhibited significantly higher levels of glucose, alanine, and gluconolactone than tropical ones. As proline and glycerol showed higher abundances in immature females of temperate species compared to the levels exhibited by the rest of the groups, we reasoned that glucose and alanine could serve as intermediates in the synthesis of these compatible solutes. All in all, our findings suggest that cold-exposed females of temperate species accumulate energy-related and protective metabolites (e.g. glycerol and proline) while delaying reproduction, and that these metabolites are relevant to cold tolerance even at modest concentrations.

Host Plant Adaptation in Cactophilic Species of the Drosophila buzzatii Cluster: Fitness and Transcriptomics.

Host plant shifts in herbivorous insects often involve facing new environments that may speed up the evolution of oviposition behavior, performance-related traits, morphology, and, incidentally, reproductive isolation. In the genus Drosophila, cactophilic species of the repleta group include emblematic species in the study of the evolution of host plant utilization. The South American D. buzzatii and its sibling D. koepferae are a model system for the study of differential host plant use. Although these species exhibit a certain degree of niche overlap, the former breeds primarily on decaying cladodes of Opuntia cacti while D. koepferae main hosts are columnar cacti of the genus Trichocereus. Opuntia sulphurea and Trichocereus terscheckii are among the main hosts in nature. These cacti differ in ecological (spatial and temporal predictability) and chemical characteristics. Particularly relevant is the presence of toxic alkaloids in T. terscheckii. Studies of the effects of these cacti and alkaloids revealed the remarkable impact on oviposition behavior, viability, developmental time, wing morphology, mating success, and developmental stability in both species. Recent whole-genome expression studies showed that expression profiles are massively affected by the rearing cactus, and that the presence of alkaloids is the main factor modulating gene expression in D. buzzatii. Functional enrichment analysis indicated that differentially expressed genes are related to detoxification processes and stress response-though genes involved in development are an important part of the transcriptomic response. The implications of our studies in the evolution of host plant use in the repleta group are discussed.

Enhanced fertility and chill tolerance after cold-induced reproductive arrest in females of temperate species of the Drosophila buzzatii complex.

Long-term exposure to low temperatures during adult maturation might decrease fertility after cold recovery as a consequence of carry-over effects on reproductive tissues. This pattern should be more pronounced in tropical than in temperate species as protective mechanisms against chilling injuries are expected to be more effective in the latter. We initially determined the lower thermal thresholds to induce ovarian maturation in four closely related Drosophila species, two inhabiting temperate regions and the other two tropical areas of South America. As expected, only temperate species regularly experience cold-inducing conditions for reproductive arrest during winter in their natural environment. Subsequently, we exposed reproductively arrested and mature females to cold-inducing conditions for reproductive arrest over a long period. Following cold exposure, tropical species exhibited a dramatic fertility decline, irrespective of reproductive status. In contrast, not only were temperate females fecund and fertile but also fertility was superior in females that underwent cold-induced reproductive arrest, suggesting that it might act as a protecting mechanism ensuring fertility after cold recovery. Based on these findings, we decided to evaluate the extent to which reproductive status affects cold tolerance and energy metabolism at low temperature. We found a lower metabolic rate and a higher cold tolerance in reproductively arrested females, although only temperate species attained high levels of chill tolerance. These findings highlight the role of cold-induced reproductive arrest as part of an integrated mechanism of cold adaptation that could potentially contribute to the spread of temperate species into higher latitudes or altitudes.

Effect of brief exposures of anesthesia on thermotolerance and metabolic rate of the spotted-wing fly, Drosophila suzukii: Differences between sexes?

The spotted-wing fly, Drosophila suzukii, is a world-wide pest insect for which there is increasing interest in its physiological traits including metabolism and thermotolerance. Most studies focus only on survival to different time exposures to extreme temperatures, mainly in female flies. In addition, it has not been tested yet how anesthesia affects these measurements. We analyzed the effects of anesthesia by brief exposures to cold, anoxia by CO2 or N2 on three standard thermotolerance assays, as well as the aerobic metabolic rate in both sexes. For heat tolerance we measured CTmax by thermolimit respirometry, and CTmin and chill-coma recovery time for cold tolerance. Aerobic metabolism was calculated by CO2 production of individual flies in real time by open flow respirometry. Results showed that females have a significantly higher V̇CO2 for inactive (at 25 °C) and maximum metabolic rate than males. This difference is mainly explained by body mass and disappears after mass correction. Males had a more sensitive MR to temperature than females showed by a significantly higher Q10 (2.19 vs. 1.98, for males and females, respectively). We observed a significantly lower CTmin (X2 = 4.27, P = 0.03) in females (3.68 ± 0.38 °C) than males (4.56 ± 0.39 °C), although we did not find significant effects of anesthesia. In contrast, anesthesia significantly modifies CTmax for both sexes (F3,62 = 7.86, P < 0.001) with a decrease of the CTmax in cold-anesthetized flies. Finally, we found a significantly higher CTmax in females (37.87 ± 0.07 °C) than males (37.36 ± 0.09 °C). We conclude that cold anesthesia seems to have detrimental effects on heat tolerance, and females have broader thermotolerance range than males, which could help them to establish in invaded temperate regions with more variable environmental temperatures.

Genetic basis of wing morphogenesis in Drosophila: sexual dimorphism and non-allometric effects of shape variation.

BACKGROUND: The Drosophila wing represents a particularly appropriate model to investigate the developmental control of phenotypic variation. Previous studies which aimed to identify candidate genes for wing morphology demonstrated that the genetic basis of wing shape variation in D. melanogaster is composed of numerous genetic factors causing small, additive effects. In this study, we analyzed wing shape in males and females from 191 lines of D. melanogaster, homozygous for a single P-element insertion, using geometric morphometrics techniques. The analysis allowed us to identify known and novel candidate genes that may contribute to the expression of wing shape in each sex separately and to compare them to candidate genes affecting wing size which have been identified previously using the same lines. RESULTS: Our results indicate that more than 63% of induced mutations affected wing shape in one or both sexes, although only 33% showed significant differences in both males and females. The joint analysis of wing size and shape revealed that only 19% of the P-element insertions caused coincident effects on both components of wing form in one or both sexes. Further morphometrical analyses revealed that the intersection between veins showed the smallest displacements in the proximal region of the wing. Finally, we observed that mutations causing general deformations were more common than expected in both sexes whereas the opposite occurred with those generating local changes. For most of the 94 candidate genes identified, this seems to be the first record relating them with wing shape variation. CONCLUSIONS: Our results support the idea that the genetic architecture of wing shape is complex with many different genes contributing to the trait in a sexually dimorphic manner. This polygenic basis, which is relatively independent from that of wing size, is composed of genes generally involved in development and/or metabolic functions, especially related to the regulation of different cellular processes such as motility, adhesion, communication and signal transduction. This study suggests that understanding the genetic basis of wing shape requires merging the regulation of vein patterning by signalling pathways with processes that occur during wing development at the cellular level.

Increased size and energy reserves in diapausing eggs of temperate Aedes aegypti populations.

Many insects overwinter in diapause, a pre-programmed anticipated response to unfavorable environmental conditions, often induced by a short-day photoperiod. Diapause involves morphological changes and increased energy stores required for metabolic demands during winter. In diapausing mosquito eggs, the accumulation of lipids plays an important role, because these molecules are the primary fuel consumed during embryogenesis and pharate larvae metabolism, and have a key role in egg desiccation resistance. The supposed inability of the mosquito Aedes aegypti to lay diapausing eggs has been recently challenged by a study on a temperate population, which showed that the inhibition of egg hatching in response to short days is possible in this species. Thus, the aim of the present study was to assess the effects of parental photoperiod on embryonic diapause-related traits, such as the triglyceride content and size of eggs laid, of two populations whose localities of origin differ in their winter length. Two colonies were maintained for each population: one under a Short-Day Photoperiod (SD: 10 h:14 h - Light:Dark) and the other under a Long-Day Photoperiod (LD: 14 h:10 h - Light:Dark). The eggs obtained from each combination of population and light treatment were used for size measurement (length, width and volume) and for the quantification of triglyceride content. Egg size showed differences between photoperiod treatments, with larger width and volume in eggs from the SD treatment. Remarkably, eggs from the SD treatment accumulated twice as many triglycerides as those from the LD treatment. Also, the eggs derived from the population having the longer winter accumulated larger amounts of triglycerides. The higher lipid content is probably contributing to a better survival during the cold season in both populations. The photoperiod-induced response in egg size and amount of triglycerides observed in this study support the hypothesis that the Ae. aegypti populations studied are able to lay diapausing eggs, a fact that provides physiological bases for the further expansion of this species to colder regions.

What does mitogenomics tell us about the evolutionary history of the Drosophila buzzatii cluster (repleta group)?

The Drosophila repleta group is an array of more than 100 species endemic to the "New World", many of which are cactophilic. The ability to utilize decaying cactus tissues as breeding and feeding sites is a key aspect that allowed the successful diversification of the repleta group in American deserts and arid lands. Within this group, the Drosophila buzzatii cluster is a South American clade of seven closely related species in different stages of divergence, making them a valuable model system for evolutionary research. Substantial effort has been devoted to elucidating the phylogenetic relationships among members of the D. buzzatii cluster, including molecular phylogenetic studies that have generated ambiguous results where different tree topologies have resulted dependent on the kinds of molecular marker used. Even though mitochondrial DNA regions have become useful markers in evolutionary biology and population genetics, none of the more than twenty Drosophila mitogenomes assembled so far includes this cluster. Here, we report the assembly of six complete mitogenomes of five species: D. antonietae, D. borborema, D. buzzatii, two strains of D. koepferae and D. seriema, with the aim of revisiting phylogenetic relationships and divergence times by means of mitogenomic analyses. Our recovered topology using complete mitogenomes supports the hypothesis of monophyly of the D. buzzatii cluster and shows two main clades, one including D. buzzatii and D. koepferae (both strains), and the other containing the remaining species. These results are in agreement with previous reports based on a few mitochondrial and/or nuclear genes, but conflict with the results of a recent large-scale nuclear phylogeny, indicating that nuclear and mitochondrial genomes depict different evolutionary histories.

Identifying candidate genes affecting developmental time in Drosophila melanogaster: pervasive pleiotropy and gene-by-environment interaction.

BACKGROUND: Understanding the genetic architecture of ecologically relevant adaptive traits requires the contribution of developmental and evolutionary biology. The time to reach the age of reproduction is a complex life history trait commonly known as developmental time. In particular, in holometabolous insects that occupy ephemeral habitats, like fruit flies, the impact of developmental time on fitness is further exaggerated. The present work is one of the first systematic studies of the genetic basis of developmental time, in which we also evaluate the impact of environmental variation on the expression of the trait. RESULTS: We analyzed 179 co-isogenic single P[GT1]-element insertion lines of Drosophila melanogaster to identify novel genes affecting developmental time in flies reared at 25 degrees C. Sixty percent of the lines showed a heterochronic phenotype, suggesting that a large number of genes affect this trait. Mutant lines for the genes Merlin and Karl showed the most extreme phenotypes exhibiting a developmental time reduction and increase, respectively, of over 2 days and 4 days relative to the control (a co-isogenic P-element insertion free line). In addition, a subset of 42 lines selected at random from the initial set of 179 lines was screened at 17 degrees C. Interestingly, the gene-by-environment interaction accounted for 52% of total phenotypic variance. Plastic reaction norms were found for a large number of developmental time candidate genes. CONCLUSION: We identified components of several integrated time-dependent pathways affecting egg-to-adult developmental time in Drosophila. At the same time, we also show that many heterochronic phenotypes may arise from changes in genes involved in several developmental mechanisms that do not explicitly control the timing of specific events. We also demonstrate that many developmental time genes have pleiotropic effects on several adult traits and that the action of most of them is sensitive to temperature during development. Taken together, our results stress the need to take into account the effect of environmental variation and the dynamics of gene interactions on the genetic architecture of this complex life-history trait.

Genetic variation for tolerance to high temperatures in a population of Drosophila melanogaster.

The range of thermal tolerance is one of the main factors influencing the geographic distribution of species. Climate change projections predict increases in average and extreme temperatures over the coming decades; hence, the ability of living beings to resist these changes will depend on physiological and adaptive responses. On an evolutionary scale, changes will occur as the result of selective pressures on individual heritable differences. In this work, we studied the genetic basis of tolerance to high temperatures in the fly Drosophila melanogaster and whether this species presents sufficient genetic variability to allow expansion of its upper thermo-tolerance limit. To do so, we used adult flies derived from a natural population belonging to the Drosophila Genetic Reference Panel, for which genomic sequencing data are available. We characterized the phenotypic variation of the upper thermal limit in 34 lines by measuring knockdown temperature (i.e., critical thermal maximum [CTmax]) by exposing flies to a ramp of increasing temperature (0.25°C/min). Fourteen percent of the variation in CTmax is explained by the genetic variation across lines, without a significant sexual dimorphism. Through a genomewide association study, 12 single nucleotide polymorphisms associated with the CTmax were identified. In most of these SNPs, the less frequent allele increased the upper thermal limit suggesting that this population harbors raw genetic variation capable of expanding its heat tolerance. This potential upper thermal tolerance increase has implications under the global warming scenario. Past climatic records show a very low incidence of days above CTmax (10 days over 25 years); however, future climate scenarios predict 243 days with extreme high temperature above CTmax from 2045 to 2070. Thus, in the context of the future climate warming, rising temperatures might drive the evolution of heat tolerance in this population by increasing the frequency of the alleles associated with higher CTmax.

Natural Genetic Variation and Candidate Genes for Morphological Traits in Drosophila melanogaster.

Body size is a complex character associated to several fitness related traits that vary within and between species as a consequence of environmental and genetic factors. Latitudinal and altitudinal clines for different morphological traits have been described in several species of Drosophila and previous work identified genomic regions associated with such variation in D. melanogaster. However, the genetic factors that orchestrate morphological variation have been barely studied. Here, our main objective was to investigate genetic variation for different morphological traits associated to the second chromosome in natural populations of D. melanogaster along latitudinal and altitudinal gradients in Argentina. Our results revealed weak clinal signals and a strong population effect on morphological variation. Moreover, most pairwise comparisons between populations were significant. Our study also showed important within-population genetic variation, which must be associated to the second chromosome, as the lines are otherwise genetically identical. Next, we examined the contribution of different candidate genes to natural variation for these traits. We performed quantitative complementation tests using a battery of lines bearing mutated alleles at candidate genes located in the second chromosome and six second chromosome substitution lines derived from natural populations which exhibited divergent phenotypes. Results of complementation tests revealed that natural variation at all candidate genes studied, invected, Fasciclin 3, toucan, Reticulon-like1, jing and CG14478, affects the studied characters, suggesting that they are Quantitative Trait Genes for morphological traits. Finally, the phenotypic patterns observed suggest that different alleles of each gene might contribute to natural variation for morphological traits. However, non-additive effects cannot be ruled out, as wild-derived strains differ at myriads of second chromosome loci that may interact epistatically with mutant alleles.

Contrasting Plasticity in Ovariole Number Induced by A Dietary Effect of the Host Plants between Cactophilic Drosophila Species.

Under the preference-performance hypothesis, natural selection will favor females that choose oviposition sites that optimize the fitness of their offspring. Such a preference-performance relationship may entail important consequences mainly on fitness-related traits. We used the well-characterized cactus-Drosophila system to investigate the reproductive capacity in the pair of sibling species D. buzzatii and D. koepferae reared in two alternative host plants. According to our hypothesis, ovariole number (as a proxy of reproductive capacity) depends on host plant selection. Our results indicate that the capacity of D. buzzatii showed to be mild, only increasing the number of ovarioles by as much as 10% when reared in its preferred host. In contrast, D. koepferae exhibited a similar reproductive capacity across host cacti, even though it showed a preference for its primary host cactus. Our study also revealed that D. buzzatii has a larger genetic variation for phenotypic plasticity than its sibling, although ovariole number did not show clear-cut differences between species. We will discuss the weak preference-performance pattern observed in these cactophilic species in the light of nutritional and toxicological differences found between the natural host plants.

Positive selection in nucleoporins challenges constraints on early expressed genes in Drosophila development.

Developmental conservation among related species is a common generalization known as von Baer's third law and implies that early stages of development are the most refractory to change. The "hourglass model" is an alternative view that proposes that middle stages are the most constrained during development. To investigate this issue, we undertook a genomic approach and provide insights into how natural selection operates on genes expressed during the first 24 h of Drosophila ontogeny in the six species of the melanogaster group for which whole genome sequences are available. Having studied the rate of evolution of more than 2,000 developmental genes, our results showed differential selective pressures at different moments of embryogenesis. In many Drosophila species, early zygotic genes evolved slower than maternal genes indicating that mid-embryogenesis is the stage most refractory to evolutionary change. Interestingly, positively selected genes were found in all embryonic stages even during the period with the highest developmental constraint, emphasizing that positive selection and negative selection are not mutually exclusive as it is often mistakenly considered. Among the fastest evolving genes, we identified a network of nucleoporins (Nups) as part of the maternal transcriptome. Specifically, the acceleration of Nups was driven by positive selection only in the more recently diverged species. Because many Nups are involved in hybrid incompatibilities between species of the Drosophila melanogaster subgroup, our results link rapid evolution of early developmental genes with reproductive isolation. In summary, our study revealed that even within functional groups of genes evolving under strong negative selection many positively selected genes could be recognized. Understanding these exceptions to the broad evolutionary conservation of early expressed developmental genes can shed light into relevant processes driving the evolution of species divergence.

Gene-by-temperature interactions and candidate plasticity genes for morphological traits in Drosophila melanogaster.

Understanding the genetic architecture of any quantitative trait requires identifying the genes involved in its expression in different environmental conditions. This goal can be achieved by mutagenesis screens in genetically tractable model organisms such as Drosophila melanogaster. Temperature during ontogenesis is an important environmental factor affecting development and phenotypic variation in holometabolous insects. In spite of the importance of phenotypic plasticity and genotype by environment interaction (GEI) for fitness related traits, its genetic basis has remained elusive. In this context, we analyzed five different adult morphological traits (face width, head width, thorax length, wing size and wing shape) in 42 co-isogenic single P-element insertional lines of Drosophila melanogaster raised at 17°C and 25°C. Our analyses showed that all lines differed from the control for at least one trait in males or females at either temperature. However, no line showed those differences for all traits in both sexes and temperatures simultaneously. In this sense, the most pleiotropic candidate genes were CG34460, Lsd-2 and Spn. Our analyses also revealed extensive genetic variation for all the characters mostly indicated by strong GEIs. Further, our results indicate that GEIs were predominantly explained by changes in ranking order in all cases suggesting that a moderate number of genes are involved in the expression of each character at both temperatures. Most lines displayed a plastic response for at least one trait in either sex. In this regard, P-element insertions affecting plasticity of a large number of traits were associated to the candidate genes Btk29A, CG43340, Drak and jim. Further studies will help to elucidate the relevance of these genes on the morphogenesis of different body structures in natural populations of D. melanogaster.

Ontogenetic stage-dependent effect of temperature on developmental and metabolic rates in a holometabolous insect.

Different hypotheses attempt to explain how different stages of organisms with complex life cycles respond to environmental changes. Most studies have focused at the among-species level showing similar responses to temperature throughout ontogeny. However, there is no agreement about the pattern expected at the intraspecific scale where a strong selective effect is expected. In this paper, we studied the effects of thermal treatments on a life history trait (developmental rate) and a physiological trait (metabolic rate) during development in the fruitfly Drosophila buzzatii. First, we estimated the rate of development during larval life (LDR) and the pupal stage (PDR) in flies derived from two natural populations exposed to several thermal treatments. Our results showed that the developmental rate ratio, LDR/PDR, did not vary between populations, and that the effects of thermal treatments were stage specific. Second, we studied the relationship between developmental rate (DR) and metabolic rate (MR) in each life cycle stage. We found that allometric relationships between DR and MR varied throughout ontogeny, a pattern that shed light on the mechanisms responsible for thermal plasticity. We conclude that, although different populations may show developmental rate isomorphy; larvae and pupae may choose alternative "decisions" in terms of life-history evolution and physiological traits when confronted to different thermal environments.

Stage-specific effects of candidate heterochronic genes on variation in developmental time along an altitudinal cline of Drosophila melanogaster.

BACKGROUND: Previously, we have shown there is clinal variation for egg-to-adult developmental time along geographic gradients in Drosophila melanogaster. Further, we also have identified mutations in genes involved in metabolic and neurogenic pathways that affect development time (heterochronic genes). However, we do not know whether these loci affect variation in developmental time in natural populations. METHODOLOGY/PRINCIPAL FINDINGS: Here, we constructed second chromosome substitution lines from natural populations of Drosophila melanogaster from an altitudinal cline, and measured egg-adult development time for each line. We found not only a large amount of genetic variation for developmental time, but also positive associations of the development time with thermal amplitude and altitude. We performed genetic complementation tests using substitution lines with the longest and shortest developmental times and heterochronic mutations. We identified segregating variation for neurogenic and metabolic genes that largely affected the duration of the larval stages but had no impact on the timing of metamorphosis. CONCLUSIONS/SIGNIFICANCE: Altitudinal clinal variation in developmental time for natural chromosome substitution lines provides a unique opportunity to dissect the response of heterochronic genes to environmental gradients. Ontogenetic stage-specific variation in invected, mastermind, cricklet and CG14591 may affect natural variation in development time and thermal evolution.