Drosophila rely on learning while foraging under semi‐natural conditions

Learning is predicted to affect manifold ecological and evolutionary processes, but the extent to which animals rely on learning in nature remains poorly known, especially for short‐lived non‐social invertebrates. This is in particular the case for Drosophila, a favourite laboratory system to study molecular mechanisms of learning. Here we tested whether Drosophila melanogaster use learned information to choose food while free‐flying in a large greenhouse emulating the natural environment. In a series of experiments flies were first given an opportunity to learn which of two food odours was associated with good versus unpalatable taste; subsequently, their preference for the two odours was assessed with olfactory traps set up in the greenhouse. Flies that had experienced palatable apple‐flavoured food and unpalatable orange‐flavoured food were more likely to be attracted to the odour of apple than flies with the opposite experience. This was true both when the flies first learned in the laboratory and were then released and recaptured in the greenhouse, and when the learning occurred under free‐flying conditions in the greenhouse. Furthermore, flies retained the memory of their experience while exploring the greenhouse overnight in the absence of focal odours, pointing to the involvement of consolidated memory. These results support the notion that even small, short lived insects which are not central‐place foragers make use of learned cues in their natural environments.     

Evidence for complex selection on four‐fold degenerate sites in Drosophila melanogaster

We considered genome‐wide four‐fold degenerate sites from an African Drosophila melanogaster population and compared them to short introns. To include divergence and to polarize the data, we used its close relatives Drosophila simulans, Drosophila sechellia, Drosophila erecta and Drosophila yakuba as outgroups. In D. melanogaster, the GC content at four‐fold degenerate sites is higher than in short introns; compared to its relatives, more AT than GC is fixed. The former has been explained by codon usage bias (CUB) favouring GC; the latter by decreased intensity of directional selection or by increased mutation bias towards AT. With a biallelic equilibrium model, evidence for directional selection comes mostly from the GC‐rich ancestral base composition. Together with a slight mutation bias, it leads to an asymmetry of the unpolarized allele frequency spectrum, from which directional selection is inferred. Using a quasi‐equilibrium model and polarized spectra, however, only purifying and no directional selection is detected. Furthermore, polarized spectra are proportional to those of the presumably unselected short introns. As we have no evidence for a decrease in effective population size, relaxed CUB must be due to a reduction in the selection coefficient. Going beyond the biallelic model and considering all four bases, signs of directional selection are stronger. In contrast to short introns, complementary bases show strand specificity and allele frequency spectra depend on mutation directions. Hence, the traditional biallelic model to describe the evolution of four‐fold degenerate sites should be replaced by more complex models assuming only quasi‐equilibrium and accounting for all four bases.     

UCP4C mediates uncoupled respiration in larvae of Drosophila melanogaster

Larvae of Drosophila melanogaster reared at 23°C and switched to 14°C for 1 h are 0.5°C warmer than the surrounding medium. In keeping with dissipation of energy, respiration of Drosophila melanogaster larvae cannot be decreased by the F‐ATPase inhibitor oligomycin or stimulated by protonophore. Silencing of Ucp4C conferred sensitivity of respiration to oligomycin and uncoupler, and prevented larva‐to‐adult progression at 15°C but not 23°C. Uncoupled respiration of larval mitochondria required palmitate, was dependent on Ucp4C and was inhibited by guanosine diphosphate. UCP4C is required for development through the prepupal stages at low temperatures and may be an uncoupling protein.     

Geographical delimitation of a partial selective sweep in African Drosophila melanogaster

Positive selection leaves characteristic footprints on DNA variation but detecting such patterns is challenging as the age, the intensity and the mode of selection as well as demography and evolutionary parameters (mutation and recombination rates) all play roles and these are difficult to disentangle. We recorded nucleotide variation in a sample of isogenic chromosomes from a western African population of Drosophila melanogaster at a locus (Fbp2) for which a partial selective sweep had previously been reported. We compared this locus to four other genes from the same chromosomes and from a European and an East African population. Then, we assessed Fbp2 variation in a sample of 370 chromosomes covering a comprehensive geographic sampling of 16 African localities. The signature of selection was tested while accounting for the demographic history of the populations. We found a significant signal of selection in two West African localities including Ivory Coast. Variation at Fpb2 would thus represent a case of an ongoing selective sweep in the range of this species. A weaker, nonsignificant, signal of selection was, however, apparent in some other populations, thus leaving open several possibilities: (i) the selective sweep originated in Ivory Coast and has spread to the rest of the continent; (ii) several African populations report the signature of a selective event having occurred in an ancestral population; (iii) this genome region is subject to independent selective events in African populations; and (iv) A neutral scenario with population subdivision and local bottleneck cannot be fully excluded to explain the molecular patterns observed in some populations.     

Experimental evolution reveals habitat‐specific fitness dynamics among Wolbachia clades in Drosophila melanogaster

The diversity and infection dynamics of the endosymbiont Wolbachia can be influenced by many factors, such as transmission rate, cytoplasmic incompatibility, environment, selection and genetic drift. The interplay of these factors in natural populations can result in heterogeneous infection patterns with substantial differences between populations and strains. The causes of these heterogeneities are not yet understood, partly due to the complexity of natural environments. We present experimental evolution as a new approach to study Wolbachia infection dynamics in replicate populations exposed to a controlled environment. A natural Drosophila melanogaster population infected with strains of Wolbachia belonging to different clades evolved in two laboratory environments (hot and cold) for 1.5 years. In both treatments, the rate of Wolbachia infection increased until fixation. In the hot environment, the relative frequency of different Wolbachia clades remained stable over 37 generations. In the cold environment, however, we observed marked changes in the composition of the Wolbachia population: within 15 generations, one Wolbachia clade increased more than 50% in frequency, whereas the other two clades decreased in frequency, resulting in the loss of one clade. The frequency change was highly reproducible not only among replicates, but also when flies that evolved for 42 generations in the hot environment were transferred to the cold environment. These results document how environmental factors can affect the composition of Wolbachia in D. melanogaster. The high reproducibility of the pattern suggests that experimental evolution studies can efficiently determine the functional basis of habitat‐specific fitness among Wolbachia strains.     

The adaptive significance of chromosomal inversion polymorphisms in Drosophila melanogaster

Chromosomal inversions, structural mutations that reverse a segment of a chromosome, cause suppression of recombination in the heterozygous state. Several studies have shown that inversion polymorphisms can form clines or fluctuate predictably in frequency over seasonal time spans. These observations prompted the hypothesis that chromosomal rearrangements might be subject to spatially and/or temporally varying selection. Here, we review what has been learned about the adaptive significance of inversion polymorphisms in the vinegar fly Drosophila melanogaster, the species in which they were first discovered by Sturtevant in 1917. A large body of work provides compelling evidence that several inversions in this system are adaptive; however, the precise selective mechanisms that maintain them polymorphic in natural populations remain poorly understood. Recent advances in population genomics, modelling and functional genetics promise to greatly improve our understanding of this long‐standing and fundamental problem in the near future.     

Secondary contact and local adaptation contribute to genome‐wide patterns of clinal variation in Drosophila melanogaster

Populations arrayed along broad latitudinal gradients often show patterns of clinal variation in phenotype and genotype. Such population differentiation can be generated and maintained by both historical demographic events and local adaptation. These evolutionary forces are not mutually exclusive and can in some cases produce nearly identical patterns of genetic differentiation among populations. Here, we investigate the evolutionary forces that generated and maintain clinal variation genome‐wide among populations of Drosophila melanogaster sampled in North America and Australia. We contrast patterns of clinal variation in these continents with patterns of differentiation among ancestral European and African populations. Using established and novel methods we derive here, we show that recently derived North America and Australia populations were likely founded by both European and African lineages and that this hybridization event likely contributed to genome‐wide patterns of parallel clinal variation between continents. The pervasive effects of admixture mean that differentiation at only several hundred loci can be attributed to the operation of spatially varying selection using an F_ST outlier approach. Our results provide novel insight into the well‐studied system of clinal differentiation in D. melanogaster and provide a context for future studies seeking to identify loci contributing to local adaptation in a wide variety of organisms, including other invasive species as well as temperate endemics.     

Intragroup competition predicts individual foraging specialisation in a group‐living mammal

Individual foraging specialisation has important ecological implications, but its causes in group‐living species are unclear. One of the major consequences of group living is increased intragroup competition for resources. Foraging theory predicts that with increased competition, individuals should add new prey items to their diet, widening their foraging niche (‘optimal foraging hypothesis’). However, classic competition theory suggests the opposite: that increased competition leads to niche partitioning and greater individual foraging specialisation (‘niche partitioning hypothesis’). We tested these opposing predictions in wild, group‐living banded mongooses (Mungos mungo), using stable isotope analysis of banded mongoose whiskers to quantify individual and group foraging niche. Individual foraging niche size declined with increasing group size, despite all groups having a similar overall niche size. Our findings support the prediction that competition promotes niche partitioning within social groups and suggest that individual foraging specialisation may play an important role in the formation of stable social groupings.     

A test for Y‐linked additive and epistatic effects on surviving bacterial infections in Drosophila melanogaster

Y‐ and W‐chromosomes offer a theoretically powerful way for sexual dimorphism to evolve. Consistent with this possibility, Drosophila melanogaster Y‐chromosomes can influence gene regulation throughout the genome; particularly immune‐related genes. In order for Y‐linked regulatory variation (YRV) to contribute to adaptive evolution it must be comprised of additive genetic variance, such that variable Ys induce consistent phenotypic effects within the local gene pool. We assessed the potential for Y‐chromosomes to adaptively shape gram‐negative and gram‐positive bacterial defence by introgressing Ys across multiple genetic haplotypes from the same population. We found no Y‐linked additive effects on immune phenotypes, suggesting a restricted role for the Y to facilitate dimorphic evolution. We did find, however, a large magnitude Y by background interaction that induced rank order reversals of Y‐effects across the backgrounds (i.e. sign epistasis). Thus, Y‐chromosome effects appeared consistent within backgrounds, but highly variable among backgrounds. This large sign epistatic effect could constrain monomorphic selection in both sexes, considering that autosomal alleles under selection must spend half of their time in a male background where relative fitness values are altered. If the pattern described here is consistent for other traits or within other XY (or ZW) systems, then YRV may represent a universal constraint to autosomal trait evolution.     

Identification of a genetic network for an ecologically relevant behavioural phenotype in Drosophila melanogaster

Pupation site choice of Drosophila third‐instar larvae is critical for the survival of individuals, as pupae are exposed to various biotic and abiotic dangers while immobilized during the 3–4 days of metamorphosis. This singular behavioural choice is sensitive to both environmental and genetic factors. Here, we developed a high‐throughput phenotyping approach to assay the variation in pupation height in Drosophila melanogaster, while controlling for possibly confounding factors. We find substantial variation of mean pupation height among sampled natural stocks and we show that the Drosophila Genetic Reference Panel (DGRP) reflects this variation. Using the DGRP stocks for genome‐wide association (GWA) mapping, 16 loci involved in determining pupation height could be resolved. The candidate genes in these loci are enriched for high expression in the larval central nervous system. A genetic network could be constructed from the candidate loci, which places scribble (scrib) at the centre, plus other genes known to be involved in nervous system development, such as Epidermal growth factor receptor (Egfr) and p53. Using gene disruption lines, we could functionally validate several of the initially identified loci, as well as additional loci predicted from network analysis. Our study shows that the combination of high‐throughput phenotyping with a genetic analysis of variation captured from the wild can be used to approach the genetic dissection of an environmentally relevant behavioural phenotype.     

Dietary choices are influenced by genotype,mating status,and sex in Drosophila melanogaster

Mating causes many changes in physiology, behavior, and gene expression in a wide range of organisms. These changes are predicted to be sex specific, influenced by the divergent reproductive roles of the sexes. In female insects, mating is associated with an increase in egg production which requires high levels of nutritional input with direct consequences for the physiological needs of individual females. Consequently, females alter their nutritional acquisition in line with the physiological demands imposed by mating. Although much is known about the female mating‐induced nutritional response, far less is known about changes in males. In addition, it is unknown whether variation between genotypes translates into variation in dietary behavioral responses. Here we examine mating‐induced shifts in male and female dietary preferences across genotypes of Drosophila melanogaster. We find sex‐ and genotype‐specific effects on both the quantity and quality of the chosen diet. These results contribute to our understanding of sex‐specific metabolism and reveal genotypic variation that influences responses to physiological demands.     

Extensive paternal mtDNA leakage in natural populations of Drosophila melanogaster

Strict maternal inheritance is considered a hallmark of animal mtDNA. Although recent reports suggest that paternal leakage occurs in a broad range of species, it is still considered an exceptionally rare event. To evaluate the impact of paternal leakage on the evolution of mtDNA, it is essential to reliably estimate the frequency of paternal leakage in natural populations. Using allele‐specific real‐time quantitative PCR (RT‐qPCR), we show that heteroplasmy is common in natural populations with at least 14% of the individuals carrying multiple mitochondrial haplotypes. However, the average frequency of the minor mtDNA haplotype is low (0.8%), which suggests that this pervasive heteroplasmy has not been noticed before due to a lack of power in sequencing surveys. Based on the distribution of mtDNA haplotypes in the offspring of heteroplasmic mothers, we found no evidence for strong selection against one of the haplotypes. We estimated that the rate of paternal leakage is 6% and that at least 100 generations are required for complete sorting of mtDNA haplotypes. Despite the high proportion of heteroplasmic individuals in natural populations, we found no evidence for recombination between mtDNA molecules, suggesting that either recombination is rare or recombinant haplotypes are counter‐selected. Our results indicate that evolutionary studies using mtDNA as a marker might be biased by paternal leakage in this species.     

Reproductive and post‐reproductive life history of wild‐caught Drosophila melanogaster under laboratory conditions

The life history of the fruit fly (Drosophila melanogaster) is well understood, but fitness components are rarely measured by following single individuals over their lifetime, thereby limiting insights into lifetime reproductive success, reproductive senescence and post‐reproductive lifespan. Moreover, most studies have examined long‐established laboratory strains rather than freshly caught individuals and may thus be confounded by adaptation to laboratory culture, inbreeding or mutation accumulation. Here, we have followed the life histories of individual females from three recently caught, non‐laboratory‐adapted wild populations of D. melanogaster. Populations varied in a number of life‐history traits, including ovariole number, fecundity, hatchability and lifespan. To describe individual patterns of age‐specific fecundity, we developed a new model that allowed us to distinguish four phases during a female's life: a phase of reproductive maturation, followed by a period of linear and then exponential decline in fecundity and, finally, a post‐ovipository period. Individual females exhibited clear‐cut fecundity peaks, which contrasts with previous analyses, and post‐peak levels of fecundity declined independently of how long females lived. Notably, females had a pronounced post‐reproductive lifespan, which on average made up 40% of total lifespan. Post‐reproductive lifespan did not differ among populations and was not correlated with reproductive fitness components, supporting the hypothesis that this period is a highly variable, random ‘add‐on’ at the end of reproductive life rather than a correlate of selection on reproductive fitness. Most life‐history traits were positively correlated, a pattern that might be due to genotype by environment interactions when wild flies are brought into a novel laboratory environment but that is unlikely explained by inbreeding or positive mutational covariance caused by mutation accumulation.     

Population genomic analysis uncovers African and European admixture in Drosophila melanogaster populations from the south‐eastern United States and Caribbean Islands

Drosophila melanogaster is postulated to have colonized North America in the past several 100 years in two waves. Flies from Europe colonized the east coast United States while flies from Africa inhabited the Caribbean, which if true, make the south‐east US and Caribbean Islands a secondary contact zone for African and European D. melanogaster. This scenario has been proposed based on phenotypes and limited genetic data. In our study, we have sequenced individual whole genomes of flies from populations in the south‐east US and Caribbean Islands and examined these populations in conjunction with population sequences from the west coast US, Africa, and Europe. We find that west coast US populations are closely related to the European population, likely reflecting a rapid westward expansion upon first settlements into North America. We also find genomic evidence of African and European admixture in south‐east US and Caribbean populations, with a clinal pattern of decreasing proportions of African ancestry with higher latitude. Our genomic analysis of D. melanogaster populations from the south‐east US and Caribbean Islands provides more evidence for the Caribbean Islands as the source of previously reported novel African alleles found in other east coast US populations. We also find the border between the south‐east US and the Caribbean island to be the admixture hot zone where distinctly African‐like Caribbean flies become genomically more similar to European‐like south‐east US flies. Our findings have important implications for previous studies examining the generation of east coast US clines via selection.     

Phenotypic plasticity,but not adaptive tracking,underlies seasonal variation in post‐cold hardening freeze tolerance of Drosophila melanogaster

In temperate regions, an organism's ability to rapidly adapt to seasonally varying environments is essential for its survival. In response to seasonal changes in selection pressure caused by variation in temperature, humidity, and food availability, some organisms exhibit plastic changes in phenotype. In other cases, seasonal variation in selection pressure can rapidly increase the frequency of genotypes that offer survival or reproductive advantages under the current conditions. Little is known about the relative influences of plastic and genetic changes in short‐lived organisms experiencing seasonal environmental fluctuations. Cold hardening is a seasonally relevant plastic response in which exposure to cool, but nonlethal, temperatures significantly increases the organism's ability to later survive at freezing temperatures. In the present study, we demonstrate seasonal variation in cold hardening in Drosophila melanogaster and test the extent to which plasticity and adaptive tracking underlie that seasonal variation. We measured the post‐cold hardening freeze tolerance of flies from outdoor mesocosms over the summer, fall, and winter. We bred outdoor mesocosm‐caught flies for two generations in the laboratory and matched each outdoor cohort to an indoor control cohort of similar genetic background. We cold hardened all flies under controlled laboratory conditions and then measured their post‐cold hardening freeze tolerance. Comparing indoor and field‐caught flies and their laboratory‐reared G1 and G2 progeny allowed us to determine the roles of seasonal environmental plasticity, parental effects, and genetic changes on cold hardening. We also tested the relationship between cold hardening and other factors, including age, developmental density, food substrate, presence of antimicrobials, and supplementation with live yeast. We found strong plastic responses to a variety of field‐ and laboratory‐based environmental effects, but no evidence of seasonally varying parental or genetic effects on cold hardening. We therefore conclude that seasonal variation in post‐cold hardening freeze tolerance results from environmental influences and not genetic changes.     

Inbreeding reveals mode of past selection on male reproductive characters in Drosophila melanogaster

Directional dominance is a prerequisite of inbreeding depression. Directionality arises when selection drives alleles that increase fitness to fixation and eliminates dominant deleterious alleles, while deleterious recessives are hidden from it and maintained at low frequencies. Traits under directional selection (i.e., fitness traits) are expected to show directional dominance and therefore an increased susceptibility to inbreeding depression. In contrast, traits under stabilizing selection or weakly linked to fitness are predicted to exhibit little‐to‐no inbreeding depression. Here, we quantify the extent of inbreeding depression in a range of male reproductive characters and then infer the mode of past selection on them. The use of transgenic populations of Drosophila melanogaster with red or green fluorescent‐tagged sperm heads permitted in vivo discrimination of sperm from competing males and quantification of characteristics of ejaculate composition, performance, and fate. We found that male attractiveness (mating latency) and competitive fertilization success (P₂) both show some inbreeding depression, suggesting they may have been under directional selection, whereas sperm length showed no inbreeding depression suggesting a history of stabilizing selection. However, despite having measured several sperm quality and quantity traits, our data did not allow us to discern the mechanism underlying the lowered competitive fertilization success of inbred (f = 0.50) males.     

Effects of larval crowding on quantitative variation for development time and viability in Drosophila melanogaster

Competition between individuals belonging to the same species is a universal feature of natural populations and is the process underpinning organismal adaptation. Despite its importance, still comparatively little is known about the genetic variation responsible for competitive traits. Here, we measured the phenotypic variation and quantitative genetics parameters for two fitness‐related traits—egg‐to‐adult viability and development time—across a panel of Drosophila strains under varying larval densities. Both traits exhibited substantial genetic variation at all larval densities, as well as significant genotype‐by‐environment interactions (GEIs). GEI was attributable to changes in the rank order of reaction norms for both traits, and additionally to differences in the between‐line variance for development time. The coefficient of genetic variation increased under stress conditions for development time, while it was higher at both high and low densities for viability. While development time also correlated negatively with fitness at high larval densities—meaning that fast developers have high fitness—there was no correlation with fitness at low density. This result suggests that GEI may be a common feature of fitness‐related genetic variation and, further, that trait values under noncompetitive conditions could be poor indicators of individual fitness. The latter point could have significant implications for animal and plant breeding programs, as well as for conservation genetics.     

Variation in male effects on female fecundity in Drosophila melanogaster

In many species, males have the capacity to directly influence (either positively or negatively) the fitness of their mates and offspring, not only via parental care contributions and/or precopulatory resource provisioning, but also via the post‐copulatory activity of those substances passed on to their mates in their ejaculates. Here, we examine how an individual male's identity may be related to phenotypic variation in short‐term female fecundity in the model species, Drosophila melanogaster. The effect of male identity on short‐term fecundity stimulation of females was repeatable across time and accounted for over a fifth of the total observed phenotypic variation in fecundity in two independent populations. The functional explanations for these results and the implications for our understanding of the factors that contribute to the adaptive significance of mating preferences and/or sexual conflict are discussed.     

Aging impairs double‐strand break repair by homologous recombination in Drosophila germ cells

Aging is characterized by genome instability, which contributes to cancer formation and cell lethality leading to organismal decline. The high levels of DNA double‐strand breaks (DSBs) observed in old cells and premature aging syndromes are likely a primary source of genome instability, but the underlying cause of their formation is still unclear. DSBs might result from higher levels of damage or repair defects emerging with advancing age, but repair pathways in old organisms are still poorly understood. Here, we show that premeiotic germline cells of young and old flies have distinct differences in their ability to repair DSBs by the error‐free pathway homologous recombination (HR). Repair of DSBs induced by either ionizing radiation (IR) or the endonuclease I‐SceI is markedly defective in older flies. This correlates with a remarkable reduction in HR repair measured with the DR‐white DSB repair reporter assay. Strikingly, most of this repair defect is already present at 8 days of age. Finally, HR defects correlate with increased expression of early HR components and increased recruitment of Rad51 to damage in older organisms. Thus, we propose that the defect in the HR pathway for germ cells in older flies occurs following Rad51 recruitment. These data reveal that DSB repair defects arise early in the aging process and suggest that HR deficiencies are a leading cause of genome instability in germ cells of older animals.     

Effects of age,sex, and genotype on high‐sensitivity metabolomic profiles in the fruit fly,Drosophila melanogaster

Researchers have used whole‐genome sequencing and gene expression profiling to identify genes associated with age, in the hope of understanding the underlying mechanisms of senescence. But there is a substantial gap from variation in gene sequences and expression levels to variation in age or life expectancy. In an attempt to bridge this gap, here we describe the effects of age, sex, genotype, and their interactions on high‐sensitivity metabolomic profiles in the fruit fly, Drosophila melanogaster. Among the 6800 features analyzed, we found that over one‐quarter of all metabolites were significantly associated with age, sex, genotype, or their interactions, and multivariate analysis shows that individual metabolomic profiles are highly predictive of these traits. Using a metabolomic equivalent of gene set enrichment analysis, we identified numerous metabolic pathways that were enriched among metabolites associated with age, sex, and genotype, including pathways involving sugar and glycerophospholipid metabolism, neurotransmitters, amino acids, and the carnitine shuttle. Our results suggest that high‐sensitivity metabolomic studies have excellent potential not only to reveal mechanisms that lead to senescence, but also to help us understand differences in patterns of aging among genotypes and between males and females.