Daily increasing or decreasing photoperiod affects stress resistance and life history traits in four Drosophila species.

Photoperiod is one of the most reliable seasonal cues that organisms can use to prepare for upcoming environmental changes. Evidence suggests that exposure to different photoperiod can activate plastic responses in stress resistance traits, while there is limited evidence on the plastic response induced by daily progressive cumulative changes in photoperiod. In this study, we assayed the effect of within generation daily uni-directional and cumulative changes in photoperiod on stress resistance and life history traits in four Drosophila species. We predicted that daily increasing photoperiod, mimicking upcoming summer conditions, should lead to an increase in heat resistance and establish trade-offs with other fitness related traits. On the other hand, we predicted that daily decreasing photoperiod should reflect upcoming winter conditions leading to an increase in cold resistance. We found that within genreation changes in photoperiod had a significant effect on life history and stress resistance traits in the four Drosophila species. The observed response was different across species, with D. melanogaster showing five out of six studied traits affected, while in D. mercatorum only one trait was significantly affected. The exposure to changing photoperiod led to an increased upper thermal resistance in D. melanogaster and D. mercatorum and a decreased lower thermal resistance in D. melanogaster and D. simulans, as well as a decreased starvation and desiccation resistance in D. virilis. The developmental time was shorter when flies were exposed to the two photoperiod regimes compared to constant day length control in D. melanogaster and D. simulans. A limited effect was observed on egg-to-adult-viability and desiccation resistance. The results of this study show that daily change in photoperiod induced a plastic response in different traits of drosophilids, suggesting that this environmental parameter needs to be carefully considered in evolutionary studies.

Satyrization in Drosophila fruitflies.

The satyr of Greek mythology was half-man, half-goat, with an animal persona signifying immoderate sexual appetites. In biology, satyrization is the disruption of reproduction in matings between closely related species. Interestingly, its effects are often reciprocally asymmetric, manifesting more strongly in one direction of heterospecific mating than the other. Heterospecific matings are well known to result in female fitness costs due to the production of sterile or inviable hybrid offspring and can also occur due to reduced female sexual receptivity, lowering the likelihood of any subsequent conspecific matings. Here we investigated the costs and mechanisms of satyrization in the Drosophila melanogaster species subgroup of fruitflies. The results showed that D. simulans females experienced higher fitness costs from a loss of remating opportunities due to significantly reduced post-mating sexual receptivity than did D. melanogaster females, as a result of reciprocal heterospecific matings. Reciprocal tests of the effects of male reproductive accessory gland protein (Acp) injections on female receptivity in pairwise comparisons between D. melanogaster and five other species within the melanogaster species subgroup revealed significant post-mating receptivity asymmetries. This was due to variation in the effects of heterospecific Acps within species with which D. melanogaster can mate, and significant but nonasymmetric Acp effects in species with which it cannot. We conclude that asymmetric satyrization due to post-mating effects of Acps may be common among diverging and hybridising species. The findings are of interest in understanding the evolution of reproductive isolation and species divergence.

Phenotype-dependent habitat choice is too weak to cause assortative mating between Drosophila melanogaster strains differing in light sensitivity.

Matching habitat choice is gaining attention as a mechanism for maintaining biodiversity and driving speciation. It revolves around the idea that individuals select the habitat in which they perceive to obtain greater fitness based on a prior evaluation of their local performance across heterogeneous environments. This results in individuals with similar ecologically relevant traits converging to the same patches, and hence it could indirectly cause assortative mating when mating occurs in those patches. White-eyed mutants of Drosophila fruit flies have a series of disadvantages compared to wild type flies, including a poorer performance under bright light. It has been previously reported that, when given a choice, wild type Drosophila simulans preferred a brightly lit habitat while white-eyed mutants occupied a dimly lit one. This spatial segregation allowed the eye color polymorphism to be maintained for several generations, whereas normally it is quickly replaced by the wild type. Here we compare the habitat choice decisions of white-eyed and wild type flies in another species, D. melanogaster. We released groups of flies in a light gradient and recorded their departure and settlement behavior. Departure depended on sex and phenotype, but not on the light conditions of the release point. Settlement depended on sex, and on the interaction between phenotype and light conditions of the point of settlement. Nonetheless, simulations showed that this differential habitat use by the phenotypes would only cause a minimal degree of assortative mating in this species.

Offspring sex ratios are stable across the life course in Drosophila simulans.

Within populations, adult sex ratios influence population growth and extinction risk, mating behaviours and parental care. Sex ratio adjustment can also have pronounced effects on individual fitness. Accordingly, it is important that we understand how often, and why, offspring sex ratios deviate from parity. In Drosophila melanogaster, females appear to improve their fitness by producing fewer sons when paired with older males. However, facultative sex ratio adjustment in D. melanogaster is controversial, and our understanding of how sex ratio skew affects fitness is hampered by pronounced sexual conflict in this species. Additionally, it is unclear whether maternal age or quality interacts with paternal age to influence offspring sex ratios. Here, we test whether offspring sex ratios vary as a function of maternal quality, and maternal and paternal age in Drosophila simulans, a sister species of D. melanogaster that lacks overt sexual conflict. We find that offspring sex ratios are slightly male-biased overall, but constant across the female life course, and independent of female quality, or paternal age. To really understand if, how and when females skew offspring sex ratios, we need studies linking offspring sex ratios to paternal and maternal phenotypes that are predicted to shift optimal investment in sons and daughters.

Does Divergence in Habitat Breadth Associate with Species Differences in Decision Making in Drosophila Sechellia and Drosophila Simulans?

Decision making is involved in many behaviors contributing to fitness, such as habitat choice, mate selection, and foraging. Because of this, high decision-making accuracy (i.e., selecting the option most beneficial for fitness) should be under strong selection. However, decision making is energetically costly, often involving substantial time and energy to survey the environment to obtain high-quality information. Thus, for high decision making accuracy to evolve, its benefits should outweigh its costs. Inconsistency in the net benefits of decision making across environments is hypothesized to be an important means for maintaining variation in this trait. However, very little is known about how environmental factors influence the evolution of decision making to produce variation among individuals, genotypes, and species. Here, we compared two recently diverged species of Drosophila differing substantially in habitat breadth and degree of environmental predictability and variability: Drosophilasechellia and Drosophilasimulans. We found that the species evolving under higher environmental unpredictability and variability showed higher decision-making accuracy, but not higher environmental sampling.

Olfactory receptor and circuit evolution promote host specialization.

The evolution of animal behaviour is poorly understood1,2. Despite numerous correlations between interspecific divergence in behaviour and nervous system structure and function, demonstrations of the genetic basis of these behavioural differences remain rare3-5. Here we develop a neurogenetic model, Drosophila sechellia, a species that displays marked differences in behaviour compared to its close cousin Drosophila melanogaster6,7, which are linked to its extreme specialization on noni fruit (Morinda citrifolia)8-16. Using calcium imaging, we identify olfactory pathways in D. sechellia that detect volatiles emitted by the noni host. Our mutational analysis indicates roles for different olfactory receptors in long- and short-range attraction to noni, and our cross-species allele-transfer experiments demonstrate that the tuning of one of these receptors is important for species-specific host-seeking. We identify the molecular determinants of this functional change, and characterize their evolutionary origin and behavioural importance. We perform circuit tracing in the D. sechellia brain, and find that receptor adaptations are accompanied by increased sensory pooling onto interneurons as well as species-specific central projection patterns. This work reveals an accumulation of molecular, physiological and anatomical traits that are linked to behavioural divergence between species, and defines a model for investigating speciation and the evolution of the nervous system.

The Loci of Behavioral Evolution: Evidence That Fas2 and tilB Underlie Differences in Pupation Site Choice Behavior between Drosophila melanogaster and D. simulans.

The behaviors of closely related species can be remarkably different, and these differences have important ecological and evolutionary consequences. Although the recent boom in genotype-phenotype studies has led to a greater understanding of the genetic architecture and evolution of a variety of traits, studies identifying the genetic basis of behaviors are, comparatively, still lacking. This is likely because they are complex and environmentally sensitive phenotypes, making them difficult to measure reliably for association studies. The Drosophila species complex holds promise for addressing these challenges, as the behaviors of closely related species can be readily assayed in a common environment. Here, we investigate the genetic basis of an evolved behavioral difference, pupation site choice, between Drosophila melanogaster and D. simulans. In this study, we demonstrate a significant contribution of the X chromosome to the difference in pupation site choice behavior between these species. Using a panel of X-chromosome deficiencies, we screened the majority of the X chromosome for causal loci and identified two regions associated with this X-effect. We then collect gene disruption and RNAi data supporting a single gene that affects pupation behavior within each region: Fas2 and tilB. Finally, we show that differences in tilB expression correlate with the differences in pupation site choice behavior between species. This evidence associating two genes with differences in a complex, environmentally sensitive behavior represents the first step toward a functional and evolutionary understanding of this behavioral divergence.

Complex interactions between sperm viability and female fertility.

Sperm viability is a major male fitness component, with higher sperm viability associated with enhanced sperm competitiveness. While many studies have focussed on sperm viability from the male fitness standpoint, its impact on female fitness is less clear. Here we used a panel of 32 isogenic Drosophila simulans lines to test for genetic variation in sperm viability (percentage of viable cells). We then tested whether sperm viability affected female fitness by mating females to males from low or high sperm viability genotypes. We found significant variation in sperm viability among genotypes, and consistent with this, sperm viability was highly repeatable within genotypes. Additionally, females mated to high sperm viability males laid more eggs in the first seven hours after mating, and produced more offspring in total. However, the early increase in oviposition did not result in more offspring in the 8 hours following mating, suggesting that mating with high sperm-viability genotypes leads to egg wastage for females shortly after copulation. Although mating with high sperm-viability males resulted in higher female fitness in the long term, high quality ejaculates would result in a short-term female fitness penalty, or at least lower realised fitness, potentially generating sexual conflict over optimal sperm viability.

Sexual selection drives the evolution of male wing interference patterns.

The seemingly transparent wings of many insects have recently been found to display unexpected structural coloration. These structural colours (wing interference patterns: WIPs) may be involved in species recognition and mate choice, yet little is known about the evolutionary processes that shape them. Furthermore, to date investigations of WIPs have not fully considered how they are actually perceived by the viewers' colour vision. Here, we use multispectral digital imaging and a model of Drosophila vision to compare WIPs of male and female Drosophila simulans from replicate populations forced to evolve with or without sexual selection for 68 generations. We show that WIPs modelled in Drosophila vision evolve in response to sexual selection and provide evidence that WIPs correlate with male sexual attractiveness. These findings add a new element to the otherwise well-described Drosophila courtship display and confirm that wing colours evolve through sexual selection.

Evolution of Mechanisms that Control Mating in Drosophila Males.

Genetically wired neural mechanisms inhibit mating between species because even naive animals rarely mate with other species. These mechanisms can evolve through changes in expression or function of key genes in sensory pathways or central circuits. Gr32a is a gustatory chemoreceptor that, in D. melanogaster, is essential to inhibit interspecies courtship and sense quinine. Similar to D. melanogaster, we find that D. simulans Gr32a is expressed in foreleg tarsi, sensorimotor appendages that inhibit interspecies courtship, and it is required to sense quinine. Nevertheless, Gr32a is not required to inhibit interspecies mating by D. simulans males. However, and similar to its function in D. melanogaster, Ppk25, a member of the Pickpocket family, promotes conspecific courtship in D. simulans. Together, we have identified distinct evolutionary mechanisms underlying chemosensory control of taste and courtship in closely related Drosophila species.

Genetic redundancy fuels polygenic adaptation in Drosophila.

The genetic architecture of adaptive traits is of key importance to predict evolutionary responses. Most adaptive traits are polygenic-i.e., result from selection on a large number of genetic loci-but most molecularly characterized traits have a simple genetic basis. This discrepancy is best explained by the difficulty in detecting small allele frequency changes (AFCs) across many contributing loci. To resolve this, we use laboratory natural selection to detect signatures for selective sweeps and polygenic adaptation. We exposed 10 replicates of a Drosophila simulans population to a new temperature regime and uncovered a polygenic architecture of an adaptive trait with high genetic redundancy among beneficial alleles. We observed convergent responses for several phenotypes-e.g., fitness, metabolic rate, and fat content-and a strong polygenic response (99 selected alleles; mean s = 0.059). However, each of these selected alleles increased in frequency only in a subset of the evolving replicates. We discerned different evolutionary paradigms based on the heterogeneous genomic patterns among replicates. Redundancy and quantitative trait (QT) paradigms fitted the experimental data better than simulations assuming independent selective sweeps. Our results show that natural D. simulans populations harbor a vast reservoir of adaptive variation facilitating rapid evolutionary responses using multiple alternative genetic pathways converging at a new phenotypic optimum. This key property of beneficial alleles requires the modification of testing strategies in natural populations beyond the search for convergence on the molecular level.

The genetic basis of female pheromone differences between Drosophila melanogaster and D. simulans.

Chemical signals are one means by which many insect species communicate. Differences in the combination of surface chemicals called cuticular hydrocarbons (CHCs) can influence mating behavior and affect reproductive isolation between species. Genes influencing three CHC compounds have been identified in Drosophila melanogaster. However, the genetic basis of other CHC compounds, whether these genes affect species differences in CHCs, and the genes' resulting effect on interspecies mating, remains unknown. We used fine-scale deficiency mapping of the third chromosome to identify 43 genomic regions that influence production of CHCs in both D. melanogaster and Drosophila simulans females. We identified an additional 23 small genomic regions that affect interspecies divergence in CHCs between females of these two species, one of which spans two genes known to influence the production of multiple CHCs within D. melanogaster. By testing these genes individually, we determined that desat1 also affects interspecific divergence in one CHC compound, while desat2 has no effect on interspecific divergence. Thus, some but not all genes affecting intraspecific amounts of CHCs also affect interspecific divergence, but not all genes or all CHCs. Lastly, we find no evidence of a relationship between the CHC profile and female attractiveness or receptivity towards D. melanogaster males.

Wolbachia infection may improve learning and memory capacity of Drosophila by altering host gene expression through microRNA.

Wolbachia are endosymbiotic bacteria present in a wide range of invertebrates. Although their dramatic effects on host reproductive biology have been well studied, little is known about the effects of Wolbachia on the learning and memory capacity (LMC) of hosts, despite their distribution in the host nervous system, including brain. In this study, we found that Wolbachia infection significantly enhanced LMC in both Drosophila melanogaster and D. simulans. Expression of LMC-related genes was significantly increased in the head of D. melanogaster infected with the wMel strain, and among these genes, crebA was up-regulated the most. Knockdown of crebA in Wolbachia-infected flies significantly decreased LMC, while overexpression of crebA in Wolbachia-free flies significantly enhanced the LMC of flies. More importantly, a microRNA (miRNA), dme-miR-92b, was identified to be complementary to the 3'UTR of crebA. Wolbachia infection was correlated with reduced expression of dme-miR-92b in D. melanogaster, and dme-miR-92b negatively regulated crebA through binding to its 3'UTR region. Overexpression of dme-miR-92b in Wolbachia-infected flies by microinjection of agomirs caused a significant decrease in crebA expression and LMC, while inhibition of dme-miR-92b in Wolbachia-free flies by microinjection of antagomirs resulted in a significant increase in crebA expression and LMC. These results suggest that Wolbachia may improve LMC in Drosophila by altering host gene expression through a miRNA-target pathway. Our findings help better understand the host-endosymbiont interactions and, in particular, the impact of Wolbachia on cognitive processes in invertebrate hosts.

Tissue-Specific cis-Regulatory Divergence Implicates eloF in Inhibiting Interspecies Mating in Drosophila.

Reproductive isolation is a key component of speciation. In many insects, a major driver of this isolation is cuticular hydrocarbon pheromones, which help to identify potential intraspecific mates [1-3]. When the distributions of related species overlap, there may be strong selection on mate choice for intraspecific partners [4-9] because interspecific hybridization carries significant fitness costs [10]. Drosophila has been a key model for the investigation of reproductive isolation; although both male and female mate choices have been extensively investigated [6, 11-16], the genes underlying species recognition remain largely unknown. To explore the molecular mechanisms underlying Drosophila speciation, we measured tissue-specific cis-regulatory divergence using RNA sequencing (RNA-seq) in D. simulans × D. sechellia hybrids. By focusing on cis-regulatory changes specific to female oenocytes, the tissue that produces cuticular hydrocarbons, we rapidly identified a small number of candidate genes. We found that one of these, the fatty acid elongase eloF, broadly affects the hydrocarbons present on D. sechellia and D. melanogaster females, as well as the propensity of D. simulans males to mate with them. Therefore, cis-regulatory changes in eloF may be a major driver in the sexual isolation of D. simulans from multiple other species. Our RNA-seq approach proved to be far more efficient than quantitative trait locus (QTL) mapping in identifying candidate genes; the same framework can be used to pinpoint candidate drivers of cis-regulatory divergence in traits differing between any interfertile species.

Evolution of a central neural circuit underlies Drosophila mate preferences.

Courtship rituals serve to reinforce reproductive barriers between closely related species. Drosophila melanogaster and Drosophila simulans exhibit reproductive isolation, owing in part to the fact that D. melanogaster females produce 7,11-heptacosadiene, a pheromone that promotes courtship in D. melanogaster males but suppresses courtship in D. simulans males. Here we compare pheromone-processing pathways in D. melanogaster and D. simulans males to define how these sister species endow 7,11-heptacosadiene with the opposite behavioural valence to underlie species discrimination. We show that males of both species detect 7,11-heptacosadiene using homologous peripheral sensory neurons, but this signal is differentially propagated to P1 neurons, which control courtship behaviour. A change in the balance of excitation and inhibition onto courtship-promoting neurons transforms an excitatory pheromonal cue in D. melanogaster into an inhibitory cue in D. simulans. Our results reveal how species-specific pheromone responses can emerge from conservation of peripheral detection mechanisms and diversification of central circuitry, and demonstrate how flexible nodes in neural circuits can contribute to behavioural evolution.

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.

Redefining reproductive dormancy in Drosophila as a general stress response to cold temperatures.

Organisms regularly encounter unfavorable conditions and the genetic adaptations facilitating survival have been of long-standing interest to evolutionary biologists. Winter is one particularly stressful condition for insects, during which they encounter low temperatures and scarcity of food. Despite dormancy being a well-studied adaptation to facilitate overwintering, there is still considerable controversy about the distribution of dormancy among natural populations and between species in Drosophila. The current definition of dormancy as developmental arrest of oogenesis at the previtellogenic stage (stage 7) distinguishes dormancy from general stress related block of oogenesis at early vitellogenic stages (stages 8 - 9). In an attempt to resolve this, we scrutinized reproductive dormancy in D. melanogaster and D. simulans. We show that dormancy shows the same hallmarks of arrest of oogenesis at stage 9, as described for other stressors and propose a new classification for dormancy. Applying this modified classification, we show that both species express dormancy in cosmopolitan and African populations, further supporting that dormancy uses an ancestral pathway induced by environmental stress. While we found significant differences between individuals and the two Drosophila species in their sensitivity to cold temperature stress, we also noted that extreme temperature stress (8 °C) resulted in very strong dormancy incidence, which strongly reduced the differences seen at less extreme temperatures. We conclude that dormancy in Drosophila should not be considered a special trait, but is better understood as a generic stress response occurring at low temperatures.

Constitutive up-regulation of Turandot genes rather than changes in acclimation ability is associated with the evolutionary adaptation to temperature fluctuations in Drosophila simulans.

Most research on thermal adaptation of ectotherms is based on experiments performed at constant temperatures. However, for short-lived insects daily fluctuations of temperature could be an important environmental parameter involved in evolutionary adaptation to thermal heterogeneity. In this study we investigated the mechanisms underlying evolutionary adaptation to daily fluctuating temperatures. We studied replicated selection lines of Drosophila simulans evolved in a constant or a daily fluctuating thermal regime. Previous studies of these lines have shown clear acclimation benefits to heat tolerance induced by the fluctuating regime. First, we tested the existence of an evolved circadian controlled adjustment of heat resistance in selected flies. This was done by investigating the daily variation in time to heat knockdown in flies from both selection regimes when exposed to either a constant or a daily fluctuating thermal regime for a single generation. While daily variation in heat resistance was found, the results suggest that there was neither an evolved adaptive circadian controlled adjustment of heat resistance nor a continuous acclimation response induced by fluctuating temperatures in these lines. Second, in order to reveal functional candidates for adaptation to the fluctuating thermal regime, we investigated the global transcriptomic response to a high temperature exposure in flies from both regimes. We found that flies selected both in constant and fluctuating thermal regimes responded similarly to increasing temperature. However, we found that evolutionary adaptation to the fluctuating thermal regime led to transcriptional enrichment of the GO terms eggshell chorion assembly and cellular response to heat. The latter category was constituted by a constitutive up-regulation of four Turandot genes and not heat shock protein genes, suggesting that Turandot genes could play a prominent role for adaptation to daily fluctuating thermal conditions.

Is diapause an ancient adaptation in Drosophila?

D. melanogaster enters a state of reproductive arrest when exposed to low temperatures (12°C) and shorter photoperiods. A number of studies have suggested that diapause has recently evolved in European D. melanogaster populations, that it is not present in the sibling species D. simulans, that it is non-photoperiodic in American D. melanogaster populations, and that it spontaneously terminates after 6-8weeks. We have studied the overwintering phenotype under different conditions and observe that American, European and, surprisingly, African D. melanogaster populations can show photoperiodic diapause, as can European, but not African D. simulans. Surprisingly other Drosophila species from pan-tropical regions can also show significant levels of photoperiodic diapause. We observe that spontaneous termination of diapause after a few weeks can be largely avoided with a more realistic winter simulation for D. melanogaster, but not D. simulans. Examining metabolite accumulation during diapause reveals that the shallow diapause of D. melanogaster has similar features to that of other more robustly-diapausing species. Our results suggest that diapause may be an ancient character that emerged in the tropics to resist unfavourable seasonal conditions and which has been enhanced during D. melanogaster's colonisation of temperate regions. Our results also highlight how different methodologies to quantify diapause can lead to apparently conflicting results that we believe can now largely be resolved.

Differences in larval nutritional requirements and female oviposition preference reflect the order of fruit colonization of Zaprionus indianus and Drosophila simulans.

Species coexist using the same nutritional resource by partitioning it either in space or time, but few studies explore how species-specific nutritional requirements allow partitioning. Zaprionus indianus and Drosophila simulans co-exist in figs by invading the fruit at different stages; Z. indianus colonizes ripe figs, whereas D. simulans oviposits in decaying fruit. Larvae feed on yeast growing on the fruit, which serves as their primary protein source. Because yeast populations increase as fruit decays, we find that ripe fruit has lower protein content than rotting fruit. Therefore, we hypothesized that Z. indianus and D. simulans larvae differ in their dietary requirements for protein. We used nutritional geometry to assess the effects of protein and carbohydrate concentration in the larval diet on life history characters in both species. Survival, development time, and ovariole number respond differently to the composition of the larval diet, with Z. indianus generally performing better across a wider range of protein concentrations. Correspondingly, we found that Z. indianus females preferred to lay eggs on low protein foods, while D. simulans females chose higher protein foods for oviposition when competing with Z. indianus. We propose the different nutritional requirements and oviposition preference of these two species allows them to temporally partition their habitat.