Genetic basis and repeatability for desiccation resistance in Drosophila melanogaster (Diptera: Drosophilidae).

Dehydration is a stress factor for organisms inhabiting natural habitats where water is scarce. Thus, it may be expected that species facing arid environments will develop mechanisms that maximize resistance to desiccation. Insects are excellent models for studying the effects of dehydration as well as the mechanisms and processes that prevent water loss since the effect of desiccation is greater due to the higher area/volume ratio than larger animals. Even though physiological and behavioral mechanisms to cope with desiccation are being understood, the genetic basis underlying the mechanisms related to variation in desiccation resistance and the context-dependent effect remain unsolved. Here we analyze the genetic bases of desiccation resistance in Drosophila melanogaster and identify candidate genes that underlie trait variation. Our quantitative genetic analysis of desiccation resistance revealed sexual dimorphism and extensive genetic variation. The phenotype-genotype association analyses (GWAS) identified 71 candidate genes responsible for total phenotypic variation in desiccation resistance. Half of these candidate genes were sex-specific suggesting that the genetic architecture underlying this adaptive trait differs between males and females. Moreover, the public availability of desiccation data analyzed on the same lines but in a different lab allows us to investigate the reliability and repeatability of results obtained in independent screens. Our survey indicates a pervasive micro-environment lab-dependent effect since we did not detect overlap in the sets of genes affecting desiccation resistance identified between labs.

Concerted evolution of metabolic rate, economics of mating, ecology, and pace of life across seed beetles.

Male-female coevolution has taken different paths among closely related species, but our understanding of the factors that govern its direction is limited. While it is clear that ecological factors, life history, and the economics of reproduction are connected, the divergent links are often obscure. We propose that a complete understanding requires the conceptual integration of metabolic phenotypes. Metabolic rate, a nexus of life history evolution, is constrained by ecological factors and may exert important direct and indirect effects on the evolution of sexual dimorphism. We performed standardized experiments in 12 seed beetle species to gain a rich set of sex-specific measures of metabolic phenotypes, life history traits, and the economics of mating and analyzed our multivariate data using phylogenetic comparative methods. Resting metabolic rate (RMR) showed extensive evolution and evolved more rapidly in males than in females. The evolution of RMR was tightly coupled with a suite of life history traits, describing a pace-of-life syndrome (POLS), with indirect effects on the economics of mating. As predicted, high resource competition was associated with a low RMR and a slow POLS. The cost of mating showed sexually antagonistic coevolution, a hallmark of sexual conflict. The sex-specific costs and benefits of mating were predictably related to ecology, primarily through the evolution of male ejaculate size. Overall, our results support the tenet that resource competition affects metabolic processes that, in turn, have predictable effects on both life history evolution and reproduction, such that ecology shows both direct and indirect effects on male-female coevolution.

Superinfection exclusion studies using West Nile virus and Culex flavivirus strains from Argentina.

In Argentina, many Flavivirus were recognised including West Nile virus (WNV). During 2009 several strains of Culex Flavivirus (CxFV), an insect-specific flavivirus, were isolated in the same region where circulation of WNV was detected. Hence, the objective of this study was to analyse the effect of co-infection in vitro assays using CxFV and WNV Argentinean strains in order to evaluate if CxFV could affect WNV replication. Our results showed that WNV replication was suppressed when multiplicity of infection (MOI) for CxFV was 10 or 100 times higher than WNV. Nevertheless, in vivo assays are necessary in order to evaluate the superinfection exclusion potential.

Superinfection exclusion studies using West Nile virus and Culex flavivirus strains from Argentina

In Argentina, many Flavivirus were recognised including West Nile virus (WNV). During 2009 several strains of Culex Flavivirus (CxFV), an insect-specific flavivirus, were isolated in the same region where circulation of WNV was detected. Hence, the objective of this study was to analyse the effect of co-infection in vitro assays using CxFV and WNV Argentinean strains in order to evaluate if CxFV could affect WNV replication. Our results showed that WNV replication was suppressed when multiplicity of infection (MOI) for CxFV was 10 or 100 times higher than WNV. Nevertheless, in vivo assays are necessary in order to evaluate the superinfection exclusion potential.

Within-species divergence in the seminal fluid proteome and its effect on male and female reproduction in a beetle.

BACKGROUND: Male seminal fluid proteins (SFPs), transferred to females during mating, are important reproductive proteins that have multifarious effects on female reproductive physiology and that often show remarkably rapid and divergent evolution. Inferences regarding natural selection on SFPs are based primarily on interspecific comparative studies, and our understanding of natural within-species variation in SFPs and whether this relates to reproductive phenotypes is very limited. Here, we introduce an empirical strategy to study intraspecific variation in and selection upon the seminal fluid proteome. We then apply this in a study of 15 distinct populations of the seed beetle Callosobruchus maculatus. RESULTS: Phenotypic assays of these populations showed significant differences in reproductive phenotypes (male success in sperm competition and male ability to stimulate female fecundity). A quantitative proteomic study of replicated samples of male accessory glands revealed a large number of potential SFPs, of which ≥127 were found to be transferred to females at mating. Moreover, population divergence in relative SFP abundance across populations was large and remarkably multidimensional. Most importantly, variation in male SFP abundance across populations was associated with male sperm competition success and male ability to stimulate female egg production. CONCLUSIONS: Our study provides the first direct evidence for postmating sexual selection on standing intraspecific variation in SFP abundance and the pattern of divergence across populations in the seminal fluid proteome match the pattern predicted by the postmating sexual selection paradigm for SFP evolution. Our findings provide novel support for the hypothesis that sexual selection on SFPs is an important engine of incipient speciation.

Male seminal fluid substances affect sperm competition success and female reproductive behavior in a seed beetle.

Male seminal fluid proteins are known to affect female reproductive behavior and physiology by reducing mating receptivity and by increasing egg production rates. Such substances are also though to increase the competitive fertilization success of males, but the empirical foundation for this tenet is restricted. Here, we examined the effects of injections of size-fractioned protein extracts from male reproductive organs on both male competitive fertilization success (i.e., P2 in double mating experiments) and female reproduction in the seed beetle Callosobruchus maculatus. We found that extracts of male seminal vesicles and ejaculatory ducts increased competitive fertilization success when males mated with females 1 day after the females' initial mating, while extracts from accessory glands and testes increased competitive fertilization success when males mated with females 2 days after the females' initial mating. Moreover, different size fractions of seminal fluid proteins had distinct and partly antagonistic effects on male competitive fertilization success. Collectively, our experiments show that several different seminal fluid proteins, deriving from different parts in the male reproductive tract and of different molecular weight, affect male competitive fertilization success in C. maculatus. Our results highlight the diverse effects of seminal fluid proteins and show that the function of such proteins can be contingent upon female mating status. We also document effects of different size fractions on female mating receptivity and egg laying rates, which can serve as a basis for future efforts to identify the molecular identity of seminal fluid proteins and their function in this model species.

Intralocus sexual conflict and environmental stress.

Intralocus sexual conflict (IaSC) occurs when selection at a given locus favors different alleles in males and females, placing a fundamental constraint on adaptation. However, the relative impact of IaSC on adaptation may become reduced in stressful environments that expose conditionally deleterious mutations to selection. The genetic correlation for fitness between males and females (rMF ) provides a quantification of IaSC across the genome. We compared IaSC at a benign (29°C) and a stressful (36°C) temperature by estimating rMF s in two natural populations of the seed beetle Callosobruchus maculatus using isofemale lines. In one population, we found substantial IaSC under benign conditions signified by a negative rMF (-0.51) and, as predicted, a significant reduction of IaSC under stress signified by a reversed and positive rMF (0.21). The other population displayed low IaSC at both temperatures (rMF : 0.38; 0.40). In both populations, isofemale lines harboring alleles beneficial to males but detrimental to females at benign conditions tended to show overall low fitness under stress. These results offer support for low IaSC under stress and suggest that environmentally sensitive and conditionally deleterious alleles that are sexually selected in males mediate changes in IaSC. We discuss implications for adaptive evolution in sexually reproducing populations.

A quantitative genetic study of starvation resistance at different geographic scales in natural populations of Drosophila melanogaster.

Food shortage is a stress factor that commonly affects organisms in nature. Resistance to food shortage or starvation resistance (SR) is a complex quantitative trait with direct implications on fitness. However, surveys of natural genetic variation in SR at different geographic scales are scarce. Here, we have measured variation in SR in sets of lines derived from nine natural populations of Drosophila melanogaster collected in western Argentina. Our study shows that within population variation explained a larger proportion of overall phenotypic variance (80%) than among populations (7·2%). We also noticed that an important fraction of variation was sex-specific. Overall females were more resistant to starvation than males; however, the magnitude of the sexual dimorphism (SD) in SR varied among lines and explained a significant fraction of phenotypic variance in all populations. Estimates of cross-sex genetic correlations suggest that the genetic architecture of SR is only partially shared between sexes in the populations examined, thus, facilitating further evolution of the SD.

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.