Experience matters: genetic variation affects male reproductive success across sequential mating events in Drosophila melanogaster.

The cost of reproduction is well studied in females but only recently have the costs of mating been investigated in males. Research suggests that males allocate resources between subsequent mating events, resulting in differential success across mating bouts. Selection should favor allocation strategies that match the likelihood of successive matings. The complexity of the system, however, suggests that one fixed strategy is unlikely to be universally favored and thus I predict that genetic variation for different allocation strategies will be segregating in natural populations. To test this, I measured several components of reproductive performance in eight inbred genotypes of Drosophila melanogaster across three sequential mating events. As predicted, there was genetic variation for how previous experience affected a male's reproductive performance for both the proportion of matings that produced offspring and the proportion of offspring sired (P1). Some genotypes had the highest success in their first matings and declined in successive matings while other genotypes did best in later matings. Mating experience had consistent effects across genotypes on fertility and induced refractoriness to remating. On average, virgin matings produced the highest fertility and third matings most effectively induced refractoriness. Genotype also had a significant effect on fertility. These results have important implications for understanding how selection may be acting on males when there is variation in the likelihood of multiple mating events and could affect the evolution of male allocation strategies in the face of perceived competitors.

Mapping mitonuclear epistasis using a novel recombinant yeast population.

Genetic variation in mitochondrial and nuclear genomes can perturb mitonuclear interactions and lead to phenotypic differences between individuals and populations. Despite their importance to most complex traits, it has been difficult to identify the interacting mitonuclear loci. Here, we present a novel advanced intercrossed population of Saccharomyces cerevisiae yeasts, called the Mitonuclear Recombinant Collection (MNRC), designed explicitly for detecting mitonuclear loci contributing to complex traits. For validation, we focused on mapping genes that contribute to the spontaneous loss of mitochondrial DNA (mtDNA) that leads to the petite phenotype in yeast. We found that rates of petite formation in natural populations are variable and influenced by genetic variation in nuclear DNA, mtDNA and mitonuclear interactions. We mapped nuclear and mitonuclear alleles contributing to mtDNA stability using the MNRC by integrating a term for mitonuclear epistasis into a genome-wide association model. We found that the associated mitonuclear loci play roles in mitotic growth most likely responding to retrograde signals from mitochondria, while the associated nuclear loci with main effects are involved in genome replication. We observed a positive correlation between growth rates and petite frequencies, suggesting a fitness tradeoff between mitotic growth and mtDNA stability. We also found that mtDNA stability was correlated with a mobile mitochondrial GC-cluster that is present in certain populations of yeast and that selection for nuclear alleles that stabilize mtDNA may be rapidly occurring. The MNRC provides a powerful tool for identifying mitonuclear interacting loci that will help us to better understand genotype-phenotype relationships and coevolutionary trajectories.

Genetic basis of susceptibility to low-dose paraquat and variation between the sexes in Drosophila melanogaster.

Toxicant resistance is a complex trait, affected both by genetics and the environment. Like most complex traits, it can exhibit sexual dimorphism, yet sex is often overlooked as a factor in studies of toxicant resistance. Paraquat, one such toxicant, is a commonly used herbicide and is known to produce mitochondrial oxidative stress, decrease dopaminergic neurons and dopamine (DA) levels, and decrease motor ability. While the main effects of paraquat are well-characterized, less is known about the naturally occurring variation in paraquat susceptibility. The purpose of this study was to map the genes contributing to low-dose paraquat susceptibility in Drosophila melanogaster, and to determine if susceptibility differs between the sexes. One hundred of the Drosophila Genetic Reference Panel (DGRP) lines were scored for susceptibility via climbing ability and used in a genome-wide association study (GWAS). Variation in seventeen genes in females and thirty-five genes in males associated with paraquat susceptibility. Only two candidate genes overlapped between the sexes despite a significant positive correlation between male and female susceptibilities. Many associated polymorphisms had significant interactions with sex, with most having conditionally neutral effects. Conditional neutrality between the sexes probably stems from sex-biased expression which may result from partial resolution of sexual conflict. Candidate genes were verified with RNAi knockdowns, gene expression analyses, and DA quantification. Several of these genes are novel associations with paraquat susceptibility. This research highlights the importance of assessing both sexes when studying toxicant susceptibility.

Effects of Dual Exposure to the Herbicides Atrazine and Paraquat on Adult Climbing Ability and Longevity in Drosophila melanogaster.

Anthropomorphic effects are changing the planet, and therefore, organisms are being exposed to many new biotic and abiotic stressors. Exposure to multiple stressors can affect organisms in ways that are different than the sum of their individual effects, and these interactions are often difficult to predict. Atrazine and paraquat are two of the most widely used herbicides in the United States, and are individually known to increase oxidative damage, affect dopaminergic functioning, reduce longevity, and alter motor ability in non-target organisms. We measured the effects of individual and combined exposure to low doses of atrazine and paraquat on climbing ability and longevity of Drosophila melanogaster. Atrazine and paraquat interact to affect D. melanogaster climbing ability and longevity in different ways. Atrazine appeared to have a weak mitigative effect against the decrease in climbing ability caused by paraquat. In contrast, combined exposure to atrazine and paraquat had detrimental synergistic effects on female longevity. Overall, this study shows that atrazine and paraquat can interact and that it is important to measure several traits when assessing the consequences of exposure to multiple stressors. Future studies should continue to assess the impacts of stressor interactions on organisms, as many combinations have never been examined.

Mitochondrial Recombination Reveals Mito-Mito Epistasis in Yeast.

Genetic variation in mitochondrial DNA (mtDNA) provides adaptive potential although the underlying genetic architecture of fitness components within mtDNAs is not known. To dissect functional variation within mtDNAs, we first identified naturally occurring mtDNAs that conferred high or low fitness in Saccharomyces cerevisiae by comparing growth in strains containing identical nuclear genotypes but different mtDNAs. During respiratory growth under temperature and oxidative stress conditions, mitotype effects were largely independent of nuclear genotypes even in the presence of mito-nuclear interactions. Recombinant mtDNAs were generated to determine fitness components within high- and low-fitness mtDNAs. Based on phenotypic distributions of isogenic strains containing recombinant mtDNAs, we found that multiple loci contributed to mitotype fitness differences. These mitochondrial loci interacted in epistatic, nonadditive ways in certain environmental conditions. Mito-mito epistasis (i.e., nonadditive interactions between mitochondrial loci) influenced fitness in progeny from four different crosses, suggesting that mito-mito epistasis is a widespread phenomenon in yeast and other systems with recombining mtDNAs. Furthermore, we found that interruption of coadapted mito-mito interactions produced recombinant mtDNAs with lower fitness. Our results demonstrate that mito-mito epistasis results in functional variation through mitochondrial recombination in fungi, providing modes for adaptive evolution and the generation of mito-mito incompatibilities.

Titanium dioxide nanoparticle exposure alters metabolic homeostasis in a cell culture model of the intestinal epithelium and Drosophila melanogaster.

Nanosized titanium dioxide (TiO2) is a common additive in food and cosmetic products. The goal of this study was to investigate if TiO2 nanoparticles affect intestinal epithelial tissues, normal intestinal function, or metabolic homeostasis using in vitro and in vivo methods. An in vitro model of intestinal epithelial tissue was created by seeding co-cultures of Caco-2 and HT29-MTX cells on a Transwell permeable support. These experiments were repeated with monolayers that had been cultured with the beneficial commensal bacteria Lactobacillus rhamnosus GG (L. rhamnosus). Glucose uptake and transport in the presence of TiO2 nanoparticles was assessed using fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG). When the cell monolayers were exposed to physiologically relevant doses of TiO2, a statistically significant reduction in glucose transport was observed. These differences in glucose absorption were eliminated in the presence of beneficial bacteria. The decrease in glucose absorption was caused by damage to intestinal microvilli, which decreased the surface area available for absorption. Damage to microvilli was ameliorated in the presence of L. rhamnosus. Complimentary studies in Drosophila melanogaster showed that TiO2 ingestion resulted in decreased body size and glucose content. The results suggest that TiO2 nanoparticles alter glucose transport across the intestinal epithelium, and that TiO2 nanoparticle ingestion may have physiological consequences.

Estimating mating rates in wild Drosophila melanogaster females by decay rates of male reproductive proteins in their reproductive tracts.

Female Drosophila melanogaster frequently mate with multiple males in nature as shown through parentage analysis. Although polyandry is well documented, we know little about the timing between mating events in wild Drosophila populations due to the challenge of following behaviours of individual females. In this study, we used the presence of a male reproductive protein that is transferred to the female during mating (Sex Peptide, SP) to determine whether she had recently mated. We sampled females throughout the day, conducted control matings to determine the decay rate of SP within the female reproductive tract and performed computer simulations to fit the observed proportion of mated females to a nonhomogenous Poisson process that defined the expected time between successive matings for a given female. In our control matings, 100% of mated females tested positive for SP 0.5 h after the start of mating (ASM), but only 24% tested positive 24 h ASM. Overall, 35% of wild-caught females tested positive for the presence of SP. Fitting our observed data to our simple nonhomogenous Poisson model provided the inference that females are mating, on average, approximately every 27 h (with 95% credibility interval 23-31 h). Thus, it appears that females are mating a bit less frequently that once per day in this natural population and that mating events tend to occur either early in the morning or late in the afternoon.

Atrazine exposure affects longevity, development time and body size in Drosophila melanogaster.

Atrazine is the one of the most widely used herbicides in the United States and non-target organisms may encounter it in the environment. Atrazine is known to affect male reproduction in both vertebrates and invertebrates but less is known about its effects on other fitness traits. Here we assessed the effects of five different chronic exposure levels on a variety of fitness traits in Drosophila melanogaster. We measured male and female longevity, development time, proportion pupated, proportion emerged, body size, female mating rate, fertility and fecundity. Atrazine exposure decreased the proportion pupated, the proportion emerged and adult survival. Development time was also affected by atrazine and exposed flies pupated and emerged earlier than controls. Although development time was accelerated, body size was actually larger in some of the exposures. Atrazine exposure had no effect on female mating rate and the effects on female fertility and fecundity were only observed in one of the two independent experimental blocks. Many of the traits showed non-monotonic dose response curves, where the intermediate concentrations showed the largest effects. Overall this study shows that atrazine influences a variety of life history traits in the model genetic system, D. melanogaster, and future studies should aim to identify the molecular mechanisms of toxicity.

Effects of atrazine exposure on male reproductive performance in Drosophila melanogaster.

Atrazine is a commonly utilized herbicide to control broadleaf weeds in the agricultural setting. It can, however, have negative effects on male reproductive performance in a variety of vertebrate species. Much less is known, however, about the effects of atrazine on invertebrates. In this study, we investigated the effects of several different concentrations of larval atrazine exposure on measures of reproductive performance in adult male Drosophila melanogaster. Atrazine exposure had significant effects on a male's mating ability and the number of eggs his partner laid when he was successful at mating. Exposed males also sired a smaller proportion of the offspring under competitive conditions when they were the first male to mate to a doubly mated female. Atrazine exposure had no measurable effect on a male's ability to prevent a mated female from mating to another male or on the proportion of offspring sired when the exposed males were the second male to mate. Exposure upregulated expression of one male reproductive gene, ovulin, but had no effect on expression of another, sex peptide. Exposed males produced and transferred more sex peptide protein to the female during mating but ovulin protein levels were not affected. In general, we observed non-monotonic responses such that the intermediate exposure levels showed the largest reduction in male reproductive performance. This study suggests that atrazine exposure affects male reproductive performance in insects and future studies should aim to understand the molecular mechanisms underlying the fitness effects of exposure.

Characterizing male-female interactions using natural genetic variation in Drosophila melanogaster.

Drosophila melanogaster females commonly mate with multiple males establishing the opportunity for pre- and postcopulatory sexual selection. Traits impacting sexual selection can be affected by a complex interplay of the genotypes of the competing males, the genotype of the female, and compatibilities between the males and females. We scored males from 96 2nd and 94 3rd chromosome substitution lines for traits affecting reproductive success when mated with females from 3 different genetic backgrounds. The traits included male-induced female refractoriness, male remating ability, the proportion of offspring sired under competitive conditions and male-induced female fecundity. We observed significant effects of male line, female genetic background, and strong male by female interactions. Some males appeared to be "generalists" and performed consistently across the different females; other males appeared to be "specialists" and performed very well with a particular female and poorly with others. "Specialist" males did not, however, prefer to court those females with whom they had the highest reproductive fitness. Using 143 polymorphisms in male reproductive genes, we mapped several genes that had consistent effects across the different females including a derived, high fitness allele in Acp26Aa that may be the target of adaptive evolution. We also identified a polymorphism upstream of PebII that may interact with the female genetic background to affect male-induced refractoriness to remating. These results suggest that natural variation in PebII might contribute to the observed male-female interactions.

Mitochondrial-nuclear epistasis contributes to phenotypic variation and coadaptation in natural isolates of Saccharomyces cerevisiae.

Mitochondria are essential multifunctional organelles whose metabolic functions, biogenesis, and maintenance are controlled through genetic interactions between mitochondrial and nuclear genomes. In natural populations, mitochondrial efficiencies may be impacted by epistatic interactions between naturally segregating genome variants. The extent that mitochondrial-nuclear epistasis contributes to the phenotypic variation present in nature is unknown. We have systematically replaced mitochondrial DNAs in a collection of divergent Saccharomyces cerevisiae yeast isolates and quantified the effects on growth rates in a variety of environments. We found that mitochondrial-nuclear interactions significantly affected growth rates and explained a substantial proportion of the phenotypic variances under some environmental conditions. Naturally occurring mitochondrial-nuclear genome combinations were more likely to provide growth advantages, but genetic distance could not predict the effects of epistasis. Interruption of naturally occurring mitochondrial-nuclear genome combinations increased endogenous reactive oxygen species in several strains to levels that were not always proportional to growth rate differences. Our results demonstrate that interactions between mitochondrial and nuclear genomes generate phenotypic diversity in natural populations of yeasts and that coadaptation of intergenomic interactions likely occurs quickly within the specific niches that yeast occupy. This study reveals the importance of considering allelic interactions between mitochondrial and nuclear genomes when investigating evolutionary relationships and mapping the genetic basis underlying complex traits.

Natural genetic variation in male reproductive genes contributes to nontransitivity of sperm competitive ability in Drosophila melanogaster.

Female Drosophila melanogaster frequently mate with multiple males, and the success of a given male depends not only on his genotype but also on the genotype of his competitor. Here, we assess how natural genetic variation affects male-male interactions for traits influencing pre- and postcopulatory sexual selection. Males from a set of 66 chromosome substitution lines were competed against each other in a 'round-robin' design, and paternity was scored using bulk genotyping. We observed significant effects of the genotype of the first male to mate, the second male to mate and an interaction between the males for measures of male mating rate and sperm utilization. We also identified specific combinations of males who show nontransitive patterns of reproductive success and engage in 'rock-paper-scissors' games. We then tested for associations between 245 polymorphisms in 32 candidate male reproductive genes and male reproductive success. We identified eight polymorphisms in six reproductive genes that associate with male reproductive success independent of the competitor (experimentwise P < 0.05). We also identified four SNPs in four different genes where the relative reproductive success of the alternative alleles changes depending on the competing males' genetic background (experimentwise P < 0.05); two of these associations include premature stop codons. This may be the first study that identifies the genes contributing to nontransitivity among males and further highlights that 'rock-paper-scissors' games could be an important evolutionary force maintaining genetic variation in natural populations.

Female influence on pre- and post-copulatory sexual selection and its genetic basis in Drosophila melanogaster.

Genetic variation among females is likely to influence the outcome of both pre- and post-copulatory sexual selection in Drosophila melanogaster. Here we use association testing to survey natural variation in 10 candidate female genes for their effects on female reproduction. Females from 91 chromosome two substitution lines were scored for phenotypes affecting pre- and post-copulatory sexual selection such as mating and remating rate, propensity to use sperm from the second male to mate, and measures of fertility. There were significant genetic contributions to phenotypic variation for all the traits measured. Resequencing of the 10 candidate genes in the 91 lines yielded 68 non-synonymous polymorphisms which were tested for associations with the measured phenotypes. Twelve significant associations (markerwise P<0.01) were identified. Polymorphisms in the putative serine protease homolog CG9897 and the putative odorant binding protein CG11797 associated with female propensity to remate and met an experimentwise significance of P<0.05. Several other associations, including those impacting both fertility and female remating rate suggest that sperm storage might be an important factor mitigating female influence on sexual selection.

Seminal fluid protein depletion and replenishment in the fruit fly, Drosophila melanogaster: an ELISA-based method for tracking individual ejaculates.

In many species, seminal fluid proteins (SFPs) affect female post-mating behavioral patterns, including sperm storage, egg laying, feeding, and remating. Yet, few studies have investigated the patterns of allocation, depletion, and replenishment of SFPs in male animals, despite the importance of these proteins to male and female reproductive success. To investigate such SFP dynamics, it is necessary to have a sensitive method for quantifying SFP levels in males and mated females. We developed such a method by adapting the enzyme-linked immunosorbent assay (ELISA) using anti-SFP antibodies. Here, we first use two Drosophila melanogaster SFPs (ovulin and sex peptide) to demonstrate that ELISAs provide accurate measures of SFP levels. We find that, consistent with previous data from Western blotting or immunofluorescence studies, levels of both ovulin and sex peptide decline in the mated female with time since mating, but they do so at different rates. We then use ELISAs to show that males become depleted of SFPs with repeated matings, but that previously mated males are able to transfer "virgin" levels of SFPs after 3 days of sexual inactivity. Finally, we demonstrate that ELISAs can detect SFPs from wild-caught D. melanogaster males and, thus, potentially can be used to track mating patterns in the wild. This method of measuring SFP dynamics can be used in a wide range of species to address questions related to male reproductive investment, female mating history, and variation in female post-mating behavioral changes.

A role for Acp29AB, a predicted seminal fluid lectin, in female sperm storage in Drosophila melanogaster.

Females of many animal species store sperm for taxon-specific periods of time, ranging from a few hours to years. Female sperm storage has important reproductive and evolutionary consequences, yet relatively little is known of its molecular basis. Here, we report the isolation of a loss-of-function mutation of the Drosophila melanogaster Acp29AB gene, which encodes a seminal fluid protein that is transferred from males to females during mating. Using this mutant, we show that Acp29AB is required for the normal maintenance of sperm in storage. Consistent with this role, Acp29AB localizes to female sperm storage organs following mating, although it does not appear to associate tightly with sperm. Acp29AB is a predicted lectin, suggesting that sugar-protein interactions may be important for D. melanogaster sperm storage, much as they are in many mammals. Previous association studies have found an effect of Acp29AB genotype on a male's sperm competitive ability; our findings suggest that effects on sperm storage may underlie these differences in sperm competition. Moreover, Acp29AB's effects on sperm storage and sperm competition may explain previously documented evidence for positive selection on the Acp29AB locus.

Autosomal variation for male body size and sperm competition phenotypes is uncorrelated in Drosophila melanogaster.

Correlations between male body size and phenotypes impacting post-copulatory sexual selection are commonly observed during the manipulation of male body size by environmental rearing conditions. Here, we control for environmental influences and test for genetic correlations between natural variation in male body size and phenotypes affecting post-copulatory sexual selection in Drosophila melanogaster. Dry weights of virgin males from 90 second-chromosome and 88 third-chromosome substitution lines were measured. Highly significant line effects (p<0.001) documented a genetic basis to variation in male body size. No significant correlations were identified between male body size and the components of sperm competitive ability. These results suggest that natural autosomal variation for male body size has little impact on post-copulatory sexual selection. If genetic correlations exist between male body size and post-copulatory sexual selection then variation in the sex chromosomes are likely candidates, as might be expected if sexually antagonistic coevolution was responsible.

Larval rearing environment affects several post-copulatory traits in Drosophila melanogaster.

In Drosophila melanogaster, accessory gland proteins (Acps) that a male transfers during mating affect his reproductive success by altering the female's behaviour and physiology. To test the role of male condition in the expression of Acps, we manipulated the pre-adult environment and examined adult males for relative transcript abundance of nine Acps, and for post-copulatory traits that Acps influence. Larval culture density had no effect on any measured trait. Larval nutrient availability impacted the number of sperm transferred and stored, the male's ability to induce refractoriness in his mate, but relative transcript abundance of only a single Acp (Acp36DE). Reduced male body size due to low yeast levels affected sperm competition. Our data indicate that some female-mediated post-copulatory traits (induced refractoriness and sperm transfer and storage) might be influenced by the male's developmental environment, but relative expression of most Acps and some traits they influence (P1') are not.

Estimating differential reproductive success from nests of related individuals, with application to a study of the mottled sculpin, Cottus bairdi.

Understanding how variation in reproductive success is related to demography is a critical component in understanding the life history of an organism. Parentage analysis using molecular markers can be used to estimate the reproductive success of different groups of individuals in natural populations. Previous models have been developed for cases where offspring are random samples from the population but these models do not account for the presence of full- and half-sibs commonly found in large clutches of many organisms. Here we develop a model for comparing reproductive success among different groups of individuals that explicitly incorporates within-nest relatedness. Inference for the parameters of the model is done in a Bayesian framework, where we sample from the joint posterior of parental assignments and fertility parameters. We use computer simulations to determine how well our model recovers known parameters and investigate how various data collection scenarios (varying the number of nests or the number of offspring) affect the estimates. We then apply our model to compare reproductive success among different age groups of mottled sculpin, Cottus bairdi, from a natural population. We demonstrate that older adults are more likely to contribute to a nest and that females in the older age groups contribute more eggs to a nest than younger individuals.

Associations between sperm competition and natural variation in male reproductive genes on the third chromosome of Drosophila melanogaster.

We applied association analysis to elucidate the genetic basis for variation in phenotypes affecting postcopulatory sexual selection in a natural population of Drosophila melanogaster. We scored 96 third chromosome substitution lines for nine phenotypes affecting sperm competitive ability and genotyped them at 72 polymorphisms in 13 male reproductive genes. Significant heterogeneity among lines (P < 0.01) was detected for all phenotypes except male-induced refractoriness (P = 0.053). We identified 24 associations (8 single-marker associations, 12 three-marker haplotype associations, and 4 cases of epistasis revealed by single-marker interactions). Fewer than 9 of these associations are likely to be false positives. Several associations were consistent with previous findings [Acp70A with the male's influence on the female's refractoriness to remating (refractory), Esterase-6 with a male's remating probability (remating) and a measure of female offspring production (fecundity)], but many are novel associations with uncharacterized seminal fluid proteins. Four genes showed evidence for pleiotropic effects [CG6168 with a measure of sperm competition (P2') and refractory, CG14560 with a defensive measure of sperm competition (P1') and a measure of female fecundity, Acp62F with P2' and a measure of female fecundity, and Esterase-6 with remating and a measure of female fecundity]. Our findings provide evidence that pleiotropy and epistasis are important factors in the genetic architecture of male reproductive success and show that haplotype analyses can identify associations missed in the single-marker approach.

Natural variation in male-induced 'cost-of-mating' and allele-specific association with male reproductive genes in Drosophila melanogaster.

One of the most sharply defined sexual conflicts arises when the act of mating is accompanied by an inflated risk of death. Several reports have documented an increased death rate of female Drosophila as a result of recurrent mating. Transgenic and mutation experiments have further identified components of seminal fluid that are at least in part responsible for this toxicity. Variation among males in their tendency for matings to be toxic to their partners has also been documented, but here for the first time we identify polymorphism within particular genes conferring differential post-mating female mortality. Such polymorphism is important, as it raises the challenge of whether sexual conflict models can provide means for maintenance of polymorphism. Using a set of second chromosome extraction lines, we scored differences in post-mating female fecundity and longevity subsequent to mating, and identified significant among-line differences. Seventy polymorphisms in ten male reproductive genes were scored and permutation tests were used to identify significant associations between genotype and phenotype. One polymorphism upstream of PEBII and an amino acid substitution in CG17331 were both associated with male-induced female mortality. The same allele of CG17331 that is toxic to females also induces greater refractoriness to remating in the females, providing an example of an allele-specific sexual conflict. Postcopulatory sexual selection could lead to sexual conflict by favouring males that prevent their mates from mating, even when there is a viability cost to those females.