Dengue and chikungunya virus loads in the mosquito Aedes aegypti are determined by distinct genetic architectures.

Aedes aegypti is the primary vector of the arboviruses dengue (DENV) and chikungunya (CHIKV). These viruses exhibit key differences in their vector interactions, the latter moving more quicky through the mosquito and triggering fewer standard antiviral pathways. As the global footprint of CHIKV continues to expand, we seek to better understand the mosquito's natural response to CHIKV-both to compare it to DENV:vector coevolutionary history and to identify potential targets in the mosquito for genetic modification. We used a modified full-sibling design to estimate the contribution of mosquito genetic variation to viral loads of both DENV and CHIKV. Heritabilities were significant, but higher for DENV (40%) than CHIKV (18%). Interestingly, there was no genetic correlation between DENV and CHIKV loads between siblings. These data suggest Ae. aegypti mosquitoes respond to the two viruses using distinct genetic mechanisms. We also examined genome-wide patterns of gene expression between High and Low CHIKV families representing the phenotypic extremes of viral load. Using RNAseq, we identified only two loci that consistently differentiated High and Low families: a long non-coding RNA that has been identified in mosquito screens post-infection and a distant member of a family of Salivary Gland Specific (SGS) genes. Interestingly, the latter gene is also associated with horizontal gene transfer between mosquitoes and the endosymbiotic bacterium Wolbachia. This work is the first to link the SGS gene to a mosquito phenotype. Understanding the molecular details of how this gene contributes to viral control in mosquitoes may, therefore, also shed light on its role in Wolbachia.

The impacts of positive selection on genomic variation in Drosophila serrata: Insights from a deep learning approach.

This study explores the impact of positive selection on the genetic composition of a Drosophila serrata population in eastern Australia through a comprehensive analysis of 110 whole genome sequences. Utilizing an advanced deep learning algorithm (partialS/HIC) and a range of inferred demographic histories, we identified that approximately 14% of the genome is directly affected by sweeps, with soft sweeps being more prevalent (10.6%) than hard sweeps (2.1%), and partial sweeps being uncommon (1.3%). The algorithm demonstrated robustness to demographic assumptions in classifying complete sweeps but faced challenges in distinguishing neutral regions from partial sweeps and linked regions under demographic misspecification. The findings reveal the indirect influence of sweeps on nearly two-thirds of the genome through linkage, with an over-representation of putatively deleterious variants suggesting that positive selection drags deleterious variants to higher frequency due to hitchhiking with beneficial loci. Gene ontology enrichment analysis further supported our confidence in the accuracy of sweep detection as several traits expected to be under positive selection due to evolutionary arms races (e.g. immunity) were detected in hard sweeps. This study provides valuable insights into the direct and indirect contributions of positive selection in shaping genomic variation in natural populations.

Genetic and social contributions to sex differences in lifespan in Drosophila serrata.

Sex differences in lifespan remain an intriguing puzzle in evolutionary biology. While explanations range from sex differences in selection to sex differences in the expression of recessive lifespan-altering mutations (via X-linkage), little consensus has been reached. One unresolved issue is the extent to which genetic influences on lifespan dimorphism are modulated by the environment. For example, studies have shown that sex differences in lifespan can either increase or decrease depending upon the social environment. Here, we took an experimental approach, manipulating multiple axes of the social environment across inbred long- and short-lived genotypes and their reciprocal F1s in the fly Drosophila serrata. Our results reveal strong genetic effects and subtle yet significant genotype-by-environment interactions for male and female lifespan, specifically due to both population density and mating status. Further, our data do not support the idea that unconditional expression of deleterious X-linked recessive alleles in heterogametic males accounts for lower male lifespan.

Dominance reversals and the maintenance of genetic variation for fitness.

Antagonistic selection between different fitness components (e.g., survival versus fertility) or different types of individuals in a population (e.g., females versus males) can potentially maintain genetic diversity and thereby account for the high levels of fitness variation observed in natural populations. However, the degree to which antagonistic selection can maintain genetic variation critically depends on the dominance relations between antagonistically selected alleles in diploid individuals. Conditions for stable polymorphism of antagonistically selected alleles are narrow, particularly when selection is weak, unless the alleles exhibit "dominance reversals"-in which each allele is partially or completely dominant in selective contexts in which it is favored and recessive in contexts in which it is harmful. Although theory predicts that dominance reversals should emerge under biologically plausible conditions, evidence for dominance reversals is sparse. In this primer, we review theoretical arguments and data supporting a role for dominance reversals in the maintenance of genetic variation. We then highlight an illuminating new study by Grieshop and Arnqvist, which reports a genome-wide signal of dominance reversals between male and female fitness in seed beetles.

Integrating genomics and multivariate evolutionary quantitative genetics: a case study of constraints on sexual selection in Drosophila serrata.

In evolutionary quantitative genetics, the genetic variance-covariance matrix, G, and the vector of directional selection gradients, ß, are key parameters for predicting multivariate selection responses and genetic constraints. Historically, investigations of G and ß have not overlapped with those dissecting the genetic basis of quantitative traits. Thus, it remains unknown whether these parameters reflect pleiotropic effects at individual loci. Here, we integrate multivariate genome-wide association study (GWAS) with G and ß estimation in a well-studied system of multivariate constraint: sexual selection on male cuticular hydrocarbons (CHCs) in Drosophila serrata. In a panel of wild-derived re-sequenced lines, we augment genome-based restricted maximum likelihood to estimate G alongside multivariate single nucleotide polymorphism (SNP) effects, detecting 532 significant associations from 1 652 276 SNPs. Constraint was evident, with ß lying in a direction of G with low evolvability. Interestingly, minor frequency alleles typically increased male CHC-attractiveness suggesting opposing natural selection on ß. SNP effects were significantly misaligned with the major eigenvector of G, gmax, but well aligned to the second and third eigenvectors g2 and g3. We discuss potential factors leading to these varied results including multivariate stabilizing selection and mutational bias. Our framework may be useful as researchers increasingly access genomic methods to study multivariate selection responses in wild populations.

Artificial selection reveals sex differences in the genetic basis of sexual attractiveness.

Mutual mate choice occurs when males and females base mating decisions on shared traits. Despite increased awareness, the extent to which mutual choice drives phenotypic change remains poorly understood. When preferences in both sexes target the same traits, it is unclear how evolution will proceed and whether responses to sexual selection from male choice will match or oppose responses to female choice. Answering this question is challenging, as it requires understanding, genetic relationships between the traits targeted by choice, mating success, and, ultimately, fitness for both sexes. Addressing this, we applied artificial selection to the cuticular hydrocarbons of the fly Drosophila serrata that are targeted by mutual choice and tracked evolutionary changes in males and females alongside changes in mating success. After 10 generations, significant trait evolution occurred in both sexes, but intriguingly there were major sex differences in the associated fitness consequences. Sexually selected trait evolution in males led to a genetically based increase in male mating success. By contrast, although trait evolution also occurred in females, there was no change in mating success. Our results suggest that phenotypic sexual selection on females from male choice is environmentally, rather than genetically, generated. Thus, compared with female choice, male choice is at best a weak driver of signal trait evolution in this species. Instead, the evolution of apparent female ornamentation seems more likely due to a correlated response to sexual selection on males and possibly other forms of natural selection.

The Genomics of Sexual Conflict.

Sexual dimorphism is a substantial contributor to the diversity observed in nature, extending from elaborate traits to the expression level of individual genes. Sexual conflict and sexually antagonistic coevolution are thought to be central forces driving the dimorphism of the sexes and its diversity. We have substantial data to support this at the phenotypic level but much less at the genetic level, where distinguishing the role of conflict from other forms of sex-biased selection and from other processes is challenging. Here we discuss the powerful effects sexual conflict may have on genome evolution and critically evaluate the supporting evidence. Although there is much potential for sexual conflict to affect genome evolution, we have relatively little compelling evidence of a genomic signature of sexual conflict. A central obstacle is the mismatch between taxa in which we understand sexually antagonistic selection and those in which we understand genetics.

Sex-biased transcriptome divergence along a latitudinal gradient.

Sex-dependent gene expression is likely an important genomic mechanism that allows sex-specific adaptation to environmental changes. Among Drosophila species, sex-biased genes display remarkably consistent evolutionary patterns; male-biased genes evolve faster than unbiased genes in both coding sequence and expression level, suggesting sex differences in selection through time. However, comparatively little is known of the evolutionary process shaping sex-biased expression within species. Latitudinal clines offer an opportunity to examine how changes in key ecological parameters also influence sex-specific selection and the evolution of sex-biased gene expression. We assayed male and female gene expression in Drosophila serrata along a latitudinal gradient in eastern Australia spanning most of its endemic distribution. Analysis of 11 631 genes across eight populations revealed strong sex differences in the frequency, mode and strength of divergence. Divergence was far stronger in males than females and while latitudinal clines were evident in both sexes, male divergence was often population specific, suggesting responses to localized selection pressures that do not covary predictably with latitude. While divergence was enriched for male-biased genes, there was no overrepresentation of X-linked genes in males. By contrast, X-linked divergence was elevated in females, especially for female-biased genes. Many genes that diverged in D. serrata have homologs also showing latitudinal divergence in Drosophila simulans and Drosophila melanogaster on other continents, likely indicating parallel adaptation in these distantly related species. Our results suggest that sex differences in selection play an important role in shaping the evolution of gene expression over macro- and micro-ecological spatial scales.

The phenome-wide distribution of genetic variance.

A general observation emerging from estimates of additive genetic variance in sets of functionally or developmentally related traits is that much of the genetic variance is restricted to few trait combinations as a consequence of genetic covariance among traits. While this biased distribution of genetic variance among functionally related traits is now well documented, how it translates to the broader phenome and therefore any trait combination under selection in a given environment is unknown. We show that 8,750 gene expression traits measured in adult male Drosophila serrata exhibit widespread genetic covariance among random sets of five traits, implying that pleiotropy is common. Ultimately, to understand the phenome-wide distribution of genetic variance, very large additive genetic variance-covariance matrices (G) are required to be estimated. We draw upon recent advances in matrix theory for completing high-dimensional matrices to estimate the 8,750-trait G and show that large numbers of gene expression traits genetically covary as a consequence of a single genetic factor. Using gene ontology term enrichment analysis, we show that the major axis of genetic variance among expression traits successfully identified genetic covariance among genes involved in multiple modes of transcriptional regulation. Our approach provides a practical empirical framework for the genetic analysis of high-dimensional phenome-wide trait sets and for the investigation of the extent of high-dimensional genetic constraint.

Interspecific divergence of transcription networks along lines of genetic variance in Drosophila: dimensionality, evolvability, and constraint.

Change in gene expression is a major facilitator of phenotypic evolution. Understanding the evolutionary potential of gene expression requires taking into account complex systems of regulatory networks, the structure of which could potentially bias evolutionary trajectories. We analyzed the evolutionary potential and divergence of multigene expression in three well-characterized signaling pathways in Drosophila, the mitogen-activated protein kinase (MapK), the Toll, and the insulin receptor/Foxo (InR/Foxo or InR/TOR) pathways in a multivariate quantitative genetic framework. Gene expression data from a natural population of D. melanogaster were used to estimate the genetic variance-covariance matrices (G) for each network. Although most genes within each pathway exhibited significant genetic variance, the number of independent dimensions of multivariate genetic variance was fewer than the number of genes analyzed. However, for expression, the reduction in dimensionality was not as large as seen for other trait types such as morphology. We then tested whether gene expression divergence between D. melanogaster and an additional six species of the Drosophila genus was biased along the major axes of standing variation observed in D. melanogaster. In many cases, divergence was restricted to directions of phenotypic space harboring above average levels of genetic variance in D. melanogaster, indicating that genetic covariances between genes within pathways have biased interspecific divergence. We tested whether co-expression of genes in both sexes has also biased the pattern of divergence. Including cross-sex genetic covariances increased the degree to which divergence was biased along major axes of genetic variance, suggesting that the co-expression of genes in males and females can generate further constraints on divergence across the Drosophila phylogeny. In contrast to patterns seen for morphological traits in vertebrates, transcriptional constraints do not appear to break down as divergence time between species increases, instead they persist over tens of millions of years of divergence.

Misalignment of plastic and evolutionary responses of lifespan to novel carbohydrate diets.

Diet elicits varied effects on longevity across a wide range of animal species where dietary discordance between an organisms' evolutionary and developmental dietary history is increasingly recognized to play a critical role in shaping lifespan. However, whether such changes, predominantly assessed in a single generation, lead to evolutionary shifts in lifespan remains unclear. In this study, we used an experimental evolution approach to test whether changes in an organisms' evolutionary and developmental dietary history, specifically carbohydrate content, causes lifespan evolution in Drosophila serrata. After 30 generations, we investigated the evolutionary potential of lifespan in response to four novel diets that varied systematically in their ratio of carbohydrate-protein content. We also examined developmental plasticity effects using a set of control populations that were raised on the four novel environments allowing us to assess the extent to which plastic responses of lifespan mirrored adaptive responses observed following experimental evolution. Both high- and low-carbohydrate diets elicited plastic effects on lifespan; however, the plastic responses for lifespan to developmental diets bore little resemblance to the evolved responses on evolutionary diets. Understanding the dietary conditions regulating the match/mismatch of plastic and evolved responses will be important in determining whether a particular match/mismatch combination is adaptive for lifespan. While the differences in evolutionary diet by developmental diet interactions are only beginning to be elucidated, this study lays the foundation for future investigations of carbohydrate contributions to evolved and plastic effects on health and lifespan.

Sex-specific fitness consequences of nutrient intake and the evolvability of diet preferences.

The acquisition of nutrients is fundamental for the maintenance of bodily functions, growth, and reproduction in animals. As a result, fitness can be maximized only when animals are able to direct their attention to foods that reflect their current nutritional needs. Despite significant literature documenting the fitness consequences of nutrient composition and preference, less is known about the underlying genetic architecture of the dietary preferences themselves, specifically, the degree to which they can respond to selection. We addressed this by integrating evolutionary quantitative genetics and nutritional geometry to examine the shape of the sex-specific fitness surfaces and the availability of genetic variance for macronutrient preferences in the fruit fly Drosophila melanogaster. Combining these analyses, we found that the microevolutionary potential of carbohydrate and protein preference was above average in this population, because the expected direction of selection was relatively well aligned with the major axis of the genetic variance-covariance matrix, G. We also found that potential exists for sexually antagonistic genetic constraint in this system; macronutrient blends maximizing fitness differed between the sexes, and cross-sex genetic correlations for their consumption were positive. However, both sexes were displaced from their feeding optima, generating similar directional selection on males and females, with the combined effect being that minimal sex-specific genetic constraints currently affect dietary preferences in this population.

Environmental dependence of mutational (co)variances of adaptive traits.

Standing genetic variation, and capacity to adapt to environment change, will ultimately depend on the fitness effects of mutations across the range of environments experienced by contemporary, panmictic, populations. We investigated how mild perturbations in diet and temperature affect mutational (co)variances of traits that evolve under climatic adaptation, and contribute to individual fitness in Drosophila serrata. We assessed egg-to-adult viability, development time and wing size of 64 lines that had diverged from one another via spontaneous mutation over 30 generations of brother-sister mating. Our results suggested most mutations have directionally concordant (i.e., synergistic) effects in all environments and both sexes. However, elevated mutational variance under reduced macronutrient conditions suggested environment-dependent variation in mutational effect sizes for development time. We also observed evidence for antagonistic effects under standard versus reduced macronutrient conditions, where these effects were further contingent on temperature (for development time) or sex (for size). Diet also influenced the magnitude and sign of mutational correlations between traits, although this result was largely due to a single genotype (line), which may reflect a rare, large effect mutation. Overall, our results suggest environmental heterogeneity and environment-dependency of mutational effects could contribute to the maintenance of genetic variance.

Natural variation at a single gene generates sexual antagonism across fitness components in Drosophila.

Mutations with conflicting fitness effects in males and females accumulate in sexual populations, reducing their adaptive capacity.1,2 Although quantitative genetic studies indicate that sexually antagonistic polymorphisms are common,3-5 their molecular basis and population genetic properties remain poorly understood.6,7 Here, we show in fruit flies how natural variation at a single gene generates sexual antagonism through phenotypic effects on cuticular hydrocarbon (CHC) traits that function as both mate signals and protectors against abiotic stress8 across a latitudinal gradient. Tropical populations of Drosophila serrata have polymorphic CHCs producing sexual antagonism through opposing but sex-limited effects on these two fitness-related functions. We dissected this polymorphism to a single fatty-acyl CoA reductase gene, DsFAR2-B, that is expressed in oenocyte cells where CHCs are synthesized. RNAi-mediated disruption of the DsFAR2-B ortholog in D. melanogaster oenocytes affected CHCs in a similar way to that seen in D. serrata. Population genomic analysis revealed that balancing selection likely operates at the DsFAR2-B locus in the wild. Our study provides insights into the genetic basis of sexual antagonism in nature and connects sexually varying antagonistic selection on phenotypes with balancing selection on genotypes that maintains molecular variation.

Maintenance of quantitative genetic variance in complex, multitrait phenotypes: the contribution of rare, large effect variants in 2 Drosophila species.

The interaction of evolutionary processes to determine quantitative genetic variation has implications for contemporary and future phenotypic evolution, as well as for our ability to detect causal genetic variants. While theoretical studies have provided robust predictions to discriminate among competing models, empirical assessment of these has been limited. In particular, theory highlights the importance of pleiotropy in resolving observations of selection and mutation, but empirical investigations have typically been limited to few traits. Here, we applied high-dimensional Bayesian Sparse Factor Genetic modeling to gene expression datasets in 2 species, Drosophila melanogaster and Drosophila serrata, to explore the distributions of genetic variance across high-dimensional phenotypic space. Surprisingly, most of the heritable trait covariation was due to few lines (genotypes) with extreme [>3 interquartile ranges (IQR) from the median] values. Intriguingly, while genotypes extreme for a multivariate factor also tended to have a higher proportion of individual traits that were extreme, we also observed genotypes that were extreme for multivariate factors but not for any individual trait. We observed other consistent differences between heritable multivariate factors with outlier lines vs those factors without extreme values, including differences in gene functions. We use these observations to identify further data required to advance our understanding of the evolutionary dynamics and nature of standing genetic variation for quantitative traits.

Effective but costly, evolved mechanisms of defense against a virulent opportunistic pathogen in Drosophila melanogaster.

Drosophila harbor substantial genetic variation for antibacterial defense, and investment in immunity is thought to involve a costly trade-off with life history traits, including development, life span, and reproduction. To understand the way in which insects invest in fighting bacterial infection, we selected for survival following systemic infection with the opportunistic pathogen Pseudomonas aeruginosa in wild-caught Drosophila melanogaster over 10 generations. We then examined genome-wide changes in expression in the selected flies relative to unselected controls, both of which had been infected with the pathogen. This powerful combination of techniques allowed us to specifically identify the genetic basis of the evolved immune response. In response to selection, population-level survivorship to infection increased from 15% to 70%. The evolved capacity for defense was costly, however, as evidenced by reduced longevity and larval viability and a rapid loss of the trait once selection pressure was removed. Counter to expectation, we observed more rapid developmental rates in the selected flies. Selection-associated changes in expression of genes with dual involvement in developmental and immune pathways suggest pleiotropy as a possible mechanism for the positive correlation. We also found that both the Toll and the Imd pathways work synergistically to limit infectivity and that cellular immunity plays a more critical role in overcoming P. aeruginosa infection than previously reported. This work reveals novel pathways by which Drosophila can survive infection with a virulent pathogen that may be rare in wild populations, however, due to their cost.

The impact of artificial selection for Wolbachia-mediated dengue virus blocking on phage WO.

Wolbachia is currently at the forefront of global efforts to control arbovirus transmission from the vector Aedes aegypti. The use of Wolbachia relies on two phenotypes-cytoplasmic incompatibility (CI), conferred by cifA and cifB genes in prophage WO, and Wolbachia-mediated pathogen blocking (WMPB). These traits allow for local, self-sustaining reductions in transmission of dengue (DENV) following release of Wolbachia-infected A. aegypti. Here, aided by previous artificial selection experiment that generated Low and High pathogen blocking lines, we examined the potential link between WMPB and phage WO. We found no evidence that Wolbachia or phage WO relative densities predict DENV blocking strength across selected lines. However, selection resulted in reduced phage WO relative density for the Low WMPB line. The Low blocking line was previously shown to have reduced fitness as a result of selection. Through subsequent genomic analyses, we demonstrate that SNP variation underpinning selection for low blocking led to elevated frequency of potential deleterious SNPs on chromosome 1. The key region on chromosome 1 contains genes relating to cell cycle regulation, oxidative stress, transcriptional pausing, among others, that may have cascading effects on Wolbachia intracellular environment. We hypothesize that reduction in phage WO may be driven by changes in the loci directly under selection for blocking, or by the accumulation of predicted deleterious alleles in linkage disequilibrium with blocking loci resulting from hitchhiking. For the Low line with fewer phage WO, we also detected reduced expression of cifA and cifB CI genes, with patterns of expression varying between somatic and reproductive tissues. In conclusion, we propose that artificial selection for WMPB trait had corresponding impacts on phage WO densities, and also the transcription of CI-causing genes. Future studies may include a more detailed analysis of the regions the A. aegypti chromosome 1's ability to affect WMPB and other Wolbachia-associated intrinsic factors such as phage WO.

The contribution of selection and genetic constraints to phenotypic divergence.

Although divergent natural selection is common in nature, the extent to which genetic constraints bias evolutionary trajectories in its presence remains largely unknown. Here we develop a general framework to integrate estimates of divergent selection and genetic constraints to estimate their contributions to phenotypic divergence among natural populations. We apply these methods to estimates of phenotypic selection and genetic covariance from sexually selected traits that have undergone adaptive divergence among nine natural populations of the fly Drosophila serrata. Despite ongoing sexual selection within populations, differences in its direction among them, and genetic variance for all traits in all populations, divergent sexual selection only weakly resembled the observed pattern of divergence. Accounting for the influence of genetic covariance among the traits significantly improved the alignment between observed and predicted divergence. Our results suggest that the direction in which sexual selection generates divergence may depend on the pattern of genetic constraint in individual populations, ultimately restricting how sexually selected traits may diversify. More generally, we show how evolution is likely to proceed in the direction of major axes of genetic variance, rather than the direction of selection itself, when genetic variance-covariance matrices are ill conditioned and genetic variance is low in the direction of selection.

Association mapping in outbred populations: power and efficiency when genotyping parents and phenotyping progeny.

We develop expressions for the power to detect associations between parental genotypes and offspring phenotypes for quantitative traits. Three different "indirect" experimental designs are considered: full-sib, half-sib, and full-sib-half-sib families. We compare the power of these designs to detect genotype-phenotype associations relative to the common, "direct," approach of genotyping and phenotyping the same individuals. When heritability is low, the indirect designs can outperform the direct method. However, the extra power comes at a cost due to an increased phenotyping effort. By developing expressions for optimal experimental designs given the cost of phenotyping relative to genotyping, we show how the extra costs associated with phenotyping a large number of individuals will influence experimental design decisions. Our results suggest that indirect association studies can be a powerful means of detecting allelic associations in outbred populations of species for which genotyping and phenotyping the same individuals is impractical and for life history and behavioral traits that are heavily influenced by environmental variance and therefore best measured on groups of individuals. Indirect association studies are likely to be favored only on purely economical grounds, however, when phenotyping is substantially less expensive than genotyping. A web-based application implementing our expressions has been developed to aid in the design of indirect association studies.

Zebrafish take their cue from temperature but not photoperiod for the seasonal plasticity of thermal performance.

Organisms adjust to seasonal variability in the environment by responding to cues that indicate environmental change. As most studies of seasonal phenotypic plasticity test only the effect of a single environmental cue, how animals may integrate information from multiple cues to fine-tune plastic responses remains largely unknown. We examined the interaction between correlated (seasonally matching) and conflicting (seasonally opposite) temperature and photoperiod cues on the acclimation of performance traits in male zebrafish, Danio rerio. We acclimated fish for 8 weeks and then tested the change in thermal dependence of maximum burst swimming and feeding rate between 8 and 38°C. We predicted that correlated environmental cues should induce a greater acclimation response than uncorrelated cues. However, we found that only temperature was important for the seasonal acclimation of performance traits in zebrafish. Thermal acclimation shifted the thermal performance curve of both traits. For maximum burst swimming, performance increased for each group near the acclimation temperature and reduced in environments that were far from their acclimation temperature. The feeding rate of cold-acclimated zebrafish was reduced across the test temperature range compared with that of warm-acclimated fish. Our study is the first that has found no effect of the covariation between temperature and photoperiod acclimation cues on locomotor performance in fishes. Our results support the intuitive idea that photoperiod may be a less important seasonal cue for animals living at lower latitudes.