Females translate male mRNA transferred during mating.

Although RNA is found in the seminal fluid of diverse organisms, it is unknown whether this RNA is functional within females. Here, we develop an experimental proteomic method called VESPA (Variant Enabled SILAC Proteomic Analysis) to test the hypothesis that Drosophila male seminal fluid RNA is translated by females. We find strong evidence for 67 male-derived, female-translated proteins (mdFTPs) in female lower reproductive tracts at six hours postmating, many with predicted functions relevant to reproduction. Gene knockout experiments indicate that genes coding for mdFTPs play diverse roles in postmating interactions, with effects on fertilization efficiency, and the formation and persistence of the insemination reaction mass, a trait hypothesized to be involved in sexual conflict. These findings advance our understanding of reproduction by revealing a novel mechanism of postmating molecular interactions between the sexes that strengthens and extends male influences on reproductive outcomes in previously unrecognized ways. Given the diverse species known to carry RNA in seminal fluid, this discovery has broad significance for understanding molecular mechanisms of cooperation and conflict during reproduction.

The identification of protein and RNA interactors of the splicing factor Caper in the adult Drosophila nervous system.

Post-transcriptional gene regulation is a fundamental mechanism that helps regulate the development and healthy aging of the nervous system. Mutations that disrupt the function of RNA-binding proteins (RBPs), which regulate post-transcriptional gene regulation, have increasingly been implicated in neurological disorders including amyotrophic lateral sclerosis, Fragile X Syndrome, and spinal muscular atrophy. Interestingly, although the majority of RBPs are expressed widely within diverse tissue types, the nervous system is often particularly sensitive to their dysfunction. It is therefore critical to elucidate how aberrant RNA regulation that results from the dysfunction of ubiquitously expressed RBPs leads to tissue specific pathologies that underlie neurological diseases. The highly conserved RBP and alternative splicing factor Caper is widely expressed throughout development and is required for the development of Drosophila sensory and motor neurons. Furthermore, caper dysfunction results in larval and adult locomotor deficits. Nonetheless, little is known about which proteins interact with Caper, and which RNAs are regulated by Caper. Here we identify proteins that interact with Caper in both neural and muscle tissue, along with neural specific Caper target RNAs. Furthermore, we show that a subset of these Caper-interacting proteins and RNAs genetically interact with caper to regulate Drosophila gravitaxis behavior.

Divergent evolutionary trajectories shape the postmating transcriptional profiles of conspecifically and heterospecifically mated cactophilic Drosophila females.

Postmating-prezygotic (PMPZ) reproductive isolation is hypothesized to result from divergent coevolutionary trajectories of sexual selection and/or sexual conflict in isolated populations. However, the genetic basis of PMPZ incompatibilities between species is poorly understood. Here, we use a comparative framework to compare global gene expression in con- and heterospecifically mated Drosophila mojavensis and D. arizonae female reproductive tracts. We find striking divergence between the species in the female postmating transcriptional response to conspecific mating, including differences in differential expression (DE), alternative splicing (AS), and intron retention (IR). As predicted, heterospecific matings produce disrupted transcriptional profiles, but the overall patterns of misregulation are different between the reciprocal crosses. Moreover, we find a positive correlation between postmating transcriptional divergence between species and levels of transcriptional disruption in heterospecific crosses. This result indicates that mating responsive genes that have diverged more in expression also have more disrupted transcriptional profiles in heterospecifically mated females. Overall, our results provide insights into the evolution of PMPZ isolation and lay the foundation for future studies aimed at identifying specific genes involved in PMPZ incompatibilities and the evolutionary forces that have contributed to their divergence in closely related species.

Roles for the RNA-Binding Protein Caper in Reproductive Output in Drosophila melanogaster.

RNA binding proteins (RBPs) play a fundamental role in the post-transcriptional regulation of gene expression within the germline and nervous system. This is underscored by the prevalence of mutations within RBP-encoding genes being implicated in infertility and neurological disease. We previously described roles for the highly conserved RBP Caper in neurite morphogenesis in the Drosophila larval peripheral system and in locomotor behavior. However, caper function has not been investigated outside the nervous system, although it is widely expressed in many different tissue types during embryogenesis. Here, we describe novel roles for Caper in fertility and mating behavior. We find that Caper is expressed in ovarian follicles throughout oogenesis but is dispensable for proper patterning of the egg chamber. Additionally, reduced caper function, through either a genetic lesion or RNA interference-mediated knockdown of caper in the female germline, results in females laying significantly fewer eggs than their control counterparts. Moreover, this phenotype is exacerbated with age. caper dysfunction also results in partial embryonic and larval lethality. Given that caper is highly conserved across metazoa, these findings may also be relevant to vertebrates.

Gene expression and alternative splicing dynamics are perturbed in female head transcriptomes following heterospecific copulation.

BACKGROUND: Despite the growing interest in the female side of copulatory interactions, the roles played by differential expression and alternative splicing mechanisms of pre-RNA on tissues outside of the reproductive tract have remained largely unknown. Here we addressed these questions in the context of con- vs heterospecific matings between Drosophila mojavensis and its sister species, D. arizonae. We analyzed transcriptional responses in female heads using an integrated investigation of genome-wide patterns of gene expression, including differential expression (DE), alternative splicing (AS) and intron retention (IR). RESULTS: Our results indicated that early transcriptional responses were largely congruent between con- and heterospecific matings but are substantially perturbed over time. Conspecific matings induced functional pathways related to amino acid balance previously associated with the brain's physiology and female postmating behavior. Heterospecific matings often failed to activate regulation of some of these genes and induced expression of additional genes when compared with those of conspecifically-mated females. These mechanisms showed functional specializations with DE genes mostly linked to pathways of proteolysis and nutrient homeostasis, while AS genes were more related to photoreception and muscle assembly pathways. IR seems to play a more general role in DE regulation during the female postmating response. CONCLUSIONS: We provide evidence showing that AS genes substantially perturbed by heterospecific matings in female heads evolve at slower evolutionary rates than the genome background. However, DE genes evolve at evolutionary rates similar, or even higher, than those of male reproductive genes, which highlights their potential role in sexual selection and the evolution of reproductive barriers.

The conserved alternative splicing factor caper regulates neuromuscular phenotypes during development and aging.

RNA-binding proteins play an important role in the regulation of post-transcriptional gene expression throughout the nervous system. This is underscored by the prevalence of mutations in genes encoding RNA splicing factors and other RNA-binding proteins in a number of neurodegenerative and neurodevelopmental disorders. The highly conserved alternative splicing factor Caper is widely expressed throughout the developing embryo and functions in the development of various sensory neural subtypes in the Drosophila peripheral nervous system. Here we find that caper dysfunction leads to aberrant neuromuscular junction morphogenesis, as well as aberrant locomotor behavior during larval and adult stages. Despite its widespread expression, our results indicate that caper function is required to a greater extent within the nervous system, as opposed to muscle, for neuromuscular junction development and for the regulation of adult locomotor behavior. Moreover, we find that Caper interacts with the RNA-binding protein Fmrp to regulate adult locomotor behavior. Finally, we show that caper dysfunction leads to various phenotypes that have both a sex and age bias, both of which are commonly seen in neurodegenerative disorders in humans.

Comparative analysis of microbiota along the length of the gastrointestinal tract of two tree squirrel species (Sciurus aberti and S. niger) living in sympatry.

Microbiota inhabiting the gastrointestinal (GI) tract of animals has important impacts on many host physiological processes. Although host diet is a major factor influencing the composition of the gut micro-organismal community, few comparative studies have considered how differences in diet influence community composition across the length of the GI tract. We used 16S sequencing to compare the microbiota along the length of the GI tract in Abert's (Sciurus aberti) and fox squirrels (S. niger) living in the same habitat. While fox squirrels are generalist omnivores, the diet of Abert's squirrels is unusually high in plant fiber, particularly in winter when they extensively consume fiber-rich inner bark of ponderosa pine (Pinus ponderosa). Consistent with previous studies, microbiota of the upper GI tract of both species consisted primarily of facultative anaerobes and was less diverse than that of the lower GI tract, which included mainly obligate anaerobes. While we found relatively little differentiation between the species in the microbiota of the upper GI tract, the community composition of the lower GI tract was clearly delineated. Notably, the Abert's squirrel lower GI community was more stable in composition and enriched for microbes that play a role in the degradation of plant fiber. In contrast, overall microbial diversity was higher in fox squirrels. We hypothesize that these disparities reflect differences in diet quality and diet breadth between the species.

Genome-wide markers reveal a complex evolutionary history involving divergence and introgression in the Abert's squirrel (Sciurus aberti) species group.

BACKGROUND: Genetic introgression between divergent lineages is now considered more common than previously appreciated, with potentially important consequences for adaptation and speciation. Introgression is often asymmetric between populations and patterns can vary for different types of loci (nuclear vs. organellar), complicating phylogeographic reconstruction. The taxonomy of the ecologically specialized Abert's squirrel species group has been controversial, and previous studies based on mitochondrial data have not fully resolved the evolutionary relationships among populations. Moreover, while these studies identified potential areas of secondary contact between divergent lineages, the possibility for introgression has not been tested. RESULTS: We used RAD-seq to unravel the complex evolutionary history of the Abert's squirrel species group. Although some of our findings reinforce inferences based on mitochondrial data, we also find significant areas of discordance. Discordant signals generally arise from previously undetected introgression between divergent populations that differentially affected variation at mitochondrial and nuclear loci. Most notably, our results support earlier claims (disputed by mitochondrial data) that S. aberti kaibabensis, found only on the north rim of the Grand Canyon, is highly divergent from other populations. However, we also detected introgression of S. aberti kaibabensis DNA into other S. aberti populations, which likely accounts for the previously inferred close genetic relationship between this population and those south of the Grand Canyon. CONCLUSIONS: Overall, the evolutionary history of Abert's squirrels appears to be shaped largely by divergence during periods of habitat isolation. However, we also found evidence for interbreeding during periods of secondary contact resulting in introgression, with variable effects on mitochondrial and nuclear markers. Our results support the emerging view that populations often diversify under scenarios involving both divergence in isolation and gene flow during secondary contact, and highlight the value of genome-wide datasets for resolving such complex evolutionary histories.

The RNA-binding protein caper is required for sensory neuron development in Drosophila melanogaster.

BACKGROUND: Alternative splicing mediated by RNA-binding proteins (RBPs) is emerging as a fundamental mechanism for the regulation of gene expression. Alternative splicing has been shown to be a widespread phenomenon that facilitates the diversification of gene products in a tissue-specific manner. Although defects in alternative splicing are rooted in many neurological disorders, only a small fraction of splicing factors have been investigated in detail. RESULTS: We find that the splicing factor Caper is required for the development of multiple different mechanosensory neuron subtypes at multiple life stages in Drosophila melanogaster. Disruption of Caper function causes defects in dendrite morphogenesis of larval dendrite arborization neurons and neuronal positioning of embryonic proprioceptors, as well as the development and maintenance of adult mechanosensory bristles. Additionally, we find that Caper dysfunction results in aberrant locomotor behavior in adult flies. Transcriptome-wide analyses further support a role for Caper in alternative isoform regulation of genes that function in neurogenesis. CONCLUSIONS: Our results provide the first evidence for a fundamental and broad requirement for the highly conserved splicing factor Caper in the development and maintenance of the nervous system and provide a framework for future studies on the detailed mechanism of Caper-mediated RNA regulation. Developmental Dynamics 246:610-624, 2017. © 2017 Wiley Periodicals, Inc.

Transcriptional variation associated with cactus host plant adaptation in Drosophila mettleri populations.

Although the importance of host plant chemistry in plant-insect interactions is widely accepted, the genetic basis of adaptation to host plants is not well understood. Here, we investigate transcriptional changes associated with a host plant shift in Drosophila mettleri. While D. mettleri is distributed mainly throughout the Sonoran Desert where it specializes on columnar cacti (Carnegiea gigantea and Pachycereus pringleii), a population on Santa Catalina Island has shifted to chemically divergent coastal prickly pear cactus (Opuntia littoralis). We compared gene expression of larvae from the Sonoran Desert and Santa Catalina Island when reared on saguaro (C. gigantea), coastal prickly pear and laboratory food. Consistent with expectations based on the complexity and toxicity of cactus relative to laboratory food, within-population comparisons between larvae reared on these food sources revealed transcriptional differences in detoxification and other metabolic pathways. The majority of transcriptional differences between populations on the cactus hosts were independent of the rearing environment and included a disproportionate number of genes involved in processes relevant to host plant adaptation (e.g. detoxification, central metabolism and chemosensory pathways). Comparisons of transcriptional reaction norms between the two populations revealed extensive shared plasticity that likely allowed colonization of coastal prickly pear on Santa Catalina Island. We also found that while plasticity may have facilitated subsequent adaptive divergence in gene expression between populations, the majority of genes that differed in expression on the novel host were not transcriptionally plastic in the presumed ancestral state.

Connecting genotypes, phenotypes and fitness: harnessing the power of CRISPR/Cas9 genome editing.

One of the fundamental goals in evolution and ecology is to identify the genetic basis of adaptive phenotypes. Unfortunately, progress towards this goal has been hampered by a lack of genetic tools available for nonmodel organisms. The exciting new development of the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated nuclease 9) genome-editing system now promises to transform the field of molecular ecology by providing a versatile toolkit for manipulating the genome of a wide variety of organisms. Here, we review the numerous applications of this groundbreaking technology and provide a practical guide to the creation of genetic knockouts, transgenics and other related forms of gene manipulation in nonmodel organisms. We also specifically discuss the potential uses of the CRISPR/Cas9 system in ecological and evolutionary studies, which will further advance the field towards the long-standing goal of connecting genotypes, phenotypes and fitness.

Host plant adaptation in Drosophila mettleri populations.

The process of local adaptation creates diversity among allopatric populations, and may eventually lead to speciation. Plant-feeding insect populations that specialize on different host species provide an excellent opportunity to evaluate the causes of ecological specialization and the subsequent consequences for diversity. In this study, we used geographically separated Drosophila mettleri populations that specialize on different host cacti to examine oviposition preference for and larval performance on an array of natural and non-natural hosts (eight total). We found evidence of local adaptation in performance on saguaro cactus (Carnegiea gigantea) for populations that are typically associated with this host, and to chemically divergent prickly pear species (Opuntia spp.) in a genetically isolated population on Santa Catalina Island. Moreover, each population exhibited reduced performance on the alternative host. This finding is consistent with trade-offs associated with adaptation to these chemically divergent hosts, although we also discuss alternative explanations for this pattern. For oviposition preference, Santa Catalina Island flies were more likely to oviposit on some prickly pear species, but all populations readily laid eggs on saguaro. Experiments with non-natural hosts suggest that factors such as ecological opportunity may play a more important role than host plant chemistry in explaining the lack of natural associations with some hosts.

Postmating transcriptional changes in reproductive tracts of con- and heterospecifically mated Drosophila mojavensis females.

In internally fertilizing organisms, mating involves a series of highly coordinated molecular interactions between the sexes that occur within the female reproductive tract. In species where females mate multiply, traits involved in postcopulatory interactions are expected to evolve rapidly, potentially leading to postmating-prezygotic (PMPZ) reproductive isolation between diverging populations. Here, we investigate the postmating transcriptional response of the lower reproductive tract of Drosophila mojavensis females following copulation with either conspecific or heterospecific (Drosophila arizonae) males at three time points postmating. Relatively few genes (15 total) were differentially regulated in the female lower reproductive tract in response to conspecific mating. Heterospecifically mated females exhibited significant perturbations in the expression of the majority of these genes, and also down-regulated transcription of a number of others, including several involved in mitochondrial function. These striking regulatory differences indicate failed postcopulatory molecular interactions between the sexes consistent with the strong PMPZ isolation observed for this cross. We also report the transfer of male accessory-gland protein (Acp) transcripts from males to females during copulation, a finding with potentially broad implications for understanding postcopulatory molecular interactions between the sexes.

Post-zygotic isolation in cactophilic Drosophila: larval viability and adult life-history traits of D. mojavensis/D. arizonae hybrids.

Drosophila mojavensis and Drosophila arizonae are cactophilic flies that have been used extensively in speciation studies. Incomplete premating isolation, evidence of reinforcement, and a lack of recent introgression between these species point to a potentially important role for post-zygotic isolating barriers in this system. Other than hybrid male sterility, however, post-zygotic isolation between D. mojavensis and D. arizonae has received little attention. In this study, we examined viability and life-history traits of D. mojavensis/D. arizonae F(1) hybrids from sympatric crosses. Specifically, we reared hybrids and purebreds on the natural host cacti of each parental species and compared viability, development time, thorax length, and desiccation resistance between hybrids and purebreds. Interestingly, hybrid females from both crosses performed similarly or even better than purebred females. In contrast, hybrid sons of D. arizonae mothers, in addition to being sterile, had shorter average thorax length than males of both parental species, and hybrid males from both crosses had substantially lower desiccation resistance than D. mojavensis males. The probable cost to hybridization for D. mojavensis females resulting from reduced desiccation resistance of hybrid sons may have been an important selective factor in the history of reinforcement for crosses involving these females.

Molecular evolution and population genetics of two Drosophila mettleri cytochrome P450 genes involved in host plant utilization.

Understanding the genetic basis of adaptation is one of the primary goals of evolutionary biology. The evolution of xenobiotic resistance in insects has proven to be an especially suitable arena for studying the genetics of adaptation, and resistant phenotypes are known to result from both coding and regulatory changes. In this study, we examine the evolutionary history and population genetics of two Drosophila mettleri cytochrome P450 genes that are putatively involved in the detoxification of alkaloids present in two of its cactus hosts: saguaro (Carnegiea gigantea) and senita (Lophocereus schottii). Previous studies demonstrated that Cyp28A1 was highly up-regulated following exposure to rotting senita tissue while Cyp4D10 was highly up-regulated following exposure to rotting saguaro tissue. Here, we show that a subset of sites in Cyp28A1 experienced adaptive evolution specifically in the D. mettleri lineage. Moreover, neutrality tests in several populations were also consistent with a history of selection on Cyp28A1. In contrast, we did not find evidence for positive selection on Cyp4D10, although this certainly does not preclude its involvement in host plant use. A surprising result that emerged from our population genetic analyses was the presence of significant genetic differentiation between flies collected from different host plant species (saguaro and senita) at Organ Pipe National Monument, Arizona, USA. This preliminary evidence suggests that D. mettleri may have evolved into distinctive host races that specialize on different hosts, a possibility that warrants further investigation.

Cofoundress relatedness and group productivity in colonies of social Dunatothrips (Insecta: Thysanoptera) on Australian Acacia.

Facultative joint colony founding by social insects provides opportunities to analyze the roles of genetic and ecological factors in the evolution of cooperation. Although cooperative nesting is observed in range of social insect taxa, the most detailed studies of this behavior have been conducted with Hymenoptera (ants, bees, and wasps). Here, we show that foundress associations in the haplodiploid social thrips Dunatothrips aneurae (Insecta: Thysanoptera) are most often comprised of close relatives (sisters), though groups with unrelated foundresses are also found. Associations among relatives appear to be facilitated by limited female dispersal, which results in viscous population structure. In addition, we found that per capita productivity declined with increasing group size, sex ratios were female-biased, and some female offspring apparently remained in their natal domicile for some time following eclosion. D. aneurae thus exhibits a suite of similarities with eusocial Hymenoptera, providing evidence for the convergent evolution of associated social and life-history traits in Hymenoptera and Thysanoptera.