BackgroundGenes in the sex determination pathway are important regulators of sexually dimorphic animal traits, including the elaborate and exaggerated male ornaments and weapons of sexual selection. In this study, we identified and functionally analyzed members of the sex determination gene family in the golden metallic stag beetle Cyclommatus metallifer, which exhibits extreme differences in mandible size between males and females.ResultsWe constructed a C. metallifer transcriptomic database from larval and prepupal developmental stages and tissues of both males and females. Using Roche 454 pyrosequencing, we generated a de novo assembled database from a total of 1,223,516 raw reads, which resulted in 14,565 isotigs (putative transcript isoforms) contained in 10,794 isogroups (putative identified genes). We queried this database for C. metallifer conserved sex determination genes and identified 14 candidate sex determination pathway genes. We then characterized the roles of several of these genes in development of extreme sexual dimorphic traits in this species. We performed molecular expression analyses with RT-PCR and functional analyses using RNAi on three C. metallifer candidate genes – Sex-lethal (CmSxl), transformer-2 (Cmtra2), and intersex (Cmix). No differences in expression pattern were found between the sexes for any of these three genes. In the RNAi gene-knockdown experiments, we found that only the Cmix had any effect on sexually dimorphic morphology, and these mimicked the effects of Cmdsx knockdown in females. Knockdown of CmSxl had no measurable effects on stag beetle phenotype, while knockdown of Cmtra2 resulted in complete lethality at the prepupal period. These results indicate that the roles of CmSxl and Cmtra2 in the sex determination cascade are likely to have diverged in stag beetles when compared to Drosophila. Our results also suggest that Cmix has a conserved role in this pathway. In addition to those three genes, we also performed a more complete functional analysis of the C. metallifer dsx gene (Cmdsx) to identify the isoforms that regulate dimorphism more fully using exon-specific RNAi. We identified a total of 16 alternative splice variants of the Cmdsx gene that code for up to 14 separate exons. Despite the variation in RNA splice products of the Cmdsx gene, only four protein isoforms are predicted. The results of our exon-specific RNAi indicated that the essential CmDsx isoform for postembryonic male differentiation is CmDsxB, whereas postembryonic female specific differentiation is mainly regulated by CmDsxD.ConclusionsTaken together, our results highlight the importance of studying the function of highly conserved sex determination pathways in numerous insect species, especially those with dramatic and exaggerated sexual dimorphism, because conservation in protein structure does not always translate into conservation in downstream function.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2522-8) contains supplementary material, which is ...

For a given gene, different mutations influence organismal phenotypes to varying degrees. However, the expressivity of these variants not only depends on the DNA lesion associated with the mutation, but also on factors including the genetic background and rearing environment. The degree to which these factors influence related alleles, genes, or pathways similarly, and whether similar developmental mechanisms underlie variation in the expressivity of a single allele across conditions and among alleles is poorly understood. Besides their fundamental biological significance, these questions have important implications for the interpretation of functional genetic analyses, for example, if these factors alter the ordering of allelic series or patterns of complementation. We examined the impact of genetic background and rearing environment for a series of mutations spanning the range of phenotypic effects for both the scalloped and vestigial genes, which influence wing development in Drosophila melanogaster. Genetic background and rearing environment influenced the phenotypic outcome of mutations, including intra-genic interactions, particularly for mutations of moderate expressivity. We examined whether cellular correlates (such as cell proliferation during development) of these phenotypic effects matched the observed phenotypic outcome. While cell proliferation decreased with mutations of increasingly severe effects, surprisingly it did not co-vary strongly with the degree of background dependence. We discuss these findings and propose a phenomenological model to aid in understanding the biology of genes, and how this influences our interpretation of allelic effects in genetic analysis.

It is widely accepted that the gut microbiome can affect various aspects of brain function, includ-2 ing anxiety, depression, learning, and memory. However, we know little about how individual microbial species contribute to communication along the gut-brain axis. Vertebrate microbiomes 4 are comprised of hundreds of species, making it difficult to systematically target individual microbes and their interactions. Here, we use Drosophila melanogaster as a simple model organism 6 to tease apart individual and combined effects of gut microbes on cognition. We used an aversive phototactic suppression assay to show that two dominant gut commensals in our lab stock, 8 Lactobacillus and Acetobacter, are necessary and sufficient for normal learning and short-term memory relative to flies with a conventional microbiome. We also demonstrate that microbes 10 did not affect their hosts' ability to detect the aversive learning stimulus (quinine), suggesting that our results were due to decreased cognition and not sensory deficits. We thus establish 12 Drosophila as a model for elucidating mechanisms of gut-brain communication at the level of individual bacterial species. 14 IntroductionWithin the last decade, there has been an explosion of studies showing that gut microbes affect 16 critical organismal functions, particularly communication along the gut-brain axis [1]. Early studies found a link between the gut microbiota and anxiety [2][3][4], depression [5], learning [6], 18 and memory [7] suggesting that gut microbes impact more than just their immediate surroundings. Since then, other studies have found that disrupting the gut microbiota through diet [7], 20 pathogens [8], antibiotics [9, 10], or probiotics [11-14] can all affect learning. Moreover, disease models for inflammatory bowel disease [15, 16], diabetes [17], and schizophrenia [18] have associ-22ated the loss of homeostasis of the gut community (dysbiosis) with learning and memory deficits in experimental animals. These deficits were ameliorated with probiotic treatment, suggesting 24 that symptoms experienced by patients may be treated with probiotics as well.If probiotic therapy is to become an effective treatment for dysbiosis of the microbial commu-26 nity, we need a better understanding of how individual community members influence cognition alone and in combination. We will then be able to target specific microbes or species combina-28 tions to more effectively address underlying issues in brain function. We know that diversity of the gut microbiota plays a role (some beneficial species have been identified), but it is not fully 30 understood how individual members of the microbiota contribute, because significantly variable communities can nonetheless be healthy [19][20][21]. 32 We chose to test learning and memory in Drosophila, because the microbiota in flies is much simpler than in vertebrates [22], allowing us to generate single and combinatorial microbial as-34 sociations that comprise the bulk of the total microbiota. In this way we can begin ...

42For a given gene, different mutations influence organismal phenotypes to varying 43 degrees. However, the expressivity of these variants not only depends on the DNA lesion 44 associated with the mutation, but also on factors including the genetic background and 45 rearing environment. The degree to which these factors influence related alleles, genes, or 46 pathways similarly, and whether similar developmental mechanisms underlie variation in the 47 expressivity of a single allele across conditions and variation across alleles is poorly 48 understood. Besides their fundamental biological significance, these questions have 49 important implications for the interpretation of functional genetic analyses, for example, if 50 these factors alter the ordering of allelic series or patterns of complementation. We 51 examined the impact of genetic background and rearing environment for a series of 52 mutations spanning the range of phenotypic effects for both the scalloped and vestigial 53 genes, which influence wing development in Drosophila melanogaster. Genetic background 54 and rearing environment influenced the phenotypic outcome of mutations, including intra-55 genic interactions, particularly for mutations of moderate expressivity. We examined whether 56 cellular correlates (such as cell proliferation during development) of these phenotypic effects 57 matched the observed phenotypic outcome. While cell proliferation decreased with 58 mutations of increasingly severe effects, surprisingly it did not co-vary strongly with the 59 degree of background dependence. We discuss these findings and propose a 60 phenomenological model to aid in understanding the biology of genes, and how this 61 influences our interpretation of allelic effects in genetic analysis. 62 63 Author Summary 64 Different mutations in a gene, or in genes with related functions, can have effects of 65 varying severity. Studying sets of mutations and analyzing how they interact are essential 66 components of a geneticist's toolkit. However, the effects caused by a mutation depend not 67 only on the mutation itself, but on additional genetic variation throughout an organism's 68 genome and on the environment that organism has experienced. Therefore, identifying how 69 the genomic and environmental context alter the expression of mutations is critical for 70 making reliable inferences about how genes function. Yet studies on this context 71 dependence have largely been limited to single mutations in single genes. We examined 72 how the genomic and environmental context influence the expression of multiple mutations 73 in two related genes affecting the fruit fly wing. Our results show that the genetic and 74 3 environmental context generally affect the expression of related mutations in similar ways. 75 However, the interactions between two different mutations in a single gene sometimes 76 depended strongly on context. In addition, cell proliferation in the developing wing and adult 77 wing size were not affected by the genetic and environmental context...

Heat resistance of ectotherms can be increased both by plasticity and evolution, but these effects may have trade-offs resulting from biotic interactions. Here, we test for predation costs in Drosophila melanogaster populations with altered heat resistance produced by adult hardening and directional selection for increased heat resistance. In addition, we also tested for genetic trade-offs by testing heat resistance in lines that have evolved under increased predation risk. We show that while 35/37 °C hardening increases heat resistance as expected, it does not increase predation risk from jumping spiders or mantids; in fact, there was an indication that survival may have increased under predation following a triple 37 °C compared to a single 35 °C hardening treatment. Flies that survived a 39 °C selection cycle showed lower survival under predation, suggesting a predation cost of exposure to a more severe heat stress. There was, however, no correlated response to selection because survival did not differ between control and selected lines after selection was relaxed for one or two generations. In addition, lines selected for increased predation risk did not differ in heat resistance. Our findings suggest independent evolutionary responses to predation and heat as measured in laboratory assays, and no costs of heat hardening on susceptibility to predation.