Project description:In Drosophila melanogaster, mating radically transforms female physiology and behavior. Post-mating responses include an increase in the oviposition rate, a reduction in female receptivity, and an activation of the immune system . The fitness consequences of mating are similarly dramatic – females must mate once in order to produce fertile eggs, but additional matings have a clear negative effect. Previously, microarrays have been used to examine gene expression of females differing in their reproductive status with the aim of identifying genes influenced by mating. However, since only virgin and single mated females were compared, transcriptional changes associated with reproduction (under natural selection) and the effects of male-induced harm (under sexually antagonistic selection) cannot be disentangled. We partitioned these fundamentally different effects by instead examining the expression profiles of virgin, single mated and double mated females. We found substantial effects relating to reproduction and further effects that are only attributable to a second mating. Immune response genes dominate this male-induced harm effect indicating that the cost of mating may be due partly to this system's activation. We propose that both sexually antagonistic and natural selection have been important in the evolution of the innate immunity genes, thereby contributing to the sexual dimorphismand rapid evolution at these loci. Keywords: Female response to mating
Project description:In Drosophila melanogaster, mating radically transforms female physiology and behavior. Post-mating responses include an increase in the oviposition rate, a reduction in female receptivity, and an activation of the immune system . The fitness consequences of mating are similarly dramatic – females must mate once in order to produce fertile eggs, but additional matings have a clear negative effect. Previously, microarrays have been used to examine gene expression of females differing in their reproductive status with the aim of identifying genes influenced by mating. However, since only virgin and single mated females were compared, transcriptional changes associated with reproduction (under natural selection) and the effects of male-induced harm (under sexually antagonistic selection) cannot be disentangled. We partitioned these fundamentally different effects by instead examining the expression profiles of virgin, single mated and double mated females. We found substantial effects relating to reproduction and further effects that are only attributable to a second mating. Immune response genes dominate this male-induced harm effect indicating that the cost of mating may be due partly to this system's activation. We propose that both sexually antagonistic and natural selection have been important in the evolution of the innate immunity genes, thereby contributing to the sexual dimorphismand rapid evolution at these loci. Keywords: Female response to mating Female flies were flash frozen in liquid nitrogen either as virgins or 6 hours after mating and stored at -80°C until RNA extraction was performed (not more than 2 days). 8 whole flies – randomly selected within each treatment – were pooled for each extraction. Total RNA was extracted using Trizol (Invitrogen) and purified with an RNeasy Mini Kit (Qiagen). RNA quantity and quality was checked with an Agilent Bioanalyzer. According to the manufacturer's instructions, samples were prepared and hybridized to Affymetrix GeneChip Drosophila Genome 2.0 (Affymetrix, Santa Clara, CA, USA) by the Uppsala Array Platform (Uppsala, Sweden). Each experimental treatment consisted of 4 independent RNA extractions and hybridizations, giving a total of 12 arrays.
Project description:Sex Peptide, a seminal fluid protein of Drosophila melanogaster males, elicits an array of post-mating responses in females, including decreased receptivity to re-mating, and increased egg laying, activity and food intake, with a preference for protein-rich food. To determine how one protein can have such widespread effects, we set out to dissect the genetic architecture of the female’s response to Sex Peptide, to determine whether Sex Peptide alters the expression of several regulators targeted to specific post-mating responses or acts on a pleiotropic regulator that controls multiple responses. We performed bulk RNA-seq of female heads at 10 time points within the first 24 hours after mating, sampling virgin females, females mated to control males and females mated to Sex Peptide-null males. Using this high-resolution time series, we identified mating- and Sex Peptide-dependent differentially expressed genes and discovered the presence of differentially used exons. We constructed gene regulatory networks using clustering and motif enrichment analyses, and identified cell types in which these changes might take place using deconvolution of our bulk RNA-seq dataset. One key network included metabolic genes which might change in expression in the female’s fat body. A second network included genes with neuronal functions, whose changes might be located in neurons or sensory organs in the female’s head. Within these networks we identified known molecular regulators of the circadian clock. Further, we found that many differentially expressed genes, and some differentially used exons, followed a circadian rhythm in virgin females, and that this rhythm was altered after mating with a Sex Peptide- male.
Project description:Seminal fluid contains some of the fastest evolving proteins currently known. These seminal fluid proteins (Sfps) play crucial roles in reproduction, such as supporting sperm function, and – particularly in insects – modifying female physiology and behaviour. Identification of Sfps in small animals is challenging, and often relies on samples taken from the female reproductive tract after mating. A key pitfall of this method is that it might miss Sfps that are of low abundance due to dilution in the female-derived sample or rapid processing in females. Here we present a new and complimentary method, which provides added sensitivity to Sfp identification. We applied label-free quantitative proteomics to Drosophila melanogaster male reproductive tissue – where Sfps are unprocessed, and highly abundant – and quantified Sfps before and immediately after mating, to infer those transferred during copulation. We also analysed female reproductive tracts immediately before and after copulation to confirm the presence and abundance of known and candidate Sfps, where possible. Results were cross-referenced with transcriptomic and sequence databases to improve confidence in Sfp detection. Our data was consistent with 124 previously reported Sfps. We found 8 high-confidence novel candidate Sfps, which were both depleted in mated versus unmated males and identified within the reproductive tract of mated but not virgin females. We also identified 31 more candidates that are likely Sfps based on their abundance, known expression and predicted characteristics, and revealed that four proteins previously identified as Sfps are at best minor contributors to the ejaculate. The estimated copy numbers for our candidate Sfps were lower than for previously identified Sfps, supporting the idea that our technique provides a deeper analysis of the Sfp proteome than previous studies. Our results demonstrate a novel, high-sensitivity approach to the analysis of seminal fluid proteomes, whose application will further our understanding of reproductive biology.
Project description:Heat shock proteins (Hsps), in particular Hsp70, play a central role in proteostasis in eukaryotic cells. Due to its chaperone properties, Hsp70 is involved in various processes both after stress and under normal physiological conditions. In contrast to mammals and many Diptera species, members of the Hsp70 family in Drosophila are constitutively synthesized at a low level and undergo dramatic induction after temperature elevation or other forms of stress. In the courtship suppression paradigm used in this study, Drosophila males that have been repeatedly rejected by mated females during courtship are less likely than naive males to court other females. Although numerous genes with known function were identified to play important roles in long-term memory, there is, to the best of our knowledge, no direct evidence implicating Hsp70 in this process. To eliminate this gap, we used D. melanogaster strains containing different hsp70 copy numbers, including strains carrying a deletion of all six hsp70 genes. Our investigations exploring the memory of courtship rejection paradigm demonstrated that a low constitutive level of Hsp70 is required for learning and the formation of short and long-term memories in males. The performed transcriptomic studies demonstrate that males with different hsp70 copy numbers differ significantly in the expression of a few definite groups of genes involved in mating and reproduction, as well as methionine metabolism and immune genes in response to rejection. Specifically, our analysis reveals several major pathways that depend on the presence of hsp70 copies in the genome and apparently participate in memory formation and consolidation, including the cAMP signalling cascade.
Project description:Purpose: Mating induces a multitude of changes in female behavior, physiology and gene expression. Interactions between female and male genotype lead to variation in post-mating phenotypes and reproductive success. So far, few female molecules responsible for these interactions have been identified. Methods: We used Drosophila melanogaster from five geographically dispersed populations to investigate such female x male genotypic interactions at the female transcriptomic and phenotypic levels. Methods: Females from each line were singly-mated to males from the same five lines, for a total of 25 combinations. To assess whether female x male genotypic interactions affect the female post-mating transcriptome, next-generation RNA sequencing was performed on virgin and mated females at 5 to 6 hours post-mating. Results: Seventy-seven genes showed strong variation in mating-induced expression changes in a female x male genotype-dependent manner. These genes were enriched for immune response and odorant-binding functions, and for expression exclusively in the head. Conclusions: The transcriptional variation found in specific functional classes of genes might be a read-out of female x male compatibility at a molecular level. Understanding the roles these genes play in the female post-mating response will be crucial to better understand the evolution of post-mating responses and related conflicts between the sexes.
Project description:By combining an experimental evolution approach with genomic techniques, we investigated the effects of seminal fluid on female gene expression. In our study, we experimentally manipulated the mating system in replicate populations of D. melanogaster, by removing post-copulatory sexual selection, with the aim of testing differences in short term post-mating reaction of females evolved under different mating strategies. We show that monogamous females suffer decreased fecundity, regardless of the type of male they were mated with, and that their post-mating gene expression profiles differ significantly from promiscuous females, involving 1141 transcripts (9% of the genes tested). These transcripts are active in several tissues, mainly ovaries, neural tissues, midgut and spermathecae, and are involved in metabolic processes, reproduction and signaling pathways. Our results provide a list of candidate genes responsible for the decrease in female fecundity in the absence of post-copulatory sexual selection, and demonstrate how the female post-mating response can evolve under different mating systems over relatively short time frames. From an LHM base population, we created 8 replicate populations and maintained them under experimental evolution: 4 populations were allowed to mate only once every generation (monogamy), and the other 4 were kept under the standard mating protocol (promiscuous). After 46 generations, we crossed males and females within the same population and with individuals of the opposite treatment. Mated female flies were frozen 6 h after mating and RNA extracted. Two biological replicates per cross per population (2x2x8=32 samples).
Project description:Seminal fluid plays an essential role in promoting male reproductive success and modulating female physiology and behaviour. In the fruit fly, Drosophila melanogaster, Sex Peptide (SP) is the best-characterised protein mediator of these effects. It is secreted from the paired male accessory glands (AGs), which, like the mammalian prostate and seminal vesicles, generate most of the seminal fluid contents. After mating, SP binds to spermatozoa and is retained in the female sperm storage organs. It is gradually released by proteolytic cleavage and induces several long-term post-mating responses including increased ovulation, elevated feeding and reduced receptivity to remating, primarily signalling through the SP receptor (SPR). We demonstrate a previously unsuspected SPR-independent function for SP. We show that, in the AG lumen, SP and secreted proteins with membrane-binding anchors are carried on abundant, large neutral lipid-containing microcarriers, also found in other SP-expressing Drosophila species. These microcarriers are transferred to females during mating, where they rapidly disassemble. Remarkably, SP is a key microcarrier assembly and disassembly factor. Its absence leads to major changes in the seminal proteome transferred to females upon mating. Males expressing non-functional SP mutant proteins that affect SP binding to and release from sperm in females also do not produce normal microcarriers, suggesting that this male-specific defect contributes to the resulting widespread abnormalities in ejaculate function. Our data reveal a novel role for SP in formation of seminal macromolecular assemblies, which may explain the presence of SP in Drosophila species that lack the signalling functions seen in D. melanogaster. In this experiment we assessed the effect of SP loss-of-function on the transferred seminal proteome.