Microguards and micromessengers of the genome

The regulation of gene expression is of fundamental importance to maintain organismal function and integrity and requires a multifaceted and highly ordered sequence of events. The cyclic nature of gene expression is known as ‘transcription dynamics’. Disruption or perturbation of these dynamics can result in significant fitness costs arising from genome instability, accelerated ageing and disease. We review recent research that supports the idea that an important new role for small RNAs, particularly microRNAs (miRNAs), is in protecting the genome against short-term transcriptional fluctuations, in a process we term ‘microguarding’. An additional emerging role for miRNAs is as ‘micromessengers’—through alteration of gene expression in target cells to which they are trafficked within microvesicles. We describe the scant but emerging evidence that miRNAs can be moved between different cells, individuals and even species, to exert biologically significant responses. With these two new roles, miRNAs have the potential to protect against deleterious gene expression variation from perturbation and to themselves perturb the expression of genes in target cells. These interactions between cells will frequently be subject to conflicts of interest when they occur between unrelated cells that lack a coincidence of fitness interests. Hence, there is the potential for miRNAs to represent both a means to resolve conflicts of interest, as well as instigate them. We conclude by exploring this conflict hypothesis, by describing some of the initial evidence consistent with it and proposing new ideas for future research into this exciting topic

The Drosophila melanogaster Seminal Fluid Protease “Seminase” Regulates Proteolytic and Post-Mating Reproductive Processes

Proteases and protease inhibitors have been identified in the ejaculates of animal taxa ranging from invertebrates to mammals and form a major protein class among Drosophila melanogaster seminal fluid proteins (SFPs). Other than a single protease cascade in mammals that regulates seminal clot liquefaction, no proteolytic cascades (i.e. pathways with at least two proteases acting in sequence) have been identified in seminal fluids. In Drosophila, SFPs are transferred to females during mating and, together with sperm, are necessary for the many post-mating responses elicited in females. Though several SFPs are proteolytically cleaved either during or after mating, virtually nothing is known about the proteases involved in these cleavage events or the physiological consequences of proteolytic activity in the seminal fluid on the female. Here, we present evidence that a protease cascade acts in the seminal fluid of Drosophila during and after mating. Using RNAi to knock down expression of the SFP CG10586, a predicted serine protease, we show that it acts upstream of the SFP CG11864, a predicted astacin protease, to process SFPs involved in ovulation and sperm entry into storage. We also show that knockdown of CG10586 leads to lower levels of egg laying, higher rates of sexual receptivity to subsequent males, and abnormal sperm usage patterns, processes that are independent of CG11864. The long-term phenotypes of females mated to CG10586 knockdown males are similar to those of females that fail to store sex peptide, an important elicitor of long-term post-mating responses, and indicate a role for CG10586 in regulating sex peptide. These results point to an important role for proteolysis among insect SFPs and suggest that protease cascades may be a mechanism for precise temporal regulation of multiple post-mating responses in females

Divergence in transcriptional and regulatory responses to mating in male and female fruitflies

Mating induces extensive physiological, biochemical and behavioural changes in female animals of many taxa. In contrast, the overall phenotypic and transcriptomic consequences of mating for males, hence how they might differ from those of females, are poorly described. Post mating responses in each sex are rapidly initiated, predicting the existence of regulatory mechanisms in addition to transcriptional responses involving de novo gene expression. That post mating responses appear different for each sex also predicts that the genome-wide signatures of mating should show evidence of sex-specific specialisation. In this study, we used high resolution RNA sequencing to provide the first direct comparisons of the transcriptomic responses of male and female Drosophila to mating, and the first comparison of mating-responsive miRNAs in both sexes in any species. As predicted, the results revealed the existence of sex- and body part-specific mRNA and miRNA expression profiles. More genes were differentially expressed in the female head-thorax than the abdomen following mating, whereas the opposite was true in males. Indeed, the transcriptional profile of male head-thorax tissue was largely unaffected by mating, and no differentially expressed genes were detected at the most stringent significance threshold. A subset of ribosomal genes in females were differentially expressed in both body parts, but in opposite directions, consistent with the existence of body part-specific resource allocation switching. Novel, mating-responsive miRNAs in each sex were also identified, and a miRNA-mRNA interactions analysis revealed putative targets among mating-responsive genes. We show that the structure of genome-wide responses by each sex to mating is strongly divergent, and provide new insights into how shared genomes can achieve characteristic distinctiveness

Male Mating Rate Is Constrained by Seminal Fluid Availability in Bedbugs, Cimex lectularius

Sexual selection, differences in reproductive success between individuals, continues beyond acquiring a mating partner and affects ejaculate size and composition (sperm competition). Sperm and seminal fluid have very different roles in sperm competition but both components encompass production costs for the male. Theoretical models predict that males should spend ejaculate components prudently and differently for sperm and seminal fluid but empirical evidence for independent variation of sperm number and seminal fluid volume is scarce. It is also largely unknown how sperm and seminal fluid variation affect future mating rate. In bedbugs we developed a protocol to examine the role of seminal fluids in ejaculate allocation and its effect on future male mating rate. Using age-related changes in sperm and seminal fluid volume we estimated the lowest capacity at which mating activity started. We then showed that sexually active males allocate 12% of their sperm and 19% of their seminal fluid volume per mating and predicted that males would be depleted of seminal fluid but not of sperm. We tested (and confirmed) this prediction empirically. Finally, the slightly faster replenishment of seminal fluid compared to sperm did not outweigh the faster decrease during mating. Our results suggest that male mating rate can be constrained by the availability of seminal fluids. Our protocol might be applicable to a range of other organisms. We discuss the idea that economic considerations in sexual conflict research might benefit from distinguishing between costs and benefits that are ejaculate dose-dependent and those that are frequency-dependent on the mating rate per se

Sperm-Storage Defects and Live Birth in Drosophila Females Lacking Spermathecal Secretory Cells

Transgenic Drosophila are used to identify the functions of a small set of secretory cells that are typically associated with the sperm-storage organs of female insects

Expression in Antennae and Reproductive Organs Suggests a Dual Role of an Odorant-Binding Protein in Two Sibling Helicoverpa Species

Odorant-binding proteins (OBPs) mediate both perception and release of semiochemicals in insects. These proteins are the ideal targets for understanding the olfactory code of insects as well as for interfering with their communication system in order to control pest species. The two sibling Lepidopteran species Helicoverpa armigera and H. assulta are two major agricultural pests. As part of our aim to characterize the OBP repertoire of these two species, here we focus our attention on a member of this family, OBP10, particularly interesting for its expression pattern. The protein is specifically expressed in the antennae of both sexes, being absent from other sensory organs. However, it is highly abundant in seminal fluid, is transferred to females during mating and is eventually found on the surface of fertilised eggs. Among the several different volatile compounds present in reproductive organs, OBP10 binds 1-dodecene, a compound reported as an insect repellent. These results have been verified in both H. armigera and H. assulta with no apparent differences between the two species. The recombinant OBP10 binds, besides 1-dodecene, some linear alcohols and several aromatic compounds. The structural similarity of OBP10 with OBP1 of the mosquito Culex quinquefasciatus, a protein reported to bind an oviposition pheromone, and its affinity with 1-dodecene suggest that OBP10 could be a carrier for oviposition deterrents, favouring spreading of the eggs in these species where cannibalism is active among larvae

Transglutaminase-Mediated Semen Coagulation Controls Sperm Storage in the Malaria Mosquito

The mating plug is a key regulator of mosquito fertility

Evolutionary Rate Covariation Identifies New Members of a Protein Network Required for Drosophila melanogaster Female Post-Mating Responses

Seminal fluid proteins transferred from males to females during copulation are required for full fertility and can exert dramatic effects on female physiology and behavior. In Drosophila melanogaster, the seminal protein sex peptide (SP) affects mated females by increasing egg production and decreasing receptivity to courtship. These behavioral changes persist for several days because SP binds to sperm that are stored in the female. SP is then gradually released, allowing it to interact with its female-expressed receptor. The binding of SP to sperm requires five additional seminal proteins, which act together in a network. Hundreds of uncharacterized male and female proteins have been identified in this species, but individually screening each protein for network function would present a logistical challenge. To prioritize the screening of these proteins for involvement in the SP network, we used a comparative genomic method to identify candidate proteins whose evolutionary rates across the Drosophila phylogeny co-vary with those of the SP network proteins. Subsequent functional testing of 18 co-varying candidates by RNA interference identified three male seminal proteins and three female reproductive tract proteins that are each required for the long-term persistence of SP responses in females. Molecular genetic analysis showed the three new male proteins are required for the transfer of other network proteins to females and for SP to become bound to sperm that are stored in mated females. The three female proteins, in contrast, act downstream of SP binding and sperm storage. These findings expand the number of seminal proteins required for SP's actions in the female and show that multiple female proteins are necessary for the SP response. Furthermore, our functional analyses demonstrate that evolutionary rate covariation is a valuable predictive tool for identifying candidate members of interacting protein networks. © 2014 Findlay et al

Dengue Virus Infection of the Aedes aegypti Salivary Gland and Chemosensory Apparatus Induces Genes that Modulate Infection and Blood-Feeding Behavior

The female Aedes aegypti salivary gland plays a pivotal role in bloodmeal acquisition and reproduction, and thereby dengue virus (DENV) transmission. It produces numerous immune factors, as well as immune-modulatory, vasodilatory, and anti-coagulant molecules that facilitate blood-feeding. To assess the impact of DENV infection on salivary gland physiology and function, we performed a comparative genome-wide microarray analysis of the naïve and DENV infection-responsive A. aegypti salivary gland transcriptomes. DENV infection resulted in the regulation of 147 transcripts that represented a variety of functional classes, including several that are essential for virus transmission, such as immunity, blood-feeding, and host-seeking. RNAi-mediated gene silencing of three DENV infection-responsive genes - a cathepsin B, a putative cystatin, and a hypothetical ankyrin repeat-containing protein - significantly modulated DENV replication in the salivary gland. Furthermore, silencing of two DENV infection-responsive odorant-binding protein genes (OBPs) resulted in an overall compromise in blood acquisition from a single host by increasing the time for initiation of probing and the probing time before a successful bloodmeal. We also show that DENV established an extensive infection in the mosquito's main olfactory organs, the antennae, which resulted in changes of the transcript abundance of key host-seeking genes. DENV infection, however, did not significantly impact probing initiation or probing times in our laboratory infection system. Here we show for the first time that the mosquito salivary gland mounts responses to suppress DENV which, in turn, modulates the expression of chemosensory-related genes that regulate feeding behavior. These reciprocal interactions may have the potential to affect DENV transmission between humans

Alternative patterns of sex chromosome differentiation in Aedes aegypti (L).

BACKGROUND: Some populations of West African Aedes aegypti, the dengue and zika vector, are reproductively incompatible; our earlier study showed that divergence and rearrangements of genes on chromosome 1, which bears the sex locus (M), may be involved. We also previously described a proposed cryptic subspecies SenAae (PK10, Senegal) that had many more high inter-sex FST genes on chromosome 1 than did Ae.aegypti aegypti (Aaa, Pai Lom, Thailand). The current work more thoroughly explores the significance of those findings. RESULTS: Intersex standardized variance (FST) of single nucleotide polymorphisms (SNPs) was characterized from genomic exome capture libraries of both sexes in representative natural populations of Aaa and SenAae. Our goal was to identify SNPs that varied in frequency between males and females, and most were expected to occur on chromosome 1. Use of the assembled AaegL4 reference alleviated the previous problem of unmapped genes. Because the M locus gene nix was not captured and not present in AaegL4, the male-determining locus, per se, was not explored. Sex-associated genes were those with FST values ≥ 0.100 and/or with increased expected heterozygosity (H exp , one-sided T-test, p < 0.05) in males. There were 85 genes common to both collections with high inter-sex FST values; all genes but one were located on chromosome 1. Aaa showed the expected cluster of high inter-sex FST genes proximal to the M locus, whereas SenAae had inter-sex FST genes along the length of chromosome 1. In addition, the Aaa M-locus proximal region showed increased H exp levels in males, whereas SenAae did not. In SenAae, chromosomal rearrangements and subsequent suppressed recombination may have accelerated X-Y differentiation. CONCLUSIONS: The evidence presented here is consistent with differential evolution of proto-Y chromosomes in Aaa and SenAae