Drosophila Sex Research Articles

Homeotic transformations suggest mechanisms for rapid evolution diversification in Drosophila sex combs.

Evolutionary innovations refer to the emergence of new traits, functions, or behaviors in organisms and lineages over time. Although research has demonstrated that such innovations can arise gradually or through small steps (Chouard 2010), the mechanisms by which rapid morphological diversification takes place remain poorly understood (Bailey et al. 2019). To explore this question, we used the evolution of sex combs, as a system (Ho et al. 2018). We used this male-specific row of leg bristles, comprising sex combs as a system, because it displays spectacular morphological diversification in a short time (Kopp 2011). Homeotic mutations in the fruit fly, Drosophila melanogaster, are those which create modifications in one part of a fly to resemble another region. Here we describe effects of some of these mutations which transform the D. melanogaster fly sex comb morphology to closely resemble sex comb morphology in other species. These findings support previous research indicating that minor alterations to regulatory elements can play a significant role in explaining morphological evolution (Atallah et al. 2004). Thus, our results suggest that rapid diversification may not require starting from scratch, but rather may require minor modifications to the sex comb ground plan, which may account for its rapid morphological evolution (Lee et al. 2011).

Effects of genistein, genistin, and daidzein on preterm Drosophila model and human cervical epithelial cells

Sex hormones play an important role in reproduction. Our previous study confirmed that the ratio of ER and PR was increased in the cervix of mouse preterm birth model. Genistein, genistin, and daidzein have been reported to regulate ER and PR expression. In this study, the effect of genistein, genistin, and daidzein on Drosophila sex hormone level, egg production and gestational period as well as the effect on ER and PR expression in the cervix epithelial cell line ME-180, were investigated.

Drosophila Sex Peptide controls the assembly of lipid microcarriers in seminal fluid

Seminal fluid plays an essential role in promoting male reproductive success and modulating female physiology and behavior. In the fruit fly, Drosophila melanogaster, Sex Peptide (SP) is the best-characterized 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 postmating responses, including increased ovulation, elevated feeding, and reduced receptivity to remating, primarily signaling through the SP receptor (SPR). Here, 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 nonfunctional SP mutant proteins that affect SP's 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 therefore reveal a role for SP in formation of seminal macromolecular assemblies, which may explain the presence of SP in Drosophila species that lack the signaling functions seen in Dmelanogaster.

Drosophila melanogaster sex peptide regulates mated female midgut morphology and physiology

Drosophila melanogaster females experience a large shift in energy homeostasis after mating to compensate for nutrient investment in egg production. To cope with this change in metabolism, mated females undergo widespread physiological and behavioral changes, including increased food intake and altered digestive processes. The mechanisms by which the female digestive system responds to mating remain poorly characterized. Here, we demonstrate that the seminal fluid protein Sex Peptide (SP) is a key modulator of female post-mating midgut growth and gene expression. SP is both necessary and sufficient to trigger post-mating midgut growth in females under normal nutrient conditions, and likely acting via its receptor, Sex Peptide Receptor (SPR). Moreover, SP is responsible for almost the totality of midgut transcriptomic changes following mating, including up-regulation of protein and lipid metabolism genes and down-regulation of carbohydrate metabolism genes. These changes in metabolism may help supply the female with the nutrients required to sustain egg production. Thus, we report a role for SP in altering female physiology to enhance reproductive output: Namely, SP triggers the switch from virgin to mated midgut state.

The structure of the Drosophila melanogaster sex peptide: Identification of hydroxylated isoleucine and a strain variation in the pattern of amino acid hydroxylation

In Drosophila melanogaster mating triggers profound changes in the behaviour and reproductive physiology of the female. Many of these post-mating effects are elicited by sex peptide (SP), a 36-mer pheromone made in the male accessory gland and passed to the female in the seminal fluid. The peptide comprises several structurally and functionally distinct domains, one of which consists of five 4-hydroxyprolines and induces a female immune response. The SP gene predicts an isoleucine (Ile14) sandwiched between two of the hydroxyprolines of the mature secreted peptide, but the identity of this residue was not established by peptide sequencing and amino acid analysis, presumably because of modification of the side chain. Here we have used matrix-assisted laser desorption ionisation mass spectrometry together with Fourier-transform ion cyclotron resonance mass spectrometry to show that Ile14 is modified by oxidation of the side chain - a very unusual post-translational modification. Mass spectrometric analysis of glands from different geographical populations of male D. melanogaster show that SP with six hydroxylated side chains is the most common form of the peptide, but that a sub-strain of Canton-S flies held at Leeds only has two or three hydroxylated prolines and an unmodified Ile14. The D. melanogaster genome has remarkably 17 putative hydroxylase genes that are strongly and almost exclusively expressed in the male accessory gland, suggesting that the gland is a powerhouse of protein oxidation. Strain variation in the pattern of sex peptide hydroxylation might be explained by differences in the expression of individual hydroxylase genes.

Layered roles of fruitless isoforms in specification and function of male aggression-promoting neurons in Drosophila.

Inter-male aggressive behavior is a prominent sexually dimorphic behavior. Neural circuits that underlie aggressive behavior are therefore likely under the control of sex-determining genes. However, the neurogenetic mechanism that generates sex-specific aggressive behavior remains largely unknown. Here, we found that a neuronal class specified by one of the Drosophila sex determining genes, fruitless (fru), belongs to the neural circuit that generates male-type aggressive behavior. This neuronal class can promote aggressive behavior independent of another sex determining gene, doublesex (dsx), although dsx is involved in ensuring that aggressive behavior is performed only toward males. We also found that three fru isoforms with different DNA binding domains show a division of labor on male aggressive behaviors. A dominant role of fru in specifying sex-specific aggressive behavior may underscore a genetic mechanism that allows male-type aggressive behavior to evolve at least partially independently from courtship behavior, which is under different selective pressures.

A Feedforward Circuit Regulates Action Selection of Pre-mating Courtship Behavior in Female Drosophila.

In the early phase of courtship, female fruit flies exhibit an acute rejection response to avoid unfavorable mating. This pre-mating rejection response is evolutionarily paralleled across species, but the molecular and neuronal basis of that behavior is unclear. Here, we show that a putative incoherent feedforward circuit comprising ellipsoid body neurons, cholinergic R4d, and its repressor GABAergic R2/R4m neurons regulates the pre-mating rejection response in the virgin female Drosophila melanogaster. Both R4d and R2/R4m are positively regulated, via specific dopamine receptors, by a subset ofneurons in the dopaminergic PPM3 cluster. Genetic deprivation of GABAergic signal via GABAA receptor RNA interference in this circuit induces a massive rejection response, whereas activation of GABAergic R2/R4m or suppression of cholinergic R4d increases receptivity. Moreover, glutamatergic signaling via N-methyl-d-aspartate receptors induces NO-mediated retrograde regulation potentially from R4d to R2/R4m, likely providing flexible control of the behavioral switching from rejection to acceptance. Our study elucidates the molecular and neural mechanisms regulating the behavioral selection process of the pre-mating female.

Recurrent gene co-amplification on Drosophila X and Y chromosomes.

Y chromosomes often contain amplified genes which can increase dosage of male fertility genes and counteract degeneration via gene conversion. Here we identify genes with increased copy number on both X and Y chromosomes in various species of Drosophila, a pattern that has previously been associated with sex chromosome drive involving the Slx and Sly gene families in mice. We show that recurrent X/Y co-amplification appears to be an important evolutionary force that has shaped gene content evolution of sex chromosomes in Drosophila. We demonstrate that convergent acquisition and amplification of testis expressed gene families are common on Drosophila sex chromosomes, and especially on recently formed ones, and we carefully characterize one putative novel X/Y co-amplification system. We find that co-amplification of the S-Lap1/GAPsec gene pair on both the X and the Y chromosome occurred independently several times in members of the D. obscura group, where this normally autosomal gene pair is sex-linked due to a sex chromosome—autosome fusion. We explore several evolutionary scenarios that would explain this pattern of co-amplification. Investigation of gene expression and short RNA profiles at the S-Lap1/GAPsec system suggest that, like Slx/Sly in mice, these genes may be remnants of a cryptic sex chromosome drive system, however additional transgenic experiments will be necessary to validate this model. Regardless of whether sex chromosome drive is responsible for this co-amplification, our findings suggest that recurrent gene duplications between X and Y sex chromosomes could have a widespread effect on genomic and evolutionary patterns, including the epigenetic regulation of sex chromosomes, the distribution of sex-biased genes, and the evolution of hybrid sterility.

Sex and the Single Fly: A Perspective on the Career of Bruce S. Baker.

Bruce Baker, a preeminent Drosophila geneticist who made fundamental contributions to our understanding of the molecular genetic basis of sex differences, passed away July 1, 2018 at the age of 72. Members of Bruce's laboratory remember him as an intensely dedicated, rigorous, creative, deep-thinking, and fearless scientist. His trainees also remember his strong commitment to teaching students at every level. Bruce's career studying sex differences had three major epochs, where the laboratory was focused on: (1) sex determination and dosage compensation, (2) the development of sex-specific structures, and (3) the molecular genetic basis for sex differences in behavior. Several members of the Baker laboratory have come together to honor Bruce by highlighting some of the laboratory's major scientific contributions in these areas.

Long-term interaction between Drosophila sperm and sex peptide is mediated by other seminal proteins that bind only transiently to sperm

Seminal fluid proteins elicit several post-mating physiological changes in mated Drosophila melanogaster females. Some of these changes persist for over a week after mating because the seminal protein that causes these changes, the Sex Peptide (SP), binds to sperm that are stored in the female reproductive tract. SP's sperm binding is mediated by a network of at least eight seminal proteins. We show here that some of these network proteins (CG1656, CG1652, CG9997 and Antares) bind to sperm within 2 h of mating, like SP. However, while SP remains bound to sperm at 4 days post-mating, none of the other network proteins are detectable at this time. We also observed that the same network proteins are detectable at 2 h post-mating in seminal receptacle tissue from which sperm have been removed, but are no longer detectable there by 4 days post-mating, suggesting short-term retention of these proteins in this female sperm storage organ. Our results suggest that these network proteins act transiently to facilitate the conditions for SP's binding to sperm, perhaps by modifying SP or the sperm surface, but are not part of a long-acting complex that stably attaches SP to sperm.

Peptides in insect oogenesis.

The physiological control of reproduction in insects depends on a combination of environmental and internal cues. In the adult stage, insects become sexually mature and generate gametes. In females, the latter process is designated as oogenesis. Peptides are a versatile class of extracellular signalling molecules that regulate many processes, including oogenesis. At present, the best documented physiological control mechanism of insect oogenesis is the insulin-related peptide signalling pathway. It regulates different stages of the process and provides a functional link between nutritional status and reproduction. Several other peptides have been shown to exert gonadoregulatory activities, but in most cases their exact mode of action still has to be unravelled and their effects on oogenesis could be direct or indirect. Some regulatory peptides, such as the Drosophila sex peptide, are being transferred from the male to the female during the mating process.

HITS-CLIP reveals sex-differential RNA binding and alterative splicing regulation of SRm160 in Drosophila.

Serine/arginine (SR)-rich proteins are critical for the regulation of alternative splicing (AS), which generates multiple mRNA isoforms from one gene and provides protein diversity for cell differentiation and tissue development. Genetic evidence suggests that Drosophila genital-specific overexpression of SR-related nuclear matrix protein of 160 kDa (SRm160), an SR protein with a PWI RNA-binding motif, causes defective development only in male flies and results in abnormal male genital structures and abnormal testis. However, the molecular characterization of SRm160 is limited. Using the high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) method in two sex-specific embryonic cell lines, S2 from the male and Kc from the female, we first identified the genome-wide RNA-binding characteristics of SRm160, which preferred binding to the exonic tri-nucleotide repeats GCA and AAC. We then validated this binding through both in vitro gel-shift assay and in vivo splicing of minigenes and found that SRm160 level affects AS of many transcripts. Furthermore, we identified 492 differential binding sites (DBS) of SRm160 varying between the two sex-specific cell lines. Among these DBS-containing genes, splicing factors were highly enriched, including transformer, a key regulator in the sex determination cascade. Analyses of fly mutants demonstrated that the SRm160 level affects AS isoforms of transformer. These findings shed crucial light on SRm160's RNA-binding specificity and regulation of AS in Drosophila sex determination and development.

Xio is a component of the Drosophila sex determination pathway and RNA N6-methyladenosine methyltransferase complex

N6-methyladenosine (m6A), the most abundant chemical modification in eukaryotic mRNA, has been implicated in Drosophila sex determination by modifying Sex-lethal (Sxl) pre-mRNA and facilitating its alternative splicing. Here, we identify a sex determination gene, CG7358, and rename it xio according to its loss-of-function female-to-male transformation phenotype. xio encodes a conserved ubiquitous nuclear protein of unknown function. We show that Xio colocalizes and interacts with all previously known m6A writer complex subunits (METTL3, METTL14, Fl(2)d/WTAP, Vir/KIAA1429, and Nito/Rbm15) and that loss of xio is associated with phenotypes that resemble other m6A factors, such as sexual transformations, Sxl splicing defect, held-out wings, flightless flies, and reduction of m6A levels. Thus, Xio encodes a member of the m6A methyltransferase complex involved in mRNA modification. Since its ortholog ZC3H13 (or KIAA0853) also associates with several m6A writer factors, the function of Xio in the m6A pathway is likely evolutionarily conserved.

Evolving doublesex expression correlates with the origin and diversification of male sexual ornaments in the Drosophila immigrans species group.

Male ornaments and other sex-specific traits present some of the most dramatic examples of evolutionary innovations. Comparative studies of similar but independently evolved traits are particularly important for identifying repeated patterns in the evolution of these traits. Male-specific modifications of the front legs have evolved repeatedly in Drosophilidae and other Diptera. The best understood of these novel structures is the sex comb of Drosophila melanogaster and its close relatives. Here, we examine the evolution of another male foreleg modification, the sex brush, found in the distantly related Drosophila immigrans species group. Similar to the sex comb, we find that the origin of the sex brush correlates with novel, spatially restricted expression of the doublesex (dsx) transcription factor, the primary effector of the Drosophila sex determination pathway. The diversity of Dsx expression patterns in the immigrans species group closely reflects the differences in the presence, position, and size of the sex brush. Together with previous work on sex comb evolution, these observations suggest that tissue-specific activation of dsx expression may be a common mechanism responsible for the evolution of sexual dimorphism and particularly for the origin of novel male-specific ornaments.

Sexually Antagonistic Male Signals Manipulate Germline and Soma of C. elegans Hermaphrodites

Males and females pursue different reproductive strategies, which often bring them into conflict-many traits exist that benefit one sex at a cost to another [1]. Decreased female survival following mating dramatically demonstrates one aspect of this phenomenon [2-5]. Particularly intriguing is the evidence that secreted compounds can shorten lifespan of members of the opposite sex in Drosophila [6] and Caenorhabditid nematodes [7] even without copulation taking place. The purpose of such signals is not clear, however. While it is possible that they could limit subsequent mating with competitors or hasten post-reproductive demise, thus decreasing competition for resources, they are also likely to harm unmated individuals. Why would a system exist that reduces the vigor of potential mates prior to mating? Addressing this question could provide insights into mechanisms and evolution of sexual conflict and reveal sensory inputs that regulate aging. Here, we describe two distinct ways in which Caenorhabditis elegans males cause faster somatic aging of hermaphrodites but also manipulate different aspects of their reproductive physiology. The first, mediated by conserved ascaroside pheromones, delays the loss of germline progenitor cells. The second accelerates development, resulting in faster sexual maturation. These signals promote male reproductive strategy and the effects harmful to hermaphrodites appear to be collateral damage rather than the goal.

Genetic dissection of intraspecific variation in a male-specific sexual trait in Drosophila melanogaster.

An open question in evolutionary biology is the relationship between standing variation for a trait and the variation that leads to interspecific divergence. By identifying loci underlying phenotypic variation in intra- and interspecific crosses we can determine the extent to which polymorphism and divergence are controlled by the same genomic regions. Sexual traits provide abundant examples of morphological and behavioral diversity within and among species, and here we leverage variation in the Drosophila sex comb to address this question. The sex comb is an array of modified bristles or ‘teeth' present on the male forelegs of several Drosophilid species. Males use the comb to grasp females during copulation, and ablation experiments have shown that males lacking comb teeth typically fail to mate. We measured tooth number in >700 genotypes derived from a multiparental advanced-intercross population, mapping three moderate-effect loci contributing to trait heritability. Two quantitative trait loci (QTLs) coincide with previously identified intra- and interspecific sex comb QTL, but such overlap can be explained by chance alone, in part because of the broad swathes of the genome implicated by earlier, low-resolution QTL scans. Our mapped QTL regions encompass 70–124 genes, but do not include those genes known to be involved in developmental specification of the comb. Nonetheless, we identified plausible candidates within all QTL intervals, and used RNA interference to validate effects at four loci. Notably, TweedleS expression knockdown substantially reduces tooth number. The genes we highlight are strong candidates to harbor segregating, functional variants contributing to sex comb tooth number.

Inbreeding removes sex differences in lifespan in a population of Drosophila melanogaster

Sex differences in ageing rates and lifespan are common in nature, and an enduring puzzle for evolutionary biology. One possibility is that sex-specific mortality rates may result from recessive deleterious alleles in ‘unguarded’ heterogametic X or Z sex chromosomes (the unguarded X hypothesis). Empirical evidence for this is, however, limited. Here, we test a fundamental prediction of the unguarded X hypothesis in Drosophila melanogaster, namely that inbreeding shortens lifespan more in females (the homogametic sex in Drosophila) than in males. To test for additional sex-specific social effects, we studied the lifespan of males and females kept in isolation, in related same-sex groups, and in unrelated same-sex groups. As expected, outbred females outlived outbred males and inbreeding shortened lifespan. However, inbreeding-mediated reductions in lifespan were stronger for females, such that lifespan was similar in inbred females and males. We also show that the social environment, independent of inbreeding, affected male, but not female lifespan. In conjunction with recent studies, the present results suggest that asymmetric inheritance mechanisms may play an important role in the evolution of sex-specific lifespan and that social effects must be considered explicitly when studying these fundamental patterns.

Homology modeling and molecular docking studies of Drosophila and Aedes sex peptide receptors

The Drosophila melanogaster sex peptide receptor (DrmSPR), which is a G protein-coupled receptor (GPCR), is known as the specific receptor for sex peptide (SP). It is responsible for the reproductive behavior in the Drosophila model system; in particular, it is involved in the post-mating responses such as the increase in egg-laying ability and decrease in receptivity in females. In a previous study, we discovered a small molecule agonist of DrmSPR for the first time, which could not, however, activate Aedes aegypti SPR (AedesSPR). To investigate the binding mechanism of the small molecule agonist of DrmSPR, the ensemble structures of low-lying packing structures of DrmSPR and AedesSPR were assembled using the GEnSeMBLE (GPCR Ensemble of Structures in Membrane BiLayer Environment) method. The generated homology models exhibited the typical pattern of inter-helical interactions of the class A GPCRs. The docking experiments of the small molecule agonist suggest that Tyr5.35 and Phe2.67 residues may be involved in a hydrophobic interaction and that Ser3.25 forms a hydrogen bond with the agonist. Additionally, we found that the docking results were consistent with the experimental data of the reference compounds with variable agonistic activities. Moreover, a potential distinction of the putative binding sites in two GPCR models of DrmSPR and AedesSPR, which was determined in this study, can explain the selective action of the agonist for DrmSPR but not for AedesSPR.

The Wright stuff: reimagining path analysis reveals novel components of the sex determination hierarchy in Drosophila melanogaster.

BackgroundThe Drosophila sex determination hierarchy is a classic example of a transcriptional regulatory hierarchy, with sex-specific isoforms regulating morphology and behavior. We use a structural equation modeling approach, leveraging natural genetic variation from two studies on Drosophila female head tissues – DSPR collection (596 F1-hybrids from crosses between DSPR sub-populations) and CEGS population (75 F1-hybrids from crosses between DGRP/Winters lines to a reference strain w1118) – to expand understanding of the sex hierarchy gene regulatory network (GRN). This approach is completely generalizable to any natural population, including humans.ResultsWe expanded the sex hierarchy GRN adding novel links among genes, including a link from fruitless (fru) to Sex-lethal (Sxl) identified in both populations. This link is further supported by the presence of fru binding sites in the Sxl locus. 754 candidate genes were added to the pathway, including the splicing factors male-specific lethal 2 and Rm62 as downstream targets of Sxl which are well-supported links in males. Independent studies of doublesex and transformer mutants support many additions, including evidence for a link between the sex hierarchy and metabolism, via Insulin-like receptor.ConclusionsThe genes added in the CEGS population were enriched for genes with sex-biased splicing and components of the spliceosome. A common goal of molecular biologists is to expand understanding about regulatory interactions among genes. Using natural alleles we can not only identify novel relationships, but using supervised approaches can order genes into a regulatory hierarchy. Combining these results with independent large effect mutation studies, allows clear candidates for detailed molecular follow-up to emerge.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-015-0200-0) contains supplementary material, which is available to authorized users.

Discovery and structure–activity relationships of pyrazolodiazepine derivatives as the first small molecule agonists of the Drosophila sex peptide receptor

In behavioral research, the sex peptide receptor in Drosophila melanogaster (DrmSPR) is the most interesting G protein-coupled receptor (GPCR) and is involved in post-mating responses such as increased egg-laying and decreased receptivity of the female; during these responses, the receptors are activated by a specific natural peptide agonist (sex peptide, SP). To discover small molecule agonists for DrmSPR, a compound library based on a pyrazolodiazepine scaffold, which was previously reported as a potential privileged structure, was screened. Structure–activity relationship (SAR) studies of the hit compounds, which exhibited weak agonistic effects (69–72% activation at 100μM), were explored through the synthesis of various analogs with substituents at the R1, R2, R3 and R4 positions of the pyrazolodiazepine skeleton. As a result, compounds 21 and 31 of the 6-benzyl pyrazolodiazepine derivative series were found to be small molecule agonists for DrmSPR with EC50 values of 3–4μM.