Visualizing Molecular Functions and Cross-Species Activity of Sex-Peptide in Drosophila

The Drosophila melanogaster sex-peptide (melSP) is a seminal fluid component that induces postmating responses (PMR) of females via the sex-peptide receptor (SPR) . Although SP orthologs are found in many Drosophila species, their functions remain poorly characterized. It is unknown whether SP functions are conserved across species or rather specific to each species. Here, we developed a GFP-tagged melSP (G-SP) and used it to visualize cross-species binding activity to the female reproductive system of various species. First we demonstrated that ectopically expressed G-SP induced PMR in D. melanogaster females and bound to the female reproductive system, most notably to the common oviduct. No binding occurred in the females lacking SPR, indicating that G-SP binding was dependent on SPR. Next we tested whether G-SP binds to the common oviducts from 11 Drosophila species using dissected reproductive tracts. The binding was observed in six species belonging to the D. melanogaster species group, but not to those outside the group. Injection of melSP reduced the receptivity of females belonging to the D. melanogaster species group, but not of those outside the group, being consistent with the ability to bind G-SP. Thus the SP-mediated PMR appears to be limited to this species group. SPR was expressed in the oviducts at high levels in this group; therefore, we speculate that an enhanced expression of SPR in the oviduct was critical to establish the SP-mediated PMR during evolution.     

Sex peptide receptor is required for the release of stored sperm by mated Drosophila melanogaster females

The storage of sperm in mated females is important for efficient reproduction. After sperm are transferred to females during mating, they need to reach and enter into the site(s) of storage, be maintained viably within storage, and ultimately be released from storage to fertilize eggs. Perturbation of these events can have drastic consequences on fertility. In Drosophila melanogaster, females store sperm for up to 2 weeks after a single mating. For sperm to be released normally from storage, Drosophila females need to receive the seminal fluid protein (SFP) sex peptide (SP) during mating. SP, which binds to sperm in storage, signals through the sex peptide receptor (SPR) to elicit two other effects on mated females: the persistence of egg laying and a reduction in sexual receptivity. However, it is not known whether SPR is also needed to mediate SP’s effect on sperm release. By phenotypic analysis of flies deleted for SPR, and of flies knocked down for SPR, ubiquitously or in specific tissues, we show that SPR is required to mediate SP’s effects on sperm release from storage. We show that SPR expression in ppk⁺ neurons is needed for proper sperm release; these neurons include those that mediate SP’s effect on receptivity and egg laying. However, we find that SPR is also needed in the spermathecal secretory cells of the female reproductive tract for efficient sperm release. Thus, SPR expression is necessary in both the nervous system and in female reproductive tract cells to mediate the release of stored sperm.     

Reduced female mating receptivity and activation of oviposition in two Callosobruchus species due to injection of biogenic amines

Analyses of proximate mechanisms that control mating and oviposition behaviours in insects are important because they link behavioural ecology and physiology. Recently, seed beetles have been used as models to study evolution of female multiple mating and cost of reproduction including mating. In the present study, we investigated the effects of biogenic amines into the abdomens of females of two Callosobruchus species, Callosobruchus chinensis and Callosobruchus maculatus, on mating receptivity and oviposition behaviour. In C. chinensis, injection of octopamine and tyramine reduced receptivity to mating and tyramine and serotonin increased the number of eggs laid. Similarly, injection of tyramine reduced the receptivity of females and increased the number of eggs laid by females of C. maculatus. These results show the possibility that biogenic amines control mating receptivity and oviposition behaviour in females of two Callosobruchus species.     

MicroRNAs Influence Reproductive Responses by Females to Male Sex Peptide in Drosophila melanogaster

Across taxa, female behavior and physiology change significantly following the receipt of ejaculate molecules during mating. For example, receipt of sex peptide (SP) in female Drosophila melanogaster significantly alters female receptivity, egg production, lifespan, hormone levels, immunity, sleep, and feeding patterns. These changes are underpinned by distinct tissue- and time-specific changes in diverse sets of mRNAs. However, little is yet known about the regulation of these gene expression changes, and hence the potential role of microRNAs (miRNAs), in female postmating responses. A preliminary screen of genomic responses in females to receipt of SP suggested that there were changes in the expression of several miRNAs. Here we tested directly whether females lacking four of the candidate miRNAs highlighted (miR-279, miR-317, miR-278, and miR-184) showed altered fecundity, receptivity, and lifespan responses to receipt of SP, when mated once or continually to SP null or control males. The results showed that miRNA-lacking females mated to SP null males exhibited altered receptivity, but not reproductive output, in comparison to controls. However, these effects interacted significantly with the genetic background of the miRNA-lacking females. No significant survival effects were observed in miRNA-lacking females housed continually with SP null or control males. However, continual exposure to control males that transferred SP resulted in significantly higher variation in miRNA-lacking female lifespan than did continual exposure to SP null males. The results provide the first insight into the effects and importance of miRNAs in regulating postmating responses in females.     

Gradual release of sperm bound sex-peptide controls female postmating behavior in Drosophila

Background: In many female insects, peptides transferred in the seminal fluid induce postmating responses (PMR), such as a drastic increase of egg laying and reduction of receptivity (readiness to mate). In Drosophila melanogaster, sex-peptide (SP) elicits short- and long-term PMR, but only the latter in the presence of stored sperm (sperm effect).Results: Here, we elucidate the interaction between SP and sperm by immunofluorescence microscopy. Transgenic males were used to study the effects of SP modification on the PMR of females in vivo. We report that SP binds to sperm with its N-terminal end. In females, the C-terminal part of SP known to be essential to induce the PMR is gradually released from stored sperm by cleavage at a trypsin cleavage site, thus prolonging the PMR. These findings are confirmed by analyzing the PMR elicited by males containing transgenes encoding modified SPs. SP lacking the N-terminal end cannot bind, and SP without the trypsin cleavage site binds permanently to sperm.Conclusion: By binding to sperm tails, SP prolongs the PMR. Thus, besides a carrier for genetic information, sperm is also the carrier for SP. Binding to sperm may protect the peptide from degradation by proteases in the hemolymph and, thus, prolong its half-life. Longer sperm tails may transfer more SP and thus increase the reproductive fitness of the male. We suggest that this could explain the excessive length of sperm tails in some Drosophila species.     

Mating-induced loss of sex pheromone and sexual receptivity in insects with emphasis on Helicoverpa zea and Lymantria dispar

Mating in most species of insects leads to a transient or permanent loss in sexual receptivity of the females. Among moths, this loss of receptivity is often accompanied with a loss of the sex pheromone in the absence of calling, which also could be temporary or permanent. Most of the earlier work on changes in reproductive behavior after mating was done with Diptera in which sperm and/or male accessory gland secretions were shown to be responsible for termination of receptivity. In the corn earworm moth, Helicoverpa zea, mated females become depleted of pheromone and become nonreceptive to further mating attempts, but only for the remainder of the night of mating. A pheromonostatic peptide isolated from the accessory glands of males may be responsible for the depletion of pheromone, while the termination of receptivity is independently controlled. In the gypsy moth, Lymantria dispar, the changes in behavior following mating are permanent. In this species, the switch from virgin to mated behavior involves three steps: a physical stimulation associated with mating, transfer of viable sperm to the spermatheca, and commencement of oviposition. Signals generated by these factors operate through neural pathways and, unlike in H. zea, accessory gland factors seem not to be involved. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Sex‐peptides bind to two molecularly different targets in Drosophila melanogaster females

Sex‐Peptide (SP) and the peptide DUP99B elicit two postmating responses in Drosophila melanogaster females: receptivity is reduced and oviposition is increased. Both are synthesized in the male genital tract and transferred into the female during copulation. To elucidate their function, we characterized the binding properties of SP and DUP99B in females. Cryostat sections of adult females were incubated with alkaline phosphatase (AP)‐tagged peptides. In virgin females, both peptides have specific target sites in the nervous system and in the genital tract. The binding pattern is almost identical for both peptides. Incubation of sections of mated females confirm that some of these target sites correspond to the in vivo targets of the two peptides. Neuronal binding is dependent on an intact C‐terminal sequence of SP, binding in the genital tract is less demanding in terms of amino acid sequence requirement. On affinity blots the AP–SP probe binds to membrane proteins extracted from abdomen and head plus thorax, respectively. The binding proteins in the nervous system and the genital tract differ in their molecular properties. Calculation of dissociation constants (K_d), and also determination of the minimal peptide concentrations necessary for binding, indicate that SP is the more important peptide inducing the postmating responses. Our results suggest that binding of SP in the nervous system is responsible for eliciting the postmating responses, whereas binding in the genital tract reflects the presence of a peptide transporter. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 372–384, 2003     

Retention of Ejaculate by Drosophila melanogaster Females Requires the Male-Derived Mating Plug Protein PEBme

Within the mated reproductive tracts of females of many taxa, seminal fluid proteins (SFPs) coagulate into a structure known as the mating plug (MP). MPs have diverse roles, including preventing female remating, altering female receptivity postmating, and being necessary for mated females to successfully store sperm. The Drosophila melanogaster MP, which is maintained in the mated female for several hours postmating, is comprised of a posterior MP (PMP) that forms quickly after mating begins and an anterior MP (AMP) that forms later. The PMP is composed of seminal proteins from the ejaculatory bulb (EB) of the male reproductive tract. To examine the role of the PMP protein PEBme in D. melanogaster reproduction, we identified an EB GAL4 driver and used it to target PEBme for RNA interference (RNAi) knockdown. PEBme knockdown in males compromised PMP coagulation in their mates and resulted in a significant reduction in female fertility, adversely affecting postmating uterine conformation, sperm storage, mating refractoriness, egg laying, and progeny generation. These defects resulted from the inability of females to retain the ejaculate in their reproductive tracts after mating. The uncoagulated MP impaired uncoupling by the knockdown male, and when he ultimately uncoupled, the ejaculate was often pulled out of the female. Thus, PEBme and MP coagulation are required for optimal fertility in D. melanogaster. Given the importance of the PMP for fertility, we identified additional MP proteins by mass spectrometry and found fertility functions for two of them. Our results highlight the importance of the MP and the proteins that comprise it in reproduction and suggest that in Drosophila the PMP is required to retain the ejaculate within the female reproductive tract, ensuring the storage of sperm by mated females.     

Factors involved in the post-copulatory neural inhibition of pheromone production in Choristoneura fumiferana and C. rosaceana females

Normal mating lasts approximately 3 h in Choristoneura fumiferana and C. rosaceana. Data generated from interrupted matings showed that the act of mating did not suppress pheromone production (pheromonostasis) in either species although, in C. rosaceana, pheromone titre declined slightly the night following mating. In both species the migration of sperm to the spermatheca (SP) occurred several hours after mating, and coincided with a significant and permanent depression in pheromone titre, as well as egg fertilisation and oviposition. However, disrupting matings within 2 h of the onset resulted in oviposition patterns similar to virgins in both species, with mostly infertile eggs being laid by C. fumiferana females while oviposition was totally inhibited in C. rosaceana. The transection of the ventral nerve cord (VNC) 1 h post-mating did not result in the depression of pheromone titres the following night in either species but if the VNC was transected 3 h post-mating, pheromonostasis was observed. While 25% of C. fumiferana females had sperm in their SP 2 h after mating, it took at least 4 h in C. rosaceana. This suggests that while the physical presence of sperm in the SP may play some role in the termination of pheromone production in C. fumiferana, other factors must trigger the neural signal that elicits pheromonostasis in both species. A better understanding of the temporal dynamics of both apyrene and eupyrene sperm within the different parts of the female reproductive system might clarify these interspecific differences.     

New reproductive anomalies in fruitless‐mutant Drosophila males: Extreme lengthening of mating durations and infertility correlated with defective serotonergic innervation of reproductive organs

Several features of male reproductive behavior are under the neural control of fruitless (fru) in Drosophila melanogaster. This gene is known to influence courtship steps prior to mating, due to the absence of attempted copulation in the behavioral repertoire of most types of fru‐mutant males. However, certain combinations of fru mutations allow for fertility. By analyzing such matings and their consequences, we uncovered two striking defects: mating times up to four times the normal average duration of copulation; and frequent infertility, regardless of the time of mating by a given transheterozygous fru‐mutant male. The lengthened copulation times may be connected with fru‐induced defects in the formation of a male‐specific abdominal muscle. Production of sperm and certain seminal fluid proteins are normal in these fru mutants. However, analysis of postmating qualities of females that copulated with transheterozygous mutants strongly implied defects in the ability of these males to transfer sperm and seminal fluids. Such abnormalities may be associated with certain serotonergic neurons in the abdominal ganglion in which production of 5HT is regulated by fru. These cells send processes to contractile muscles of the male's internal sex organs; such projection patterns are aberrant in the semifertile fru mutants. Therefore, the reproductive functions regulated by fruitless are expanded in their scope, encompassing not only the earliest stages of courtship behavior along with almost all subsequent steps in the behavioral sequence, but also more than one component of the culminating events. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 121–149, 2001     

Drosophila Pheromone-Sensing Neurons Expressing the ppk25 Ion Channel Subunit Stimulate Male Courtship and Female Receptivity

As in many species, gustatory pheromones regulate the mating behavior of Drosophila. Recently, several ppk genes, encoding ion channel subunits of the DEG/ENaC family, have been implicated in this process, leading to the identification of gustatory neurons that detect specific pheromones. In a subset of taste hairs on the legs of Drosophila, there are two ppk23-expressing, pheromone-sensing neurons with complementary response profiles; one neuron detects female pheromones that stimulate male courtship, the other detects male pheromones that inhibit male-male courtship. In contrast to ppk23, ppk25, is only expressed in a single gustatory neuron per taste hair, and males with impaired ppk25 function court females at reduced rates but do not display abnormal courtship of other males. These findings raised the possibility that ppk25 expression defines a subset of pheromone-sensing neurons. Here we show that ppk25 is expressed and functions in neurons that detect female-specific pheromones and mediates their stimulatory effect on male courtship. Furthermore, the role of ppk25 and ppk25-expressing neurons is not restricted to responses to female-specific pheromones. ppk25 is also required in the same subset of neurons for stimulation of male courtship by young males, males of the Tai2 strain, and by synthetic 7-pentacosene (7-P), a hydrocarbon normally found at low levels in both males and females. Finally, we unexpectedly find that, in females, ppk25 and ppk25-expressing cells regulate receptivity to mating. In the absence of the third antennal segment, which has both olfactory and auditory functions, mutations in ppk25 or silencing of ppk25-expressing neurons block female receptivity to males. Together these results indicate that ppk25 identifies a functionally specialized subset of pheromone-sensing neurons. While ppk25 neurons are required for the responses to multiple pheromones, in both males and females these neurons are specifically involved in stimulating courtship and mating.     

Prior oviposition, female receptivity and last-male sperm precedence in the cosmopolitan pest Callosobruchus maculatus (Coleoptera: Bruchidae)

Sperm competition studies have shown that P₂ (the proportion of ova fertilized by the last male to mate) increases as the interval between inseminations is experimentally increased. Variation in the number of sperm in storage is associated with sperm use (or loss) from the female's sperm stores between copulations (fewer sperm from previous mates at the time of the last copulation) and with the extent of prior oviposition and female receptivity to further copulation: females that lay many eggs tend to have few remaining sperm in storage and to be more receptive to further copulation. Using the bruchid beetle Callosobruchus maculatus, we examined the effect of prior oviposition and female receptivity to further copulation on the extent of last-male sperm precedence (measured as P₂). Extent of prior oviposition was experimentally manipulated independently of the intermating interval by altering the availability of oviposition sites between inseminations. Females given few or no oviposition sites laid fewer eggs, were less receptive and had a lower P₂ than females given abundant oviposition sites. To examine the effect of female receptivity on P₂ independently of prior oviposition, we examined the outcome of sperm competition experiments using (1) females from lines that had been selected for different latencies to copulation and (2) natural variation in female latency to receptivity. Female receptivity to further copulation had no detectable effect on P₂. When oviposition resource is abundant, female receptivity may be a poor predictor of current sperm load.     

Evolution of sex-peptide in Drosophila

The Drosophila sex-peptide (SP) has been identified as a seminal fluid component that induces post-mating responses (PMRs) in the inseminated females, such as inhibition of remating and stimulation of egg-laying. SP has been thought to play a central role in sexual conflict and sexually antagonistic co-evolution. Most of the sequenced Drosophila genomes contain SP orthologs, but their functions have been poorly characterized. Recently, we have investigated cross-species activity of D. melanogaster SP by means of injection into virgin females of other species. Among 11 species examined, SP response was observed in 6 species belonging to the D. melanogaster species group only. These species females express SP receptor (SPR) in their oviducts at relatively high levels, which was visualized by using a GFP-tagged SP. Furthermore, females of this species group responded to their own SP orthologs. However, females of the species outside the group did not respond to their own SP orthologs, even though all of them were potent inducers of SP-response in D. melanogaster. Our results suggested that the SP/SPR-mediated PMR was established in the lineage of the D. melanogaster species group.     

The subgenus Persicargas (Ixodoidea: Argasidae: Argas), 26. Argas (P.) arboreus: Effect of photoperiod on diapause induction and termination

Prefeeding exposure of female Argas (Persicargas) arboreus to short photoperiod (SP) induces diapause, expressed as oviposition delay for more than 40 days postfeeding. Diapause incidence increases as prefeeding SP exposure periods increase. Postfeeding SP exposure is not essential to maintain diapause. Postfeeding exposure to SP without prefeeding SP exposure does not induce diapause. A 2 week prefeeding exposure to long photoperiod (LP) terminates diapause in two-thirds of the females exposed to LP after feeding. The remaining one-third, which may differ genetically, require a 6 week LP prefeeding exposure to terminate diapause. An 8 week prefeeding exposure to LP is required to terminate diapause in females exposed to SP after feeding.     

Genome assembly,sex-biased gene expression and dosage compensation in the damselfly Ischnura elegans

The evolution of sex chromosomes, and patterns of sex-biased gene expression and dosage compensation, are poorly known among early winged insects such as odonates. We assembled and annotated the genome of Ischnura elegans (blue-tailed damselfly), which, like other odonates, has a male-hemigametic sex-determining system (X0 males, XX females). By identifying X-linked genes in I. elegans and their orthologs in other insect genomes, we found homologies between the X chromosome in odonates and chromosomes of other orders, including the X chromosome in Coleoptera. Next, we showed balanced expression of X-linked genes between sexes in adult I. elegans, i.e. evidence of dosage compensation. Finally, among the genes in the sex-determining pathway only fruitless was found to be X-linked, while only doublesex showed sex-biased expression. This study reveals partly conserved sex chromosome synteny and independent evolution of dosage compensation among insect orders separated by several hundred million years of evolutionary history.     

Sex-peptides bind to two molecularly different targets in Drosophila melanogaster females

Sex-Peptide (SP) and the peptide DUP99B elicit two postmating responses in Drosophila melanogaster females: receptivity is reduced and oviposition is increased. Both are synthesized in the male genital tract and transferred into the female during copulation. To elucidate their function, we characterized the binding properties of SP and DUP99B in females. Cryostat sections of adult females were incubated with alkaline phosphatase (AP)-tagged peptides. In virgin females, both peptides have specific target sites in the nervous system and in the genital tract. The binding pattern is almost identical for both peptides. Incubation of sections of mated females confirm that some of these target sites correspond to the in vivo targets of the two peptides. Neuronal binding is dependent on an intact C-terminal sequence of SP, binding in the genital tract is less demanding in terms of amino acid sequence requirement. On affinity blots the AP-SP probe binds to membrane proteins extracted from abdomen and head plus thorax, respectively. The binding proteins in the nervous system and the genital tract differ in their molecular properties. Calculation of dissociation constants (K(d)), and also determination of the minimal peptide concentrations necessary for binding, indicate that SP is the more important peptide inducing the postmating responses. Our results suggest that binding of SP in the nervous system is responsible for eliciting the postmating responses, whereas binding in the genital tract reflects the presence of a peptide transporter.     

Flipping the doublesex switch with a piRNA

Recent work in the silkworm Bombyx mori has uncovered a novel Piwi-interacting RNA regulator of the sex determination switch doublesex.