InteractiveFly: GeneBrief

Sex Peptide: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

Conflicts between females and males over reproductive decisions are common. In Drosophila, as in many other organisms, there is often a conflict over how often to mate. The mating frequency that maximizes male reproductive success is higher than that which maximizes female reproductive success. In addition, frequent mating reduces female lifespan and reproductive success, a cost that is mediated by male ejaculate accessory gland proteins (Acps). This study demonstrates that a single Acp, the Sex peptide (SP or Acp70A; Chen, 1988), that decreases female receptivity and stimulates egg production in the first matings of virgin females, is a major contributor to Acp-mediated mating costs in females (Chapman, 2003; Liu, 2003). Females continuously exposed to SP-deficient males, which produce no detectable SP, have significantly higher fitness and higher lifetime reproductive success than control females. Hence, rather than benefiting both sexes, receipt of SP decreases female fitness, making SP the first identified gene that is likely to play a central role in sexual conflict (Wigby, 2005).

In many species, there is a potential for disparity in the optimum mating frequency of males and females. Selection for frequent matings is predicted to be stronger in males than in females; males gain fitness from each extra mating they obtain, whereas female fitness gains may cease as mating frequency increases. Hence, the presence of female mating costs may reflect sexual conflict over mating. In such conflicts, males may evolve traits that increase their fitness relative to other males but that decrease the fitness of the females with which they mate or attempt to mate (Wigby, 2005).

In Drosophila, the proximate mechanism underlying mating costs in females has been explored. Females that mate at high frequencies suffer fitness costs (reduced longevity and reproductive success) as a result of the actions of male seminal fluid accessory gland proteins (Acps). This Acp-mediated mating cost is potentially large and is incurred in addition to reproduction costs, such as those that result from egg production and other nonmating activities. Acps mediate a variety of effects that benefit males; such effects include stimulation of female egg production, reduction of female receptivity, ensuring effective sperm storage, and promotion of male success in sperm competition. The female mating cost that arises from Acp transfer by males may be a side effect of Acp function or a direct effect that is selected to reduce the likelihood of female re-mating and/or to increase current investment in reproduction (Wigby, 2005 and references therein).

This study investigated whether a single Acp, the sex peptide (SP or Acp70A), is responsible for mating costs in females. SP decreases female receptivity and stimulates egg production following the first matings of virgin females; it has been generally assumed to benefit both sexes by acting as a signal to initiate high reproductive rates in successfully mated females and as a mechanism for increasing paternity in males. Wld-type females were exposed throughout life either to SP-knockdown males (which produced no detectable SP; Chapman, 2003) or to control males (which were matched for autosomal genetic background). Two independent replicate pairs of SP-knockdown and control male lines (SP1 knockdown and C1, and SP2 knockdown and C2) were used. One hundred and ten females for each treatment of each line were kept in groups of five, and five males were added to each group. Female survival was measured, female mating frequency, egg production, and egg-adult viability were sampled throughout the experiments. Female survival and age-specific offspring-production data were used to calculate fitness (an index of r, the intrinsic rate of population increase for each treatment of each line), and indices of lifetime egg production per female and lifetime offspring production per female were calculated (Wigby, 2005).

It was predicted that females continuously exposed to SP-knockdown males would mate significantly more frequently than females continuously exposed to control males because SP-induced receptivity inhibition would be absent in mates of SP-knockdown males. It was predicted that this difference in mating frequency would lead to higher survival mating costs in females mated to SP-knockdown males, provided there was a difference in mating frequency of a least 2.2-fold (previously shown to be sufficient to cause mating costs in females over that of the control females). It was also predicted that females exposed to SP-knockdown males would produce fewer eggs than controls because SP stimulates egg production in first matings of virgin females (Chapman, 2003; Liu, 2003). To check that the survival of females continuously exposed to males was determined by male-derived reproductive costs, the survival of females exposed to SP-knockdown or control males was measured for just 48 hr (Wigby, 2005).

The results show that, as expected, females continuously exposed to SP-knockdown males mate significantly more often than females continuously exposed to control males. Females continuously exposed to SP-knockdown males were also courted significantly more often than control females. However, despite mating more than 12 times as frequently and receiving significantly elevated levels of courtship, females continuously exposed to SP-knockdown males did not have reduced survival in comparison to controls (contrary to the prediction that substances other than SP contribute to mating costs). Instead, it was found that mates of SP-knockdown males lived at least as long as, or even significantly longer than, females continuously exposed to control males [median survival, in days, from the first day of exposure to males]. The difference in mating rates between females exposed to SP-knockdown males and those exposed to control males far exceeded that previously shown to increase female survival mating costs. The results therefore indicate that, in terms of female survival, matings with SP-knockdown males were largely free of mating costs. Females exposed to control males mated at a lower frequency than was observed in similar assays of mating frequency in a previous study of female mating costs. This would have led to relatively low mating costs in the control females in this study; however, females mated to SP-knockdown males mated at much higher frequencies than did the high-mating females from the previous study, in which significant mating costs were observed. Hence, chances of detecting survival mating costs in females exposed to SP-knockdown males, had such costs been present, were maximized. Of course, survival measures alone do not necessarily indicate the existence of reproductive costs, and to address whether SP contributes to Acp-mediated mating costs, survival was considered together with reproductive success (Wigby, 2005).

In further contrast to predictions, females continuously exposed to SP-knockdown males had significantly higher egg production in the first three (line 2) or in two of the first three (line 1) of the nine egg-production samples taken during the experiments. Furthermore, females continuously exposed to SP-knockdown males had marginally significantly higher indices of lifetime egg production than control females. Previous work has shown that virgin females that were mated for the first time to SP-knockdown males show significantly lower egg production than females that were mated once to control males (Chapman, 2003; Liu, 2003). Therefore significantly higher early egg production in females continuously exposed to SP-deficient males was unexpected in this study. This observation is not attributable to a low stimulation of egg production in females mated to the control males. The same control male genotype stimulates egg production more than that of the SP-knockdown males both after single matings (Chapman, 2003) and in assays in which males and females are housed in individual pairs. The increased early egg production in females continuously exposed to SP-knockdown males is consistent with a gene × mating frequency interaction. At low mating frequencies, the receipt of other ovulation- and oviposition-stimulating seminal fluid proteins, such as Acp26Aa (Herndon, 1995) and possibly Dup99B (Saudan, 2002), may be insufficient to offset the lack of SP, leading to low egg production in mates of SP-knockdown males. However, at higher mating frequencies the receipt of Acp26Aa and Dup99B may be at a level sufficiently high enough to result in increased egg production relative to that of control females (which receive lower levels of these other Acps). This is consistent with functional redundancy among Acps that stimulate egg production. An alternative explanation is that the higher egg production in females continuously exposed to SP-knockdown males is the result of an improvement, arising from the absence of SP, in female health. Because egg production is known to contribute to reproductive costs, the finding that the magnitude of differences in egg production was lower and occurred over a shorter time in line 1 than in line 2 might explain why females continuously exposed to SP-knockdown males lived significantly longer than their controls in line 1 but not line 2. The eggs laid by females mated to males of both lines generally showed no differences in egg-adult viability, although mates of SP-knockdown males had significantly higher egg-adult viability in one of the later samples of the experiment (Wigby, 2005).

The most striking effect in this study was that females continuously exposed to SP-knockdown males had significantly higher indices of lifetime offspring production and fitness, as well as marginally significantly higher indices of lifetime egg production, than controls. Fitness (r) was calculated from age-specific progeny and survival values. Measures based on r are more directly related to fitness than to lifetime reproductive success, particularly with D. melanogaster, which probably does much of its reproduction in expanding populations. Nevertheless, the measures of lifetime egg production and reproductive success are entirely consistent with the fitness measures; they all indicate that females exposed to SP-knockdown males had higher fitness and higher lifetime reproductive success than did females mated to control males. Significant Acp-mediated survival costs of mating can be observed in females even when other costly activities, such as egg production and exposure to courting males, are held constant. In this study, females exposed to SP-knockdown males had significantly higher exposure to courtship and significantly higher early egg production than did control females. Despite this, these females mated at least 12 times as often as control females and still had significantly higher fitness and lifetime reproductive success.It is conclude that SP is therefore responsible for at least a major part of the Acp-mediated female mating costs in D. melanogaster (Wigby, 2005).

As expected, the survival of females exposed to males for 48 hr only was significantly higher than the survival of females continuously exposed to males for both treatments of both lines. The fact that females continuously exposed to males had lower survival than females exposed to males for 48 hr is likely to be due to higher reproductive costs, such as those arising from egg production and the receipt of courtship. In addition, other potentially harmful Acps (such as Acp62F, which reduces female survival when ectopically expressed; Lung, 2002) could also contribute to reproductive and mating costs. Therefore, it is not possible to exclude the possibility that Acps other than the SP contribute to reproductive costs. As expected, because mating costs are detectable only against a background of frequent mating in this species, there were no differences in the survival of females exposed to SP-knockdown or control males for 48 hr in either line (Wigby, 2005).

X chromosome differences between SP-knockdown and control males could have contributed to differences in male behavior (e.g., courtship and mating frequency) and hence in female reproductive success. However, differences in X chromosome constitution are not likely to confound the results through any potential effects on Acp levels because the genes encoding all the Acps responsible for mating costs in females are autosomal. However, the possibility that there are X-linked, trans-acting genes that modulate Acp function (e.g., genes that encode for enzymes that regulate Acp potency) cannot be excluded (Wigby, 2005).

Mating and courtship rates of the control males in this experiment are broadly comparable to the range seen in the wild-type cage populations from which the experimental females were drawn. Even in the wild, females are subject to very intense bombardment from males, and multiple mating is common. The mating and courtship rates observed were also comparable to those seen in previous experiments. If mating and courtship are artificially high in the experimental setup, the lack of cost seen in females mating with SP-knockdown males would be all the more remarkable (Wigby, 2005).

Males gain from SP transfer because, even though it ultimately reduces the fitness of their mates, SP also induces a refractory period that significantly increases 'per-mating' paternity levels. The results indicate that, rather than benefiting both sexes, the receipt of SP decreases female fitness. It would therefore be predicted that females with elevated SP resulting from ectopic SP-induction or from matings with males that produce and transfer elevated levels of SP, should incur increased mating costs. The results are also consistent with the finding, from a large-scale study of the effects of variation in male-sperm competitive ability on females, of positive correlations between the length of female refractoriness (i.e., re-mating interval) and early female mortality. This finding may suggest that males that can induce longer re-mating intervals can impair female survival. The study highlights SP as an obvious candidate mechanism (Wigby, 2005).

Females could gain indirect genetic benefits from mating with SP-transferring males if their male offspring had higher reproductive success. However, such benefits are likely to be small in comparison to the direct costs incurred by the receipt of SP. Females could also benefit directly, through increased egg production, from the receipt of SP if mating opportunities were limited to one or a very few matings. However, multiple mating is the norm in D. melanogaster both in the laboratory and in the wild, and as shown in this study, fecundity benefits through receipt of SP may not occur with frequent mating. It is therefore unlikely that females often benefit from the receipt of SP. Consequently, the SP gene is likely to play a role in sexual conflict rather than in cooperation (Wigby, 2005).

Initially, natural selection may have caused females to evolve a sensitivity to substances such as SP and allowed them to adaptively modulate egg production and receptivity after sperm transfer. The demonstration of direct costs that result from the receipt of SP is, however, consistent with a scenario in which SP is under the influence of sexual selection and sexual conflict. Such a scenario may have selected for SP activity that increased male reproductive success regardless of the effect upon females. If SP is subject to sexual conflict, then theory predicts that it should show relatively rapid evolutionary change. Although the SP C terminus appears relatively conserved in the melanogaster species subgroups, D. subobscura and D. suzukii, the N terminal region is somewhat divergent, and significant departures from neutrality have been detected in the region flanking the 5′ end of the SP gene. It is not clear whether SP alone is responsible for female mating costs or whether harm is caused by the interaction of SP with other ejaculate molecules. SP binds to sperm and can be detected on sperm heads several days after its deposition in the female reproductive tract. There is no reduction in the cost of mating in females continuously exposed to spermless males, which suggests that the SP that is harming females must be free from association with sperm. SP appears to stimulate egg production by causing the release of juvenile hormone (JH) BIII from the Corpora allata (Moshitzky, 1996), and this release stimulates oocyte progression in the ovary. Increased JH levels are negatively associated with lifespan in other insects. Hence, costs, such as immunity suppression, that result from the effects of increased JH are candidate mechanisms for future study (Wigby, 2005).

A receptor that mediates the post-mating switch in Drosophila reproductive behaviour

Mating in many species induces a dramatic switch in female reproductive behaviour. In most insects, this switch is triggered by factors present in the male's seminal fluid. How these factors exert such profound effects in females is unknown. Identified here is a receptor for the Drosophila melanogaster Sex peptide (SP, also known as Acp70A), the primary trigger of post-mating responses in this species. Females that lack the sex peptide receptor (SPR, also known as CG16752), either entirely or only in the nervous system, fail to respond to SP and continue to show virgin behaviours even after mating. SPR is expressed in the female's reproductive tract and central nervous system. The behavioural functions of SPR map to the subset of neurons that also express the fruitless gene, a key determinant of sex-specific reproductive behaviour. SPR is highly conserved across insects, opening up the prospect of new strategies to control the reproductive and host-seeking behaviours of agricultural pests and human disease vectors (Yapici, 2008).

At various stages in their lifespan, animals can undergo marked switches in their innate behavioural patterns. Such behavioural switches are attractive models to explore the genetic and neural control of innate behaviours more generally, and are particularly apparent in the dimorphic behaviours involved in mating and reproduction. For example, males and females of most species have distinct mating behaviours that are usually specified during development, but in some species these can also be switched in the adult. In Drosophila melanogaster, the switch that specifies male or female mating behaviour is thought to be set during development by the sex-specific transcripts of the fruitless (fru) gene (Yapici, 2008).

Another type of behavioural switch found in many species is the marked change in female behaviour that occurs on mating. For example, in many insect species, virgin females are receptive to courting males and retain their eggs, whereas those that have recently mated are unreceptive and lay eggs. These changes in female behaviour are induced by factors present in the male seminal fluid. In Drosophila, the primary trigger of this behavioural switch is the sex peptide (SP), a 36-amino-acid peptide produced in the male accessory gland. How SP exerts its effects on female behaviour is unknown, although it has been suggested that the SP might act in part by modulating the activity of neurons that express fru. This study has identified a SP receptor, SPR, and shows that it is specifically required in the fru neurons for the post-mating switch in female reproductive behaviour (Yapici, 2008).

The gene CG16752, henceforth referred to as SPR, was identified in a genome-wide transgenic RNA interference (RNAi) screen for genes required in the female nervous system for post-mating reproductive behaviour. Specifically, it was found that pan-neuronal expression of an RNAi transgene targeting SPR (elav-GAL4 UAS-SPR-IR1) led to a marked reduction in egg laying. To examine this egg-laying phenotype more carefully, and to assess other reproductive behaviours, a protocol was used in which individual virgin females were first tested for receptivity towards a naive male. Those females that mated were then allowed to lay eggs for 48 h before being retested for receptivity with a second naive male. In the initial mating assays, virgin SPR RNAi females were as receptive as the control females. However, in contrast to control females, SPR RNAi females laid very few eggs after mating, mated again at high frequency, and did not actively reject the second male. In all these assays, mated SPR RNAi females behaved indistinguishably from wild-type virgin females, as well as from females previously mated to SP null males (Yapici, 2008).

To control for potential off-targeting effects of the initial RNAi transgene, a second independent line, UAS-SPR-IR2, was generated that targets a different region of the SPR gene. In all four assays, this new RNAi line gave results similar to those obtained with the original line from the genome-wide library. A molecularly defined deficiency, Df(1)Exel6234, was identified that removes 88 kilobases (kb) from the X-chromosomal region that includes SPR and four other annotated genes. The molecular breakpoints of this deficiency were verified and it was confirmed to delete the entire SPR gene. Females homozygous for this deficiency were fully viable and had no obvious defects in the gross anatomy of their nervous system or reproductive organs. When tested in parallel in the same series of receptivity and egg-laying assays, Df(1)Exel6234 homozygous females showed the same post-mating defects as observed on RNAi knockdown of SPR (Yapici, 2008).

By mating SPR RNAi or deficiency females to males with sperm labelled by GFP (green fluorescent protein), it was confirmed that sperm were transferred and stored normally in these animals. The few eggs laid by these females are also fertilized and develop normally. It is therefore postulated that the abnormal post-mating behaviours of these females could be due to a lack of sensitivity to SP, rather than due to a more general defect in reproductive physiology. To test this, SP was injected into the haemolymph of SPR RNAi or deficiency virgin females. The receptivity of these females was then tested 5 h later in pairings with naive wild-type males. As controls, it was confirmed that wild-type virgins injected with SP were unreceptive, whereas those injected with buffer alone were just as receptive as uninjected virgins. In contrast, SPR RNAi and deficiency virgins remained receptive even after injection with SP. These genetic data demonstrate that SPR is required in the nervous system for the behavioural switch triggered by SP (Yapici, 2008).

The SPR gene is predicted to encode a G-protein-coupled receptor (GPCR). To test whether this GPCR might be the SP receptor itself, SPR complementary DNA was expressed in mammalian Chinese hamster ovary (CHO) cells together with the Ca²⁺ reporter aequorin. In this assay, ligand-mediated GPCR activation triggers a luminescent flash by means of the Gα_q- or Gα ₁₁-dependent Ca²⁺ pathway. Only a very weak response to SP was detected in these cells, even at concentrations as high as 10 microM. It has been suggested that SP responses might involve the cAMP rather than the Ca²⁺ pathway (Harshman, 1999), and so one reason for this poor response might be that SPR normally couples to G proteins other than Gα_q/11. Accordingly, these cells were cotransfected with constructs encoding one of three different chimaeric G proteins (Gα_qs, Gα_qi or Gα_qo) designed to divert Gα_s-, Gα_i- or Gα_o-dependent signals, respectively, from the cAMP pathway into the Ca²⁺ pathway. Indeed, co-expression of Gα_qi or Gα_qo, but not Gα_qs, resulted in robust Ca²⁺ responses to SP (Yapici, 2008).

The response to SP is highly specific, because comparable levels of activation by any of eight other Drosophila peptides, even at 10 microM, were not detected. Among the closest relatives of SPR in Drosophila are CG2114 (also known as Fmrf Receptor) and CG8784, receptors for FMRFamides and hugin-gamma, respectively. Neither FMRFamide nor hugin-gamma activated SPR, and, conversely, expression of CG2114 or CG8784 in CHO cells conferred sensitivity to their respective ligands, but not to SP. In a dose-response assay, it was determined that SP activates SPR with an effector concentration for half-maximum response (EC₅₀) of 1.3 nM. The closely related peptide, DUP99B, which induces the same post-mating responses as SP in injection assays, activates SPR with an EC₅₀ of 7.3 nM. Thus, both SP and DUP99B specifically activate SPR at physiological concentrations, with EC₅₀ values in the low nanomolar range typical for such peptide-GPCR interactions. It is concluded that SPR encodes a functional receptor for SP that couples to Gα_i and/or Gα_o to regulate cAMP levels (Yapici, 2008).

To define the cellular targets of SP, antisera were generated against an amino-terminal region of SPR. These antisera revealed high levels of SPR expression in the female reproductive organs, in particular in the spermathecae, the primary sites for long-term sperm storage, and the lower oviduct. Staining with the anti-SPR antisera was restricted to the cell membrane and was absent in Df(1)Exel6234 homozygous females, confirming the specificity of the antisera. SPR could not be detected in the male reproductive organs (Yapici, 2008).

SP is thought to pass from the reproductive tract into the haemolymph, and ultimately to act directly on targets in the central nervous system (CNS). Indeed, staining the adult female CNS with anti-SPR revealed a broad expression on the surface regions of both the brain and the CNS. This staining was absent or greatly reduced in SPR deficiency or RNAi females. Expression was most prominent in ventral regions of the subesophageal ganglion (SOG), the cervical connective and many nerve roots in the brain and VNC. The restricted staining on the surface of the CNS is consistent with SPR detecting a ligand that circulates in the haemolymph and crosses the blood-brain barrier. It is unlikely to be an artefact caused by poor antibody penetration, because SPR was reliably detected in central brain regions on ectopic expression of a UAS-SPR transgene. Overall, the distribution of SPR concords remarkably well with the reported binding sites of radiolabelled SP applied to whole-female-tissue sections in vitro. Intriguingly, a very similar distribution was also observed in the male CNS, although at this point no function can be ascribed to SPR in males (Yapici, 2008).

Post-mating responses can be induced in virgin females not only by injection of SP but also by blocking synaptic transmission of neurons that express the sex-specific transcripts of the fru gene. It was also found that some of the central neurons that express SPR are also positive for fru, as revealed by the fru^GAL4 driver. In particular, SPR seemed to be expressed in many fru^GAL4-positive neurons in the SOG and throughout the VNC. To test whether SPR function is specifically required in fru neurons, the fru^GAL4 driver and UAS-SPR-IR1 were used to knockdown SPR only in these cells. Just like the SPR-deficiency mutants, these females showed normal receptivity as virgins, but then laid very few eggs and re-mated at high frequency (Yapici, 2008).

To test whether expression in fru neurons is also sufficient for the post-mating switch, fru^GAL4 and UAS-SPR were introduced into SPR-deficient females. In these females, SPR is expressed only in the fru neurons, yet complete rescue of the re-mating phenotype and partial but significant rescue of the egg-laying phenotype were observed. Together, these RNAi and rescue experiments strongly support the notion that SP triggers the post-mating behavioural switch primarily by modulating the activity of a subset of the fru neurons (Yapici, 2008).

The post-mating switch in female behaviour is not unique to D. melanogaster, but is common to most insect species. Although SP genes are difficult to identify outside the Drosophilidae, perhaps because of their small size, putative SPR orthologues in most sequenced insect genomes were readily identifiable, including Drosophila pseudoobscura, the mosquitos Aedes aegypti and Anopheles gambiae, the moth Bombyx mori and the beetle Tribolium castaneum. More distant relatives can also be detected in Caenorhabditis elegans, but potential vertebrate orthologues are less apparent (Yapici, 2008).

To test for functional conservation of the insect SPR family, SPR cDNAs were isolated from each of these five other insect species and tested for responses to D. melanogaster SP in the CHO cell assay. SP was shown to be a potent activator of the D. pseudoobscura, A. aegypti and B. mori receptors, with EC₅₀ values of 4.3 nM, 167 nM and 63 nM, respectively. These receptors also responded to DUP99B, but not to any of the eight control peptides, including FMRFamide and hugin-gamma. The A. gambiae and T. castaneum receptors were not activated by either SP or DUP99B, possibly because they do not bind the Drosophila ligands or were not functionally expressed in CHO cells. Nonetheless, the structural and functional conservation of SPR genes from Drosophila, Aedes and Bombyx, together with the observation that D. melanogaster SP can induce post-mating responses in the moth Helicoverpa armigera, indicates that the family of receptors identified are likely to mediate post-mating changes in female reproductive behaviour in many different insect orders (Yapici, 2008).

These data provide strong evidence that SPR is a receptor for SP, and that activation of SPR in fru neurons induces the switch to post-mating reproductive behaviour. The identification of SPR is the critical first step in explaining this behavioural switch at the molecular, cellular and circuit levels. Furthermore, because SPR is highly conserved across insect species, it provides the basis for cellular assays to identify SP-like activities in other species, and to develop new approaches for controlling the reproductive or host-seeking behaviours of various agricultural pests and human disease vectors (Yapici, 2008).

Neural circuitry underlying Drosophila female postmating behavioral responses

After mating, Drosophila females undergo a remarkable phenotypic switch resulting in decreased sexual receptivity and increased egg laying. Transfer of male sex peptide (SP) during copulation mediates these postmating responses via sensory neurons that coexpress the sex-determination gene fruitless (fru) and the proprioceptive neuronal marker pickpocket (ppk) in the female reproductive system. Little is known about the neuronal pathways involved in relaying SP-sensory information to central circuits and how these inputs are processed to direct female-specific changes that occur in response to mating. This study demonstrates an essential role played by neurons expressing the sex-determination gene doublesex (dsx) in regulating the female postmating response. Shared circuitry was uncovered between dsx and a subset of the previously described SP-responsive fru⁺/ppk⁺-expressing neurons in the reproductive system. In addition, sexually dimorphic dsx circuitry was identified within the abdominal ganglion (Abg) that was critical for mediating postmating responses. Some of these dsx neurons target posterior regions of the brain while others project onto the uterus. It is proposed that dsx-specified circuitry is required to induce female postmating behavioral responses, from sensing SP to conveying this signal to higher-order circuits for processing and through to the generation of postmating behavioral and physiological outputs (Rezával, 2012).

These results show that in the female, dsx neurons associated with the internal genitalia not only form a component part of the previously described fru⁺/ppk⁺ network, but in fact define a more minimal SP-responsive neural circuit capable of inducing postmating changes, such as reduced receptivity, increased levels of rejection, and egg deposition (Rezával, 2012).

In addition to these 'classic' postmating behavioral responses, it was also noted that SP signaling to dsx neurons induces postmating changes in locomotor activity between unmated and mated females. Studies have shown that Drosophila males court immobilized females less than moving females; essentially, males react to changes in female locomotion, suggesting a causal link between female locomotion and increased courtship levels. It has been proposed that males are 'acoustically tuned' to signals generated by active females, stimulating increased courtship by changing the attention state of the male. Therefore, female mobility appears to contribute to her 'sex appeal' and decreased locomotion in mated females is likely to affect the male's willingness to copulate (Rezával, 2012).

The female's nervous system must have the capacity to receive, and interpret, postcopulatory signals derived from the male seminal package to direct physiological and behavioral responses required for successful deposition of fertilized eggs. It was demonstrated that two dsx clusters, composed of three bilateral neurons of the uterus, comprise a more defined component of the SP-responsive sensory circuit. In addition, the majority of other dsx neurons originating on the internal genitalia were shown to coexpress ppk. As ppk neurons are mechanosensory, these may be acting as uterine stretch receptors, facilitating sperm and egg transport, fertilization, and oviposition. Silencing neural function of ppk neurons appears to inhibit egg deposition, presumably by impeding egg transport along the oviducts. Similarly, in dsx^Gal4 females expressing TNT no egg deposition is ever observed, with unfertilized eggs atrophying in the lateral oviducts. In contrast, when fru⁺ neurons are silenced, deposition of successfully fertilized eggs is still observed, suggesting that different subsets of the dsx⁺/fru⁺/ppk⁺ SP-responsive sensory circuit may direct distinct postmating behavioral responses. As SP has been detected in the hemolymph of mated females, it has been suggested that this peptide could pass from the reproductive tract into the hemolymph to reach CNS targets. The fact that neither receptivity nor oviposition was restored to control levels when ppk-Gal80 (or Cha-Gal80) was expressed in dsx^Gal4/UAS-mSP flies opens the possibility that SP expression might affect additional dsx neurons in the CNS (Rezával, 2012).

Triggering of postmating responses via SP reception appears to occur via a small number of neurons expressing SPR on the female reproductive tract; however, SPR is also found on surface regions of the CNS as well as in endocrine glands and other reproductive tissues. Surprisingly, SPR may even be detected in the Drosophila male CNS, where no exposure to SP would be expected, and in insects that apparently lack SP-like. SPRs are therefore potentially responsive to other ligands, performing functions other than those associated with postmating responses in the diverse tissues in which SPRs are expressed (Rezával, 2012).

Extensive coexpression was found of dsx-expressing cells and SPR in the epithelium of the lower oviduct and spermathecae in females. However, mSP expression (or SPR downregulation) specifically in spermathecal secretory cells (SSC) or oviduct epithelium cells had no effect on receptivity or egg laying. In agreement with rescue experiments using neuronal Gal80 drivers to intersect Gal4-responsive UAS expression in dsx cells, this suggests that these cells are neither neuronal nor directly involved in SP-mediated postmating behaviors. SPR staining in the CNS was more difficult to determine given the limitations of the antibody; while no colocalization in the brain was observed, apparent coexpression was observed between SPR and a small subset of ventral (Rezával, 2012).

The results indicate that dsx-Abg neurons are required for the induction and regulation of specific components of the postmating response. It has been shown that inhibition of neurotransmission in apterous-expressing Abg neurons impairs SP-mediated postmating changes in receptivity and oviposition, emphasizing the importance of these neurons in the modulation of postmating responses (Rezával, 2012).

The level of dsx neuronal expression within the Abg and their associated fascicles projecting to the brain, where they form extensive presynaptic arborizations within the SOG, coupled with the effects that impairment of function in these neurons has on postmating responses, speaks to the involvement of these neurons in relaying information from the reproductive tract to the brain. That dsx-Abg neurons also project, and form presynaptic arborizations on the uterus, and that the effects on postmating responses when their function is impaired again argue that these neurons play a direct role in mediating processes such as egg fertilization and oviposition. Interestingly, most dsx intersecting neurons are specific to females. Sex-specific behaviors can arise from either shared circuits between males and females that operate differently and/or sex-specific circuits that result from the presence/absence of unique circuit components in one sex versus the other. The results support the latter (Rezával, 2012).

The VNC has been implicated in the modulation of postmating responses, with an identified focus specifically involved in ovulation and transfer of eggs into the uterus for fertilization. Octopaminergic modulatory neurons located at the distal tip of the VNC projecting to the reproductive tract are required for triggering ovulation, possibly by regulating muscle contractions in the ovaries and oviducts. Since earlier studies have shown that ablation of the pars intercerebralis revealed an additional focus for egg laying in the head, and the brain appears to be required for sexual behaviors, such that decapitated virgin females neither mate nor lay eggs, it seems likely that neurons in the Abg also require signals from the brain to regulate postmating responses such as egg transport, fertilization, and deposition (Rezával, 2012 and references therein).

Higher-order circuits in the female brain must be capable of integrating sensory inputs from the olfactory, auditory, and reproductive systems to decide between the alternative actions of acceptance or rejection of the male. Early gynandromorph studies mapped a region of the dorsal brain that must be female for an animal to be receptive; it has been recently shown that the majority of dsx neuronal clusters are located in this region. While neurons coexpressing dsx and fru in male brains define a more restricted circuitry for determining male mating decisions, in females no overlap between dsx⁺ and fru⁺ neurons is observable in the brain. It is also important to note that the sex-specific Fru isoform is absent in females; thus any circuits that are actively specified in the female are likely to depend on the female isoform Dsx^F. Most dsx neurons in the brain are found in the lateral protocerebrum, a region where multiple sensory inputs are thought to be integrated and discrete motor actions selected and coordinated. Further high-resolution functional and connectivity mapping will help to define which neurons participate in specific pre- and postmating behaviors in the female, allowing circuit architecture to be integrated with underlying cellular and synaptic properties. Future experiments will define what activity patterns trigger these behaviors and what activity patterns correlate with these behaviors (Rezával, 2012).

Postmating circuitry modulates salt taste processing to increase reproductive output in Drosophila

To optimize survival and reproduction, animals must match their nutrient intake to their current needs. Reproduction profoundly changes nutritional requirements, with many species showing an appetite for sodium during reproductive periods. How this internal state modifies neuronal information processing to ensure homeostasis is not understood. This study shows that dietary sodium levels positively affect reproductive output in Drosophila melanogaster; to satisfy this requirement, females develop a strong, specific appetite for sodium following mating. This study shows that mating modulates gustatory processing to increase the probability of initiating feeding on salt. This postmating effect is not due to salt depletion by egg production, since abolishing egg production leaves the sodium appetite intact. Rather, the salt appetite is induced need-independently by male-derived Sex Peptide acting on the Sex Peptide Receptor in female reproductive tract neurons. It was further demonstrated that postmating appetites for both salt and yeast are driven by the resultant silencing of downstream SAG neurons. Surprisingly, unlike the postmating yeast appetite, the salt appetite does not require octopamine, suggesting a divergence in the postmating circuitry. These findings demonstrate that the postmating circuit supports reproduction by increasing the palatability of specific nutrients. Such a feedforward regulation of sensory processing may represent a common mechanism through which reproductive state-sensitive circuits modify complex behaviors across species (Walker, 2015).

Animals' nutritional requirements vary over their life cycle, and this necessitates specific behavioral mechanisms to adapt their food choices to their current internal state. This study shows that similarly to the previously characterized switch in feeding preference toward high-protein yeast, Drosophila also develop a specific appetite for sodium following mating. This appetite is adaptive for the female since, like protein, salt is important for reproductive success: this study demonstrates that dietary sodium levels positively impact the rate of offspring production. Salt could increase reproductive output in two ways: it could support egg production by providing ions required for the osmotic balance within the newly created eggs, or the phagostimulatory power of sodium could result in increased total food intake and hence an increase in egg production. Irrespective of the exact mechanisms, the results show that dietary sodium clearly affects the rate of offspring production. The postmating salt appetite is due primarily to an increase in the probability of initiating feeding from salt, which can be attributed to an increased gustatory attraction to sodium. Mating not only elevates the gustatory response to all concentrations of salt, but also results in a shift in the peak response toward higher concentrations. This shift would allow mated females to regulate their salt consumption to a different intake target from virgins, without requiring nutrient-specific feedback to operate within the fly. Indeed, neither the postmating salt nor yeast appetites are driven by feedback from depletion of internal nutrient stores by egg production. While it cannot be exclude that physiological processes induced by mating, other than egg production, could consume salt or protein, the data indicate that a feedforward signal in the male seminal fluid, Sex Peptide, directly drives salt and yeast appetites. Sex Peptide binds to SPR in SPSNs, whose silencing results in silencing of SAG neurons. This leads to appetites for both salt and yeast, in addition to the previously described changes in receptivity and egg laying. These results suggest that the intake of reproductive nutritional resources is controlled by a common regulatory logic, whereby the signal of mating is detected by local uterine neurons and changes nutrition in a feedforward, anticipatory manner. It will be interesting to explore to what extent feedforward regulation is employed to control specific behavioral strategies used to acquire nutrients depending on different internal state signals (Walker, 2015).

The data are consistent with the current view that the signal of mating status is brought into the central brain through a common pathway, the SPSN-SAG axis, to regulate the full set of postmating responses including egg laying, remating, and nutrition. Given the diverse set of behaviors regulated by mating, one would expect the circuit to diverge downstream. However, the point of divergence is currently unknown. Octopamine is known to be required for ovulation and is required for the full reduction in receptivity that normally follows mating. In agreement with these results, it as found that octopamine is also required for the postmating increase in yeast intake in protein-deprived females, while it is dispensable for sensing internal amino acid deficiency. However, while octopamine does influence the overall level of salt responses, the results show that it is not necessary for the postmating change in salt response. These data suggest that octopamine may represent such a divergence point in the postmating circuit, with the previously characterized dsx+Tdc2+ neurons being likely neuronal candidates mediating this divergence. It has, however, been proposed that octopamine may act genetically upstream of SP; this could be compatible with tje results if the salt appetite is relatively insensitive to small changes in SP levels. Regardless, this result hints at distinct circuitry controlling the different behavioral changes elicited by mating, which could aid in the future elucidation of how a specific internal state signal could coordinate changes in a wide range of different behaviors (Walker, 2015).

Salt has been shown to be one of the most limiting nutritional resources in many ecosystems. The results provide insights into the physiological regulation of salt intake, which until now has remained unexplored in Drosophila. The postmating sodium appetite demonstrated in this study is intriguing in the light of the specific sodium appetite seen during pregnancy and lactation in various mammalian herbivores, and even humans. As in Drosophila, these species show an increased gustatory attraction to salt following mating. While the mechanism used to detect mating in these species is different, the feedforward, need-independent nature of the salt appetite is likely to be similar. In rats, this appetite is induced within a few days after mating and is present even if the animal has access to sufficient salt in its diet; furthermore, a salt appetite can be induced in rabbits by administration of a mixture of reproductive hormones in the absence of mating. Thus, the detection of mating by the nervous system and the subsequent feedforward increase in response to salt taste is likely to be a common feature of many non-carnivorous species, making alliesthesia a likely universal mechanism driving reproductive salt appetites. While much is known about the regulation of salt intake in mammals, the mechanisms through which reproduction affects salt appetite remain poorly understood in any species. Functional genetic circuit analysis combined with activity imaging in Drosophila offer the unique opportunity to understand the circuit mechanisms through which this internal state signal can modulate taste processing in the brain, and thus bring about an adaptive change in food preference. To achieve this, three possibilities exist. Mating could modulate the response of sensory neurons to salt taste, as demonstrated in the olfactory pheromone system of moths. In a similar way, GRN responses are modulated by starvation, and the sensitivity of pheromone-sensitive olfactory receptor neurons in mice is modulated across the estrus cycle. Alternatively, mating could alter higher-order taste processing. Finally, mating state could lead to a combination of modulation at the receptor neuron level and modification of higher-order processing. Identifying how alliesthesia is implemented at the circuit level will represent a unique opportunity to understand how internal state changes affect sensory processing to mediate adaptive behaviors (Walker, 2015).

Neural circuitry linking mating and egg laying in Drosophila females

Mating and egg laying are tightly cooordinated events in the reproductive life of all oviparous females. Oviposition is typically rare in virgin females but is initiated after copulation. This study identified the neural circuitry that links egg laying to mating status in Drosophila melanogaster. Activation of female-specific oviposition descending neurons (oviDNs) is necessary and sufficient for egg laying, and is equally potent in virgin and mated females. After mating, sex peptide-a protein from the male seminal fluid-triggers many behavioural and physiological changes in the female, including the onset of egg laying. Sex peptide is detected by sensory neurons in the uterus, and silences these neurons and their postsynaptic ascending neurons in the abdominal ganglion. This study shows that these abdominal ganglion neurons directly activate the female-specific pC1 neurons. GABAergic (gamma-aminobutyric-acid-releasing) oviposition inhibitory neurons (oviINs) mediate feed-forward inhibition from pC1 neurons to both oviDNs and their major excitatory input, the oviposition excitatory neurons (oviENs). By attenuating the abdominal ganglion inputs to pC1 neurons and oviINs, sex peptide disinhibits oviDNs to enable egg laying after mating. This circuitry thus coordinates the two key events in female reproduction: mating and egg laying (Wang, 2020).

It was reasoned that egg laying is likely to depend on cell types that are female-specific and hence express one or both of the sex-determination genes fruitless (fru) and doublesex (dsx). In particular, egg laying is blocked by either silencing or masculinizing all fru+ neurons. Some of these fru+ neurons are descending interneurons, which project from the brain to the ventral nerve cord and are thought to convey high-level motor commands. This study therefore focused on female-specific fru+ descending neurons and used the split-GAL4 technique to obtain two driver lines that label two female-specific fru+dsx- cholinergic descending neurons per brain hemisphere. In optogenetic activation experiments using Chrimson, both split-GAL4 driver lines reliably induced oviposition behaviour in mated females, with most but not all females also depositing an egg (it is presumed that not all females had an egg in the uterus at the time of neuronal activation). Accordingly, these neurons are referred to as oviposition descending neurons (oviDNs), and to the two split-GAL4 driver lines that label them as oviDN-SS1 and oviDN-SS2 (in which SS denotes stable split-GAL4). Stochastic labelling of single neurons resolved two morphologically distinct types of oviDN, which are refered to as oviDNa and oviDNb cells. In an electron microscopy volume of a full adult female brain (FAFB15), two oviDNa-like cells and one oviDNb-like cell were identified in each hemisphere (Wang, 2020).

Egg laying by mated females was completely blocked by genetic ablation of oviDNs, and markedly reduced by their chronic silencing. Virgin females in which oviDNs were ablated were as receptive to mating as control females. Several days after mating, the ovaries of oviDN-ablated females contained many mature eggs, and most carried either a fertilized egg or a first-instar larva in the uterus. It is concluded that oviDNs are essential for oviposition, but dispensable for mating, ovulation and fertilization (Wang, 2020).

It was not possible to generate driver lines that specifically target oviDNa or oviDNb cells. To determine which oviDN subtype is involved in oviposition, a stochastic 'unsilencing' experiment was performed, in which a tdTomato-tagged silencing transgene was targeted to all oviDNs, but stochastically replaced in some of these cells with GFP. Individual females were assayed for egg laying over five days after mating, then dissected and stained to determine their complement of red (tdTomato; silenced) and green (GFP; unsilenced) oviDNs. Females with no unsilenced cells laid no or very few eggs, whereas those with just a single functional oviDN cell generally laid large numbers of eggs. The number of eggs laid per female was variable in these cases, but there was no appreciable difference between females in which an oviDNa cell was unsilenced and those in which an oviDNb cell was unsilenced, nor between females in which either one or two cells of either type were functional. Although the oviDNa and oviDNb subtypes differ in their morphology-and probably their connectivity and physiology-these data suggest that they nonetheless have similar functions in oviposition (Wang, 2020).

Oviposition involves a coordinated and highly stereotyped sequence of motor actions that progresses from abdomen bending to ovipositor extrusion and egg deposition. Abdomen bending, ovipositor extrusion and egg deposition were all eliminated in females in which oviDNs were ablated. Conversely, abdomen bending and ovipositor extrusion were reliably triggered by strong photoactivation of oviDNs in either virgin or mated females. Egg deposition was also induced, but only in mated females (presumably because mating is required to stimulate ovulation). In all of these oviDN activation experiments, the sequence of motor actions was the same as that in natural egg laying. By varying the stimulus intensity, it was found that egg deposition has a higher activation threshold than abdomen bending and ovipositor extrusion, and that action latencies were shorter at higher stimulus intensities. Moreover, at low stimulus intensities, the oviposition sequence was often truncated, but an action was never skipped, and only once was a single action occurring out of order observed (in a total of 38 flies at each of 3 intensities). These data suggest that oviDNs may use a ramp-to-threshold mechanism to elicit the successive motor actions of oviposition. Notably, the activation thresholds and action latencies were indistinguishable between virgins and mated females, indicating that mating status regulates egg laying through the brain circuits upstream of oviDNs rather than through downstream motor circuits (Wang, 2020).

The onset of egg laying after mating is induced by sex peptide, a protein of the male seminal fluid that is detected by sex-peptide sensory neurons (SPSNs) of the uterus. Sex peptide silences both SPSNs and their postsynaptic targets in the abdominal ganglion, the SP abdominal ganglion (SAG) neurons. Artificially activating either SPSNs or SAG neurons suppressed egg laying in mated females. Conversely, ablating or silencing these cells increased the number of eggs laid by virgin females. Virgin egg laying as a result of SPSN or SAG ablation depended on oviDNs, as egg laying was prevented if these cells were co-ablated. SPSN and SAG activity is thus critical in keeping oviDNs inactive until after mating. This inhibition is most likely to be indirect, because the SAGs are cholinergic and hence probably excitatory. This study identified and extensively traced the ascending projections of the two SAG neurons in the FAFB volume and found just a single synapse from SAG neurons to oviDNs (Wang, 2020).

The targets of SAG neurons in the brain have not been identified. Because SAG neurons regulate female receptivity as well as egg laying, it is speculated that their targets could include the female-specific fru-dsx+ pC1 neurons in the protocerebrum, which are known to regulate receptivity. Within the FAFB volume five morphologically distinct pC1 cells were identified in each hemisphere, which are referred to as pC1a-pC1e. Extensive tracing of single pC1a, pC1c and pC1e cells, as well as more limited tracing of pC1b and pC1d cells, suggests that the SAG neurons provide numerous synaptic inputs to the pC1a, pC1b and pC1c cells, with fewer if any direct inputs to pC1d and pC1e cells. Whole-cell recordings were performed from individual pC1 neurons while photoactivating the SAGs; pC1a cells were strongly depolarized, pC1b cells were weakly depolarized and pC1c, pC1d and pC1e cells showed little or no response upon SAG activation. There were numerous synaptic connections amongst all five pC1 subtypes, however, suggesting that any information on mating status that is obtained from SAG neurons by pC1a and pC1b cells is potentially shared across the entire set of pC1 cells (Wang, 2020).

Two split-GAL4 driver lines were obtained for pC1 neurons: pC1-SS1, which labels pC1a, pC1c and pC1e, and pC1-SS2, which labels all five pC1 cells. Ablation of pC1 cells using either driver resulted in an increase in egg laying in virgin females that was dependent on oviDN function, whereas mated females in which pC1 neurons were chronically activated laid fewer eggs. Brief optogenetic silencing of pC1 neurons in virgins did not acutely trigger egg laying, as would be expected if pC1-inactivated virgins (like pC1-intact mated females) rely on additional substrate-borne cues for the induction of egg laying (Wang, 2020).

These behavioural data indicate that-similar to SPSNs and SAG neurons-pC1 neurons suppress the function of oviDNs and therefore suppress egg laying in virgin females. Consistent with this interpretation, it was found by in vivo imaging that basal calcium levels in pC1 neurons, although variable, are generally higher in virgin than mated females. Moreover, whole-cell recordings from oviDNs revealed that both oviDNa and oviDNb cells are hyperpolarized after photoactivation of pC1 neurons, and that this effect is sensitive to picrotoxin, a chloride channel blocker. This inhibition is probably indirect, because pC1 neurons are cholinergic and have very few synapses onto the oviDNs (Wang, 2020).

To look for inhibitory intermediates from pC1 to oviDN cells-as well as excitatory inputs that might stimulate egg laying upon detection of a preferred substrate- the synaptic inputs to oviDNa and oviDNb cells were reconstructed in the Full Adult Fly Brain (FAFB) volume. Sparse split-GAL4 driver lines were obtained for the two cell types with the largest numbers of oviDN input synapses. Whole-cell recordings reliably showed changes in membrane potential in oviDNs after photoactivation of either of these two cell types. The cell type with the most oviDN input synapses is cholinergic, and activation of these cells depolarized oviDNs. These cells were therefore named oviposition excitatory neurons (oviENs). The cell type with the second-highest number of oviDN input synapses is GABAergic, and activation of these cells hyperpolarized oviDNs. Accordingly, these cells were named oviposition inhibitory neurons (oviINs). There is a single oviEN and a single oviIN per hemisphere, and they are reciprocally connected. The oviINs are also reciprocally connected with pC1 cells, and calcium-imaging experiments showed that photoactivation of pC1 cells elicits an excitatory response in oviINs. The pC1 cells have few direct synaptic connections with oviENs, and no connections were detected between SAG neurons and either oviINs or oviENs (Wang, 2020).

Silencing oviENs in mated females strongly suppressed egg laying, similarly to the effect observed when oviDNs were silenced. By contrast, potentiating oviENs in virgin females caused them to lay significantly more eggs than control virgins, albeit not as many as mated females (presumably because ovulation remains infrequent). Manipulating oviIN activity had the opposite effects: silencing oviINs caused virgins to lay significantly more eggs, whereas depolarizing oviINs reduced the number of eggs laid by mated females. Thus, as expected from the sign of their inputs to oviDNs (that is, excitatory for oviENs; inhibitory for oviINs), oviENs promote egg laying, whereas oviINs inhibit it (Wang, 2020).

It was hypothesized that oviENs could mediate the external sensory signals that trigger egg laying in mated females, which are likely to include both gustatory and mechanosensory cues from the substrate. When provided with a choice of substrates, females lay more eggs on agarose medium than on a hard surface or a substrate of agarose and sucrose. Therefore in vivo calcium imaging was performed to determine the responses of oviDNs, oviENs and oviINs to the presentation of each of these substrates to the legs. In oviDNs, an increase was observed in calcium levels only upon contact with the agarose substrate. This response was stronger in mated females than in virgins. The agarose-and-sucrose substrate elicited a small reduction in calcium levels, which was more pronounced in virgin females. The oviENs showed a positive calcium response to agarose but to neither of the other two substrates, and this response was indistinguishable between virgins and mated females. The oviINs responded to all three substrates, but more strongly to agarose and sucrose than to agarose alone, and only weakly to the hard surface. Regardless of substrate, oviIN responses were stronger in virgins than in mated females (Wang, 2020).

In conclusion, these findings support the following model for the neural coordination of mating and egg laying in Drosophila. The oviDNs control the entire oviposition motor programme. They receive excitatory input from oviENs, which respond to stimulatory cues from the substrate, and inhibitory input from oviINs, which convey information about mating status from pC1 cells. In virgins, increased activity of pC1 neurons potentiates oviIN-mediated inhibition of both oviDNs and oviENs, which suppresses egg laying. After mating, sex peptide silences SAG inputs onto pC1 neurons, thereby decreasing the activity of pC1 neurons and oviINs to facilitate egg laying when a preferred substrate is encountered. Reciprocal connections between oviINs and oviENs might ensure that oviDNs respond to oviEN activation with the appropriate temporal pattern and dynamic range, through feed-forward and feedback inhibition, respectively. The oviDNs, oviENs and oviINs all have numerous synaptic inputs in addition to those that have been described in this stduy-all of which remain functionally uncharacterized. These inputs may mediate other controls on the egg-laying process, such as the presence of an egg in the uterus and the nutritional state of the female. The pC1 neurons might also regulate other female behaviours that switch after mating, perhaps through different sets of output neurons. Notably, the male counterparts of pC1 neurons are thought to encode an analogous state of courtship arousal that modulates command pathways for specific motor actions such as courtship song and 'licking'. Thus, functionally analogous but anatomically divergent circuits-shaped during development by fru and dsx-could account for the distinct reproductive behaviours of Drosophila males and females (Wang, 2020).

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. This study demonstrates 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, this study reports a role for SP in altering female physiology to enhance reproductive output: Namely, SP triggers the switch from virgin to mated midgut state (White, 2021).

Production of progeny requires a significant female energy investment. Consequently, mated females alter aspects of nutrient intake and digestion to maintain energy homeostasis. In Drosophila, such changes help sustain egg production. Given the important role of these responses in supporting reproduction, understanding their mechanisms is crucial to understanding the determinants of reproductive success. This study identified female receipt of the seminal SP as a central signal that triggers post-mating shifts in mated female midgut size and gene expression (White, 2021).

Previous studies have shown that males can modulate female physiology and behavior to enhance reproductive output via SP. These SP-induced post-mating responses that influence female nutrition include increasing female food intake, shifting nutrient preference, and concentrating female excreta. In mated females, SP also regulates levels of JHB3 and 20E, both of which are necessary for midgut resizing. This study shows that SP received during mating is both necessary and sufficient for enlargement of the mated female's midgut and thus that SP is the sole male-derived signal needed to mediate the switch between a female's virgin and mated midgut size. Other SP-related ligands likely play a minimal role in the initiation of post-mating midgut growth. The SP paralog Dup99B is transferred at normal levels within the seminal fluid of SP0 males. Since the midgut lengths and transcriptomes of SP0 mated females were no different from those of virgin females, it is unlikely that Dup99B contributes significantly to stimulating midgut growth. For similar reasons, while any post-mating midgut growth role of myoinhibitory peptides (MIPs), the ancestral ligands of SPR, is unknown, any effect that MIPs might have is likely downstream of SP (White, 2021).

This study shows several additional ways that SP modifies the midgut, beyond the roles that were described above. Midgut size peaks at 6 d post-mating and persists for at least 15 d after mating. This is consistent with the time frame of other SP-mediated long-term post-mating responses, such as reduced receptivity to remating, which persists for ~10 d. Additionally, these experiments utilizing males that transfer SP defective for either sperm binding or release from sperm demonstrate that post-mating midgut resizing requires both long-term storage of SP and the gradual release of its C-terminal domain from sperm (White, 2021).

Consistent with the finding that release of SP's C terminus is necessary for post-mating midgut growth, its receptor SPR was found to play a role in post-mating midgut growth as well. The midguts of females homozygous for a deletion that removes SPR and four other genes did not exhibit post-mating midgut growth. To determine whether their lack of post-mating midgut growth was due to loss of SPR, rather than to loss of any of the other four genes, post-mating midgut grown was also examined in SPR-knockdown females. Although the knockdown females still had small amounts of residual SPR expression, they exhibited suppressed post-mating midgut growth. Taken together, these data indicate that SPR is needed for post-mating midgut growth. SP acting via SPR neuronal signaling has previously been linked to midgut post-mating responses, such as stimulating neuropeptide F release from EEs and increasing intestinal transit time. Additionally, the RNA-seq analysis identified SPR expression in the midgut, suggesting the possibility that SP could act directly on the gut to stimulate growth. Investigating where SPR acts to stimulate post-mating gut growth is an intriguing avenue for future research (White, 2021).

The mechanisms by which SP stimulates post-mating midgut growth are important areas for future investigation. For example, SP could act indirectly, through its regulation of the hormones JHB3 and 20E. SP acts via neuronal SPR to increase 20E synthesis in the ovaries, and ovarian 20E is necessary for post-mating gut growth, together suggesting that SP could stimulate midgut growth by raising ovarian 20E levels. SP's stimulation of JHB3 could also contribute to post-mating midgut growth. During the early hours after mating (short-term response), SP's N-terminal domain (unbound to sperm) can stimulate JHB3 release from corpora allata, potentially inducing midgut growth. However, while JHB3 release may initiate growth in the midgut, this is not sufficient for SP's persistent, long-term effect on midgut growth because no midgut growth was observedafter 3 d in females that had mated to SP-TGQQ or spermless males, both of which deposit SP with an intact N terminus and thus should initiate SP's short-term responses. Moreover, the requirement for SPR implicates SP's C terminus (as opposed to its JHB3-inducing N terminus) in the extended post-mating midgut growth. Thus, any early effects of SP on JHB3 that potentially impacted midgut growth cannot be sustained without activity of SP's C-terminal region, released long-term from sperm (White, 2021).

Additionally, SP-induced gut morphological changes are dependent on the female's nutritional state. Previous studies have shown that sterile OvoD females, who do not increase food intake post-mating, still undergo post-mating gut growth. This study shows that post-mating gut growth is not entirely independent of female nutrition. Midguts of mated females fed a nutrient-poor diet do not grow after mating, despite receiving WT SP from males. This could be the result of nutritional deficiencies negatively regulating production of JHB3 in the corpora allata. Under stress conditions, such as starvation, increased levels of 20E have been proposed to negatively regulate juvenile hormones. Additionally, arrested vitellogenesis of nutritionally deprived females can be rescued by treating them with the juvenile hormone analog methoprene, and insulin receptor mutants exhibit reduced JHB3 synthesis. These findings suggest that signals of nutritional state can affect JHB3 synthesis, and poor nutritional state may suppress SP's effect on JHB3 (White, 2021).

In addition to the effects of SP on post-mating midgut morphology, SP alters midgut physiology by reshaping the intestinal transcriptome. Gioti (2012) and Domanitskaya (2007) profiled transcriptomic responses to SP using microarrays to assay the effects of SP on the head and abdomen at 3 to 6 h after mating. Both studies demonstrated SP-induced changes in the transcriptome, such as induction of genes involved in immune responses. This study shows that SP modulates the messenger RNA (mRNA) complement of a single tissue and demonstrates that SP's effect on midgut transcription can persist for at least 2 d after mating, consistent with the timescale of SP's long-term response. SP was found to underly the vast majority of transcriptional changes in the midgut: Only 11 genes that were significantly differentially expressed between the guts of virgin females and females mated to SP0 males, and none of those genes exhibited a greater than twofold difference in expression between virgin and SP0-mated females. Additionally, the genes differentially regulated between females mated to SPWT and those mated to SP0 males largely overlap with those differentially expressed between virgin and mated females. In other words, the switch from a virgin to a mated state at the RNA level does not occur without SP, analogous to the finding that post-mating midgut size is regulated by SP (White, 2021).

Male proteins transferred during mating may also trigger enteric changes in other insects. In mosquitoes, extracts from male accessory glands can stimulate post-mating responses in females. In the dengue vector Aedes aegypti, male-derived substances can increase blood meal size and promote blood meal digestion in the female. Additionally, post-mating transcriptional changes have been observed in the guts of Anopheles gambiae females. In Anopheles coluzzii females, post-mating transcriptomic changes in the gut are evoked by male transfer of the ecdysteroid hormone 20E, suggesting that male-derived molecules triggering gut remodeling may be common in insects. Given the link between nutrition and egg production, understanding how male mosquitoes influence the female midgut may lead to new strategies for vector control (White, 2021).

The observed post-mating changes in midgut transcriptome parallel post-mating dietary shifts. Mated females showed down-regulation of genes involved in carbohydrate metabolism, such as the maltase-encoding genes and those linked to galactose and glucose metabolism, in the midgut. In conjunction, there was up-regulation of genes required for protein digestion and lipid metabolism. Despite differences in experimental design and execution, other studies examining mating-induced transcriptional changes in whole females or female abdomens have detected similar gene expression changes to those that reported in this study. Down-regulation has been observed of maltase genes in the whole-organism transcriptome of 3- to 5-d-old mated females, and another study also found down-regulation of carbohydrate metabolic genes in female abdomens at 3 h after mating. Two other studies found up-regulation of several proteases, and another study found up-regulation of proteolysis-related genes in female abdomens 3 h after mating. qPCR has been used to detect up-regulation of several fatty acid metabolic genes in the midgut after mating. The results confirm that bgm expression is induced upon mating and add bmm to the list although the current study did not find induction of some other genes (SREBP, Acsl, FAS, and ACC) detected by previously as up-regulated after mating. Discrepancies in exact complements of differentially expressed genes likely reflect methodological differences. Down-regulation was found of genes with GST activity involved in detoxification. Their down-regulation after mating could have consequences for the female's ability to deal with toxic dietary compounds and oxidative stress. Indeed, post-mating gut growth increases a female's propensity to develop life span-shortening gut dysplasia. This may reflect potential trade-offs between the demands of reproduction and somatic maintenance (White, 2021).

These results may reflect a post-mating increase in protein and lipid digestion to help sustain egg production. Previous studies have shown that food intake increases after mating, and sufficient dietary protein and lipids are essential for yolk protein production and female fecundity. However, the up-regulation of protein and lipid metabolic genes is likely not simply a consequence of increased food intake since this study saw a coincident down-regulation of carbohydrate metabolic genes. Rather, it is probable that this study observed the mated female midgut altering digestive parameters to adapt to new nutritional demands (White, 2021).

Although the molecular pathways underlying post-mating midgut transcriptomic changes remain unknown, this study detected enrichment of several transcription factor binding motifs among genes regulated by mating, including those for caudal and GATAe. GATAe has been previously linked to regulating ISC maintenance and differentiation and also plays a key role in maintaining gut homeostasis, suggesting that GATAe could play a role in the increase in ISC proliferation observed after mating, as well as the altered expression of digestive enzymes (White, 2021).

In addition to observing mating-induced changes in the midgut transcriptome, this study found that regions of the midgut display varying degrees of morphological plasticity. Although all regions of the midgut increase in length after mating,this study found that the length of the posterior midgut, a region involved in nutrient absorption, grows more in proportion to total midgut length than any other region. This may be the result of egg production increasing nutrient demand. Alternatively, it could be an indirect result of the proximity of the posterior midgut to the ovaries, or mechanical stress exerted from the ovaries toward the midgut (White, 2021).

This study found that the seminal peptide SP is a key regulator of both female post-mating midgut size and transcription of metabolic pathways. The data show that, in well-nourished females, SP is both necessary and sufficient for post-mating midgut enlargement. Additionally, post-mating midgut growth is a component of SP's long-term response, requiring both long-term SP storage and release from sperm, as well as SPR. Mating also causes a shift in the transcriptome of the midgut, a change due almost completely to SP. The post-mating midgut increased transcription of genes involved in lipid and protein metabolism, while decreasing mRNA levels of sugar metabolic genes, and genes involved in detoxification. These results provide insight into SP's role as a regulator of mated female nutritional homeostasis, by helping the female meet the energetic demands of egg production. Overall, these findings illustrate the dynamic nature of the Drosophila midgut, demonstrating how the male can alter female internal morphology and physiology to enhance reproduction (White, 2021).

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). This study demonstrates a previously unsuspected SPR-independent function for SP. In the AG lumen, SP and secreted proteins with membrane-binding anchors are carried on abundant, large neutral lipid-containing microcarriers. These microcarriers are transferred to females during mating where they rapidly disassemble. Remarkably, SP is a key microcarrier assembly and disassembly factor. Males expressing nonfunctional SP mutant proteins that affect SP's binding to and release from sperm in females also do not produce normal microcarriers. These 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 D melanogaster (Wainwright, 2021).

Seminal fluid plays an essential role in male reproductive success. In D. melanogaster, SP, produced from the male AG, has been highlighted as a central player in this process, acting via receptors in the female to stimulate changes that increase fecundity and prevent remating. This study demonstrates that SP has an additional, unsuspected role in males in the assembly of neutral lipid-containing microcarriers in the AG lumen. These microcarriers store SP and can carry other proteins with lipid anchors. Furthermore, proteomics analysis reveals that the normal delivery of subgroups of SFPs to females during mating requires SP, potentially because these subgroups interact differently with microcarriers. Microcarrier interactions are likely to also affect dispersal of these proteins in the female reproductive tract. This analysis of microcarriers in other Drosophila species reveals that SP's microcarrier assembly function may exist in species in which SP has more limited roles in modulating the PMR, suggesting that the former function might have been critical in the evolution of this molecule (Wainwright, 2021).

Seminal proteins are produced throughout adult life, but these proteins are only transferred to females sporadically. Some of these proteins are then rapidly activated via mechanisms that are thought to include proteolytic cleavage and pH changes in the female reproductive tract (discussed in Wilson, 2017). The data suggest that microcarriers could contribute to this activation process. They are repositories for main cell-derived seminal proteins, which presumably partition from the aqueous phase of the AG's secretions, either because of their lipophilicity or because they have binding partners on the microcarrier surface. In the male, molecules like SP bind specifically to microcarriers and not to AG epithelial cells, strongly suggesting that these surfaces are structurally distinct. Subsequent microcarrier dissipation in the female reproductive tract provides a mechanism for dispersing proteins like SP so they can associate with receptors and cell membranes following mating (Wainwright, 2021).

Although both staining of normal microcarriers with lipophilic dyes and the homogeneous internal structure of large defective SP¹/Df(SP) null 'microcarriers' observed with DIC strongly suggest that neutral lipids are a major component of these structures, their precise composition remains unclear. In addition, their nonspherical shape in wild-type males suggests that architectural proteins are highly likely to be involved in establishing their final structure, a proposal supported by the SP mutant phenotype. It will now be important to identify these other structural constituents and to establish whether any of these, unlike SP, play evolutionarily conserved roles in seminal fluid production outside the Drosophila genus (Wainwright, 2021).

Analysis of transcriptomics data from adult Drosophila organs reveals high level expression in the AG of multiple lipases that are predicted to be secreted (e.g., CG5162, CG11598, CG11600, CG11608, CG13034, CG18258, CG18284, CG31872, and CG34447), with all having been detected in proteomics analyses of seminal fluid. These include proteins sharing homology with triacylglycerol lipases (e.g., CG5162, CG13034, CG18258, and CG34447). These lipases provide a potential mechanism to break down neutral lipid transferred in microcarriers to females so the products can be used as fuel. Mammalian seminal fluid also contains lipases and triacylglycerides, suggesting that the latter may be required, perhaps as a male-derived nutrient source, in the reproductive system of all higher organisms (Wainwright, 2021).

Identification of extracellular neutral lipid microcarriers as accessible stores of specific seminal proteins is reminiscent of the role of intracellular lipid droplets in storage of cytoplasmic and nuclear proteins. Lipid droplets are able to dock with specific intracellular organelles to mediate their functions and deliver their cargos. It will be interesting to investigate whether the remnants of microcarriers, such as the microdomains observed with SP-GFP, are in any way targeted to specific cells or structures after transfer to females as these storage vehicles break down (Wainwright, 2021).

It has previously been reported in Drosophila that males can adaptively modulate the relative balance of seminal proteins, including SP, in the ejaculate, depending on female mating status and the presence of rival males. Loading of selected proteins onto microcarriers might provide a simple mechanism to control such rapid changes if the transfer of these large structures can be differentially regulated compared to soluble proteins: for example, by controlling the opening of the sphincters through which seminal fluid passes from the AGs to the ejaculatory duct (Wainwright, 2021).

This study reveals a previously unsuspected male-specific, SPR-independent role for SP in regulating microcarrier shape and size. SP mutants in D. melanogaster still have neutral lipid-containing structures, but they appear to aggregate and fuse, particularly after mating, to generate large lipid droplet-like structures that no longer retain molecules like SP at their surface. To date, it has not been possible to separate the different activities of SP in males and females through expression of different mutants or altered SP levels, making it difficult to fully gauge the importance of the male-specific microcarrier function. However, the observation that SP mutants, which were known to affect binding of SP to the surface of sperm or its subsequent release, also fail to rescue the microcarrier defect in SP null males suggests that the interpretation of the phenotypes associated with these mutants requires some reevaluation (Wainwright, 2021).

The data suggest that both the C-terminal and N-terminal domains of SP can interact with microcarriers even though they share no structural similarity. This may involve direct binding to the outer surface of the microcarrier or, because both domains contain charged residues, more indirect associations via other molecules attached to microcarriers. The multidomain interaction contrasts with sperm binding and may underlie why SP can transfer to sperm in the female reproductive tract (Wainwright, 2021).

It has been suggested that SP is likely to have roles in addition to its effects mediated via SPR signaling in the female reproductive tract, which include induction of a female sexual refractory period. This is because some SP-expressing species, like D. pseudoobscura and D. persimilis, do not appear to express SPR in this location and additionally show much less female postmating refractoriness relative to other SP-producing species. The current data suggest that microcarrier assembly may be this additional function, with the shape of microcarriers rapidly coevolving with SP. An absence of microcarriers in species with a highly divergent (D. virilis) or no (D. mojavensis) SP homolog, as evidenced by two different staining methods, supports the hypothesis. Not unexpectedly, DIC microscopy suggested that the luminal content of these latter two species is not homogenous, but it is clearly different from the other Drosophila species that ware studied. Interestingly, D. virilis expresses SPR in the female reproductive tract so, unlike in other species, its SP protein may be specifically involved in activating the female PMR, rather than microcarrier formation (Wainwright, 2021).

In light of these findings, it will now be important to investigate whether other proteins with fundamental roles in packaging and storing seminal fluid components have also evolved signaling roles in animals (Wainwright, 2021).

An important conclusion from this study is that the normal transfer of different subgroups of SFPs is dependent on SP. One simple explanation is that this reflects differences in their interactions with microcarriers. Having shown that main cell-expressed GFP-GPI binds to microcarriers, it was interesting to identify the GPI-anchored junctional protein Contactin as one of the proteins, which appears to be retained more in the AGs of SP null males. Furthermore, preferential retention of Dup99B in SP null males is consistent with the idea that this SP-like protein might bind to microcarriers even though it is primarily expressed in the adjacent ejaculatory duct epithelium (Wainwright, 2021).

Elegant studies by Wolfner and coworkers have identified several long-term response (LTR) network genes expressed in the AG that are interdependent and required either in the male or female for SP to be retained in the sperm storage organs. It was noticed that several of these proteins appear to be expressed at higher levels in SP nulls and also that a greater proportion is transferred from mutant males to females upon mating. A previous study has suggested that two of these proteins, CG1652 and CG1656, are present at similar levels in the female reproductive tract 1 h ASM to SP null and SP rescue males (40). This difference cannot be explained, but it is important to emphasize that this study measures the relative quantity of these proteins that leaves the male AG, not what remains in the female reproductive tract some time later. Overall, the proteomics analysis clearly shows that SP modulates the transfer of specific subclasses of SFPs to females, and, particularly in the case of proteins that are retained in SP nulls, this could result from the disruption of microcarriers (Wainwright, 2021).

It will now be important to investigate whether any of the network genes is involved in loading or unloading SP from microcarriers or, indeed, whether they play a role in microcarrier assembly, particularly since they appear to be present in species where SP does not seem to be involved in signaling. The role of secondary cells in microcarrier morphology also needs to be examined in more detail. Furthermore, confirming that other SFPs identified in the proteomics analysis or main cell-expressed GPI-anchored proteins are microcarrier cargos should allow the functions of these structures to be assessed more extensively and may suggest molecular tools that could be used to screen for similar processes in higher organisms (Wainwright, 2021).

REGULATION

Drosophila Sex-peptide stimulates female innate immune system after mating via the Toll and Imd pathways

Insect immune defense is mainly based on humoral factors like antimicrobial peptides (AMPs) that kill the pathogens directly or is based on cellular processes involving phagocytosis and encapsulation by hemocytes. In Drosophila, the Toll pathway (activated by fungi and gram-positive bacteria) and the Imd pathway (activated by gram-negative bacteria) leads to the synthesis of AMPs. But AMP genes are also regulated without pathogenic challenge, e.g., by aging, circadian rhythms, and mating. This study shows that AMP genes are differentially expressed in mated females. Metchnikowin (Mtk) expression is strongly stimulated in the first 6 hr after mating. Sex-peptide (SP), a male seminal peptide transferred during copulation, is the major agent eliciting transcription of Mtk and of other AMP genes. Both pathways are needed for Mtk induction by SP. Furthermore, SP induces additional AMP genes via the Toll (Drosomycin) and the Imd (Diptericin) pathways. SP affects the Toll pathway at or upstream of the gene spätzle, and the Imd pathway at or upstream of the gene imd. Mating may physically damage females and pathogens may be transferred. Thus, endogenous stimulation of AMP transcription by SP at mating might be considered as a preventive step to encounter putative immunogenic attacks (Peng, 2005b).

Mating in D. melanogaster and in many other insects elicits various postmating responses (PMR) in females, e.g., enhanced ovulation and oviposition, reduced receptivity (willingness to remate) (Gillott, 2003) and stimulation of the innate immune system. The PMR are mainly elicited by seminal fluid transferred during copulation. One of its components, Sex-peptide (SP; a 36 amino acid long peptide synthesized in the male accessory glands (Chen, 1988), is the major agent eliciting oviposition and reduction of receptivity (Chapman, 2003; Liu, 2003). This study investigates the time course of AMP induction after copulation; SP has been determined to be one of the major seminal components eliciting transcription of AMP genes after mating (Wigby, 2005).

D. melanogaster produces about 20 different antimicrobial peptides. The AMPs can be classified into seven distinct families: Attacins, Cecropins, Diptericins, Drosocins (against gram-negative bacteria), Defensins, Metchnikowin (against gram-positive bacteria), and Drosomycins (against fungi). Mating induces transcription of all probed AMP genes: Attacin A and C, Cecropin B, Diptericin, Drosocin, Defensin, Metchnikowin, and Drosomycin, thus confirming the data obtained by microarray analysis (Peng, 2005b).

To cover the whole spectrum of AMPs induced by different types of pathogens, focus was placed on the transcription of the Metchnikowin (Mtk; induced by both pathways), Drosomycin (Drs; induced by the Toll pathway), and Diptericin (Dipt; induced by the Imd pathway) genes, respectively. In mated females Mtk, Drs, and Dipt induction are observed as early as 1 hr after mating. Expression peaks between 2 and 4 hr and fades away after 8 hr, again reaching the virgin level. However, the degree of upregulation varies, Mtk showing the strongest response (Peng, 2005b).

To identify the elements responsible for the elevated transcription of the AMP genes, males without functional SP (SP⁰ males [Liu, 2003]) and males without germline (germline-less [GLL] males; sons of tropomyosinII mutant [TmII^gs1/TmII^gs1] females lacking the germline) were mated with virgin wt females. 2 hr after mating, RNA was extracted from the mated females and analyzed by Northern blots and quantitative PCR. SP⁰ males fail to induce the transcription of AMP genes, whereas GLL males induce AMP genes at about 4/5^th of the level of wt males. The latter finding indicates that sperm plays a minor role in eliciting Mtk transcription and is consistent with the findings of McGraw (2004) (Peng, 2005b).

To confirm the capability of SP to induce the transcription of the Mtk gene, transgenic females expressing SP ectopically and constitutively (driven by the promoter of the Yp1 gene; i.e., Yp-SP females; Aigaki, 1991) were analyzed for Mtk expression. In contrast to the very low level of Mtk expression in virgin control females, Mtk expression in virgin Yp-SP transgenic females is already high, even higher than in mated control females. It is not further increased by mating, i.e., Mtk in Yp-SP virgins is already transcribed at a maximal rate. It is concluded that SP is the major agent eliciting Mtk expression after mating and that constitutive expression of SP leads to permanent high levels of Mtk transcription. Furthermore, since SP concentration in the hemolymph of transgenic Yp-SP females is higher than in the hemolymph of mated females and Mtk transcription is statistically significantly higher in Yp-SP transgenic females than in wt females mated with wt males, the level of transcription of AMP genes is very likely dependent on SP concentration (Peng, 2005b).

The SP-induced expression of two additional AMPs dependent on the Toll (Drosomycin) or the Imd (Diptericin) pathways, respectively, was investigated. Expression of Drs and Dipt was monitored in virgin wt females, in wt females mated with wt or SP⁰ males, respectively, and in virgin and mated Yp-SP transgenic females (the latter mated with wt males). Sex-peptide also induces the transcription of Drs and Dipt, but the induction of Drs and Dipt is weaker by orders of magnitude than that of Mtk. Constitutive expression of SP in Yp-SP transgenic females leads to continuous expression of Drs and Dipt and elevates the expression of Dipt statistically significantly above the level induced by mating. It is concluded that specific AMP genes respond differentially to SP induction (Peng, 2005b).

The Toll and Imd signaling cascades are the major and best-characterized pathways involved in the activation of AMPs after pathogenic challenges. The effect of SP on AMP expression was studied by comparing the expression of Mtk, Drs, and Dipt in wt females or in females mutant in the Toll and Imd pathways, respectively, before and after mating with wt males. RNA was extracted from virgin and mated females and analyzed by quantitative PCR (Peng, 2005b).

With the exception of dorsal (dl), all loss-of-function mutants of the Toll and Imd pathways abolish or strongly reduce Mtk expression after mating. Thus, Mtk expression induced by SP is dependent on both pathways. Furthermore, since spz and imd females fail to induce Mtk transcription after mating, SP must act on or upstream of spz and imd. dl and its functional homolog dif have been reported to be involved in AMP gene transcription under pathogenic challenge in the larval stage, but not functional in the adult immune defense. A partial response is observed in dl females, indicating that dl may be partially involved in the innate immune response elicited by SP in adult females (Peng, 2005b).

Drs expression, controlled by the Toll pathway, is completely abolished in spz and Tl mutants. Correspondingly, Dipt expression, which is controlled by the Imd pathway, is completely abolished in the Imd pathway loss-of-function mutants imd, Tak1, and rel. It is concluded that SP can activate the Toll and the Imd pathways. The Toll pathway is essential for Drs expression, whereas the Imd pathway is essential for Dipt expression (Peng, 2005b).

The SP-induced immune response activates the transcription of all three AMP genes studied. After pathogenic infections, Drs is induced by the Toll pathway and Dipt by the Imd pathway, whereas both pathways induce Mtk expression. The results obtained with the loss-of-function mutants follow this scheme. Whereas loss-of-function mutants of both pathways reduce or abolish Mtk expression after mating, induction of Drs expression is only abolished by loss-of-function mutants of the Toll pathway, whereas induction of Dipt expression is only lost in mutants of the Imd pathway. In sum, the classical pathways are activated to induce the transcription of AMP genes after mating as after microbial or fungal infections (Peng, 2005b).

Detection of microorganisms and triggering the appropriate pathway is achieved by pattern recognition receptors (PRRs), immune proteins recognizing general microbial components. Two families of PRRs have been identified in Drosophila: the peptidoglycan recognition proteins (PGRPs) and the gram-negative binding proteins (GNBPs). Some of the 13 PGRPs encoded in the D. melanogaster genome have been implicated in the activation of specific immune responses. However, the signaling cascades between the PRRs and the Toll and the Imd pathways are not well characterized. Since in spz and imd null mutants AMP induction by SP is specifically abolished, the inducing signals must affect the signaling cascades at or upstream of those genes. At this stage, it cannot be determined whether SP enters the pathways at the PRR level or at an intermediate level between the PRRs and spz or imd, respectively. Furthermore, the induction of AMPs may occur systemically (e.g., in the fat body) or locally in the reproductive tract. Microarray analysis of AMP expression after mating of wt females with either wt or SP⁰ males, respectively, suggests that AMPs are mainly induced in the abdomen, but it does not discriminate between a systematic response in the abdomen and a specific response in the genital tract (Peng, 2005b).

Drosophila females undergo dramatic physiological changes after mating, predominantly induced by SP. Mating may also physically damage females and may expose the female to pathogens transferred by the male as shown for the milkweed leaf beetle. Thus, the activation of the innate immune system to encounter putative immunogenic attacks during this sensitive phase of the life history of females makes biological sense. The signal is plausibly coupled to copulation in the form of SP transferred in the seminal fluid. Such a mechanism might allow the female to respond preventively to potential threats. In sum, these findings may describe the result of an optimal economical balance between spending costly energy for the innate immune response and preventive measures to fight a putative pathogenic attack (Peng, 2005b).

Gradual release of sperm bound Sex-Peptide, regulated by enzymatic cleavage, controls female postmating behavior in Drosophila

A set of synthetic (Sex-Peptide) SP fragments was used to determine the part of SP interacting with sperm. The fragment SP_1-11 competes for binding with full length SP_1-36. Thus, SP binds to sperm with the N-terminal end. This part of SP stimulates juvenile hormone synthesis in vitro (Moshitzky, 1996), but the C-terminal part is essential to elicit the post-mating response (PMR) (Schmidt, 1993). Thus, binding to sperm does not block the part of SP that is necessary to elicit the PMR, full size SP bound to sperm is not able to elicit the PMR. How then is this achieved (Peng, 2005a)?

Sex-peptide is eventually lost from the tail. Hence, reincubation of sperm isolated 5 days after mating with SP should yield labeled sperm tails. However, this is not the case; only the head is labeled. 'Sex-peptide-null' sperm isolated from females 5 days after mating with SP⁰ males does not produce any signal, as expected, but incubation of such SP⁰ sperm with SP in vitro results in fully labeled sperm. Thus, once sperm has been in contact with SP, binding of additional SP is not possible anymore, but the mere storage of sperm in the genital tract in the absence of SP is not responsible for this effect. The antibody used in the experiments reported thus far is specific for the fragment SP_6-20 (AB SP_6-20); hence it does not detect other parts of SP. If, for example, SP would be cleaved between the N-terminal part (site of SP attachment to sperm) and the part of SP recognized by AB SP_6-20, the N-terminal part of SP remaining on sperm could not be detected by this antibody. Indeed, a putative trypsin cleavage site is localized near the N terminus of SP (R₇K₈). An antibody specific for the fragment SP_1-7 (AB SP_1-7) reveals that this fragment is still bound to the sperm tail even 5 days after mating. These findings show that the N-terminal end of SP remains and binds to sperm (thus blocking full-length SP_1-36 binding upon reincubation). The C-terminal part, known to be essential to elicit the PMR, is cleaved off and released (Peng, 2005a).

The signal on the sperm head remains strong even after storage for 5 d in the female genital tract. The label cannot be due to another molecule that, for example, shares sequence identity with SP. The only compound missing in the seminal fluid of SP⁰ males is SP, but incubation of sperm derived from a SP⁰ male with the antibody AB SP_6-20 does not yield any signal. Hence, the labeling is due to SP (Peng, 2005a).

To confirm the above findings, three transgenes were introduced into flies with a SP⁰ background: (1) the wild-type SP gene (control, transgenic wild-type = TG^WT males); (2) a modified SP gene coding for a SP mutated at a putative trypsin cleavage site (R₇-Q₇, K₈-Q₈) near the N terminus of SP (TG^QQ males; Q maintains the polarity of the replaced amino acids); (3) a truncated SP gene with a deletion comprising the codons of the amino acids E₂-R₇ (TG^Δ2-7 males; to allow appropriate processing of the signal peptide, W₁ was not removed). The DNA fragment -210 to +1 of the SP promoter that was used to drive the expression of the constructs induces expression of a lacZ reporter exclusively in the accessory glands of the male. The transgenic males used for the experiments reported below contain one copy of the respective transgene in a SP⁰ background. The amount of SP produced in their accessory glands is about the same (Peng, 2005a).

Females mated with TG^WT males show normal PMR. Thus, the amount of SP produced by one copy of the wild-type rescue construct is sufficient to support the PMR, as observed after mating with an Ore R wild-type male. Because the accessory glands of all transgenic males produce the same amount of SP, the results presented below are due to the modification of the SPs and not to varying amounts of SP production in the transgenic males. Females mated with TG^QQ or with TG^Δ2-7 males show only short-term PMR. After 2 days, the PMR are lacking, as was also observed after matings with a SP⁰ male. These short-term responses are due to the transfer of free functional SP not bound to sperm. (In a mating with a wild-type male, about 3 pM SP are transferred per copulation; only part of it is bound to sperm, the rest is free SP, which elicits a short-term PMR). Only the wild-type rescue males, TG^WT, elicit PMR indistinguishable from the PMR elicited from an Ore R wild-type male. However, all males transfer and store sperm, and sperm of all transgenic males fertilize eggs. Sperm isolated from females 5 hr after mating with TG^WT or TG^QQ males show labeling of the tail, but no labeling is observed after mating with TG^Δ2-7 males. After 5 days, there is no labeling of the tail after mating with TG^WT males, but still full labeling of sperm after mating with TG^QQ males. It is concluded that full-length SP_1-36 bound to the tail is subsequently cleaved at the trypsin cleavage site R₇K₈, thus releasing the C-terminal fragment SP_8-36 and that this fragment is sufficient to elicit the PMR. Furthermore, these results confirm that unmodified SP binds to sperm with its N-terminal end (Peng, 2005a).

Allocrine modulation of feeding behavior by the Sex Peptide of Drosophila

Mating elicits a dramatic reprogramming of female behavior in numerous insect species. In Drosophila, this postmating response (PMR) comprises increased egg-laying rate and reduced sexual receptivity and is controlled by the products of the male accessory glands, a family of 80 small peptides transferred in the male seminal fluid. Copulation strongly stimulates female food intake. Remarkably, this change is abolished if the males lack a single, small seminal protein, the Sex Peptide (SP). Ectopic expression of SP in virgin females mimics the effect of mating on feeding behavior, demonstrating that SP is the main agent controlling this behavioral paradigm. These observations identify enhanced feeding behavior as a novel component of the Drosophila PMR and suggest that SP represents a molecular link between energy acquisition and reproductive investment. Since SP acts on the corpus allatum to stimulate the secretion of Juvenile Hormone (JH), which plays a crucial role in sexual maturation and oogenesis in Drosophila females, induction of oogenesis and vitellogenesis by JH may may explain the increase in female food intake (Carvalho, 2006).

Nutrient availability plays a critical role in reproductive success. Accordingly, changes in patterns of feeding behavior correlate with reproductive status in a wide range of organisms. However, the mechanisms regulating this vital process are not well understood. To investigate this issue, adult food intake was recorded by allowing flies to feed on medium colored with a nonabsorbable, nonmetabolizable dye. Visual inspection revealed a striking effect of mating status on female abdominal food accumulation. Mated females ingested substantially larger meals than age-matched virgins. This disparity was both accentuated and accelerated if a 12 hr starvation period preceded the feeding trial. Spectrophotometric quantitation showed that, in these conditions, mated females consumed ~2.3 times as much food as virgins (Carvalho, 2006).

Drosophila feeding behavior can be monitored by radioactive labeling of the medium. An essential advantage of this method lies in its enhanced specificity and sensitivity, which make it possible to record steady-state food consumption in nonstarved flies. In addition, food intake can be measured over longer periods, avoiding short-term fluctuations and circadian variation. Adult food ingestion was recorded over a 24 hr period by using food labeled with [α-³²P]dCTP. Averaged across multiple, independent trials, ad-libitum-fed, mated females showed a 56% elevation in radioactive tracer level when compared to virgins. Together with the results obtained with dye-colored food, these findings strongly suggest that the measurements reflect bona fide differences in volume of food ingestion between the virgin and mated states. In contrast to the situation in females, male feeding was not affected by mating status. These results identify enhanced feeding behavior as a novel component of the Drosophila PMR (Carvalho, 2006).

Both previously described elements of the behavioral PMR—egg laying and rejection of secondary copulation—are regulated by the products of the male accessory gland. It was therefore asked whether the accessory-gland proteins (Acps) are also responsible for the feeding-behavior changes in mated females. Genetic ablation of the accessory-gland main cells can be achieved through expression of a modified form of diphtheria toxin subunit A (DTA) under the control of the main cell-specific promoter Acp95EF. These DTA-expressing males produce only vestigial amounts of Acps (~1% of wild-type) and induce no egg-laying and only a slight, transient reduction of female receptivity. Females mated to DTA males displayed no elevation of food intake, whereas isogenic control males lacking the DTA construct induced a normal response, indicating that the physiological stimulation of feeding behavior requires the Acps (Carvalho, 2006).

One Acp in particular, the Sex Peptide (SP), is both necessary and sufficient to induce the PMR in virgins. It was therefore asked whether SP is the particular Acp responsible for stimulating female food intake. SP⁰ males, which specifically lack SP as a result of a targeted chromosomal deletion, but normally express and transfer all remaining Acps and sperm, failed to significantly induce feeding in females. Both DTA and SP⁰ males showed courtship and mating rates similar to those of the respective controls and successfully fertilized all females they were kept with, as assayed by scoring viable progeny of females kept in individual vials. These results demonstrate that the main-cell Acps, and SP in particular, are required for stimulation of postcopulatory food ingestion in females (Carvalho, 2006).

Next the action of SP in regulating female feeding behavior was directly tested. Ectopic expression of SP in the adult fat body of virgin females by means of a yolk protein 1 enhancer (yp1) has been shown to be sufficient to induce the two classical components of the PMR. Females bearing the yp1-SP fusion construct exhibit a constitutively increased feeding rate that is not further elevated by mating, suggesting that SP can, by itself, elicit a mated-like appetite in virgins. This hypothesis was tested further by expressing SP under the control of a UAS promoter. Previous work has identified several independent GAL4 insertion lines that, when used to drive SP, can elicit the PMR in virgin females. Indeed, expression of SP under the control of either the 9Y- or C370-GAL4 driver lines markedly stimulates virgin feeding rate. Importantly, in neither case does copulation further increase food ingestion. Three additional GAL4 drivers gave identical results. Although the central nervous system is the only area in common among the expression patterns of the five driver lines, the fact that SP is expressed as a secreted, diffusible molecule precludes a definite conclusion concerning its site of action. These findings demonstrate that SP modulates postcopulatory feeding in females, whereas sperm and the act of copulation per se do not play substantial roles (Carvalho, 2006).

In numerous animal species, including humans, enhancing nutrient acquisition is a common strategy accompanying reproductive effort, and its pivotal role in ensuring reproductive success is well established. Drosophila has found an elegant and effective way to couple reproductive investment to increased acquisition of energy resources—a single, small peptide transferred in the male ejaculate. Peptides play a central role in appetite control, both in insects and in higher organisms. Remarkably, in this case, the molecule is produced by and regulates the feeding behavior of two separate individuals. Sexual allocrine mechanisms have also been described in vertebrates. For example, prostaglandins secreted in human semen can modulate female immune response, a role that has also been attributed to the SP of Drosophila. How does SP orchestrate such a dramatic behavioral and physiological reprogramming? In the case of appetite modulation, a possible mechanism is suggested by the fact that SP binds to the subesophageal ganglion, a neuronal center previously implicated in taste recognition and feeding. Alternatively, SP may regulate food intake indirectly. Ex vivo, SP acts on the corpus allatum to stimulate the secretion of Juvenile Hormone (JH), which plays a crucial role in sexual maturation and oogenesis in Drosophila females. Induction of oogenesis and vitellogenesis by JH may in turn induce female food intake. In this regard, it will be interesting to investigate whether appetite modulation requires intact reproductive activity (Carvalho, 2006).

These findings raise another intriguing question. Mating drastically reduces the lifespan of Drosophila females, a phenomenon that has been attributed to the action of the Acps, and to SP in particular. Given the link between increased food consumption and shortened lifespan in many organisms, it is conceivable that the reduced longevity of mated females may somehow relate to their accrued nutrient ingestion. Further study on the biology of Acps should help elucidate this intriguing aspect of animal reproduction (Carvalho, 2006).

Acp70A regulates Drosophila pheromones through juvenile hormone induction

Mated Drosophila melanogaster females show a decrease in mating receptivity, enhanced ovogenesis, egg-laying and activation of juvenile hormone (JH) production. Components in the male seminal fluid, especially the sex peptide ACP70A stimulate these responses in females. This study demonstrates that ACP70A is involved in the down-regulation of female sex pheromones and hydrocarbon (CHC) production. Drosophila G10 females which express Acp70A under the control of the vitellogenin gene yp1, produced fewer pheromones and CHCs. There was a dose-dependent relationship between the number of yp1-Acp70A alleles and the reduction of these compounds. Similarly, a decrease in CHCs and diene pheromones was observed in da > Acp70A flies that ubiquitously overexpress Acp70A. Quantitative-PCR experiments showed that the expression of Acp70A in G10 females was the same as in control males and 5 times lower than in da > Acp70A females. Three to four days after injection with 4.8 pmol ACP70A, females from two different strains, exhibited a significant decrease in CHC and pheromone levels. Similar phenotypes were observed in ACP70A injected flies whose ACP70A receptor expression was knocked-down by RNAi and in flies which overexpress ACP70A N-terminal domain. These results suggest that the action of ACP70A on CHCs could be a consequence of JH activation. Female flies exposed to a JH analog had reduced amounts of pheromones, whereas genetic ablation of the corpora allata or knock-down of the JH receptor Met, resulted in higher amounts of both CHCs and pheromonal dienes. Mating had negligible effects on CHC levels, however pheromone amounts were slightly reduced 3 and 4 days post copulation. The physiological significance of ACP70A on female pheromone synthesis is discussed (Bontonou, 2014).

Protein Interactions

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, the binding properties of SP and DUP99B were characterized 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 (Kd), and also determination of the minimal peptide concentrations necessary for binding, indicate that SP is the more important peptide inducing the postmating responses. These 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 (Ding, 2003).

In Drosophila females Sex-peptides elicit two major responses: receptivity is reduced and egg laying increases. Control of receptivity and egg laying are very likely achieved via the nervous system. These results are in accord with findings that ¹²⁵I-iodinated Sps and alkaline phosphatase-tagged peptides label specific parts of the nervous system and the female genital tract (Ottiger, 2000). This approach has identified the presence of Sp-binding proteins in adult females for the following reasons. (1) Strong binding is only observed with fusion proteins containing a SP sequence that has been shown to be biologically active as a peptide fragment or as a fusion protein. (2) The calculated dissociation constants (Kds) are of the order of magnitude expected for hormone-receptor interactions. (3) Both probes label the same sites. (4) The appearance of the binding proteins during development in the nervous system and the genital tract is independent of the labeling method of the probe (Ding, 2003).

The binding sites observed on cryostat tissue sections of virgin females do not necessarily reflect the sites of interaction of the ligand with functional proteins in vivo. By incubating sections of mated females with the AP-SP probe, attempts were made to identify the in vivo targets of Sps after mating. Only the uterus was blocked by previously transferred peptides. Neither the target sites in the upper part of the genital tract nor those in the nervous system were blocked. Thus, from these results one might conclude that only the uterus is an in vivo target. However, it is thought that at least for the labeling of the nervous system, that this is not the case. Both SP and DUP99B are transferred in picomolar amounts to the female during copulation (Chen, 1988). However, the amount of SP in the hemolymph is very small and barely detectable by Western blotting. This is very likely due to rapid uptake of SP by the pericardial cells resulting in a low concentration of SP in the hemolymph. It is reasonable to assume that these small amounts of peptide can only partially block the many target sites accessible via hemolymph. Hence, most binding sites in the nervous system may still be available for binding (Ding, 2003).

Only the first contact site of the peptides after mating, the uterus, was blocked by the transferred peptides, and this only for the first few hours. Most of the transferred peptides are lost from the genital tract by the expulsion of the first egg. The concentration of the peptides in the female decreases drastically within the first 2 h after mating. Because binding of the two peptides occurs with the almost identical C-terminal parts, the proportion of SP and/or DUP99B binding to the uterus cannot be determined with this approach (Ding, 2003).

The two Sex-peptides differ partially in their amino acid sequence. Whereas the amino acid sequences encoded by the first exons are different, the sequences encoded by the second exons are almost identical (Saudan, 2002). Thus, one would expect functional redundancy for the C-terminal parts of the peptides, but not for the N-terminal parts. Indeed Sex-Peptide stimulates juvenile hormone synthesis in isolated corpora cardiaca/corpora allata complexes, whereas DUP99B does not (Moshitzky, 1996; Fan, 2000). It has been shown that the N-terminal amino acids of SP are responsible for this stimulation (Fan, 2000). The almost identical C-terminal parts are known to be responsible for eliciting the two postmating responses. Thus, they seem to be functionally redundant. Nevertheless, the import of the two peptides in inducing the two responses in vivo may not be the same (Ding, 2003).

The Kds of the two peptides indicate that SP has an about 20 times higher binding affinity than DUP99B in the genital tract. The data show that SP has a higher binding affinity than DUP99B in the nervous system and in the genital tract. This interpretation is also supported by the ovulation response measured at different time points after mating, SP, or DUP99B injections, respectively. With native DUP99B the time span needed to obtain a maximal response of ovulation is about twice the time span observed after a normal mating or a SP injection. These results suggest that the efficiency of DUP99B in inducing the postmating responses is lower than that of SP. Taken together, these results suggest that SP is the key player in eliciting the two postmating responses (Ding, 2003).

Although the differences in the binding properties to the nervous system and the genital tract of each of both peptides are only two- to four-fold, this is a first indication that there may exist two molecular types of binding proteins. The other results reported in this article confirm this hypothesis. The sequence requirements for binding to the two tissues are also different. Whereas an intact C-terminal part is needed for binding to the nervous system, binding to the genital tract is less stringent. Thus, the properties for binding to the nervous system are identical to the properties for eliciting the two postmating responses. Furthermore, these results indicate that the Kds and the minimal concentrations needed for binding as determined for the antennal nerve and the uterus, reflect the binding properties for the whole nervous system or the whole genital tract, respectively (Ding, 2003).

The Drosophila G protein-coupled receptor, Methuselah, exhibits a promiscuous response to peptides

Methuselah (Mth) is a G protein-coupled receptor (GPCR) associated with longevity in Drosophila melanogaster. Previously, Stunted (Sun) was identified as a peptide agonist of Mth. This study identified two additional activators of Mth signaling: Drosophila Sex Peptide (SP) and a novel peptide (Serendipitous Peptide Activator of Mth, SPAM). Minimal functional sequences and key residues were identified from Sun and SPAM by studying truncation and alanine-scanning mutations. These peptide agonists share little sequence homology and illustrate the promiscuity of Mth for activation. mth mutants exhibit no defects in behaviors controlled by SP, casting doubt on the biological significance of Mth activation by any of these agonists, and illustrating the difficulty in applying in vitro studies to their relevance in vivo. Future studies of Mth ligands will help further understanding of the functional interaction of agonists and GPCRs (Ja, 2009).

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 100mμM), 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 3-4mμM (Kim, 2015).

DEVELOPMENTAL BIOLOGY

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, sex-peptide (SP) elicits short- and long-term PMR, but only the latter in the presence of stored sperm (sperm effect). This study elucidates 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. SP is shown to bind 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. It is concluded that 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. It is suggested that this could explain the excessive length of sperm tails in some Drosophila species (Peng, 2005a).

Despite their amazing species diversity, some reproductive traits are common to most insects. For example, in many higher insects, mating elicits a drastic increase in egg laying and a reduction of receptivity (readiness to copulate). Thus, the postmating responses (PMR) are either evolutionarily old, or they evolve fast. They are induced by components of the seminal fluid transferred into the female during copulation and are stored in the female genital tract. Due to its well-known genetics, combined with its amenability for biochemical, physiological, and genomic analysis, Drosophila has become the best-studied species with respect to seminal peptides influencing postmating behavior in insects (Peng, 2005a).

In D. melanogaster, the PMR are elicited by three male peptides. Sex-peptide (SP) and Ovulin (Herndon, 1995) are products of the accessory glands, and the Ductus ejaculatorius peptide (DUP) is synthesized in the ejaculatory duct. They are transferred together with sperm into the female during mating. SP and DUP induce both PMR. Ovulin elicits ovulation and oviposition on the first day after mating, but does not affect receptivity. Mating with wild-type males induces the PMR for about 1 week; however, mating with males that do not transfer sperm elicits PMR only for one day (long-term and short-term PMR, respectively). (This phenomenon has been described as 'sperm effect' by Manning (1962, 1967) Males lacking functional SP (SP0 males) elicit only weak PMR lasting about one day (Chapman, 2003: Liu, 2003). Hence, in vivo SP is the major agent eliciting both the short- and the long-term PMR; Ovulin and DUP play only minor roles during the first day after copulation. Because SP0 males transfer and store sperm (Liu, 2003), sperm alone cannot induce the PMR, but stored sperm are essential for their persistence (Manning, 1962; Manning, 1967). Without sperm transfer, or injection of physiological amounts of the peptide, SP elicits only short-term PMR (Kubli, 2003, Manning, 1996 and Schmidt, 1993). How do SP and sperm interact to support the long-term PMR? A possible mechanism could be binding of SP to sperm (Kubli, 2003). This study shows that SP binds to sperm with the N-terminal end. During storage, SP is cleaved off from the tail, and the released C-terminal part elicits the long-term PMR (Peng, 2005a).

Immunofluorescence was used to visualize putative binding of peptides to sperm. Sperm were isolated from females at different time intervals after mating and were then incubated with antibodies specific for SP, DUP, or Ovulin, respectively. DUP binds only to the head and only in the first few hours, whereas Ovulin does not bind to sperm at all. Furthermore, sperm is not autofluorescent at the wavelength used for this study. Sperm isolated 5 hr after mating show binding of SP along the entire length of sperm (The antibody used for these experiments is specific for the fragment SP_6-20.) During storage in the female genitalia, SP is lost from the tail. Two days after mating, the signal intensity along the entire length of the tail, in comparison with the head, is weaker and spottier than immediately after mating. Five days after mating, SP is almost absent from the tail. However, the signal is still strong on the head in all stages. A quantitative analysis reveals that SP is gradually lost from the sperm tail. Thus, SP indeed binds to sperm during several days, but it is lost from the tail while sperm are stored in the female genital tract. In contrast, DUP binds to the sperm head only for a few hours, and Ovulin does not bind at all. This is consistent with the finding that DUP and Ovulin play only minor roles in the short-term PMR. Thus, focus will be placed on the fate of SP bound to sperm (Peng, 2005a).

This study shows that SP binds to sperm with its N-terminal end and that the C-terminal part of SP is gradually cleaved from the sperm tail. Labeled SP binds with its C-terminal region to specific sites in the female nervous system and the genital tract. Binding to the central and peripheral nervous systems is dependent upon an intact C-terminal part, whereas binding to the genital tract is less demanding in terms of amino acid sequence (Ding, 2003). These findings suggest that binding in the nervous system is responsible for eliciting the PMR, whereas binding in the genital tract may reflect the presence of a peptide transporter (Kubli, 2003). This interpretation is supported by the fact that the free (non-bound) modified SP_Q7Q8 of TG^QQ males elicits the short-term responses, but the same SP_Q7Q8 bound irreversibly to the sperm tail cannot elicit the long-term responses. Hence, replacement of R₇ by Q₇ and K₈ by Q₈ does not affect SP function, but mutant SP_Q7Q8 permanently bound to the tail cannot elicit the PM responses via the SP binding proteins present in the genital tract, i.e., these SP binding proteins are probably not receptors involved in the PMR. Furthermore, the results strongly suggest that the released C-terminal part of SP enters the hemolymph to reach its targets in the nervous system. Apparently the sperm tail is the only source of SP to sustain the long-term PMR. These results are also in accord with the findings that the brain is the site of action of SP (Nakayama, 1997) and that ectopic expression of SP in the fat body of virgin females or injection of SP into the hemolymph does elicit the PMR (Schmidt, 1993; Aigaki, 1993; Peng, 2005a and references therein).

Cooperative reproductive behavior of the two sexes promotes the evolutionary success of a species, but males and females also compete to control the number and genetic diversity of their offspring. Because D. melanogaster females are polyandrous, it is in the interest of the female to eliminate surplus SP, at the latest when sperm has been used up. In contrast, it is in the male's interest to keep his mating partner monogamous. Thus, sexual conflict arises. Binding of SP to the sperm tail substantially increases the functional half-life of SP from 1 day to about 1 week, probably by hiding it from the hemolymph proteases. It is also in the male's interest to transfer as much SP as possible. Because sperm serves as a carrier to transport and stabilize SP, selection may have favored long sperm tails binding more SP and thereby increasing the reproductive fitness of the male. This could explain the excessive length of sperm tails in some Drosophila species (D. melanogaster males produce sperm of 1.8 mm, and D. bifurca males produce sperm of 58 mm!). Association of components of the male seminal fluid with sperm has also been reported for other insects, mammals, and birds. Binding of proteins that enhance male fitness to sperm may be a novel mechanism of general importance in insects and beyond. Selection experiments involving males either lacking functional SP completely (Liu, 2003; Schmidt, 1993) or producing modified SPs should enable testing of the putative influence of SP on sperm tail length in D. melanogaster. Such experiments may lead to an understanding of male-male and male-female competition at a molecular level (Peng, 2005a).

In sum, the PMR of D. melanogaster females can be divided into two phases: the short-term PMR and the long-term PMR, respectively. The short-term PMR are induced immediately after mating mainly by free SP; the long-term PMR, lasting about one week, by the C-terminal SP fragment cleaved from SP bound to the sperm tail. Both responses likely elicit the PMR by binding of SP to specific sites in the central and peripheral nervous systems. In addition, immediately after mating, the free SP probably also stimulates juvenile hormone synthesis because it contains the N terminus known to be essential for the stimulation of the corpus allatum in (Moshitzky, 1996). The elucidation of the molecular mechanism supporting the persistence of the PMR in D. melanogaster may shed light on a fundamental aspect of insect reproduction in general (Peng, 2005a).

Drosophila melanogaster sex peptide stimulates juvenile hormone synthesis and depresses sex pheromone production in Helicoverpa armigera

Virgin female adult Helicoverpa armigera (Lepidoptera: Noctuidae) moths exhibit calling behaviour and produce sex pheromone in scotophase from the day after emergence; mating turns off both of these pre-mating activities. In the fruit fly Drosophila melanogaster, a product of the male accessory glands, termed sex peptide (SP), has been identified as being responsible for suppressing female receptivity after transfer to the female genital tract during mating. Juvenile hormone (JH) production is activated in the D. melanogaster corpus allatum (CA) by SP in vitro. Cross-reactivity of D. melanogaster SP has been demonstrated in the H. armigera moth: JH production in photophase virgin female moth CA in vitro is directly activated in a dose-dependent manner by synthetic D. melanogaster SP, and concurrently inhibits pheromone biosynthesis activating neuropeptide (PBAN)-activated pheromone production by isolated pheromone glands of virgin females. Control peptides (locust adipokinetic hormone, AKH-I, and human corticotropin, ACTH) do not inhibit in vitro pheromone biosynthesis. Moreover, SP injected into virgin H. armigera females, decapitated 24 h after eclosion, or into scotophase virgin females, suppresses pheromone production. In the light of these results, the existence is hypothesized of a SP-like factor among the peptides transmitted to female H. armigera during copulation, inducing an increased level of JH production and depressing the levels of pheromone produced thereafter (Fan, 2000).

The homeodomain protein defective proventriculus is essential for male accessory gland development to enhance fecundity in Drosophila

The Drosophila male accessory gland has functions similar to those of the mammalian prostate gland and the seminal vesicle, and secretes accessory gland proteins into the seminal fluid. Each of the two lobes of the accessory gland is composed of two types of binucleate cell: about 1,000 main cells and 40 secondary cells. A well-known accessory gland protein, sex peptide, is secreted from the main cells and induces female postmating response to increase progeny production, whereas little is known about physiological significance of the secondary cells. The homeodomain transcriptional repressor Defective proventriculus (Dve) is strongly expressed in adult secondary cells, and its mutation resulted in loss of secondary cells, mononucleation of main cells, and reduced size of the accessory gland. dve mutant males had low fecundity despite the presence of sex peptide, and failed to induce the female postmating responses of increased egg laying and reduced sexual receptivity. RNAi-mediated dve knockdown males also had low fecundity with normally binucleate main cells. This study provides evidence that secondary cells are crucial for male fecundity, and also that Dve activity is required for survival of the secondary cells. These findings provide new insights into a mechanism of fertility/fecundity (Minami, 2012).

Mutant males for the longer transcript, dve-A, showed low fecundity together with loss of secondary cells and reduced size of accessory glands. It has been reported that greatly reduced size of accessory glands results in sterility, and also suggested that there is a minimum size to maintain fertility. If a male is selected for larger size of accessory glands with 16 generations, the selected males have good fecundity. However, they have only about 1.4-fold larger size compared to the control males, suggesting that there is also a maximal size of accessory gland not to waste energy. Thus, the size of accessory glands should be controlled in an appropriate range and binucleation seems to be the best strategy to provide highly plastic change of the siz. Although reduced size of dve-A mutant accessory glands may have some effects on fecundity, dve KD males had similar size of accessory glands to the dve-A heterozygous controls and dve KD males showed low fecundity with loss of secondary cells. Thus, it is most likely that the low fecundity in dve-A mutant males is due to the absence of mature secondary cells. This is consistent with independent findings that Abd-B is required for maturation of secondary cells and for maintaining female postmating response (personal communication to Minami from M. F. Wolfner and F. Karch). Although it cannot be excluded that some defects in dve mutant main cells affect the fecundity, SP was normally expressed and transferred into the female reproductive tract. Thus, the following mechanisms should be considered for SP activation to induce long-term postmating response; (1) secondary cell products cooperate in parallel with SP-mediated signaling; (2) secondary cell products enhance SP binding to its receptor; (3) secondary cell products stabilize SP binding to sperm; and (4) secondary cell products are involved in modification and/or stabilization of SP secreted from main cells. Interestingly, egg laying of females mated with dve mutant or dve KD males was gradually reduced over time, suggesting that the last two interpretations, stabilization of SP by secondary cell products, are more plausible. Identification of unknown factors secreted from secondary cells will provide new insights into a mechanism that is crucial for activation of seminal fluid functions (Minami, 2012).

It seems likely that Dve functions are crucial to inhibit cell death of secondary cells, and an intriguing possibility is that inactivation of the Dve activity is closely linked to the regulated cell death to adjust the number of secondary cells. Dve and the special AT-rich sequence binding proteins (SATBs) belong to the cut superclass of homeobox genes and have an evolutionarily conserved compass domain. It is reported that SATB1 is cleaved and inactivated by Caspase 6 in response to the apoptotic signaling pathway. Expression of the BCL2 gene, which is a key regulator to inhibit apoptosis, is finely tuned by a variety of stimuli and activated through SATB1-mediated chromatin looping. Thus, SATB1 is required for cell survival through inhibition of programmed cell death. The functional similarity between Dve and SATB1 for inhibition of cell death raises a possibility that an evolutionarily conserved CMP plays important roles to inhibit cell death. The CMP of SATB1 is characterized as a PDZ-like domain (amino acids 90 to 204) involved in protein-protein interaction, and the Caspase 6-dependent cleavage of SATB1 at amino acid position 254 disrupts the dimerization of SATB1. Further characterization of CMP-interacting proteins will clarify the underlying mechanism and provide new insights into a regulatory mechanism of fecundity/fertility (Minami, 2012).

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, a comparative genomic method was used 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, these functional analyses demonstrate that evolutionary rate covariation is a valuable predictive tool for identifying candidate members of interacting protein networks (Findlay, 2014).

REFERENCES

Search PubMed for articles about Drosophila Sex Peptide

Aigaki, T., Fleischmann, I., Chen, P. S. and Kubli, E. (1993). Ectopic expression of sex peptide alters reproductive behavior of female D. melanogaster. Neuron 7: 557-563. 1931051

Bontonou, G., Shaik, H. A., Denis, B. and Wicker-Thomas, C. (2014). Acp70A regulates Drosophila pheromones through juvenile hormone induction. Insect Biochem Mol Biol 56:36-49. PubMed ID: 25484200

Carvalho, G. B., Kapahi, P., Anderson, D. J. and Benzer, S. (2006). Allocrine modulation of feeding behavior by the Sex Peptide of Drosophila. Curr. Biol. 16(7): 692-6. 16581515

Chapman, T., et al. (2003). The Sex Peptide of Drosophila melanogaster: investigation of post-mating responses of females using RNA interference. Proc. Natl. Acad. Sci. 100: 9923-9928. 12893873

Chen, P. S. et al. (1988). A male accessory gland peptide that regulates reproductive behavior of female D. melanogaster. Cell 54: 291-29. 3135120

Ding, Z., Haussmann, I., Ottiger, M. and Kubli, E. (2003). Sex-peptides bind to two molecularly different targets in Drosophila melanogaster females. J. Neurobiol. 55: 372-384. 12717705

Domanitskaya, E. V., Liu, H., Chen, S. and Kubli, E. (2007). The hydroxyproline motif of male sex peptide elicits the innate immune response in Drosophila females. FEBS J 274(21): 5659-5668. PubMed ID: 17922838

Fan, Y., et al. (1999). Drosophila melanogaster sex peptide stimulates juvenile hormone synthesis and depresses sex pheromone production in Helicoverpa armigera. J. Insect Physiology 45: 127-133. 12770380

Fan, Y., Rafaeli, A., Moshitzky, P., Kubli, E., Choffat, Y. and Applebaum, S. W. (2000). Common functional elements of Drosophila melanogaster seminal peptides involved in reproduction of Drosophila melanogaster and Helicoverpa armigera females. Insect Biochem Mol Biol 30: 805- 812. 12770380

Findlay, G. D., Sitnik, J. L., Wang, W., Aquadro, C. F., Clark, N. L. and Wolfner, M. F. (2014). Evolutionary Rate Covariation Identifies New Members of a Protein Network Required for Drosophila melanogaster Female Post-Mating Responses. PLoS Genet 10: e1004108. PubMed ID: 24453993

Gillott, C. (2003). Male accessory gland secretions: modulators of female reproductive physiology and behavior. Annu. Rev. Entomol. 48: 163-184. 12208817

Gioti, A., Wigby, S., Wertheim, B., Schuster, E., Martinez, P., Pennington, C. J., Partridge, L. and Chapman, T. (2012). Sex peptide of Drosophila melanogaster males is a global regulator of reproductive processes in females. Proc Biol Sci 279(1746): 4423-4432. PubMed ID: 22977156

Häsemeyer, M., Yapici, N., Heberlein, U. and Dickson, B. J. (2009). Sensory neurons in the Drosophila genital tract regulate female reproductive behavior. Neuron 61(4): 511-8. PubMed Citation: 19249272

Herndon, L. A. and Wolfner, M. F. (1995). A Drosophila seminal fluid protein, Acp26Aa, stimulates egg laying in females for 1 day after mating. Proc. Natl. Acad. Sci. 92: 10114-10118. 7479736

Ja, W. W., Carvalho, G. B., Madrigal, M., Roberts, R. W. and Benzer, S. (2009). The Drosophila G protein-coupled receptor, Methuselah, exhibits a promiscuous response to peptides. Protein Sci 18: 2203-2208. Pubmed: 19672878

Kim, J. H., Jeong, P. H., Lee, J. Y., Lee, J. H., Kim, Y. J. and Kim, Y. C. (2015). Discovery and structure-activity relationships of pyrazolodiazepine derivatives as the first small molecule agonists of the Drosophila sex peptide receptor. Bioorg Med Chem 23: 1808-1816. PubMed ID: 25797164

Kubli, E. (2003). Sex-peptides: seminal peptides of the Drosophila male. Cell. Mol. Life Sci. 60: 1689-1704. 14504657

Liu, H. and Kubli, E. (2003). Sex-peptide is the molecular basis of the sperm effect in Drosophila melanogaster. Proc. Natl. Acad. Sci. 100: 9929-9933. 12897240

Lung, O., et al. (2002). The Drosophila melanogaster seminal fluid protein Acp62F is a protease inhibitor that is toxic upon ectopic expression. Genetics 160: 211-224. 11805057

Manning, A. (1962). A sperm factor affecting the receptivity of Drosophila melanogaster females. Nature 194: 252-253

Manning, A. (1967). The control of sexual receptivity in Drosophila. Animal Behav. 15: 239-250

McGraw, L. A., Gibson, G., Clark, A. G. and Wolfner, M. F. (2004). Genes regulated by mating, sperm, or seminal proteins in mated female Drosophila melanogaster. Curr. Biol. 14(16): 1509-14. 15324670

Minami, R., et al. (2012). The homeodomain protein defective proventriculus is essential for male accessory gland development to enhance fecundity in Drosophila. PLoS One 7(3): e32302. PubMed Citation: 22427829

Moshitzky, P., et al. (1996). SP activates juvenile hormone biosynthesis in the Drosophila melanogaster corpus allatum. Arch. Insect Biochem. Physiol. 32: 363-374. 8756302

Nakayama, S., Kaiser, K. and Aigaki, T. (1997). Ectopic expression of SP in a variety of tissues in Drosophila females using the P(GAL4) enhancer-trap system. Mol. Gen. Genet. 254: 449-55. 9180699

Ottiger, M., Soller, M., Stocker, R. F. and Kubli, E. (2000). Binding sites of Drosophila melanogaster sex peptide pheromones. J. Neurobiol. 44: 57-71. 10880132

Peng, J., et al. (2005a). Gradual release of sperm bound sex-Peptide controls female postmating behavior in Drosophila. Curr. Biol. 15: 207-213. 15694303

Peng, J., Zipperlen, P. and Kubli, E. (2005b). Drosophila Sex-peptide stimulates female innate immune system after mating via the Toll and Imd pathways. Curr. Biol. 15: 1690-1694. 16169493

Rezával, C., et al. (2012). Neural circuitry underlying Drosophila female postmating behavioral responses. Curr. Biol. 22(13): 1155-65. PubMed Citation: 22658598

Saudan, P., et al. (2002). Ductus ejaculatorius peptide 99B (DUP99B), a novel Drosophila melanogaster sex-peptide pheromone. Eur. J. Biochem. 269: 989-997. 11846801

Schmidt, T., Choffat, Y., Klauser, S., Kubli, E. (1993). The Drosophila melanogaster sex-peptide: a molecular analysis of structure-function relationships. J. Insect Physiol. 39(5): 361-368

Wainwright, S. M., Hopkins, B. R., Mendes, C. C., Sekar, A., Kroeger, B., Hellberg, J., Fan, S. J., Pavey, A., Marie, P. P., Leiblich, A., Sepil, I., Charles, P. D., Thezenas, M. L., Fischer, R., Kessler, B. M., Gandy, C., Corrigan, L., Patel, R., Wigby, S., Morris, J. F., Goberdhan, D. C. I. and Wilson, C. (2021). Drosophila Sex Peptide controls the assembly of lipid microcarriers in seminal fluid. Proc Natl Acad Sci U S A 118(5). PubMed ID: 33495334

Walker, S. J., Corrales-Carvajal, V. M. and Ribeiro, C. (2015). Postmating circuitry modulates salt taste processing to increase reproductive output in Drosophila. Curr Biol. PubMed ID: 26412135

Wang, F., Wang, K., Forknall, N., Patrick, C., Yang, T., Parekh, R., Bock, D. and Dickson, B. J. (2020). Neural circuitry linking mating and egg laying in Drosophila females. Nature 579(7797): 101-105. PubMed ID: 32103180

White, M. A., Bonfini, A., Wolfner, M. F. and Buchon, N. (2021). Drosophila melanogaster sex peptide regulates mated female midgut morphology and physiology. Proc Natl Acad Sci U S A 118(1):e2018112118. PubMed ID: 33443193

Wigby, S. and Chapman, T. (2005). Sex peptide causes mating costs in female Drosophila melanogaster. Curr. Biol. 15: 316-321. 15723791

Wilson, C., Leiblich, A., Goberdhan, D. C. and Hamdy, F. (2017). The Drosophila accessory gland as a model for prostate cancer and other pathologies. Curr Top Dev Biol 121: 339-375. PubMed ID: 28057306

Yapici, N., Kim, Y. J., Ribeiro, C. and Dickson, B. J. (2008). A receptor that mediates the post-mating switch in Drosophila reproductive behaviour. Nature 451(7174): 33-7. PubMed citation: 18066048

date revised: 25 September 2023

Home page: The Interactive Fly © 2017 Thomas Brody, Ph.D.