InteractiveFly: GeneBrief

sex peptide receptor: Biological Overview | References

BIOLOGICAL OVERVIEW

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 (Chen, 1988; Chapman, 2003; Liu, 2003). 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 (Kvitsiani, 2006). 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 (Saudan, 2002), 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) (Kubli, 2003; Peng, 2005). 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 (Ottiger, 2000). 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).

Sensory neurons in the Drosophila genital tract regulate female reproductive behavior

Females of many animal species behave very differently before and after mating. In Drosophila, changes in female behavior upon mating are triggered by the sex peptide (SP), a small peptide present in the male's seminal fluid. SP activates a specific receptor, the sex peptide receptor (SPR), which is broadly expressed in the female reproductive tract and nervous system. This study pinpoints the action of SPR to a small subset of internal sensory neurons that innervate the female uterus and oviduct. These neurons express both fruitless (fru), a marker for neurons likely to have sex-specific functions, and pickpocket (ppk), a marker for proprioceptive neurons. SPR expression in these fru⁺ ppk⁺ neurons is both necessary and sufficient for behavioral changes induced by mating. These neurons project to regions of the central nervous system that have been implicated in the control of reproductive behaviors in Drosophila and other insects (Häsemeyer, 2009).

SPR was initially identified in a genome-wide pan-neuronal RNAi screen. In this screen, the panneuronal elav-GAL4 driver was crossed to a genome-wide collection of RNAi transgenes, and female progeny were scored for egg-laying defects. Mated elav-GAL4 UAS-SPR-IR females lay very few eggs and remain sexually receptive, and thus, like SPR null mutants, behave as though they were still virgins. To define the cellular requirement for SPR function, the logic of this screen was inverted, crossing the UAS-SPR-IR transgene to a collection of 998 GAL4 lines and scoring the female progeny for egg-laying defects in the same fashion. In each of these lines, the GAL4 transcriptional activator is expressed in a random but stereotyped subset of cells, in which SPR function should now be inhibited by the UAS-SPR-IR transgene (Häsemeyer, 2009).

Fifty-nine lines were identified that resulted in a strong and reproducible egg-laying defect. Many of these lines were found to be broadly expressed, as revealed with a UAS-mCD8-GFP reporter. These lines were not examined further. More restricted neuronal expression was observed in seven lines, and for each of these a series of secondary assays was performed to confirm the egg-laying defect and to assess the receptivity of both virgin and mated females. For all seven GAL4 lines, SPR knockdown resulted in reduced egg laying and increased remating of mated females, but little if any change in the receptivity of virgin females. These defects were indistinguishable from those observed upon panneuronal SPR knockdown with the elav-GAL4 driver, or in SPR null mutant females. For the most restricted of the positive GAL4 lines, ppk-GAL4, it was confirmed that these defects can indeed be attributed to a diminished response to SP. It was then determined that SPR is required in ppk⁺ sensory neurons in the female reproductive tract (Häsemeyer, 2009).

This study describes a set of internal ppk⁺ fru⁺ sensory neurons in the female reproductive tract and provides evidence that SPR functions in these neurons to trigger the behavioral changes induced by SP upon mating. This conclusion rests on two complementary sets of observations. First, SPR is required in both ppk⁺ and fru⁺ cells, because postmating responses are eliminated upon knockdown of SPR in either cell population. Second, SPR is sufficient in either ppk⁺ or fru⁺ cells alone, as expression in either restores the postmating response in SPR null mutant females. This forces the conclusion that SPR acts exclusively in cells that are both ppk⁺ and fru⁺. The sensory neurons innervating the uterus are the only cells that were identified that express both of these markers. There are typically four to six such cells, and it is not yet known if they are functionally equivalent, or if egg laying and receptivity are regulated by two distinct cell subtypes (Häsemeyer, 2009).

Silencing synaptic transmission of ppk⁺ fru⁺ neurons mimics the activity of SP, in that they both cause virgin females to become unreceptive and initiate egg laying. Thus, an attractive hypothesis is that activation of SPR by SP reduces the synaptic output of these neurons. Like other ppk⁺ neurons, the ppk⁺ fru⁺ uterus neurons are probably mechanosensory. They may therefore have an important function as uterus stretch receptors in the coordination of sperm transfer, fertilization, and egg release. They may have two distinct functional states, depending on the presence or absence of SP. Because receptivity can be genetically uncoupled from egg production and egg laying, it is inferred that SP can also act independently of any stretch signal in the uterus. Modulation of receptivity and egg laying might be mediated through either distinct ppk⁺ fru⁺ subtypes or distinct central synapses (Häsemeyer, 2009).

How might SP regulate these sensory neurons? Two possibilities are envisioned. First, the ppk⁺ fru⁺ neurons may detect SP in the reproductive tract and alter their firing rate accordingly. In this model, passage of SP into the hemolymph would not be required to induce the postmating response. A second possibility is that SP enters the circulatory system and acts presynaptically to modulate the release of these neurons at their central targets. The fact that SP can indeed be detected in the hemolymph of mated females does not in itself exclude the former possibility. At least some effects of SP, such as stimulating juvenile hormone synthesis in the corpus allatum, probably do require SP to enter the hemolymph. Similarly, the fact that SP triggers a postmating response even when injected directly into the hemolymph is also consistent with either model. The somata and some processes of the ppk⁺ fru⁺ neurons lie outside the uterus and would be readily accessible to factors in the hemolymph. A neural rather than a circulatory route has been proposed to mediate postmating responses in several species of moths. However, this conclusion is based upon the loss of this response upon nerve cord transection, a result predicted by both of these models. Thus, both models are consistent with currently available evidence from studies in Drosophila and other species, and distinguishing between them will require detailed studies of the physiological properties of the ppk⁺ fru⁺ neurons in response to SP (Häsemeyer, 2009).

The central targets of the ppk⁺ fru⁺ sensory neurons include the abdominal and/or subesophageal ganglia -- regions of the CNS likely to contain circuits that mediate behavioral responses to mating. The abdominal ganglion houses the octopaminergic neurons that are believed to regulate the release and passage of mature eggs from the ovary to the uterus. It is suspected that these neurons are direct or indirect targets of the ppk⁺ fru⁺ sensory neurons and that these circuits serve to ensure that ovulation and oviposition are coordinated with the presence of sperm (Häsemeyer, 2009).

Some ppk⁺ fibers project from the abdominal trunk nerve right through to the SOG, potentially forming a direct neural connection from the reproductive tract to the brain. It is suspected that these projections may feed into circuits that regulate female receptivity and other postmating behaviors. Virgin females are enticed to mate by the male's courtship song. Most auditory sensory neurons project to the mechanosensory neuropil in the lateral SOG, close to the terminal arborizations of the ppk⁺ neurons. The proximity of the auditory processing centers and the ascending ppk⁺ projections raises the attractive possibility that mating modulates an early step in song processing. The SOG also contains processes of the Ilp7 neurons, which function in egg-laying site selection after mating. Direct evidence for mating-induced changes in SOG circuit function is lacking in flies but has been obtained in other insects. In some species of moth, mating induces a long-term inhibition of the SOG neurosecretory cells that regulate female pheromone biosynthesis, making mated females less attractive to other males (Häsemeyer, 2009).

Having identified sensory neurons that detect SP in the reproductive tract, it will now be important to characterize the central pathways that process these signals to regulate female behavior. In the olfactory system, sensory neurons that detect pheromones are fru⁺, as are their postsynaptic partners in the brain. Given that the sensory neurons that detect SP are also fru⁺, and many fru⁺ neurons are also located in both the abdominal and subesophageal ganglia, it is enticing to think that a similar logic may apply in these pathways too. Elucidating the operation of these circuits should reveal how the female CNS integrates both external and internal information to switch between two very different behavioral patterns (Häsemeyer, 2009).

Neuronal octopamine signaling regulates mating-induced germline stem cell increase in female Drosophila melanogaster

Stem cells fuel the development and maintenance of tissues. Many studies have addressed how local signals from neighboring niche cells regulate stem cell identity and their proliferative potential. However, the regulation of stem cells by tissue-extrinsic signals in response to environmental cues remains poorly understood. This study reports that efferent octopaminergic neurons projecting to the ovary are essential for germline stem cell (GSC) increase in response to mating in female Drosophila. The neuronal activity of the octopaminergic neurons is required for mating-induced GSC increase as they relay the mating signal from sex peptide receptor-positive cholinergic neurons. Octopamine and its receptor Oamb are also required for mating-induced GSC increase via intracellular Ca(2+) signaling. Moreover, Matrix metalloproteinase-2 was identified as a downstream component of the octopamine-Ca(2+) signaling to induce GSC increase. This study provides a mechanism describing how neuronal system couples stem cell behavior to environmental cues through stem cell niche signaling (Yoshinari, 2020).

This study reports that the mating-induced GSC increase in female D. melanogaster is regulated by octopaminergic neurons directly projecting to the ovary. From in vivo and ex vivo experiments, the following model is proposed to explain the mating-induced GSC increase. After mating, the male seminal fluid SP is transferred into the female uterus, stimulating SPR-positive neurons. As the liganded SPR silences the neuronal activity of SPR-positive sensory neurons (SPSNs), the acetylcholine released from SPSNs is suppressed. As SPSNs and dsx+ Tdc2+ neurons are directly connected, this suppression directly modulates dsx+ Tdc2+ neuronal activity. Because this study has shown that nAChRs in dsx+ Tdc2+ neurons exhibit an inhibitory effect with an unknown mechanism, the inactivation of nAChRs in the absence of acetylcholine results in the activation of dsx+ Tdc2+ neurons in mated females. As a consequence, octopamine is released from dsx+ Tdc2+ neurons, received by Oamb, induces [Ca2+]i in the escort cells, and finally activates the Mmp2 enzymatic activity. The activity of Mmp2 positively regulates the Dpp-mediated niche signaling, thereby leading to mating-induced GSC increase. (Yoshinari, 2020).

The proposed model is that the OA from dsx+ Tdc2+ neurons is directly received by the escort and follicle cells in the germarium. This model is supported by two observations. First, mating-induced GSC increase is impaired by Oamb RNAi using a GAL4 driver that is active specifically in the germarium cells but not mature follicle cells. Second, OA treatment evokes [Ca2+]i elevation in these germarium cells in an Oamb-dependent manner. However, this study did not address whether the escort cells and/or the follicle cells in the germarium express Oamb, as no clear GAL4 expression was observed in two independent Oamb-T2A-GAL4 drivers. It is surmised that this may be due to lower amounts of Oamb transcript in the germarium (Yoshinari, 2020).

It was shown that the activation of the ovary-projecting dsx+ Tdc2+ neurons is necessary and sufficient to induce GSC increase. However, from an anatomical point of view, the dsx+ Tdc2+ neurons project to the distal half of the ovary but not to the germarium. Considering the model described above, this disagreement can be attributed to the characteristic volume transmission of monoamine neurotransmitters. In other words, neurotransmitters act at a distance well beyond their release sites from cells or synapses. Therefore, the OA secreted from the terminals of dsx+ Tdc2+ neurons could reach the germarium located at the most proximal part of the ovary (Yoshinari, 2020).

Several previous studies have revealed that OA signaling has a pivotal role in reproductive tissues other than germarium, such as mature follicle cells, oviduct, and ovarian muscle, to promote ovulation, oviduct remodeling, and ovarian-muscle contraction, respectively. Therefore, it is likely that the dsx+ Tdc2+ neurons orchestrate multiple different events during oogenesis in response to mating stimulus. Because a mated female needs to activate oogenesis to continuously produce eggs in concert with sperm availability, it is reasonable that the ovary-projecting neurons switch on the activity of the entire process of reproduction (Yoshinari, 2020).

Based on the present study and several previous studies, the OA-Oamb-Ca2+-Mmp2 axis is required for GSC increase and follicle rupture, both of which are induced by mating stimuli in D. melanogaster. In both cases, Mmp2 enzymatic activity is likely to be essential, as the overexpression of Timp encoding a protein inhibitor of Mmp2 suppresses GSC increase, as well as follicle rupture. Mmp2 in mature follicle cells cleaves and downregulates Viking/collagen VI. In fact, several previous studies have revealed that Viking/collagen VI is required for GSC maintenance in female D. melanogaster. However, no significant change was observed in Viking/Collagen VI levels in the germarium between the control and Mmp2 RNAi flies. Therefore, it is concluded that Viking/collagen VI is not a substrate of Mmp2 in the regulation of mating-induced GSC increase. Besides Viking/Collagen VI, Dally-like (Dlp) is another basement membrane protein associated with extracellular matrix and known as the Mmp2 substrate. Interestingly, dlp is expressed in the escort cells. Moreover, Dlp controls the distribution of Dpp and Wnts, both of which significantly affect GSC self-renewal and differentiation. Future research should decipher the exact substrate by which Mmp2 controls Dpp and/or Wnts to modulate GSC behavior in response to mating stimulus (Yoshinari, 2020).

Another remaining question to be addressed is how Mmp2 function is regulated in GSC increase. Ecdysteroid biosynthesis and signaling in the ovary are necessary but not sufficient for the OA-Oamb-Ca2+-mediated GSC increase and follicle rupture. This study found that in the regulation of mating-induced GSC increase, ecdysteroid signaling acts downstream of Ca2+ signaling. On the other hand, in the follicle rupture process, ecdysteroid signaling either acts downstream, upstream, or both, of Ca2+ signaling. Further, the precise action of ecdysteroid has yet to be elucidated. The Mmp2-GFP fusion protein level in the follicle cells is not changed in the loss-of-Ecdysone receptor-function flies, implying that ecdysteroid signaling might regulate Mmp2 enzymatic activity by an unknown mechanism. Considering the involvement of both the OA-Oamb-Ca2+-Mmp2 axis and ecdysteroid biosynthesis, it is very likely that the Mmp2 enzymatic activity is also regulated by the same, unknown mechanism not only in the mature follicle cells to control follicle rupture, but also in the germarium to control mating-induced GSC increase (Yoshinari, 2020).

In many animals, reproduction involves significant behavioral and physiological shifts in response to mating. In female D. melanogaster, several post-mating responses are coordinated by SPSNs and their downstream afferent neuronal circuit, including Stato-Acoustic Ganglion neurons, the ventral abdominal lateral Myoinhibitory peptide neurons, and the efferent dsx+ Tdc2+ neurons. GRASP analysis indicates a direct synaptic connection between cholinergic SPSNs and OAergic neurons. Moreover, this study demonstrated that nAChRs in dsx+ Tdc2+ neurons are responsible for the suppression of their neuronal activity in virgin females. However, nAChRs are the cation channels leading to depolarization upon acetylcholine binding, and therefore usually activate neurons. How is the opposite role of nAChRs in dsx+ Tdc2+ neuronal activity achieved? One possibility is that acetylcholine-nAChR signaling does not evoke a simple depolarization but rather generates a virgin-specific temporal spike pattern in dsx+ Tdc2+ neurons. Interestingly, recent studies demonstrated that the pattern, instead of the frequency, of neuronal firing is significant in adjusting the neuronal activity of clock neurons in D. melanogaster. The firing pattern relies on control of ionic flux by the modulation of Ca2+-activated potassium channel and Na+/K+ ATPase activity. Because whether mating changes the firing pattern of dsx+ Tdc2+ neurons remains to be examined, the neuronal activity in SPSNs and the dsx+ Tdc2+ neuronal circuit between virgin and mated females are future research areas (Yoshinari, 2020).

In the last decades, there is growing evidence that GSCs and their niche are influenced by multiple humoral factors. Based on the data from the current study and previous studies, there are at least four crucial humoral factors for regulating the increase and/or maintenance of D. melanogaster female GSCs, including DILPs, ecdysteroids, Neuropeptide F (NPF), and OA. Notably, all of these come from different sources: DILPs are from the insulin-producing cells located in the pars intercerebralis of the central brain; ecdysteroids from the ovary; NPF from the midgut; and OA from the neurons located in the abdominal ganglion. In addition to these identified humoral factors, recent studies also imply that adiponectin and unknown adipocyte-derived factor(s) are essential for GSC maintenance. These data clearly indicate that D. melanogaster female GSCs are systemically regulated by interorgan communication involving multiple organs. The additional interorgan communication mechanisms that ensure the faithful coupling of the increase and maintenance of GSC to the organism's external and physiological environments are essential to be investigated in future studies (Yoshinari, 2020).

To modulate the increase and maintenance of GSC, ecdysteroids are received by both GSCs and niche cells, whereas DILPs, NPF, and OA are received by niche cells. A major signal transduction mechanism of each of these humoral factors have been well characterized, namely phosphoinositide 3-kinase pathway for DILPs-InR signaling, EcR/Ultraspiracle-mediated pathway for ecdysteroid signaling, cAMP pathway for NPF-NPFR signaling, and Ca2+ pathway for OA-Oamb signaling. However, it remains unclear whether and how each of these signaling pathways control the production and secretion of the niche signal, as well as its distribution and transduction. In addition, it is important to understand whether and how the multiple system signals are integrated to control the mating-induced increase and maintenance of GSCs (Yoshinari, 2020).

In recent years, many studies have revealed that not only local niche signals but also systemic and neuronal factors play indispensable roles in regulating GSC behavior. In D. melanogaster, ecdysteroid signaling is essential for the proliferation and maintenance of GSCs and neural stem cells. This study has identified the ovary-projecting OAergic neurons as new regulators of stem cell homeostasis. Both steroid hormones and OA-like monoamines, such as noradrenaline, are also involved in stem cell regulation in mammals. For example, the mammalian steroid hormone, estrogen, is important in regulating cell division and/or maintenance of hematopoietic stem cells, mammary stem cell, neural stem cells, and hematopoietic stem cells. Moreover, noradrenergic neurons, which directly project to the bone marrow, regulate the remodeling of hematopoietic stem cells niche. Therefore, the steroid hormone- and noradrenergic nerve-dependent control of stem cell homeostasis are likely conserved across animal species. In this regard, the D. melanogaster reproductive system will further serve as a powerful model to unravel the conserved systemic and neuronal regulatory mechanisms for stem cell homeostasis in animals (Yoshinari, 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).

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).

A homeostatic sleep-stabilizing pathway in Drosophila composed of the Sex Peptide receptor and its ligand, the myoinhibitory peptide

Sleep, a reversible quiescent state found in both invertebrate and vertebrate animals, disconnects animals from their environment and is highly regulated for coordination with wakeful activities, such as reproduction. The fruit fly, Drosophila melanogaster, has proven to be a valuable model for studying the regulation of sleep by circadian clock and homeostatic mechanisms. This study demonstrates that the Sex peptide receptor (SPR) of Drosophila, known for its role in female reproduction, is also important in stabilizing sleep in both males and females. Mutants lacking either the SPR or its central ligand, myoinhibitory peptide (MIP), fall asleep normally, but have difficulty in maintaining a sleep-like state. This analyses have mapped the SPR sleep function to pigment dispersing factor (pdf) neurons, an arousal center in the insect brain. MIP downregulates intracellular cAMP levels in pdf neurons through the SPR. MIP is released centrally before and during night-time sleep, when the sleep drive is elevated. Sleep deprivation during the night facilitates MIP secretion from specific brain neurons innervating pdf neurons. Moreover, flies lacking either SPR or MIP cannot recover sleep after the night-time sleep deprivation. These results delineate a central neuropeptide circuit that stabilizes the sleep state by feeding a slow-acting inhibitory input into the arousal system and plays an important role in sleep homeostasis (Oh, 2014: PubMed).

Ionotropic chemosensory receptors mediate the taste and smell of polyamines: Neuropeptides modulate female chemosensory processing upon mating in Drosophila

A female's reproductive state influences her perception of odors and tastes along with her changed behavioral state and physiological needs. The mechanism that modulates chemosensory processing, however, remains largely elusive. Using Drosophila, this study has identified a behavioral, neuronal, and genetic mechanism that adapts the senses of smell and taste, the major modalities for food quality perception, to the physiological needs of a gravid female. Pungent smelling polyamines, such as putrescine and spermidine, are essential for cell proliferation, reproduction, and embryonic development in all animals. A polyamine-rich diet increases reproductive success in many species, including flies. Using a combination of behavioral analysis and in vivo physiology, this study shows that polyamine attraction is modulated in gravid females through a G-protein coupled receptor, the sex peptide receptor (SPR), and its neuropeptide ligands, MIPs (myoinhibitory peptides), which act directly in the polyamine-detecting olfactory and taste neurons. This modulation is triggered by an increase of SPR expression in chemosensory neurons, which is sufficient to convert virgin to mated female olfactory choice behavior. Together, these data show that neuropeptide-mediated modulation of peripheral chemosensory neurons increases a gravid female's preference for important nutrients, thereby ensuring optimal conditions for her growing progeny (Hussain, 2016b).

The behavior of females in most animal species changes significantly as a consequence of mating. Those changes are interpreted from an evolutionary standpoint as the female's preparation to maximize the fitness of her offspring. In general, they entail a qualitative and quantitative change in her diet, as well as the search for an optimal site where her progeny will develop. In humans, the eating behavior and perception of tastes and odors of a pregnant woman are modulated in concert with altered physiology and the specific needs of the embryo. While several neuromodulatory molecules such as noradrenaline are found in the vertebrate olfactory and gustatory systems, little is known about how reproductive state and pregnancy shape a female's odor and taste preferences. Very recent work in the mouse showed that olfactory sensory neurons (OSNs) are modulated during the estrus cycle. Progesterone receptor expressed in OSNs decreases the sensitivity of pheromone-detecting OSNs and thereby reduces the non-sexually receptive female's interest in male pheromones. The mechanisms of how mating, pregnancy, and lactation shape the response of the female olfactory and gustatory systems remain poorly understood (Hussain, 2016b).

The neuronal underpinnings of mating and its consequences on female behaviors have arguably been best characterized in Drosophila. Shortly after copulation, female flies engage in a series of post-mating behaviors contrasting with those of virgins: their sexual receptivity decreases, and they feed to accumulate essential resources needed for the production of eggs; finally, they lay their eggs. This suite of behaviors results from a post-mating trigger located in the female's reproductive tract. Sensory neurons extending their dendrites directly into the oviduct are activated by a component of the male's ejaculate, the sex peptide (SP). Sex peptide receptor (SPR) expressed by these sensory neurons triggers the post-mating switch. Mated females mutant for SPR produce and lay fewer eggs while maintaining a high sexual receptivity. In addition to SP, male ejaculate contains more than 200 proteins, which are transferred along with SP into the female. These have been implicated in conformational changes of the uterus, induction of ovulation, and sperm storage (Hussain, 2016b).

Additional SPR ligands have been identified that are not required for the canonical post-mating switch, opening the possibility that this receptor is involved in the neuromodulation of other processes. These alternative ligands, the myoinhibitory peptides (MIPs)/allatostatin-Bs, unlike SP, have been found outside of drosophilids, in many other insect species such as the silkmoth (Bombyx mori), several mosquito species, and the red flour beetle (Tribolium castaneum). They are expressed in the brain of flies and mosquitoes, including in the centers of olfactory and gustatory sensory neuron projections, the antennal lobe (AL), and the subesophageal zone (SEZ), respectively. Although these high-affinity SPR ligands have recently been implicated in the control of sleep in Drosophila males and females, nothing thus far suggests a function in reproductive behaviors (Hussain, 2016b).

To identify optimal food and oviposition sites, female flies rely strongly on their sense of smell and taste. Drosophila females prefer to oviposit in decaying fruit and use byproducts of fermentation such as ethanol and acetic acid to choose oviposition sites. Their receptivity to these byproducts is enhanced by their internal state. It was shown, for instance, that the presence of an egg about to be laid results in increased attraction to acetic acid. Yet the mechanisms linking reproductive state to the modulation of chemosensory processing remain unknown (Hussain, 2016b).

This study has examined the causative mechanisms that integrate reproductive state into preference behavior and chemosensory processing. Focus was placed on the perception of another class of byproducts of fermenting fruits, polyamines. Polyamines such as putrescine, spermine, and spermidine are important nutrients that are associated with reproductive success across animal species. A diet high in polyamines indeed increases the number of offspring of a fly couple, and female flies prefer to lay their eggs on polyamine-rich food (Hussain, 2016a). Importantly, previous studies have characterized the chemosensory mechanisms flies use to find and evaluate polyamine-rich food sources and oviposition sites. In brief, volatile polyamines are detected by OSNs on the fly's antenna, co-expressing two ionotropic receptors (IRs), IR41a and IR76b. Interestingly, the taste of polyamines is also detected by IR76b in labellar gustatory receptor neurons (GRNs) (Hussain 2016a; Hussain, 2016b).

This beneficial role of polyamines has a well-characterized biological basis: polyamines are essential for basic cellular processes such as cell growth and proliferation, and are of specific importance during reproduction. They enhance the quality of sperm and egg and are critical during embryogenesis and postnatal development. While the organism can generate polyamines, a significant part is taken in with the diet. Moreover, endogenous synthesis of polyamines declines with ageing and can be compensated for through a polyamine-rich diet. Therefore, these compounds represent a sensory cue as well as an essential component of the diet of a gravid female fly (Hussain, 2016b and references therein).

This study shows that the olfactory and gustatory perception of polyamines is modulated by the female's reproductive state and guides her choice behavior accordingly. This sensory and behavioral modulation depends on SPR and its conserved ligands, the MIPs that act directly on the chemosensory neurons themselves. Together, these results suggest that mating-state-dependent neuropeptidergic modulation of chemosensory neurons matches the female fly's decision-making to her physiological needs (Hussain, 2016b).

Mechanistically, this study shows that virgin females, or mated females lacking the G-protein coupled receptor SPR, display reduced preference for polyamine-rich food and oviposition sites. Using targeted gene knockdown, mutant rescue, overexpression, and in vivo calcium imaging, a new role was uncovered for SPR and its conserved ligands, MIPs, in directly regulating the sensitivity of chemosensory neurons and modulating taste and odor preferences according to reproductive state. Together with recent work in the mouse, these results emphasize that chemosensory neurons are potent targets for tuning choice behavior to reproductive state (Hussain, 2016b).

Reproductive behaviors such as male courtship and female egg-laying strongly depend on the mating state. While previous work has suggested that mating modulates odor- or taste-driven choice behavior of Drosophila females, how mating changes the processing of odors and tastes remained elusive. This study shows that a female-specific neuropeptidergic mechanism acts in peripheral chemosensory neurons to enhance female preference for essential nutrients. The data suggests that this modulation is autocrine and involves the GPCR SPR and its conserved MIP ligands. Notably, MIPs are expressed in chemosensory cells in the apical organs of a distant organism, the annelid (Platynereis) larvae, in which they trigger settlement behavior via an SPR-dependent signaling cascade. Importantly, as SP and not MIP induces the SPR-dependent canonical post-mating switch, the current findings report the first gender and mating-state-dependent role of these peptides. Whether this regulation is also responsible for previously reported changes in preference behavior upon mating remains to be seen, but it is anticipated that this type of regulation is not only specific to polyamines. On the other hand, mating-dependent changes for salt preference-salt preference is also dependent on IR76b receptor but in another GRN type-might undergo a different type of regulation, as RNAi-mediated knockdown of SPR in salt receptor neurons had no effect on salt feeding. Instead, the change in salt preference is mediated by the canonical SP/SPR pathway and primarily reflects the fact that mating has taken place. The mechanism of how salt detection and/or processing are modulated is not known. In contrast to salt preference and polyamine preference, acetic acid preference is strongly modulated by egg-laying activity and not just mating. The extent to which changes in salt or acetic acid preference are similar to the modulation of behavior to polyamine that this study has described can currently not be tested, because the olfactory neurons that mediate acetic acid preference have not been determined (Hussain, 2016b).

While SPR regulates the neuronal output of both olfactory and gustatory neurons, the behavioral and physiological data surprisingly revealed that it does so through two opposite neuronal mechanisms. SPR signaling increases the presynaptic response of GRNs and decreases it in OSNs. Behaviorally, these two types of modulation produce the same effect: they enhance the female's attraction to polyamine and tune it to levels typical for decaying or fermenting fruit. How these two effects are regulated by the same receptor and ligand pair remains open. GPCRs can recruit and activate different G-proteins. SPR was previously shown to recruit the inhibitory Gα_i/o-type, thereby down-regulating cAMP levels in the cell. In the female reproductive tract, SP inhibits SPR-expressing internal sensory neurons and thereby promotes several post-mating behaviors. This type of inhibitory G-protein signaling could also explain the data in the olfactory system. Here, mating decreases the presynaptic activity of polyamine-detecting OSNs, and conversely, RNAi knockdown of SPR increases their responses strongly. This decrease in neuronal output also shifts the behavioral preference from low to high polyamine levels. While the relationship between behavior and GRN activity is much more straightforward in the gustatory system (increased neuronal response, increased preference behavior), it implies that another G-protein might be activated downstream of SPR. G-protein Gα_i/s increases cAMP levels and Gα_q enhances phospholipase C (PLC) and calcium signaling. In addition, Gβγ subunits regulate ion channels and other signaling effectors, including PLC. Future work will address the exact mechanisms of this bi-directional modulation through SPR signaling. Nonetheless, it is interesting to speculate that different cells, including sensory neurons, could be modulated differentially by the same molecules depending on cell-specific states and the availability of signaling partners (Hussain, 2016b).

While the data provides a neuronal and molecular mechanism of how chemosensory processing itself is affected by mating, it remains unclear how mating regulates MIP/SPR signaling in chemosensory neurons. The data indicates that SPR levels strongly increase in chemosensory organs upon mating. In addition, MIP levels appear to be mildly increased by mating. This suggests that mating regulates primarily the expression of the GPCR resembling the modulation of sNPFR expression during hunger states. On the other hand, MIP overexpression also induced mated-like preference behavior in virgin flies, suggesting a somewhat more complex situation. For instance, it is possible that overexpression of MIP induces the expression of SPR. Alternatively, active MIP levels might also be regulated at the level of secretion or posttranslational processing, and overexpression might override this form of regulation. In the case of hunger, sNPFR levels are increased through a reduction of insulin signaling. SP could be viewed as the possible equivalent of insulin for mating state. Females mated to SP mutant males, however, do not show a significant change in olfactory perception of polyamines. It is yet important to note that male sperm contains roughly 200 different proteins, some of which might be involved in mediating the change in MIPs/SPR signaling upon mating. In the mosquito, which does not possess SP, the steroid hormone 20E serves as the post-mating switch. Interestingly, mating or treatment with 20E induces in particular the expression of the enzymes required for the synthesis of polyamines in the female spermatheca, a tissue involved in sperm storage and egg-laying. Whether such a mechanism also exists in Drosophila is not known (Hussain, 2016b).

In addition to mating and signals transferred by mating, it is possible that egg-laying activity contributes to the regulation of MIPs/SPR signaling in chemosensory neurons through a mechanism that involves previously identified mechanosensory neurons of the female's reproductive tract; such neurons may sense the presence of an egg to be laid. Indeed, females that cease to lay eggs return to polyamine preferences as found before mating. On the other hand, SP mutant male-mated females and ovoD1 sterile females still show enhanced attraction to polyamine odor, although they lay very few or no eggs. Conversely, knockdown of SPR in IR41a neurons reduced polyamine odor attraction but had a marginal effect on the number of eggs laid. Nevertheless, somewhat reduced numbers of eggs laid were observed upon inactivation of IR76b neurons. At this point, possible reasons can only be speculated. Although IR76b receptor is not expressed in ppk-positive internal SPR neurons, no expression of IR76b-Gal4 is found in neurons innervating the rectum and possibly gut. Hence, there might be an IR76b-mediated interplay between metabolism and nutrient uptake that influences egg-laying. However, females mated to SP-mutant males do not display an increase in feeding, indicating that preference for polyamines does not depend on the metabolic cost of egg-laying. This conclusion is strengthened by the data obtained with mated ovoD1 sterile females, who show similar attraction to polyamines as compared to mated controls. Due to very few or no eggs laid by SP mutant male-mated females and ovoD1 females, respectively, it is not possible to fully exclude a contribution of egg-laying activity to taste-dependent oviposition choice behavior (Hussain, 2016b).

A further argument against an important role of egg-laying activity in the current paradigm comes from the observation that the sensory modulation of OSNs and GRNs occurs rapidly after mating and is maintained only for a few hours. Similarly, SPR expression increases within the same time window shortly after mating. Egg-laying, however, continues for several days after this. In addition, overexpression of SPR was sufficient to switch virgin OSN calcium responses and female behavioral preferences to that of mated females without increasing the number of eggs laid. All in all, these data are more consistent with the hypothesis that mating and not egg-laying activity per se is the primary inducer of sensory modulation leading to the behavioral changes of females (Hussain, 2016b).

It remains that the exact signal triggered by mating that regulates odor and taste preference for polyamines, through the mechanism identified in this study, needs to still be determined. Furthermore, the role of metabolic need and polyamine metabolism is not completely clear. This is similar to the situation found for increased salt preference after mating. While more salt is beneficial for egg-laying, sterile females still increase their preference for salt upon mating. Regardless, in the case of polyamines, it is tempting to speculate that exogenous (by feeding) and endogenous (by enzymatic activity or expression) polyamine sources are regulated by reproductive state and together contribute to reach optimal levels for reproduction in the organism. (Hussain, 2016b).

The results bear some similarities to recent work on the modulation of OSN sensitivity in hunger states (Root, 2011). sNPF/sNPFR signaling modulates the activity of OSNs in the hungry fly. MIPs/SPR might play a very similar role in the mated female. Overexpression of sNPFR in OSNs of fed flies was sufficient to trigger enhanced food search behavior. Likewise, an increase in SPR signaling in taste or smell neurons converts virgin to mated female preference behavior. Therefore, different internal states appear to recruit similar mechanisms to tune fly behavior to internal state. Furthermore, such modulation of first order sensory neurons appears not only be conserved within a species, but also for regulation of reproductive state-dependent behavior across species. For instance, a recent study in female mice showed that progesterone-receptor signaling in OSNs modulates sensitivity and behavior to male pheromones according to the estrus cycle. Also in this case, sensory modulation accounts in full for the switch in preference behavior. What is the biological significance of integrating internal state at the level of the sensory neuron? First, silencing of neurons in a state-dependent manner shields the brain from processing unnecessary information. As sensory information may not work as an on/off switch, it is possible that an early shift in neural pathway activation might reduce costly inhibitory activity to counteract activation once the sensory signal has been propagated. Second, another interesting possibility is that peripheral modulation might help to translate transient changes in internal state into longer-lasting behavioral changes that manifest in higher brain centers. This might be especially important in the case of female reproductive behaviors such as mate choice or caring for pups or babies. In contrast to hunger, which increases with time of starvation, the effect of mating decays slowly over time as the sperm stored in the female's spermatheca is used up. This study has shown that the effect of mating on chemosensory neurons mediated by MIPs/SPR signaling is strong within the first 6 h after mating and remains a trend at 1 wk post-mating. However, it triggers a long-lasting behavioral switch, which is observed for over a week. Therefore, this transient modulation and altered sensitivity to polyamines could be encoded more permanently in the brain when the animal encounters the stimulus, for instance, in the context of an excellent place to lay her eggs. Because polyamine preference continues to be high for as long as stored sperm can fertilize the eggs, it is speculated that this change in preference might be maintained by a memory mechanism in higher centers of chemosensory processing. Thus, short-term sensory enhancement not only increases perceived stimulus intensity, it may also help to associate a key sensation to a given reward or punishment. These chemosensory associations are of critical importance in parent-infant bonding in mammals, including humans, which form instantly after birth and last for months, years, or a lifetime (Hussain, 2016b).

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).

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).

Search PubMed for articles about Drosophila Sex peptide receptor

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

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. PubMed ID: 12893873

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

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

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

Harshman, L. G., Loeb, A. M., Johnson, B. A. (1999). Ecdysteroid titers in mated and unmated Drosophila melanogaster females. J. Insect Physiol. 45: 571-577 PubMed ID: 12770342

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 ID: 19249272

Hussain, A., Zhang, M., Ucpunar, H. K., Svensson, T., Quillery, E., Gompel, N., Ignell, R. and Grunwald Kadow, I. C. (2016a). Ionotropic Chemosensory Receptors Mediate the Taste and Smell of Polyamines. PLoS Biol 14: e1002454. PubMed ID: 27145030

Hussain, A., Ucpunar, H. K., Zhang, M., Loschek, L. F. and Grunwald Kadow, I. C. (2016b). Neuropeptides modulate female chemosensory processing upon mating in Drosophila. PLoS Biol 14: e1002455. PubMed ID: 27145127

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

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

Kvitsiani, D. and Dickson, B. J. (2006). Shared neural circuitry for female and male sexual behaviours in Drosophila. Curr. Biol. 16: R355-R356. PubMed ID: 16713940

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. PubMed ID: 12897240

Oh, Y., Yoon, S. E., Zhang, Q., Chae, H. S., Daubnerova, I., Shafer, O. T., Choe, J. and Kim, Y. J. (2014). A homeostatic sleep-stabilizing pathway in Drosophila composed of the Sex Peptide receptor and its ligand, the myoinhibitory peptide. PLoS Biol 12: e1001974. PubMed ID: 25333796

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

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

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

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

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

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 ID: 18066048

Yoshinari, Y., Ameku, T., Kondo, S., Tanimoto, H., Kuraishi, T., Shimada-Niwa, Y. and Niwa, R. (2020). Neuronal octopamine signaling regulates mating-induced germline stem cell increase in female Drosophila melanogaster. Elife 9. PubMed ID: 33077027

date revised: 25 April 2021

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