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 [TmIIgs1/TmIIgs1] 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 4/5th 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).
A set of synthetic (Sex-Peptide) SP fragments was used to determine the part of SP interacting with sperm. The fragment SP1-11 competes for binding with full length SP1-36. 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 SP6-20 (AB SP6-20); 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 SP6-20, 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 SP1-7 (AB SP1-7) 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 SP1-36 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 SP6-20 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 = TGWT 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 (TGQQ 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 (TGΔ2-7 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 TGWT 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 TGQQ or with TGΔ2-7 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, TGWT, 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 TGWT or TGQQ males show labeling of the tail, but no labeling is observed after mating with TGΔ2-7 males. After 5 days, there is no labeling of the tail after mating with TGWT males, but still full labeling of sperm after mating with TGQQ males. It is concluded that full-length SP1-36 bound to the tail is subsequently cleaved at the trypsin cleavage site R7K8, thus releasing the C-terminal fragment SP8-36 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).
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).
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).
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 Gαq- 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 Gαq/11. Accordingly, these cells were cotransfected with constructs encoding one of three different chimaeric G proteins (Gαqs, Gαqi or Gαqo) designed to divert Gαs-, Gαi- or Gαo-dependent signals, respectively, from the cAMP pathway into the Ca2+ pathway. Indeed, co-expression of Gαqi or Gαqo, but not Gαqs, 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 Gαi and/or Gαo 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 our 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 suboesophageal 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 fruGAL4 driver. In particular, SPR seemed to be expressed in many fruGAL4-positive neurons in the SOG and throughout the VNC. To test whether SPR function is specifically required in fru neurons, the fruGAL4 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, fruGAL4 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).
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