Control of mating plug expelling and sperm storage in Drosophila: A gynandromorph- and mutation-based dissection.

INTRODUCTION: In this study, we analyzed gynandromorphs with female terminalia, to dissect mating-related female behaviors in Drosophila. MATERIALS AND METHODS: We used gynandromorphs, experimentally modified wild-type (Oregon-R) females, and mutant females that lacked different components of the female reproductive apparatus. RESULTS: Many of the gynandromorphs mated but did not expel the mating plug (MP). Some of these - with thousands of sperm in the uterus - failed to take up sperm into the storage organs. There were gynandromorphs that stored plenty of sperm but failed to release them to fertilize eggs. Expelling the MP, sperm uptake into the storage organs, and the release of stored sperm along egg production are separate steps occurring during Drosophila female fertility. Cuticle landmarks of the gynandromorphs revealed that while the nerve foci that control MP expelling and also those that control sperm uptake reside in the abdominal, the sperm release foci derive from the thoracic region of the blastoderm. DISCUSSION AND CONCLUSION: The gynandromorph study is confirmed by analyses of (a) mutations that cause female sterility: Fs(3)Avar (preventing egg deposition), Tm2gs (removing germline cells), and iab-4DB (eliminating gonad formation) and (b) by experimentally manipulated wild-type females: decapitated or cut through ventral nerve cord.

Sexual behavior: how Sex Peptide flips the postmating switch of female flies.

Drosophila male Sex Peptide elicits an amazing variety of postmating responses in mated females, some of which are transmitted via a receptor on specific neurons of the female genital tract. New work shows that neurons expressing the sex-determination gene doublesex (dsx) play a pivotal role in the female postmating switch.

NMR studies of the solution conformation of the sex peptide from Drosophila melanogaster.

The insect sex peptide (SP) elicits a variety of biological responses upon transfer to the mated female. SP contains 36 amino acids, including a tryptophan-rich N-terminal region, a central region containing five hydroxyproline (Hyp) residues, and a C-terminal region enclosed by a disulfide bridge. The solution structure of SP, studied here using NMR spectroscopy, includes a motif WPWN that adopts a type I ß-turn in the N-terminal Trp-rich region. This turn region is connected to the central Hyp-rich region, which adopts extended and/or PPII-like conformations. The C-terminal disulfide-bonded loop populates helical turns or nascent helical structure. Overall, the results reveal a rather flexible peptide that lacks a compact folded structure in solution.

Sexual behavior: dietary food switch induced by sex.

Pregnancy in humans induces cravings for special food: the same occurs in Drosophila females. New work now shows that mating throws a nutritional switch in favor of a high-protein diet and that modulation of nutritional balance depends on the sex peptide receptor and involves neuronal TOR-S6 kinase signaling.

Sexual behaviour: a receptor for sex control in Drosophila females.

Male Drosophila manipulate the sexual behaviour of their female mating partners by release of a Sex-peptide, but how does this work? A G-protein-coupled receptor has now been identified which acts in the female flies to detect male Sex-peptide and trigger increased egg laying and reduced sexual receptivity.

The effect of mating on immunity can be masked by experimental piercing in female Drosophila melanogaster.

Mating and immunity are two major components of fitness and links between them have been demonstrated in a number of recent investigations. In Drosophila melanogaster, a seminal fluid protein, sex-peptide (SP), up-regulates a number of antimicrobial peptide (AMP) genes in females after mating but the resulting effect on pathogen resistance is unclear. In this study, we tested (1) whether SP-induced changes in gene expression affect the ability of females to kill injected non-pathogenic bacteria and (2) how the injection process per se affects the expression of AMP genes relative to SP. The ability of virgin females and females mated to SP lacking or control males to clear bacteria was assayed using an established technique in which Escherichia coli are injected directly into the fly body and the rate of clearance of the injected bacteria is determined. We found no repeatable differences in clearance rates between virgin females and females mated to SP producing or SP lacking males. However, we found that the piercing of the integument, as occurs during injection, up-regulates AMP gene expression much more strongly than SP. Thus, assays that involve piercing, which are commonly used in immunity studies, can mask more subtle and biologically relevant changes in immunity, such as those induced by mating.

The hydroxyproline motif of male sex peptide elicits the innate immune response in Drosophila females.

Seminal fluid elicits a variety of physiological and behavioral changes in insect females. In Drosophila melanogaster females, sex peptide (SP) is the major seminal agent eliciting oviposition and reduction of receptivity. But SP also has many other effects; for example, it stimulates food intake, egg production, ovulation, juvenile hormone production and antimicrobial peptide synthesis. Thus, SP very probably has several receptors. To identify putative targets and signaling cascades, we studied the genome-wide regulation of genes by microarray analysis of RNA isolated from females after mating with wild-type males or males lacking SP, respectively. In addition, we studied the effects of SP on the proteome of females. Sex peptide regulates gene activity differentially in the head and in the abdomen. Genes coding for unspecific antimicrobial peptides are specifically transcribed in the abdomen, e.g. the antimicrobial peptide drosocin in epithelial tissues of the female genital tract (oviduct and calyx). Hence, SP elicits a systemic [Peng J, Zipperlen P & Kubli E (2005) Curr Biol15, 1690-1694] and an epithelial immune response. Ectopic expression of SP in the fat body of transgenic virgin females (with subsequent secretion into the hemolymph) does not elicit drosocin synthesis in the genital tract. Thus, the receptors for the stimulation of the systemic and the epithelial responses by SP are compartmentalized. The hydroxyproline (P*) motif of SP, P*TKFP*IP*SP*NP*, is identified as a novel elicitor of the innate immune response. We suggest that SP acts by chemical mimicry of sugar components of the bacterial cell wall. Thus, SP may induce the immune system via pattern recognition receptors.

Sex-peptide-regulated female sexual behavior requires a subset of ascending ventral nerve cord neurons.

BACKGROUND: Male-derived Sex-peptide (SP) elicits egg laying and rejection of courting males in mated Drosophila females. Little is known about the genes that specify the underlying neuronal circuits and mediate this switch in female sexual behavior. RESULTS: Here we show that the egghead gene involved in glycosphingolipid biosynthesis provides an essential component to the SP response. We have isolated viable alleles of the vital egghead gene that abolish egghead expression from a distal promoter resulting in the absence of the largest transcript of this complex transcription unit. Temporally and spatially restricted expression of egghead revealed a requirement for egghead early in the development of apterous-expressing ventral nerve cord neurons to rescue the SP response. In viable egghead alleles, these ascending interneurons, three per abdominal and seven per thoracic hemisegment, fail to innervate the central brain. egghead expression in apterous neurons rescues neuronal targeting and the response to SP. Furthermore, neurotransmission in apterous neurons is required to elicit the SP response. CONCLUSION: Together with the former finding of SP binding to afferent nerves , these results suggest that SP-mediated modification of sensory input switches female sexual behavior from the virgin to the mated state.

The Drosophila calcipressin sarah is required for several aspects of egg activation.

Activation of mature oocytes initiates development by releasing the prior arrest of female meiosis, degrading certain maternal mRNAs while initiating the translation of others, and modifying egg coverings. In vertebrates and marine invertebrates, the fertilizing sperm triggers activation events through a rise in free calcium within the egg. In insects, egg activation occurs independently of sperm and is instead triggered by passage of the egg through the female reproductive tract ; it is unknown whether calcium signaling is involved. We report here that mutations in sarah, which encodes an inhibitor of the calcium-dependent phosphatase calcineurin, disrupt several aspects of egg activation in Drosophila. Eggs laid by sarah mutant females arrest in anaphase of meiosis I and fail to fully polyadenylate and translate bicoid mRNA. Furthermore, sarah mutant eggs show elevated cyclin B levels, indicating a failure to inactivate M-phase promoting factor (MPF). Taken together, these results demonstrate that calcium signaling is involved in Drosophila egg activation and suggest a molecular mechanism for the sarah phenotype. We also find the conversion of the sperm nucleus into a functional male pronucleus is compromised in sarah mutant eggs, indicating that the Drosophila egg's competence to support male pronuclear maturation is acquired during activation.

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

Insect immune defense is mainly based on humoral factors like antimicrobial peptides (AMPs) that kill the pathogens directly or 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) lead to the synthesis of AMPs. But AMP genes are also regulated without pathogenic challenge, e.g., by aging, circadian rhythms, and mating. Here, we show 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, 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.

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

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

The presence of Drosophila melanogaster sex peptide-like immunoreactivity in the accessory glands of male Helicoverpa armigera.

In this study a highly specific polyclonal antibody to DrmSP was produced and used to develop and standardize a sensitive direct ELISA. Structure-activity studies revealed that the antiserum is specific to the N-terminal of DrmSP. This ELISA was used for the detection of DrmSP-like immunoreactivity in the reproductive tissues of male Helicoverpa armigera moths at femtomole levels. Two positive immunoreactive peaks were found in HPLC purified extracts of male accessory glands. The immunoreactive peak, which contained a higher amount of immunoreactivity, was also found to be pheromonostatic in PBAN-injected decapitated females as well as in intact female moths during their peak pheromone production. Lower levels of DrmSP-like immunoreactivity were found in younger males (1-2 day-old) when compared to older males (3-7 day-old).

The sex-peptide DUP99B is expressed in the male ejaculatory duct and in the cardia of both sexes.

Mating elicits two postmating responses in many insect females: the egg laying rate increases and sexual receptivity is reduced. In Drosophila melanogaster, two peptides of the male genital tract, sex-peptide and DUP99B, elicit these postmating responses when injected into virgin females. Here we show that the gene encoding DUP99B is expressed in the male ejaculatory duct and in the cardia of both sexes. The DUP99B that is synthesized in the ejaculatory duct is transferred, during mating, into the female genital tract. Expression of the gene is first seen in a late pupal stage. Males containing an intact ejaculatory duct, but lacking accessory glands, initiate the two postmating responses in their female partners [Xue, L. & Noll, M. (2000) Proc. Natl Acad. Sci. USA97, 3272-3275]. Although such males synthesize DUP99B in wild-type quantities, they elicit only weak postmating responses in their mating partners. Males lacking the Dup99B gene elicit the two postmating responses to the same extent as wild-type males. These results suggest that both sex-peptide and DUP99B can elicit both responses in vivo. However, sex-peptide seems to play the major role in eliciting the postmating responses, while DUP99B may have specialized for other, as yet unknown, functions.

Sex-peptide is the molecular basis of the sperm effect in Drosophila melanogaster.

Mating elicits two major changes in the reproductive behavior of many insect females. The egg-laying rate increases and the readiness to accept males (receptivity) is reduced. These postmating responses last approximately 1 week in Drosophila melanogaster. Males that do not transfer sperm but transfer seminal fluid during mating induce a short-term response of 1 day. The long-term response of 1 week requires the presence of sperm (sperm effect). Hence, sperm is essential for the long-term persistence of the postmating responses. Three seminal fluid peptides elicit postmating responses: ovulin, sex-peptide (SP), and DUP99B. Using the technique of targeted mutagenesis by homologous recombination, we have produced males with mutant SP genes. Here, we report that males lacking functional SP elicit only a weak short-term response. However, these males do transfer sperm. Thus, (i) SP is the major agent eliciting the short-term and the long-term postmating responses and (ii) sperm is merely the carrier for SP. The second conclusion is supported by the finding that SP binds to sperm. The 36-aa-encoding SP gene is the first small Drosophila gene knocked out with the method of homologous recombination.

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

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

Ductus ejaculatorius peptide 99B (DUP99B), a novel Drosophila melanogaster sex-peptide pheromone.

We have characterized a glycosylated, 31 amino-acid peptide of 4932 Da isolated from Drosophila melanogaster males. The mature peptide contains a sugar moiety of 1184 Da at a NDT consensus glycosylation site and a disulfide bond. It is synthesized in the male ejaculatory duct via a 54 amino-acid precursor containing an N-terminal signal peptide and Arg-Lys at the C-terminus which is cleaved off during maturation. The gene contains an intron of 53 bp and is localized in the cytological region 99B of the D. melanogaster genome. The peptide is therefore named DUP99B (for ductus ejaculatorius peptide, cytological localization 99B). The C-terminal parts of mature DUP99B and D. melanogaster sex-peptide (ACP70A) are highly homologous. Injected into virgin females, DUP99B elicits the same postmating responses as sex-peptide (increased oviposition, reduced receptivity). These effects are also induced by de-glycosylated native peptide or synthetic DUP99B lacking the sugar moiety. Presence of the glycosyl group, however, decreases the amount needed to elicit the postmating responses. Homologies in the coding regions of the two exons of DUP99B and sex-peptide, respectively, suggest that the two genes have evolved by gene duplication. Thus, we consider these two genes to be members of the new sex-peptide gene family.

Wild-type tRNATyrG reads the TMV RNA stop codon, but Q base-modified tRNATyrQ does not.

Although protein synthesis usually terminates when a stop codon is reached along the messenger RNA sequence, there are examples, mainly in viruses, of the stop codon being suppressed by a tRNA species. A strong candidate for this phenomenon occurs in tobacco mosaic virus (TMV) in the form of two proteins (110K and 160K, of molecular weights 110,000 and 160,000, respectively)1, sharing an N-terminus sequence, which are translated in vitro from a purified species of viral RNA. We have investigated the identity of the tRNA responsible for production of the 160K protein and show here that it is one of the tyrosine tRNAs. Another tyrosine tRNA, in which the first base of the anticodon is highly modified, does not act as a suppressor, indicating the possible regulatory function of such modifications.