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Seminal fluid proteins elicit several post-mating physiological changes in mated Drosophila melanogaster females. Some of these changes persist for over a week after mating because the seminal protein that causes these changes, the Sex Peptide (SP), binds to sperm that are stored in the female reproductive tract. SP's sperm binding is mediated by a network of at least eight seminal proteins. We show here that some of these network proteins (CG1656, CG1652, CG9997 and Antares) bind to sperm within 2 h of mating, like SP. However, while SP remains bound to sperm at 4 days post-mating, none of the other network proteins are detectable at this time. We also observed that the same network proteins are detectable at 2 h post-mating in seminal receptacle tissue from which sperm have been removed, but are no longer detectable there by 4 days post-mating, suggesting short-term retention of these proteins in this female sperm storage organ. Our results suggest that these network proteins act transiently to facilitate the conditions for SP's binding to sperm, perhaps by modifying SP or the sperm surface, but are not part of a long-acting complex that stably attaches SP to sperm.

Dimerization is an important feature of the function of some proteins, including prohormones. For proteins whose amino acid sequences evolve rapidly, it is unclear how such structural characteristics are retained biochemically. Here we address this question by focusing on ovulin, a prohormone that induces ovulation in Drosophila melanogaster females after mating. Ovulin is known to dimerize, and is one of the most rapidly evolving proteins encoded by the Drosophila genome. We show that residues within a previously hypothesized conserved dimerization domain (a coiled-coil) and a newly identified conserved dimerization domain (YxxxY) within ovulin are necessary for the formation of ovulin dimers. Moreover, dimerization is conserved in ovulin proteins from non-melanogaster species of Drosophila despite up to 80% sequence divergence. We show that heterospecific ovulin dimers can be formed in interspecies hybrid animals and in two-hybrid assays between ovulin proteins that are 15% diverged, indicating conservation of tertiary structure amidst a background of rapid sequence evolution. Our results suggest that because ovulin's self-interaction requires only small conserved domains, the rest of the molecule can be relatively tolerant to mutations. Consistent with this view, in comparisons of 8510 proteins across 6 species of Drosophila we find that rates of amino acid divergence are higher for proteins with coiled-coil protein-interaction domains than for non-coiled-coil proteins.

Male reproductive gland proteins (mRGPs) impact the physiology and/or behavior of mated females in a broad range of organisms. We sought to identify mRGPs of the yellow fever mosquito, Aedes aegypti, the primary vector of dengue and yellow fever viruses. Earlier studies with Ae. aegypti demonstrated that “matrone” (a partially purified male reproductive accessory gland substance) or male accessory gland fluid injected into virgin female Ae. aegypti affect female sexual refractoriness, blood feeding and digestion, flight, ovarian development, and oviposition. Using bioinformatic comparisons to Drosophila melanogaster accessory gland proteins and mass spectrometry of proteins from Ae. aegypti male accessory glands and ejaculatory ducts (AG/ED) and female reproductive tracts, we identified 63 new putative Ae. aegypti mRGPs. Twenty-one of these proteins were found in the reproductive tract of mated females, but not of virgin females, suggesting that they are transferred from males to females during mating. Most of the putative mRGPs fall into the same protein classes as mRGPs in other organisms, although some appear to be evolving rapidly and lack identifiable homologs in Culex pipiens, Anopheles gambiae, and D. melanogaster. Our results identify candidate male-derived molecules that may have an important influence on behavior, survival and reproduction of female mosquitoes.

Male accessory gland proteins (Acps) in Drosophila are components of the seminal fluid and are transferred to females during copulation. In mated females, Acps enhance egg production, augment sperm storage, induce refractory mating behaviors, and affect the female's longevity. To address the functions of eight previously uncharacterized Acps and further analyze five others, we determined the tissues to which they target after transfer to females. Each Acp has multiple targets and is unique in its pattern of localization. Within the reproductive tract, Acps target to the uterus, oviduct, sperm storage organs, ovary and oocytes. Some Acps also leave the reproductive tract, to enter the hemolymph. Some Acps are detected on the surface of eggs laid by mated females but were not detectable within those eggs. Our results can help to identify the likely functions of these Acps as well as to create models for the mechanism of action of Acps.

Seminal fluid proteins from males of many insect species affect the behavior and physiology of their mates. In some cases, these effects result from entry of the proteins into the female's circulatory system. In the fruit fly Drosophila melanogaster, some seminal fluid proteins enter the female's circulatory system after transfer from the male while others remain confined within the reproductive tract. To address where and how seminal fluid proteins enter the hemolymph of the mated female, we compared the kinetics of transfer and localization in mated females of two seminal fluid proteins that enter the hemolymph (Acp26Aa and Acp62F) and one that does not (Acp36DE). We also generated transgenic flies that produce Acp26Aa tagged with Aequorea victoria green fluorescent protein (GFP) to monitor its transfer in vivo. We report that Acps enter the female circulatory system from the posterior vagina immediately after insemination. The ability of Acps to enter the female hemolymph correlates with their ability to cross the intima that lines the posterior vagina. The ventral posterior vagina is structurally unlike other parts of the female reproductive tract in that it lacks muscles. We hypothesize that it has higher permeability thus affording access to the female's circulatory system.

The accessory gland of male insects produces components of the seminal fluid that alter the behavior, physiology and life span of the mated female, and contribute to her efficient storage and utilization of sperm. As a step towards understanding how this occurs, we have isolated genes encoding 12 previously unreported accessory gland-specific mRNAs from the fruit fly Drosophila melanogaster. We report here the restriction maps of the new genes, the chromosome positions--which are all autosomal--of the 11 non-repetitive genes, their expression patterns, and the sequences of the accessory gland proteins (Acps) encoded by nine of the genes. Eight of the proteins predicted from these sequences begin with putative secretion signals. Following their signal sequences, three of the predicted molecules are peptides and the other five are larger polypeptides with characteristics of cleavable prohormones. The ninth molecule, which has an N-terminal hydrophobic region but no consensus signal peptide cleavage site, is predicted to be a 716 amino acid glycoprotein. Of the nine proteins, two have intriguing similarities to sequences in protein databases. Acp76A is a 388 amino acid pro-protein which contains a signature sequence for the serpin class of protease inhibitors. The 115 amino acid Acp62F has a 28 amino acid region of high sequence similarity to a neurotoxin of the Brazilian armed spider Phoneutria nigriventer. Models are discussed in which Acp76A plays a role in the observed regulation of Acp proteolysis and/or in the coagulation of seminal fluid to form a mating plug, and in which Acp62F contributes to the reported toxicity of Drosophila seminal fluid.

A male fruit fly influences the behavior and physiology of his mate via molecules that he transmits to her'in his semen. The mated female fly has an elevated rate of egg laying, a decreased receptivity to mating and a shorter life span; she also stores sperm from the mating. Molecular genetic analyses possible in this insect model system permit the dissection of seminal fluid components that cause these mating responses in the female. Studies with transgenic and mutant flies have shown that products of the male's accessory gland cause short-term changes in the female's behavior and physiology; persistence of these changes requires the storage of sperm. Further dissection of accessory gland function has defined several molecules that cause these effects. A “sex peptide” and a prohormone-like molecule (Accessory gland protein 26Aa) stimulate the female's egg-laying rate; the sex peptide also depresses her receptivity to mating. A large glycoprotein (Acp36DE) appears to function in “corralling” sperm for storage. Studies of accessory gland products and the regulation of the genes that encode them will be important in understanding insect reproduction, behavior, and speciation and ultimately in designing ways to control the impressive fertility of unwanted insects. These studies also provide excellent models to address basic questions in cell biology such as the control of genes in response to sex-specific, mating-regulated and cell type-specific cues and the function and targeting of peptide hormones.

In many insect species, sperm transferred in a single mating are stored by the female in specialized organs and are gradually used to fertilize eggs. Thus, insects must have mechanisms to ensure that substantial numbers of sperm reach and become stored in the storage organs. We report here that a glycoprotein, Acp36DE, made by the accessory glands of Drosophila melanogaster males, shows localization in the mated female suggesting a role in sperm storage. In the mated female, Acp36DE associates with the wall of the oviduct, just anterior to the openings of the sperm storage organs. Acp36DE also associates with the leading edge of the sperm mass. It does not enter the hemolymph. Complete localization of Acp36DE in the mated female requires sperm and the presence of eggs in the ovaries. We hypothesize that Acp36DE, or a complex containing it, forms a barrier that "corrals" sperm near the openings to the sperm storage organs. Concentration of sperm here could facilitate their efficient storage. Acp36DE remains in the genital tract for several hours after mating, concurrent with the time that sperm are being stored. Facilitation of sperm storage by Acp36DE may also explain the previously observed effect of this protein on sperm competition.

In many insects, semen coagulates into a mating plug at the distal part of the female's genital tract. Mating plugs have been proposed to facilitate sperm movement or to prevent subsequent matings or sperm loss. The molecular constituents of insect mating plugs have not previously been characterized. Here we report that an abundant autofluorescent protein made by the Drosophila melanogaster male's ejaculatory bulb is a major constituent of the posterior region of the mating plug. Identities in size, chromosomal location and expression pattern indicate that the autofluorescent protein is PEB-me, an abundant ejaculatory bulb protein reported by Ludwig et al. [Biochem. Genet. 29 (1991) 215]. We cloned and sequenced the RNA encoding this protein. The transcript, which is male-specific and expressed only in the ejaculatory bulb, encodes a 377 a.a. predicted secreted protein with PGG repeats similar to those in homopolymer-forming proteins found in spider silk.