A large portion of the annotated genes in Drosophila melanogaster show sex-biased expression, indicating that sex and reproduction-related genes (SRR genes) represent an appreciable component of the genome. Previous studies, in which subsets of genes were compared among few Drosophila species, have found that SRR genes exhibit unusual evolutionary patterns. Here, we have used the newly released genome sequences from 12 Drosophila species, coupled to a larger set of SRR genes, to comprehensively test the generality of these patterns. Among 2505 SRR genes examined, including ESTs with biased expression in reproductive tissues and genes characterized as involved in gametogenesis, we find that a relatively high proportion of SRR genes have experienced accelerated divergence throughout the genus Drosophila. Several testis-specific genes, male seminal fluid proteins (SFPs), and spermatogenesis genes show lineage-specific bursts of accelerated evolution and positive selection. SFP genes also show evidence of lineage-specific gene loss and/or gain. These results bring us closer to understanding the details of the evolutionary dynamics of SRR genes with respect to species divergence.

Successful reproduction requires contributions from both the male and the female. In Drosophila, contributions from the male include accessory gland proteins (Acps) that are components of the seminal fluid. Upon their transfer to the female, Acps affect the female's physiology and behavior. Although primary sequences of Acp genes exhibit variation among species and genera, the conservation of protein biochemical classes in the seminal fluid suggests a conservation of functions. Bioinformatics coupled with molecular and genetic tools available for Drosophila melanogaster has expanded the functional analysis of Acps in recent years to the genomic/proteomic scale. Molecular interplay between Acps and the female enhances her egg production, reduces her receptivity to remating, alters her immune response and feeding behavior, facilitates storage and utilization of sperm in the female and affects her longevity. Here, we provide an overview of the D. melanogaster Acps and integrate the results from several studies that bring the current number of known D. melanogaster Acps to 112. We then discuss several examples of how the female's physiological processes and behaviors are mediated by interactions between Acps and the female. Understanding how Acps elicit particular female responses will provide insights into reproductive biology and chemical communication, tools for analyzing models of sexual cooperation and/or sexual conflict, and information potentially useful for strategies for managing insect pests.

Successful reproduction is critical to pass genes to the next generation. Seminal proteins contribute to important reproductive processes that lead to fertilization in species ranging from insects to mammals. In Drosophila, the male's accessory gland is a source of seminal fluid proteins that affect the reproductive output of males and females by altering female post-mating behavior and physiology. Protein classes found in the seminal fluid of Drosophila are similar to those of other organisms, including mammals. By using RNA interference (RNAi) to knock down levels of individual accessory gland proteins (Acps), we investigated the role of 25 Acps in mediating three post-mating female responses: egg production, receptivity to remating and storage of sperm. We detected roles for five Acps in these post-mating responses. CG33943 is required for full stimulation of egg production on the first day after mating. Four other Acps (CG1652, CG1656, CG17575, and CG9997) appear to modulate the long-term response, which is the maintenance of post-mating behavior and physiological changes. The long-term post-mating response requires presence of sperm in storage and, until now, had been known to require only a single Acp. Here, we discovered several novel Acps together are required which together are required for sustained egg production, reduction in receptivity to remating of the mated female and for promotion of stored sperm release from the seminal receptacle. Our results also show that members of conserved protein classes found in seminal plasma from insects to mammals are essential for important reproductive processes. In sexually reproducing organisms, sperm enter the female in combination with seminal proteins that are critical for fertility. These proteins can activate sperm or enhance sperm storage within the female, and can improve the chance that sperm will fertilize eggs. Understanding the action of seminal proteins has potential utility in insect pest control and in the diagnosis of certain human infertilities. However, the precise function of very few seminal proteins is known. To address this, we knocked down the levels of 25 seminal proteins individually in male fruit flies, and tested the males' abilities to modulate egg production, sperm storage/release, or behavior of their mates. We found five seminal proteins that are necessary to elevate offspring production in mated females. Four of these proteins are needed for efficient release of sperm from storage to fertilize eggs, a function that had not been previously assigned to any seminal protein. All four are in biochemical classes that are conserved in seminal fluid from insects to humans, suggesting they may play similar sperm-related roles in other animals. In addition to assigning functions to particular seminal proteins, our results suggest that fruit flies can serve as a model with which to dissect the functions of conserved protein classes in seminal fluid.

Despite the importance of seminal proteins in fertility and their capacity to alter mated females' physiology, the molecular pathways and networks through which they act have not been well characterized. Drosophila seminal fluid includes proteins that fall into biochemical classes conserved from insects to mammals, making it an excellent model with which to address this question. Drosophila seminal fluid also contains a “sex peptide” (SP, Acp70A) that plays a major role in regulating egg production and mating behavior in females for several days after mating. This long-term postmating response (LTR) initially requires the association of SP with sperm. The LTR also requires members of the conserved seminal protein classes (two lectins, a protease, and a cysteine-rich secretory protein). Here, we show that these seminal proteins function interdependently, regulating a three-step cascade (first, at the level of seminal protein transfer to the female; second, at the level of stability; and third, at the level of localization within females), leading to the normal localization of SP to sperm-storage organs. This localization is, in turn, necessary for successful induction of the LTR. The requirements for manifestation of the LTR in Drosophila establish the paradigm that multiple seminal proteins can exert their actions through a multistep, multicomponent network of interactions.

Drosophila melanogaster males transfer seminal fluid proteins along with sperm during mating. Among these proteins, ACPs (Accessory gland proteins) from the male's accessory gland induce behavioral, physiological, and life span reduction in mated females and mediate sperm storage and utilization. A previous evolutionary EST screen in D. simulans identified partial cDNAs for 57 new candidate ACPs. Here we report the annotation and confirmation of the corresponding Acp genes in D. melanogaster. Of 57 new candidate Acp genes previously reported in D. melanogaster, 34 conform to our more stringent criteria for encoding putative male accessory gland extracellular proteins, thus bringing the total number of ACPs identified to 52 (34 plus 18 previously identified). This comprehensive set of Acp genes allows us to dissect the patterns of evolutionary change in a suite of proteins from a single male-specific reproductive tissue. We used sequence-based analysis to examine codon bias, gene duplications, and levels of divergence (via dN/dS values and ortholog detection) of the 52 D. melanogaster ACPs in D. simulans, D. yakuba, and D. pseudoobscura. We show that 58% of the 52 D. melanogaster Acp genes are detectable in D. pseudoobscura. Sequence comparisons of ACPs shared and not shared between D. melanogaster and D. pseudoobscura show that there are separate classes undergoing distinctly dissimilar evolutionary dynamics.