Female Drosophila melanogaster with environmentally or genetically elevated rates of mating die younger than controls. This cost of mating is not attributable to receipt of sperm. We demonstrate here that seminal fluid products from the main cells of the male accessory gland are responsible for the cost of mating in females, and that increasing exposure to these products increases female death rate. Main-cell products are also involved in elevating the rate of female egg-laying, in reducing female receptivity to further matings and in removing or destroying sperm of previous mates. The cost of mating to females may therefore represent a side-effect of evolutionary conflict between males.

Comparative analysis of multiple genomes in a phylogenetic framework dramatically improves the precision and sensitivity of evolutionary inference, producing more robust results than single-genome analyses can provide. The genomes of 12 Drosophila species, ten of which are presented here for the first time (sechellia, simulans, yakuba, erecta, ananassae, persimilis, willistoni, mojavensis, virilis and grimshawi), illustrate how rates and patterns of sequence divergence across taxa can illuminate evolutionary processes on a genomic scale. These genome sequences augment the formidable genetic tools that have made Drosophila melanogaster a pre-eminent model for animal genetics, and will further catalyse fundamental research on mechanisms of development, cell biology, genetics, disease, neurobiology, behaviour, physiology and evolution. Despite remarkable similarities among these Drosophila species, we identified many putatively non-neutral changes in protein-coding genes, non-coding RNA genes, and cis-regulatory regions. These may prove to underlie differences in the ecology and behaviour of these diverse species.

Seminal fluid proteins (SFPs) produced in reproductive tract tissues of male insects and transferred to females during mating induce numerous physiological and behavioral post-mating changes in females. These changes include decreasing receptivity to re-mating, affecting sperm storage parameters, increasing egg production, modulating sperm competition, feeding behaviors, and mating plug formation. In addition, SFPs also have anti-microbial functions and induce expression of anti-microbial peptides in at least some insects. Here, we review recent identification of insect SFPs and discuss the multiple roles these proteins play in the post-mating processes of female insects.

Sequence comparisons of genomes or expressed sequence tags (ESTs) from related organisms provide insight into functional conservation and diversification. We compare the sequences of ESTs from the male accessory gland of Drosophila simulans to their orthologs in its close relative Drosophila melanogaster, and demonstrate rapid divergence of many of these reproductive genes. Nineteen (ϳ11%) of 176 independent genes identified in the EST screen contain protein-coding regions with an excess of nonsynonymous over synonymous changes, suggesting that their divergence has been accelerated by positive Darwinian selection. Genes that encode putative accessory gland-specific seminal fluid proteins had a significantly elevated level of nonsynonymous substitution relative to nonaccessory gland-specific genes. With the 57 new accessory gland genes reported here, we predict that ϳ90% of the male accessory gland genes have been identified. The evolutionary EST approach applied here to identify putative targets of adaptive evolution is readily applicable to other tissues and organisms.positive selection ͉ accessory glands ͉ seminal fluid ͉ peptide hormones ͉ sexual conflict T he Drosophila male accessory gland is a highly specialized reproductive organ. Its function is to secrete seminal-fluid proteins. Therefore, it may be relatively easy to identify many of the proteins found in seminal fluid by sequencing expressed sequence tags (ESTs) from the accessory gland. Secreted accessory gland proteins (Acps) have diverse and important reproductive roles and interesting patterns of evolutionary change. Acps are transferred along with sperm to the female's reproductive tract and have a variety of effects on the female's reproductive physiology (1). Acps increase the egg-laying rate of mated females by inducing oogenesis (2, 3) and ovulation (4), decrease the female's propensity to remate (5), are required for sperm storage (6, 7), and influence egg hatchability (8, 9). Also, Acps may play a role in cryptic female choice (10), sperm competition (11), and intersexual genomic conflict (12)-three evolutionary scenarios thought to promote the divergence of reproductive proteins. The unique role of Acps has made them the focus of much interest by cell and evolutionary biologists, because they seem to be a currency of chemical communication between males and females (1).Two-dimensional protein electrophoresis has been used to show that male reproductive proteins (including Acps) are twice as diverse as nonreproductive proteins (13), but because the nucleotide sequences encoding these proteins remained unidentified, it was impossible to determine whether positive selection or low constraint on amino acid sequence led to the apparent high divergence of this large class of proteins. Identification of the nucleotide sequences encoding these highly variable proteins will allow for evolutionary inferences of the magnitude of forces affecting their evolution (14) and provide tools for determining the molecular function of the selected gene (2-6, 1...