For more than 100 years, the fruit fly Drosophila melanogaster has been one of the most studied model organisms. Here, we present a single-cell atlas of the adult fly, Tabula Drosophilae , that includes 580,000 nuclei from 15 individually dissected sexed tissues as well as the entire head and body, annotated to >250 distinct cell types. We provide an in-depth analysis of cell type–related gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal. Analysis of common cell types between tissues, such as blood and muscle cells, reveals rare cell types and tissue-specific subtypes. This atlas provides a valuable resource for the Drosophila community and serves as a reference to study genetic perturbations and disease models at single-cell resolution.

Summary Post-copulatory sexual selection can select for sperm allocation strategies in males [1, 2] but males should also strategically allocate non-sperm components of the ejaculate [3, 4] such as seminal fluid proteins (Sfps). Sfps can influence the extent of post-copulatory sexual selection [5–7] but little is known of the causes or consequences of quantitative variation in Sfp production and transfer. Using Drosophila melanogaster, we demonstrate that Sfps are strategically allocated to females in response to the potential level of sperm competition. We also show that males who can produce and transfer larger quantities of specific Sfps have a significant competitive advantage. When males were exposed to a competitor male, matings were longer and more of two key Sfps, sex peptide [8] and ovulin [9], were transferred, indicating strategic allocation of Sfps. Males selected for large accessory glands (AGs, a major site of Sfp synthesis) produced and transferred significantly more sex peptide, but not more ovulin. Large AG males also had significantly increased competitive reproductive success. Our results show that quantitative variation in specific Sfps is likely to play an important role in post-copulatory sexual selection and that investment in Sfp production is essential for male fitness in a competitive environment.

Egg activation is the process by which a mature oocyte becomes capable of supporting embryo development. In vertebrates and echinoderms, activation is induced by fertilization. Molecules introduced into the egg by the sperm trigger progressive release of intracellular calcium stores in the oocyte. Calcium wave(s) spread through the oocyte and induce completion of meiosis, new macromolecular synthesis, and modification of the vitelline envelope to prevent polyspermy. However, arthropod eggs activate without fertilization: in the insects examined, eggs activate as they move through the female’s reproductive tract. Here, we show that a calcium wave is, nevertheless, characteristic of egg activation in Drosophila. This calcium rise requires influx of calcium from the external environment and is induced as the egg is ovulated. Pressure on the oocyte (or swelling by the oocyte) can induce a calcium rise through the action of mechanosensitive ion channels. Visualization of calcium fluxes in activating eggs in oviducts shows a wave of increased calcium initiating at one or both oocyte poles and spreading across the oocyte. In vitro, waves also spread inward from oocyte pole(s). Wave propagation requires the IP3 system. Thus, although a fertilizing sperm is not necessary for egg activation in Drosophila, the characteristic of increased cytosolic calcium levels spreading through the egg is conserved. Because many downstream signaling effectors are conserved in Drosophila, this system offers the unique perspective of egg activation events due solely to maternal components.

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.

In many species, seminal fluid proteins (SFPs) affect female post-mating behavioral patterns, including sperm storage, egg laying, feeding, and remating. Yet, few studies have investigated the patterns of allocation, depletion, and replenishment of SFPs in male animals, despite the importance of these proteins to male and female reproductive success. To investigate such SFP dynamics, it is necessary to have a sensitive method for quantifying SFP levels in males and mated females. We developed such a method by adapting the enzyme-linked immunosorbent assay (ELISA) using anti-SFP antibodies. Here, we first use two Drosophila melanogaster SFPs (ovulin and sex peptide) to demonstrate that ELISAs provide accurate measures of SFP levels. We find that, consistent with previous data from Western blotting or immunofluorescence studies, levels of both ovulin and sex peptide decline in the mated female with time since mating, but they do so at different rates. We then use ELISAs to show that males become depleted of SFPs with repeated matings, but that previously mated males are able to transfer “virgin” levels of SFPs after 3 days of sexual inactivity. Finally, we demonstrate that ELISAs can detect SFPs from wild-caught D. melanogaster males and, thus, potentially can be used to track mating patterns in the wild. This method of measuring SFP dynamics can be used in a wide range of species to address questions related to male reproductive investment, female mating history, and variation in female post-mating behavioral changes.