ReviewFunctions and analysis of the seminal fluid proteins of male Drosophila melanogaster fruit flies
Introduction
The study of insect seminal fluid proteins provides a unique window upon adaptive evolution in action. The proteins and peptides transferred together with sperm have sites of action within and outside the female reproductive tract, and they cause a wide variety of responses in females. These responses include increased egg-laying (through increased oogenesis [101], [102] and ovulation [50]) and decreased female receptivity [21], [29], [62]. In addition, seminal fluid proteins are essential for normal sperm storage in females [78], [112], for success in sperm competition [24] and are necessary for the formation of the mating plug (a gelatinous structure which forms in the female reproductive tract during mating [67]). In addition, the seminal fluid of males contains substances with antibacterial activity [66], [89], and enzymes such as protease inhibitors, putative proteases and lipases [107], [116]. In this review, we focus on the currently known functions of the seminal fluid proteins of Drosophila melanogaster. We then address some of the interesting, unanswered questions that are provoked by these functional studies and describe techniques that can be used to investigate them.
It has long been known that mating causes two very striking changes in female D. melanogaster: they become temporarily unreceptive and increase their rate of egg-laying [27], [43], [59]. Potential candidates for inducing these responses were substances in the ejaculate (i.e. sperm or seminal fluid proteins). Bioactive molecules affecting egg-laying and receptivity were localized to the reproductive tracts of D. melanogaster males by examining the behavior and physiology of females transplanted with male reproductive tract accessory glands, or injected with accessory gland extracts [27], [43], [59]. Furthermore, experiments using XO males (produced by sex chromosomal non-disjunction), that transfer seminal fluids but no sperm at mating showed that receptivity could be reduced and egg-laying stimulated by the transfer of seminal fluid proteins alone (e.g. [53], [70]). Although these responses persisted for a shorter period (1–2 days) than those observed following matings to normal males (>5 days), the results nevertheless provided evidence that seminal fluid proteins were involved in modulating post-mating responses in females [53], [69], [70]. The very first seminal fluid protein to be identified was the ‘sex peptide’ or accessory gland protein 70A (Acp70A) [29]. Throughout the 1960s to 1980s the nature of Acp70A was gradually revealed [26], [27], [28], [29], [30], [93], [94], [106]. The Acp70A peptide was identified by injecting HPLC separated fractions of male accessory gland extracts into virgin females, to reveal the fraction that boosted egg-laying and decreased receptivity [29]. Peptide sequencing of this fraction was then employed and the Acp70A gene that encodes the 36 amino acid Acp70A peptide was identified [29]. Since then, the rate at which seminal fluid genes and their functions have been identified has increased dramatically. Rather than focusing on the identification of Acps through functional assays of single ejaculate molecules (e.g. [29]), strategies to simultaneously identify multiple Acps have increasingly been taken. These strategies involve using differential hybridization screens to identify mRNAs expressed specifically in the male accessory glands [36], [74], [91], [100], [117]. Together these screens identified 18 Acp genes. Utilizing the D. melanogaster genome sequence it was subsequently possible, using an expressed sequence tag (EST) screen, to simultaneously identify all remaining Acps, and estimate the total number of Acps to be 83 [107], [116].
The ongoing study of seminal fluid molecules is revealing that they have an unexpected variety of functions. In addition, some of the genes that encode these molecules show evidence of extremely rapid evolutionary change (e.g. [1], [2], [7], [107], [108], [113], [114]). This suggests that seminal fluid molecules may be strong targets for natural or sexual selection. It is an exciting challenge to investigate why, by revealing seminal fluid protein function, by probing the underlying mechanisms involved and by investigating the reasons for functional redundancy between seminal fluid proteins.
Section snippets
Site of synthesis of seminal fluid peptides and proteins in D. melanogaster
The non-sperm part of the male ejaculate in fruit flies mostly comprises molecules synthesized by secretory cells in the paired accessory glands (e.g. [36], [116], [117]), but also substances made in the ejaculatory duct (e.g. [66], [90]) and ejaculatory bulb (e.g. [67]) (Fig. 1a and Table 1). The male accessory glands comprise two types of secretory cells that each express separate sets of genes [8], [35], [73]. Each gland contains ∼1000 main cells, which comprise 96% of the secretory cells
The timing of ejaculate transfer
The duration of mating is typically 15–20 min in D. melanogaster. Seminal fluid molecules from the accessory glands, ejaculatory duct and ejaculatory bulb can be detected in females within the first 6 min of the start of mating (e.g. [9], [65], [73], [87], [117]). A gelatinous mating ‘plug’ is also formed early, within the first 7 min of mating [6], [67]. Sperm are transferred typically in the first 7–10 min, sometime before the midpoint of mating [4], [6], [43], [46], [67] and must therefore
Functions of seminal fluid peptides
Seminal fluid proteins have a number of striking effects on the reproductive success of males and females. Here we review their currently known functions, see Table 2 (and [19], [115], [116]).
Acps with dual functions and functional redundancy between different Acps
An interesting finding emerging from the increasing numbers of functional tests of Acps is that there are cases in which single Acps have more than one quite different function, and others in which different Acps affect the same trait (functional redundancy). Here we briefly discuss the potential reasons for, and significance of, these observations.
Techniques for studying seminal fluid protein function
The number of techniques brought to bear on the study of seminal fluid protein function has increased dramatically in recent years. The usefulness of each of them depends upon the questions being asked, and the pros and cons of each are discussed below.
Future study: the significance of natural variation in seminal fluid protein function
The increasing sophistication of techniques that can be brought to bear on the study of seminal fluid protein function represent an important advance and bring us closer to an important goal: realizing the significance of natural variation in seminal fluid protein function. For example the manipulation of Acp levels by using knock out and knock down stocks is a powerful technique, but a relatively blunt tool with which to study variation in wild-type seminal fluid protein effects. It will be
Acknowledgements
We thank the Leverhulme Trust and the Royal Society for funding, Richard Marguerie for the accessory gland images and two anonymous reviewers for helpful comments.
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