Sustained post-mating response in Drosophila melanogaster requires multiple seminal fluid proteins

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.     

Proteomic analysis of Drosophila mojavensis male accessory glands suggests novel classes of seminal fluid proteins

Fruit-flies of the genus Drosophila are characterized by overwhelming variation in fertilization traits such as copulatory plug formation, sperm storage organ use, and nutritional ejaculatory donation. Despite extensive research on the genetic model Drosophila melanogaster, little is known about the molecular underpinnings of these interspecific differences. This study employs a proteomic approach to pin-point candidate seminal fluid proteins in Drosophila mojavensis, a cactophilic fruit-fly that exhibits divergent reproductive biology when compared to D. melanogaster. We identify several classes of candidate seminal fluid proteins not previously documented in the D. melanogaster male ejaculate, including metabolic enzymes, nutrient transport proteins, and clotting factors. Conversely, we also define 29 SFPs that are conserved despite >40 million years of Drosophila evolution. We discuss our results in terms of universal processes in insect reproduction, as well as the specialized reproductive biology of D. mojavensis.     

Male seminal fluid proteins are essential for sperm storage in Drosophila melanogaster.

The seminal fluid that is transferred along with sperm during mating acts in many ways to maximize a male's reproductive success. Here, we use transgenic Drosophila melanogaster males deficient in the seminal fluid proteins derived from the accessory gland (Acps) to investigate the role of these proteins in the fate of sperm transferred to females during mating. Competitive PCR assays were used to show that while Acps contribute to the efficiency of sperm transfer, they are not essential for the transfer of sperm to the female. In contrast, we found that Acps are essential for storage of sperm by females. Direct counts of stored sperm showed that 10% of normal levels are stored by females whose mates transfer little or no Acps along with sperm.     

Circadian regulation of egg-laying behavior in fruit flies Drosophila melanogaster

Significant progress has been made in our understanding of the neurogenetics of circadian clocks in fruit flies Drosophila melanogaster. Several pacemaker neurons and clock genes have now been identified and their roles in the cellular and molecular clockwork established. Some recent findings suggest that the basic architecture of the clock is multi-oscillatory; the clock mechanisms in the ventral lateral neurons (LN(v)s) of the fly brain govern locomotor activity and adult emergence rhythms, while the peripheral oscillators located in antennal cells regulate olfactory rhythm. Among circadian phenomena exhibited by Drosophila, the egg-laying rhythm is unique in many ways: (i) this rhythm persists under constant light (LL), while locomotor activity and adult emergence become arrhythmic, (ii) its circadian periodicity is much longer than 24h, and (iii) while egg-laying is rhythmic under constant darkness, the expression of two core clock genes period (per) and timeless (tim), is non-oscillatory in the ovaries. In this paper, we review our current knowledge of the circadian regulation of egg-laying behavior in Drosophila, and provide some possible explanations for its self-sustained nature. We conclude by discussing the existing limitations in our understanding of the regulatory mechanisms and propose few approaches to address them.     

Circadian consequence of socio-sexual interactions in fruit flies Drosophila melanogaster

In fruit flies Drosophila melanogaster, courtship is an elaborate ritual comprising chasing, dancing and singing by males to lure females for mating. Courtship interactions peak in the night and heterosexual couples display enhanced nighttime activity. What we do not know is if such socio-sexual interactions (SSI) leave long-lasting after-effects on circadian clock(s). Here we report the results of our study aimed at examining the after-effects of SSI (as a result of co-habitation of males and females in groups) between males and females on their circadian locomotor activity rhythm. Males undergo reduction in the evening activity peak and lengthening of circadian period, while females show a decrease in overall activity. Such after-effects, at least in males, require functional circadian clocks during SSI as loss-of-function clock mutants and wild type flies interacting under continuous light (LL), do not display them. Interestingly, males with electrically silenced Pigment Dispersing Factor (PDF)-positive ventral lateral (LNv) clock neurons continue to show SSI mediated reduction in evening activity peak, suggesting that the LNv clock neurons are dispensable for SSI mediated after-effects on locomotor activity rhythm. Such after-effects in females may not be clock-dependent because clock manipulated females with prior exposure to males show decrease in overall activity, more or less similar to rhythmic wild type females. The expression of SSI mediated after-effects requires a functional olfactory system in males because males with compromised olfactory ability do not display them. These results suggest that SSI causes male-specific, long-lasting changes in the circadian clocks of Drosophila, which requires the presence of functional clocks and intact olfactory ability in males.     

Rhythmic egg-laying behaviour in virgin females of fruit flies Drosophila melanogaster

Fruit fly Drosophila melanogaster females display rhythmic egg-laying under 12:12?h light/dark (LD) cycles which persists with near 24?h periodicity under constant darkness (DD). We have shown previously that persistence of this rhythm does not require the neurons expressing pigment dispersing factor (PDF), thought to be the canonical circadian pacemakers, and proposed that it could be controlled by peripheral clocks or regulated/triggered by the act of mating. We assayed egg-laying behaviour of wild-type Canton S (CS) females under LD, DD and constant light (LL) conditions in three different physiological states; as virgins, as females allowed to mate with males for 1?day and as females allowed to mate for the entire duration of the assay. Here, we report the presence of a circadian rhythm in egg-laying in virgin D. melanogaster females. We also found that egg-laying behaviour of 70 and 90% females from all the three male presence/absence protocols follows circadian rhythmicity under DD and LL, with periods ranging between 18 and 30?h. The egg-laying rhythm of all virgin females synchronized to LD cycles with a peak occurring soon after lights-off. The rhythm in virgins was remarkably robust with maximum number of eggs deposited immediately after lights-off in contrast to mated females which show higher egg-laying during the day. These results suggest that the egg-laying rhythm of D. melanogaster is endogenously driven and is neither regulated nor triggered by the act of mating; instead, the presence of males results in reduction in entrainment to LD cycles.     

Targeted gene deletion and phenotypic analysis of the Drosophila melanogaster seminal fluid protease inhibitor Acp62F

Internally fertilizing organisms transfer a complex assortment of seminal fluid proteins, a substantial fraction of which are proteolysis regulators. In mammals, some seminal protease inhibitors have been implicated in male infertility and these same molecular classes of protease inhibitors are also found in Drosophila seminal fluid. Here, we tested the reproductive functions of the Drosophila melanogaster seminal fluid protease inhibitor Acp62F by generating a precise deletion of the Acp62F gene. We did not detect a nonredundant function for Acp62F in modulating the egg laying, fertility, remating frequency, or life span of mated females. However, loss of Acp62F did alter a male's defensive sperm competitive ability, consistent with the localization of Acp62F to sperm storage organs. In addition, the processing of at least one seminal protein, the ovulation hormone ovulin, is slower in the absence of Acp62F.     

Late emergence chronotypes of fruit flies Drosophila melanogaster exhibit higher accuracy of entrainment

Inter-individual variation in phase-of-entrainment (chronotype) is widely observed in many species, but the underlying mechanisms and its consequences remain largely unexplored. In light of considerable limitations of previous studies proposing that the late chronotypes exhibit weakly stable rhythms, we employed outbred Drosophila populations exhibiting early and late emergence chronotypes to re-visit such associations. Contrary to previous reports, we observed that the late chronotypes consistently exhibit higher stability in emergence and activity–rest rhythms as compared to the early chronotypes, both under laboratory and semi-natural conditions, which is not associated with higher precision of circadian clocks, thus demonstrating the existence of genetic correlations between accuracy of entrainment and chronotype. Our results, along with the previously reported clock property differences between the early and the late emergence chronotypes highlights a possible complex interplay of clock period, phase response curve and accuracy in determining phase-of-entrainment.     

Evidence for positive selection on Drosophila melanogaster seminal fluid protease homologs

Proteins present in the seminal fluid of Drosophila melanogaster (accessory gland proteins Acps) contribute to female postmating behavioral changes, sperm storage, sperm competition, and immunity. Consequently, male-female coevolution and host-pathogen interactions are thought to underlie the rapid, adaptive evolution that characterizes several Acp-encoding genes. We propose that seminal fluid proteases are likely targets of selection due to their demonstrated or potential roles in between-sex interactions and immune processes. We use within- and between-species sequence data for 5 predicted protease-encoding Acp loci to test this hypothesis. Our polymorphism-based analyses find evidence for positive selection at 2 genes, both of which encode predicted serine protease homologs. One of these genes, CG6069, also shows evidence for consistent selection on a subset of codons over a deeper evolutionary time scale. The second gene, CG9997, was previously shown to be essential for normal sperm usage, suggesting that sexual selection may underlie its history of adaptation.     

Circadian clock properties of fruit flies Drosophila melanogaster exhibiting early and late emergence chronotypes

The role of circadian clocks in timing daily behaviors is widely acknowledged, and while empirical evidence suggests that clock period is correlated with the preferred phase of a rhythmic behavior (chronotype), other clock properties have also been hypothesized to underlie chronotype variation. Here, we report that fruit fly Drosophila melanogaster populations exhibiting evening emergence chronotype (late) are characterized by higher incidence of behavioral arrhythmicity in constant dim light, wider range of entrainment, reduced rates of re-entrainment to simulated jet-lag and higher amplitude of both entrained and free-running rhythms as compared to those exhibiting morning emergence chronotype (early). Our results thus highlight the role of circadian clock properties such as zeitgeber sensitivity, amplitude and coupling in driving chronotype variation.     

Evidence for adaptive male mate choice in the fruit fly Drosophila melanogaster

Theory predicts that males will benefit when they bias their mating effort towards females of higher reproductive potential, and that this discrimination will increase as males become more resource limited. We conducted a series of experiments to test these predictions in a laboratory population of the fruitfly, Drosophila melanogaster. In this species, courtship and copulation have significant costs to males, and females vary greatly in fecundity, which is positively associated with body size. When given a simultaneous choice between small and large virgin females, males preferentially mated with larger, more fecund, females. Moreover, after males had recently mated they showed a stronger preference for larger females. These results suggest that male D. melanogaster adaptively allocate their mating effort in response to variation in female quality and provide some of the first support for the theoretical prediction that male stringency in mate choice increases as resources become more limiting.     

Proteomics reveals novel Drosophila seminal fluid proteins transferred at mating

Across diverse taxa, seminal fluid proteins (Sfps) transferred at mating affect the reproductive success of both sexes. Such reproductive proteins often evolve under positive selection between species; because of this rapid divergence, Sfps are hypothesized to play a role in speciation by contributing to reproductive isolation between populations. In Drosophila, individual Sfps have been characterized and are known to alter male sperm competitive ability and female post-mating behavior, but a proteomic-scale view of the transferred Sfps has been missing. Here we describe a novel proteomic method that uses whole-organism isotopic labeling to detect transferred Sfps in mated female D. melanogaster. We identified 63 proteins, which were previously unknown to function in reproduction, and confirmed the transfer of dozens of predicted Sfps. Relative quantification of protein abundance revealed that several of these novel Sfps are abundant in seminal fluid. Positive selection and tandem gene duplication are the prevailing forces of Sfp evolution, and comparative proteomics with additional species revealed lineage-specific changes in seminal fluid content. We also report a proteomic-based gene discovery method that uncovered 19 previously unannotated genes in D. melanogaster. Our results demonstrate an experimental method to identify transferred proteins in any system that is amenable to isotopic labeling, and they underscore the power of combining proteomic and evolutionary analyses to shed light on the complex process of Drosophila reproduction.     

Female Drosophila melanogaster suffer reduced defense against infection due to seminal fluid components

Reduced defense against infection is commonly observed as a consequence of reproductive activity, but little is known about how post-mating immunosuppression occurs. In this work, we use Drosophila melanogaster as a model to test the role of seminal fluid components and egg production in suppressing post-mating immune defense. We also evaluate whether systemic immune system activity is altered during infection in mated females. We find that post-mating reduction in female defense depends critically on male transfer of sperm and seminal fluid proteins, including the accessory gland protein known as "sex peptide." However, the effect of these male factors is dependent on the presence of the female germline. We find that mated females have lower antimicrobial peptide gene expression than virgin females in response to systemic infection, and that this lower expression correlates with higher systemic bacterial loads. We conclude that, upon receipt of sperm and seminal fluid proteins, females experience a germline-dependent physiological shift that directly or indirectly reduces their overall ability to defend against infection, at least in part through alteration of humoral immune system activity.     

Paragonial proteins of Drosophila melanogaster adult male: In vitro biosynthesis

When paragonia were incubated for 8 hr in simplified Robb's medium a continuous increase in incorporation of ¹⁴C-amino acids into TCA precipitable proteins was found. The increase is linear over the first 2 hr.Copulation provides the stimulus for an alteration in rate of in vitro incorporation: a rapid and clear increase occurs. A similar increase in rate of incorporation of ¹⁴C-uridine into RNA and ³H-ethanolamine into ‘paragonial substance’ could be found. The pattern of incorporation of ¹⁴C-amino acids into proteins, as determined by polyacrylamide gel electrophoresis, remains qualitatively unaltered. Results from transplantation experiments of single paragonia suggest that the stimulation, at least in part, is neuronal or humoral mediated.In vitro incubations with actinomycin D, under conditions of 90% inhibition, resulted in an increased rate of incorporation of ¹⁴C-amino acids into proteins.     

Early- and late-emerging Drosophila melanogaster fruit flies differ in their sensitivity to light during morning and evening

The authors derived early and late populations of fruit flies showing increased incidence of emergence during morning or evening hours by imposing selection for timing of emergence under 12:12 h light/dark (LD) cycles. From previous studies, it was clear that the increased incidence of adult emergence during morning and evening hours in early and late populations was a result of evolution of divergent and characteristic emergence waveforms in these populations. Such characteristic waveforms are henceforth referred to as "evolved emergence waveforms" (EEWs). The early and late populations also evolved different circadian clocks, which is evident from the divergence in their clock period (τ) and photic phase response curve (PRC). Although correlation between emergence waveforms and clock properties suggests functional significance of circadian clocks, τ and PRCs do not satisfactorily explain the early and late emergence phenotypes. In order to understand the functional significance of the PRC for early and late emergence phenotypes, the authors investigated whether circadian clocks of these flies exhibit any difference in photosensitivity under entrained conditions. Such differences would suggest that the light requirement for circadian entrainment of the emergence rhythm in early and late populations is different. To test this, they examined if early and late flies differ in their light utilization behavior, first by assaying their emergence rhythm under complete photoperiod and then in three different skeleton photoperiods. The results showed that early and late populations require different durations of light during the morning and evening to achieve their EEWs, suggesting that for the circadian entrainment of the emergence rhythm, early and late flies utilize light from different parts of the day.     

The Drosophila melanogaster seminal fluid protease "seminase" regulates proteolytic and post-mating reproductive processes

Proteases and protease inhibitors have been identified in the ejaculates of animal taxa ranging from invertebrates to mammals and form a major protein class among Drosophila melanogaster seminal fluid proteins (SFPs). Other than a single protease cascade in mammals that regulates seminal clot liquefaction, no proteolytic cascades (i.e. pathways with at least two proteases acting in sequence) have been identified in seminal fluids. In Drosophila, SFPs are transferred to females during mating and, together with sperm, are necessary for the many post-mating responses elicited in females. Though several SFPs are proteolytically cleaved either during or after mating, virtually nothing is known about the proteases involved in these cleavage events or the physiological consequences of proteolytic activity in the seminal fluid on the female. Here, we present evidence that a protease cascade acts in the seminal fluid of Drosophila during and after mating. Using RNAi to knock down expression of the SFP CG10586, a predicted serine protease, we show that it acts upstream of the SFP CG11864, a predicted astacin protease, to process SFPs involved in ovulation and sperm entry into storage. We also show that knockdown of CG10586 leads to lower levels of egg laying, higher rates of sexual receptivity to subsequent males, and abnormal sperm usage patterns, processes that are independent of CG11864. The long-term phenotypes of females mated to CG10586 knockdown males are similar to those of females that fail to store sex peptide, an important elicitor of long-term post-mating responses, and indicate a role for CG10586 in regulating sex peptide. These results point to an important role for proteolysis among insect SFPs and suggest that protease cascades may be a mechanism for precise temporal regulation of multiple post-mating responses in females.     

An ectopic expression screen reveals the protective and toxic effects of Drosophila seminal fluid proteins

In Drosophila melanogaster, seminal fluid regulates the reproductive and immune responses of mated females. Some seminal fluid proteins may provide protective functions to mated females, such as antimicrobial activity and/or stimulation of antimicrobial gene expression levels, while others appear to have negative effects, contributing to a "cost of mating." To identify seminal proteins that could participate in these phenomena, we used a systemic ectopic expression screen to test the effects on unmated females of proteins normally produced by the male accessory gland (Acps). Of the 21 ectopically expressed Acps that we tested for ability to assist in the clearance of a bacterial infection with Serratia marcescens, 3 Acps significantly reduced the bacterial counts of infected females, suggesting a protective role. Of the 23 Acps that we tested for toxicity, 3 were toxic, including one that has been implicated in the cost of mating in another study. We also tested ectopic expression females for other Acp-induced effects, but found no additional Acps that affected egg laying or receptivity upon ectopic expression.     

Fates and targets of male accessory gland proteins in mated female Drosophila melanogaster

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.