Quantitative trait loci and interaction effects responsible for variation in female postmating mortality in Drosophila simulans and D. sechellia introgression lines

Mating appears to inflict a cost to Drosophila females, resulting in a reduction of their lifespan shortly after mating. Males from different chromosome extracted lines differ significantly in their detrimental effects on postmating female survival, and seminal fluid proteins produced in the male accessory glands are at least partially responsible for the effect. This suggests that there is a genetic basis underlying the male inflicted effect on female's postmating mortality. However, the genes responsible for this effect remain elusive. Using males from introgression lines between D. simulans and D. sechellia genomes and a quantitative trait locus (QTL) mapping approach, we identified chromosomal regions that affect postmating mortality of females. We found a second chromosome QTL with an effect on average female lifespan after mating and a third chromosome QTL with an effect on postmating female mortality rate. Under the general observation of a faster divergence of sex-related genes among closely related species, it is predicted that genes for reproductive traits other than hybrid sterility will show evidence of epistatic effects when brought into a heterospecific background. We detected a significant epistatic genetic effect on postmating female mortality rate that supports this prediction.     

Cholinergic control of synchronized seminal emissions in Drosophila

In many animal species, copulation involves the coordinated release of both sperm and seminal fluid, including substances that change female fertility and postmating behavior. In Drosophila melanogaster, these substances increase female fertility and prevent mating with a second male. By using a PGal4 strain, we targeted together with other cells a dozen cholinergic neurons found only in the male abdominal ganglion (Abg-MAch). Genetic feminization apparently deleted these neurons in males and significantly increased their copulation duration, blocked their fertility in 60% of cases, and only weakly repressed remating in females. Genetic repression of Gal4 activity in all cholinergic neurons completely rescued copulation duration and fertility, and totally prevented remating, indicating that Abg-MAch neurons were functional. The conditional blocking of the synaptic activity of these neurons during copulation induced separate effects on the transfer of the seminal substances involved in fertilization and those involved in remating. These effects were dissociated only when Abg-MAch neurons were feminized, indicating that their presence is required to synchronize the emission of the male substance(s) that changes reproductive behaviors.     

Multiple pathways mediate the sex-peptide-regulated switch in female Drosophila reproductive behaviours

Male-derived sex-peptide (SP) induces profound changes in the behaviour of Drosophila females, resulting in decreased receptivity to further mating and increased egg laying. SP can mediate the switch in female reproductive behaviours via a G protein-coupled receptor, SPR, in neurons expressing fruitless, doublesex and pickpocket. Whether SPR is the sole receptor and whether SP induces the postmating switch in a single pathway has not, to our knowledge been tested. Here we report that the SP response can be induced in the absence of SPR when SP is ectopically expressed in neurons or when SP, transferred by mating, can access neurons through a leaky blood brain barrier. Membrane-tethered SP can induce oviposition via doublesex, but not fruitless and pickpocket neurons in SPR mutant females. Although pickpocket and doublesex neurons rely on G(o) signalling to reduce receptivity and induce oviposition, G(o) signalling in fruitless neurons is required only to induce oviposition, but not to reduce receptivity. Our results show that SP''s action in reducing receptivity and inducing oviposition can be separated in fruitless and doublesex neurons. Hence, the SP-induced postmating switch incorporates shared, but also distinct circuitry of fruitless, doublesex and pickpocket neurons and additional receptors.     

The sex-peptide DUP99B is expressed in the male ejaculatory duct and in the cardia of both sexes.

Mating elicits two postmating responses in many insect females: the egg laying rate increases and sexual receptivity is reduced. In Drosophila melanogaster, two peptides of the male genital tract, sex-peptide and DUP99B, elicit these postmating responses when injected into virgin females. Here we show that the gene encoding DUP99B is expressed in the male ejaculatory duct and in the cardia of both sexes. The DUP99B that is synthesized in the ejaculatory duct is transferred, during mating, into the female genital tract. Expression of the gene is first seen in a late pupal stage. Males containing an intact ejaculatory duct, but lacking accessory glands, initiate the two postmating responses in their female partners [Xue, L. & Noll, M. (2000) Proc. Natl Acad. Sci. USA97, 3272-3275]. Although such males synthesize DUP99B in wild-type quantities, they elicit only weak postmating responses in their mating partners. Males lacking the Dup99B gene elicit the two postmating responses to the same extent as wild-type males. These results suggest that both sex-peptide and DUP99B can elicit both responses in vivo. However, sex-peptide seems to play the major role in eliciting the postmating responses, while DUP99B may have specialized for other, as yet unknown, functions.     

Postmating reproductive barriers contribute to the incipient sexual isolation of the United States and Caribbean Drosophila melanogaster

The nascent stages of speciation start with the emergence of sexual isolation. Understanding the influence of reproductive barriers in this evolutionary process is an ongoing effort. We present a study of Drosophila melanogaster admixed populations from the southeast United States and the Caribbean islands known to be a secondary contact zone of European‐ and African‐derived populations undergoing incipient sexual isolation. The existence of premating reproductive barriers has been previously established, but these types of barriers are not the only source shaping sexual isolation. To assess the influence of postmating barriers, we investigated putative postmating barriers of female remating and egg‐laying behavior, as well as hatchability of eggs laid and female longevity after mating. In the central region of our putative hybrid zone of American and Caribbean populations, we observed lower hatchability of eggs laid accompanied by increased resistance to harm after mating to less‐related males. These results illustrate that postmating reproductive barriers act alongside premating barriers and genetic admixture such as hybrid incompatibilities and influence early phases of sexual isolation.     

Pregnancy detection and fetal monitoring via ultrasound in a giant panda (Ailuropoda melanoleuca)

Transabdominal ultrasound was used to monitor the reproductive tract of a female giant panda (Ailuropoda melanoleuca) for three consecutive breeding seasons starting in 2001. Parturition did not occur in 2001 or 2002. In 2003, uterine enlargement was noted 109 days after natural mating occurred. A twin pregnancy was detected via transabdominal ultrasound 134 days following natural mating. Four subsequent ultrasound examinations, performed at 137, 138, 140, and 142 days postmating, documented fetal development. The female gave birth to one infant 150 days postmating. Zoo Biol 23:449–461, 2004. © 2004 Wiley‐Liss, Inc.     

Gene expression changes in male accessory glands during ageing are accompanied by reproductive decline in Drosophila melanogaster

Senescence is accompanied by loss of reproductive functions. Here, we studied reproductive ageing in Drosophila melanogaster males and asked whether the expected decline in male reproductive success is due to diminished functionality of the male accessory gland (AG). The male AG produces the majority of seminal fluid proteins (SFPs) transferred to the female at mating. SFPs induce female postmating changes and are key to male reproductive success. We measured age‐dependent gene expression changes for five representative SFP genes in males from four different age groups ranging from 1 to 6 weeks after eclosion. Simultaneously, we also measured male reproductive success in postmating traits mediated by transfer of these five SFPs. We found a decreased in male SFP gene expression with advancing age and an accompanying decline in male postmating success. Hence, male reproductive senescence is associated with a decline in functionality of the male AG. While overall individual SFP genes decreased in expression, our results point towards the idea that the composition of an ejaculate might change with male age as the rate of change was variable for those five genes.     

Ultrastructural examination of the insemination reaction in Drosophila

The insemination reaction is a swelling of the female vagina caused by the male ejaculate. This postmating phenomenon is common among species in the genus Drosophila. It could act as a plug securing male paternity. It is not clear, however, what benefits it provides to the female. The structure formed in the female vagina is expelled in some species and disappears gradually in others suggesting different phenomena. Based on ultrastructural examination of the vaginal contents of five Drosophila species (D. mettleri, D. nigrospiracula, D. melanogaster, D. mojavensis, and D. hexastigma), we propose three terms to describe these vaginal structures: the sperm sac, the mating plug, and the true insemination reaction. Each term describes a distinct structure associated with a specific female postmating behavior. This study questions the concept of the insemination reaction as a single phenomenon and discusses its possible functions from an evolutionary perspective.     

Genetic divergence of the seminal signal-receptor system in houseflies: the footprints of sexually antagonistic coevolution?

To understand fully the significance of cryptic female choice, we need to focus on each of those postmating processes in females which create variance in fitness among males. Earlier studies have focused almost exclusively on the proportion of a female's eggs fertilized by different males (sperm precedence). Yet, variance in male postmating reproductive success may also arise from differences in ability to stimulate female oviposition and to delay female remating. Here, we present a series of reciprocal mating experiments among genetically differentiated wild-type strains of the housefly Musca domestica. We compared the effects of male and female genotype on oviposition and remating by females. The genotype of each sex affected both female oviposition and remating rates, demonstrating that the signal-receptor system involved has indeed diverged among these strains. Further, there was a significant interaction between the effects of male and female genotype on oviposition rate. We discuss ways in which the pattern of such interactions provides insights into the coevolutionary mechanism involved. Females in our experiments generally exhibited the weakest, rather than the strongest, response to males with which they are coevolved. These results support the hypothesis that coevolution of male seminal signals and female receptors is sexually antagonistic.     

Identification,RNAi Knockdown,and Functional Analysis of an Ejaculate Protein that Mediates a Postmating,Prezygotic Phenotype in a Cricket

Postmating, prezygotic phenotypes, especially those that underlie reproductive isolation between closely related species, have been a central focus of evolutionary biologists over the past two decades. Such phenotypes are thought to evolve rapidly and be nearly ubiquitous among sexually reproducing eukaryotes where females mate with multiple partners. Because these phenotypes represent interplay between the male ejaculate and female reproductive tract, they are fertile ground for reproductive senescence – as ejaculate composition and female physiology typically change over an individual''s life span. Although these phenotypes and their resulting dynamics are important, we have little understanding of the proteins that mediate these phenotypes, particularly for species groups where postmating, prezygotic traits are the primary mechanism of reproductive isolation. Here, we utilize proteomics, RNAi, mating experiments, and the Allonemobius socius complex of crickets, whose members are primarily isolated from one another by postmating, prezygotic phenotypes (including the ability of a male to induce a female to lay eggs), to demonstrate that one of the most abundant ejaculate proteins (a male accessory gland-biased protein similar to a trypsin-like serine protease) decreases in abundance over a male''s reproductive lifetime and mediates the induction of egg-laying in females. These findings represent one of the first studies to identify a protein that plays a role in mediating both a postmating, prezygotic isolation pathway and reproductive senescence.     

Female postmating immune responses,immune system evolution and immunogenic males

Females in many taxa experience postmating activation of their immune system, independently of any genital trauma or pathogenic attack arising from male‐female genital contact. This response has always been interpreted as a product of natural selection as it either prepares the female immune system for antigens arising from an implanted embryo (in the case of placental mammals), or is a “pre‐emptive strike” against infection or injury acquired during mating. While the first hypothesis has empirical support, the second is not entirely satisfactory. Recently, studies that have experimentally dissected the postmating responses of Drosophila melanogaster females point to a different explanation: male reproductive peptides/proteins that have evolved in response to postmating male‐male competition are directly responsible for activating particular elements of the female immune system. Thus, in a broad sense, males may be said to be immunogenic to females. Here, we discuss a possible direct role of sexual selection/sexual conflict in immune system evolution, in contrast to indirect trade‐offs with other life‐history traits, presenting the available evidence from a range of taxa and proposing ways in which the competing hypotheses could be tested. The major implication of this review is that immune system evolution is not only a product of natural selection but also that sexual selection and potentially sexual conflict enforces a direct selective pressure. This is a significant shift, and will compel researchers studying immune system evolution and ecological immunity to look beyond the forces generated by parasites and pathogens to those generated by the male ejaculate.     

BEYOND REINFORCEMENT: THE EVOLUTION OF PREMATING ISOLATION BY DIRECT SELECTION ON PREFERENCES AND POSTMATING, PREZYGOTIC INCOMPATIBILITIES

Abstract The evolution of premating isolation after secondary contact is primarily considered in the guise of reinforcement, which relies on low hybrid fitness as the driving force for mating preference divergence. Here I consider two additional forces that may play a substantial role in the adaptive evolution of premating isolation, direct selection on preferences and indirect selection against postmating, prezygotic incompatibilities. First, I argue that a combination of ecological character displacement and sensory bias can cause direct selection on preferences that results in the pattern of reproductive character displacement. Both analytical and numerical methods are then used to demonstrate that, as expected from work in single populations, such direct selection will easily overwhelm indirect selection due to low hybrid fitness as the primary determinant of preference evolution. Second, postmating, prezygotic incompatibilities are presented as a driving force in the evolution of premating isolation. Two classes of these mechanisms, those increasing female mortality after mating but before producing offspring and those reducing female fertility, are shown to be identical in their effects on preference divergence. Analytical and numerical techniques are then used to demonstrate that postmating, prezygotic factors may place strong selection on preference divergence. These selective forces are shown to be comparable if not greater than those produced by the low fitness of hybrids.     

Postmating sexual selection: the effects of male body size and recovery period on paternity and egg production rate in a water strider

The role of male body size in postmating sexual selection wasexplored in a semiaquatic insect, the water strider Gerris lateralis.To separate effects of male size per se from those due to numericsperm competition, male recovery period (shown here to be proportionalto ejaculate size) was manipulated independently of body sizein a factorial experiment where virgin females were mated firstwith sterile males and then with focal males. Both relativemale fertilization success and female reproductive rate were measured.The number of sperm transferred increased with male recoveryperiod, an effect that was mediated by longer copulation duration,but there were no effects of body size on ejaculate size. Neithermale size nor recovery period had any significant direct effectson male fertilization success. However, copulation durationinfluenced relative fertilization success, suggesting that malesable to transfer more sperm also achieved higher fertilizationsuccess. Females exercised cryptic female choice by modulatingtheir reproductive rate in a manner favoring large males andmales that were successful in terms of achieving high relativefertilization success. Thus, successful males gained a twofoldadvantage in postmating sexual selection. This study has important implicationsfor previous estimates of sexual selection in this group of insectsbecause pre- and postmating sexual selection will be antagonisticdue to limitations in male sperm production: males mating frequently(high mating success) will on average transfer fewer sperm ineach mating and will hence tend to fertilize fewer eggs permating (low fertilization success).     

橘小实蝇交配行为观察

在实验室内对橘小实蝇Bactrocera dorsalis（Hendel）的交配行为进行了观察。描述了橘小实蝇交配中雄虫的“求偶场”,雄雄相遇、雌雌相遇、雌雄相遇时的相互反应,雌雄虫的交配行为与交配后行为。     

Retention of Ejaculate by Drosophila melanogaster Females Requires the Male-Derived Mating Plug Protein PEBme

Within the mated reproductive tracts of females of many taxa, seminal fluid proteins (SFPs) coagulate into a structure known as the mating plug (MP). MPs have diverse roles, including preventing female remating, altering female receptivity postmating, and being necessary for mated females to successfully store sperm. The Drosophila melanogaster MP, which is maintained in the mated female for several hours postmating, is comprised of a posterior MP (PMP) that forms quickly after mating begins and an anterior MP (AMP) that forms later. The PMP is composed of seminal proteins from the ejaculatory bulb (EB) of the male reproductive tract. To examine the role of the PMP protein PEBme in D. melanogaster reproduction, we identified an EB GAL4 driver and used it to target PEBme for RNA interference (RNAi) knockdown. PEBme knockdown in males compromised PMP coagulation in their mates and resulted in a significant reduction in female fertility, adversely affecting postmating uterine conformation, sperm storage, mating refractoriness, egg laying, and progeny generation. These defects resulted from the inability of females to retain the ejaculate in their reproductive tracts after mating. The uncoagulated MP impaired uncoupling by the knockdown male, and when he ultimately uncoupled, the ejaculate was often pulled out of the female. Thus, PEBme and MP coagulation are required for optimal fertility in D. melanogaster. Given the importance of the PMP for fertility, we identified additional MP proteins by mass spectrometry and found fertility functions for two of them. Our results highlight the importance of the MP and the proteins that comprise it in reproduction and suggest that in Drosophila the PMP is required to retain the ejaculate within the female reproductive tract, ensuring the storage of sperm by mated females.     

Expression level of sarah, a homolog of DSCR1, is critical for ovulation and female courtship behavior in Drosophila melanogaster

To better understand the genetic bases of postmating responses in Drosophila melanogaster females, we screened a collection of P{GS} insertion lines and identified two insertions in sarah (sra), whose misexpression in the nervous system induced high levels of ovulation in virgins. The gene sra encodes a protein similar to human Down syndrome critical region 1 (DSCR1). The ovulation phenotype was reproduced in transgenic virgins expressing UAS-sra in the nervous system. The flies also extruded the ovipositor toward courting males as seen in wild-type mated females, supporting the notion that ovulation and behavioral patterns are physiologically coupled. The sra insertions were found to be hypomorphic alleles with reduced expression levels. Females homozygous for these alleles show: (1) spontaneous ovulation in virgins, (2) sterility with impaired meiotic progression, and (3) compromised postmating responses with lower ovulation level, higher remating rate, and shorter period for restoration of receptivity. No obvious defects were observed in the homozygous males. The gene sra is predominantly expressed in oocytes, nurse cells, and the nervous system. Taken together, these results indicate that the expression level of sra is critical for ovulation and female courtship behavior, including their postmating changes.     

Population density and structure drive differential investment in pre‐ and postmating sexual traits in frogs

Sexual selection theory predicts a trade‐off between premating (ornaments and armaments) and postmating (testes and ejaculates) sexual traits, assuming that growing and maintaining these traits is costly and that total reproductive investments are limited. The number of males in competition, the reproductive gains from investing in premating sexual traits, and the level of sperm competition are all predicted to influence how males allocate their finite resources to these traits. Yet, empirical examination of these predictions is currently scarce. Here, we studied relative expenditure on pre‐ and postmating sexual traits among frog species varying in their population density, operational sex ratio, and the number of competing males for each clutch of eggs. We found that the intensifying struggle to monopolize fertilizations as more and more males clasp the same female to fertilize her eggs shifts male reproductive investment toward sperm production and away from male weaponry. This shift, which is mediated by population density and the associated level of male–male competition, likely also explains the trade‐off between pre‐ and postmating sexual traits in our much broader sample of anuran species. Our results highlight the power of such a multilevel approach in resolving the evolution of traits and allocation trade‐offs.     

Patterns of divergence in the effects of mating on female reproductive performance in flour beetles

Sexual selection can lead to rapid divergence in reproductive characters. Recent studies have indicated that postmating events, such as sperm precedence, may play a key role in speciation. Here, we stress that other components of postmating sexual selection may be involved in the evolution of reproductive isolation. One of these is the reproductive investment made by females after mating (i.e., differential allocation). We performed an experiment designed to assess genetic divergence in the effects of mating on female reproductive performance in flour beetles, Tribolium castaneum. Females were mated to males of three different wild-type genotypes at two different frequencies, in all possible reciprocal combinations. Male genotype affected all aspects of female reproduction, through its effects on female longevity, total offspring production, reproductive rate, mating rate, and fertility. Moreover, male and female genotype interacted in their effects on offspring production and reproductive rate. We use the pattern of these interactions to discuss the evolutionary process of divergence and suggest that the pattern is most consistent with that expected if divergence was driven by sexually antagonistic coevolution. In particular, the fact that females exhibited a relatively weak response to males with which they were coevolved suggests that females have evolved resistance to male gonadotropic signals/stimuli.     

COPULATORY BEHAVIOR,GENITAL MORPHOLOGY,AND MALE FERTILIZATION SUCCESS IN WATER STRIDERS

Recent theoretical and empirical interest in postmating processes have generated a need for increasing our understanding of the sources of variance in fertilization success among males. Of particular importance is whether such postmating sexual selection merely reinforces the effects of premating sexual selection or whether other types of male traits are involved. In the current study, we document large intraspecific variation in last male sperm precedence in the water strider Gerris lateralis. Male relative paternity success was repeatable across replicate females, showing that males differ consistently in their ability to achieve fertilizations. By analyzing shape variation in male genital morphology, we were able to demonstrate that the shape of male intromittent genitalia was related to relative paternity success. This is the first direct experimental support for the suggestion that male genitalia evolve by postmating sexual selection. A detailed analysis revealed that different components of male genitalia had different effects, some affecting male ability to achieve sperm precedence and others affecting male ability to avoid sperm precedence by subsequent males. Further, the effects of the shape of the male genitalia on paternity success was in part dependent on female morphology, suggesting that selection on male genitalia will depend on the frequency distribution of female phenotypes. We failed to find any effects of other morphological traits, such as male body size or the degree of asymmetry in leg length, on fertilization success. Although males differed consistently in their copulatory behavior, copulation duration was the only behavioral trait that had any significant effect on paternity.