Female mating receptivity inhibited by injection of male-derived extracts in Callosobruchus chinensis

The effects of male-derived extracts on female receptivity to remating were investigated in Callosobruchus chinensis (Coleoptera: Bruchidae). Injection of aqueous extracts of male reproductive tracts into the abdomen of females reduced receptivity. When aqueous extracts of male reproductive tracts were divided to three molecular weight (MW) fractions by ultrafiltration: <3, 3-14, and >14 kDa, the filtrate containing MW substances <3 kDa reduced female receptivity 3h and 1 day after injection, whereas the fraction containing MW substances >14 kDa inhibited receptivity 2 and 4 days after injection. Finally, male reproductive tract organs were divided into accessory gland, seminal vesicle, and testis. Aqueous extracts of testis reduced receptivity of females on the second day and at 3h, and aqueous extracts of accessory gland reduced receptivity of females on the second day after injection. On the other hand, aqueous extracts of seminal vesicle did not reduce female receptivity. The results indicate that more than one mechanism may be involved in producing the effects of male-derived substances on female receptivity; low MW male-derived substances, which possibly exist in testis, cause short-term inhibition, while high MW substances, which possibly exist in the accessory gland, inhibit female mating later than low MW substances in C. chinensis.     

Induction of oviposition by injection of male-derived extracts in two Callosobruchus species

In some insect species, certain substances in the seminal fluid of males induce egg production and laying in females. We determined the effects of male-derived substances on female oviposition behaviour in two Callosobruchus species, C. chinensis and C. maculatus. Aqueous extracts of the accessory gland; testis; and seminal vesicle, including the ejaculatory duct, were prepared. The injection of these extracts into abdomen of females induced oviposition in both species. Oviposition was induced by the testis and seminal vesicle extracts in C. chinensis and by the accessory gland extracts in C. maculatus. The extracts were separated into three fractions by ultrafiltration: fractions I, molecular weight (MW) <3 kDa; fraction II, 3-14 kDa; and fraction III, >14 kDa. Fraction III induced oviposition in both species. These results suggest that in these two species, the substances that induce oviposition have similar MW but are present in different organs. Oviposition was induced by high-MW (>14 kDa) substances in the testis and seminal vesicle in C. chinensis, and by high-MW substances in accessory gland in C. maculatus. Here, we have discussed the relationship between oviposition and the abovementioned male-derived substances.     

Seminal fluids mediate sexual inhibition and short copula duration in mated female Queensland fruit flies

Molecules in male seminal fluid transferred to female insects during mating can have potent effects on their subsequent sexual and reproductive behaviour. Like many other tephritids, female Queensland fruit flies (Bactrocera tryoni) typically have diminished sexual receptivity after their first mating. Also, copulations of females that do remate tend to be shorter than those of virgins. We here find that virgin females injected with small doses (0.1, 0.2 or 0.5 male equivalents) of extracts from the male reproductive tract accessory tissues, which consist of male accessory glands, ejaculatory apodeme and ejaculatory duct (AG/EA/ED), have diminished receptivity and short copula duration very similar to naturally mated females. In contrast, virgin females injected with saline or with high doses of AG/EA/ED (1 or 2 male equivalents) that likely exceed the range of natural variation retain the higher levels of sexual receptivity and longer copulations of un-injected virgins. We conclude that reduced sexual receptivity and shorter copulations of mated female Q-flies are mediated by products in the male seminal fluid derived from the male reproductive tract accessory tissues.     

Male Seminal Fluid Substances Affect Sperm Competition Success and Female Reproductive Behavior in a Seed Beetle

Male seminal fluid proteins are known to affect female reproductive behavior and physiology by reducing mating receptivity and by increasing egg production rates. Such substances are also though to increase the competitive fertilization success of males, but the empirical foundation for this tenet is restricted. Here, we examined the effects of injections of size-fractioned protein extracts from male reproductive organs on both male competitive fertilization success (i.e., P2 in double mating experiments) and female reproduction in the seed beetle Callosobruchus maculatus. We found that extracts of male seminal vesicles and ejaculatory ducts increased competitive fertilization success when males mated with females 1 day after the females’ initial mating, while extracts from accessory glands and testes increased competitive fertilization success when males mated with females 2 days after the females’ initial mating. Moreover, different size fractions of seminal fluid proteins had distinct and partly antagonistic effects on male competitive fertilization success. Collectively, our experiments show that several different seminal fluid proteins, deriving from different parts in the male reproductive tract and of different molecular weight, affect male competitive fertilization success in C. maculatus. Our results highlight the diverse effects of seminal fluid proteins and show that the function of such proteins can be contingent upon female mating status. We also document effects of different size fractions on female mating receptivity and egg laying rates, which can serve as a basis for future efforts to identify the molecular identity of seminal fluid proteins and their function in this model species.     

Inhibition of female mating receptivity by male-derived extracts in two Callosobruchus species: Consequences for interspecific mating

We investigated the effects of injecting male-derived extracts on congeneric female receptivity in two species of Callosobruchus beetle, C. chinensis and C. maculatus. We also examined the influence of interspecific mating on female remating behaviour in these two species. Male-derived extracts reduced congeneric female receptivity in both species. As quick-acting components, extracts of C. chinensis male seminal vesicles reduced the receptivity of C. maculatus females, whereas extracts of C. maculatus male testes reduced the receptivity of C. chinensis females. As slow-acting components, extracts of male accessory glands of other species reduced the receptivity of both C. maculatus and chinensis females. After interspecific mating, the sperm of C. maculatus males were transferred to the reproductive organs of C. chinensis females, thereby reducing their receptivity. In contrast, no C. chinensis sperm were transferred to the reproductive organs of C. maculatus females; accordingly, the latter's receptivity was not reduced. Furthermore, the survival rate of C. chinensis females decreased markedly after interspecific mating. These results raise the possibility that under circumstances where populations of these two species share the same habitat, reproductive interference would occur only in the interactions between C. maculatus males and C. chinensis females.     

Inhibition of female mating by male accessory gland substances in the ground beetle Leptocarabus procerulus

The ground beetle Leptocarabus procerulus (Chaudoir) possesses seminal substances that have a physical function to form mating plugs and a physiological function to induce female refractory behaviour, which act together to hinder female remating. Little is known about the physiological properties of the substances inducing female refractory behaviour, especially with respect to their secretory organ, dose‐dependency, molecular characteristics and the effect of female maturity. By injecting male‐derived substances into females, substances that induce female refractory behaviour are shown to be produced in the male accessory gland but not in the testis. Injection of extracts from the accessory gland increases the female refractory period at moderate doses but not at lower or higher doses. By contrast, injection of extracts from the testis reduces the female refractory period at high doses. The lack of an effect of accessory gland substances at higher doses could be the result of an anomalous effect of unnaturally large doses of seminal products by direct injection, the toxicity of seminal substances that deter female responses, or counteraction by injected substances that promote female receptivity. The accessory gland substances lose their activity when heated, although the testis substances do not. Females without mature eggs tend to reject mating entirely, although variation in the number of mature eggs (one or more) is not associated with the female refractory period, indicating the limited effect of female reproductive maturity. These findings may help to clarify the physiological basis of the evolution of the elaborated male mating behaviour in L. procerulus.     

Drosophila males transfer antibacterial proteins from their accessory gland and ejaculatory duct to their mates

The male fruitfly, Drosophila melanogaster, transfers to his mate proteins that increase his reproductive success by causing changes in her behavior and physiology. Here we show that among the transferred proteins are ones with antibacterial activity. We performed Escherichia coli overlay assays of native PAGE or renatured SDS-PAGE of reproductive tissue extracts of wild-type or transgenic males deficient in accessory gland function. We detected a 28 kDa male accessory gland-derived protein and two ejaculatory duct-derived proteins all with antibacterial activity. Based on its gel mobility and tissue of synthesis, one of the ejaculatory duct proteins is likely to be andropin, a previously-reported 6 kDa antibacterial peptide. All three proteins are transferred to females during mating. Therefore, they could assist in protecting the male's reproductive tract and, after transfer to the female, the female's reproductive tract or eggs against bacterial infection. Since seminal fluid proteins are transferred before the sperm, these antibacterial proteins may also protect sperm from bacterial infection.     

The consequences of genetic variation in sex peptide expression levels for egg laying and retention in females

The accessory gland proteins (Acps) that male Drosophila melanogaster produce and transfer to females during copulation are key to male and female fitness. One Acp, the sex peptide (SP), is largely responsible for a dramatic increase in female egg laying and decrease in female receptivity after copulation. While genetic variation in male SP expression levels correlate with refractory period duration in females, it is unknown whether male SP expression influences female egg laying or if any effect of SP is mediated by SP retention in the female reproductive tract. Here we measured the amount of SP retained in the female reproductive tract after mating and female egg laying after copulating with virgin males. We found no correlation between male SP expression levels and egg laying, or the amount of SP in the female reproductive tract after mating. Additionally, the amount of SP retained in the female did not influence egg laying. These finding suggests that additional factors, such as variation in other Acps, are important for the retention of SP in females and its quantitative effects on egg laying. It also shows that egg laying and refractory period response to SP is at least partially uncoupled.     

Males of the seed bug Togo hemipterus (Heteroptera: Lygaeidae) use accessory gland substances to inhibit remating by females

In species in which females mate repeatedly, males can adopt several strategies to reduce the risk of sperm competition with future males. The refractory period of females significantly increased as the mating duration increased in the seed bug Togo hemipterus (Heteroptera: Lygaeidae). To elucidate the mechanisms by which mated females are inhibited from remating, we investigated the effects of male-derived substances on the inhibition of mating receptivity of virgin females by injecting the substances into their abdomens. The length of time from injection to mating in virgin females was significantly longer for females injected with accessory gland B solution than for those injected with seminal vesicle, accessory gland A, or control solutions. This is the first report showing that heteropteran males inhibit female remating by using substances from an accessory gland. We discuss and consider the adoption and evolution of this strategy by T. hemipterus males by focusing on female genitalia structures, oviposition habit, and paternity and comparing these traits with those of other heteropterids.     

平疣桑椹石磺生殖系统结构及精子储存场所

雌雄同体贝类精子的储存和利用规律一直是国内外贝类生物学研究的难点之一,本文利用活体解剖、显微观察、组织切片和扫描电镜技术,综合研究了平疣桑椹石磺(Platevindex mortoni)的生殖系统及精子储存场所。结果显示,其生殖系统包括生殖器本部、雌性生殖部分和雄性生殖部分。生殖器本部由两性腺、两性输送管、蛋白腺、黏液腺、支囊组成;雌性生殖部分包括输卵管、受精囊、阴道,位于身体中后方体腔内;雄性生殖部分包括输精管、刺激器、阴茎、阴茎鞘和阴茎牵引肌,位于身体前端右侧体腔内;其阴茎有阴茎鞘,阴茎表面布满倒刺。平疣桑椹石磺阴茎为直线状,无雄性附属腺。未交配的性成熟个体支囊内充满细长精子,受精囊内无精子;而交配后充当雌性个体的支囊内均为细长的自体精子,受精囊内有大量活力较强的粗短精子,其支囊为自体精子的存储场所,而受精囊为异体精子的存储场所。其精子储运情况为:两性腺内精子成熟后暂存于支囊,交配时通过输精管运输至阴茎,由阴茎输送精子至对方的阴道,异体精子进入受精囊内存储待用。     

Elucidation of the protein composition of mouse seminal vesicle fluid

The seminal vesicles are male accessory sex glands that contribute the major portion of the seminal plasma in which mammalian spermatozoa are bathed during ejaculation. In addition to conveying sperm through the ejaculatory duct, seminal vesicle secretions support sperm survival after ejaculation, and influence the female reproductive tract to promote receptivity to pregnancy. Analysis of seminal vesicle fluid (SVF) composition by proteomics has proven challenging, due to its highly biased protein signature with a small subset of dominant proteins and the difficulty of solubilizing this viscous fluid. As such, publicly available proteomic datasets identify only 85 SVF proteins in total. To address this limitation, we report a new preparative methodology involving sequential solubilization of mouse SVF in guanidine hydrochloride, acetone precipitation, and analysis by label-free mass spectrometry. Using this strategy, we identified 126 SVF proteins, including 83 previously undetected in SVF. Members of the seminal vesicle secretory protein family were the most abundant, accounting for 79% of all peptide spectrum matches. Functional analysis identified inflammation and formation of the vaginal plug as the two most prominent biological processes. Other notable processes included modulation of sperm function and regulation of the female reproductive tract immune environment. Together, these findings provide a robust methodological framework for future SVF studies and identify novel proteins with potential to influence both male and female reproductive physiology.     

Sexual receptivity in Anopheles gambiae mosquitoes: absence of control by male accessory gland substances

Although male accessory gland substances modulate female receptivity in most mosquito genera that have been studied, they are not active in the development of sexual refractoriness in the malaria vectors, Anopheles gambiae or Anopheles albimanus. Neither the implantation of male accessory glands nor the injection of gland homogenates affected the insemination rate of unmated females. Similarly, portions of the male genital tract and spermathecae from mated females were also inactive when introduced into these females. Interruption of nervous transmission from the brain reduced the tendency of females to become inseminated and removal of the terminal abdominal ganglion completely abolished insemination. Oviposition behavior did not occur unless the spermatheca containing sperm was present, suggesting that this may be the mechanism that mediates the switchover to mated behavior.     

Reduced female mating receptivity and activation of oviposition in two Callosobruchus species due to injection of biogenic amines

Analyses of proximate mechanisms that control mating and oviposition behaviours in insects are important because they link behavioural ecology and physiology. Recently, seed beetles have been used as models to study evolution of female multiple mating and cost of reproduction including mating. In the present study, we investigated the effects of biogenic amines into the abdomens of females of two Callosobruchus species, Callosobruchus chinensis and Callosobruchus maculatus, on mating receptivity and oviposition behaviour. In C. chinensis, injection of octopamine and tyramine reduced receptivity to mating and tyramine and serotonin increased the number of eggs laid. Similarly, injection of tyramine reduced the receptivity of females and increased the number of eggs laid by females of C. maculatus. These results show the possibility that biogenic amines control mating receptivity and oviposition behaviour in females of two Callosobruchus species.     

Male accessory gland proteins in Drosophila: a multifaceted field [corrected

Male accessory gland in Drosophila is a secretory tissue of the reproductive system. The proteins synthesized in the accessory gland are tissue specific, stage specific-seen only during the adult stage and sex specific in the sense of male limited expression. These secretions that form a component of the seminal fluid are transferred to the female at the time of copulation and play an important role in reproduction. In conjunction with sperm, these secretory proteins assure reproductive success by reducing the female's receptivity to mating and escalating the rate of egg laying. Some of these proteins are antibacterial in nature with a likely function of protecting the female's genital tract against microbial infection during/after mating. Most of the genes involved in the synthesis of accessory gland proteins are autosomal but a few are still X-linked. Their male specific expression is achieved at the time of sex determination. The level of expression of these genes is dose dependent and they follow Mendelian pattern of segregation. Further, majority of these proteins are rapidly evolving with high rates of non-synonymous substitutions. In this review, by considering the work carried out in different fields, we have tried to generate a comprehensive picture about the male accessory gland and the role of its proteins in the reproduction of Drosophila.     

Effects of seminal vesicle removal on fertility and uterine sperm motility in the house mouse

The relative significance of the accessory glands of the male reproductive tract in fertility is unclear. To clarify the role of the seminal vesicles, fertility and uterine sperm motility were determined before and after removal of seminal vesicles in the house mouse. After removal of seminal vesicles, the pregnancy rate (number of females pregnant/number of females X 100) was reduced and the time to birth was increased, while the average litter size was not changed. Fertilization, determined by examining the oocytes 30 h after mating, was highly variable after matings with males whose seminal vesicles were removed; in some cases none of the oocytes were fertilized. The motility of sperm recovered from the uterus 1 h after matings with males before and after seminal vesicle removal and sham operations was analyzed using a videomicrographic system. The motility of uterine sperm was less progressive with more lateral displacement of the head about the trajectory and a less linear trajectory after removal of the seminal vesicles. Sham-operated animals showed no consistent changes in motility of uterine sperm. The changes in sperm motility could contribute to the reduction in fertilization since sperm motility is necessary for transport in the female reproductive tract and interaction with the oocytes.     

Factors affecting Anastrepha fraterculus female receptivity modulation by accessory gland products

In the context of the sterile insect technique (SIT), mass-rearing and male irradiation are imperative. Post-teneral treatments such as the addition of protein in adult's male diet and male hormonal treatment are used to improve sexual performance and to accelerate sexual maturation. In this work we investigated the effect of male accessory glands products (AGPs) on female receptivity of the South American fruit fly Anastrepha fraterculus (Wiedemann), and the effect of strain rearing history, male irradiation, male diet and hormonal treatment on AGPs. Injections of aqueous extracts of male accessory glands into the abdomen of females reduced their receptivity. The AGPs from laboratory males were more effective in inhibiting female receptivity, compared to AGPs from wild males, irrespective of females' origin. The AGPs from fertile males were more effective than AGPs from sterile males. The AGPs from protein-fed males were more effective than AGPs from sugar-fed males. Finally, the AGPs of males treated with juvenile hormone were less effective in inhibiting female receptivity than AGPs of untreated males. We conclude that inhibition of sexual receptivity of A. fraterculus mated females is mediated by products in male accessory gland's and the way that these products act vary widely according to the effect of extrinsic factors. We discuss the results in the perspective of the SIT application for A. fraterculus.     

Copulation, genital damage and early death in Callosobruchus maculatus

Antagonistic sexual coevolution stems from the notion that male and female interests over reproduction are in conflict. Such conflicts appear to be particularly obvious when male genital armature inflicts damage to the female reproductive tract resulting in reduced female longevity. However, studies of mating frequency, genital damage and female longevity are difficult to interpret because females not only sustain more genital damage, but also receive more seminal fluid when they engage in multiple copulations. Here, we attempt to disentangle the effects of genital damage and seminal fluid transfer on female longevity in the beetle Callosobruchus maculatus (Coleoptera: Bruchidae). Males copulating for the sixth time in succession inflicted greater levels of genital damage, but transferred smaller ejaculates in comparison with virgin males. The number of copulations performed by males was negatively related to female fecundity and positively related to female longevity, suggesting a trade-off between fecundity and longevity. However, inclusion of fecundity as a covariate revealed sperm and/or seminal fluid transfer to have a negative impact on female longevity above that caused by the fecundity-longevity trade-off. The consequences of multiple copulations on female longevity were examined. Females that mated twice laid more eggs and died sooner than those that mated once. However, incorporation of fecundity as a covariate into our statistical model removed the effect of female mating frequency on female longevity, indicating that double-mated females suffer greater mortality owing to the trade-off between fecundity and longevity. Males of this species are known to transfer very large ejaculates (up to 8% of their body weight), which may represent a significant nutritional benefit to females. However, the receipt of large ejaculates appears to carry costs. Thus, the interpretation of multiple mating experiments on female longevity and associated functional explanations of polyandry in this species are likely to be complex.     

3种蛾类雄性内生殖系统形态学研究

为了探究蛾类昆虫新的有效分类特征,本研究对直脉青尺蛾Geometra valida、白雪灯蛾Chionarctia niveus和多斑豹蠹蛾Zeuzera multistrigata雄性内生殖系统进行了解剖观察和形态学比较。结果表明：3种蛾类雄性内生殖系统均由精巢、贮精囊（前贮精囊和后贮精囊）、输精管、复射精管、单射精管（单射精管原节和单射精管表皮节）和附腺组成;种间形态差异表现在贮精囊的形状及连接方式、复射精管的形状及长短、单射精管和附腺的长度等方面,这些鉴别特征可为蛾类分类及系统发育研究提供形态学借鉴。     

Seminal proteins but not sperm induce morphological changes in the Drosophila melanogaster female reproductive tract during sperm storage

In most insects, sperm transferred by the male to the female during mating are stored within the female reproductive tract for subsequent use in fertilization. In Drosophila melanogaster, male accessory gland proteins (Acps) within the seminal fluid are required for efficient accumulation of sperm in the female's sperm storage organs. To determine the events within the female reproductive tract that occur during sperm storage, and the role that Acps and sperm play in these events, we identified morphological changes that take place during sperm storage in females mated to wild-type, Acp-deficient or sperm-deficient males. A reproducible set of morphological changes occurs in a wild-type mating. These were categorized into 10 stereotypic stages. Sperm are not needed for progression through these stages in females, but receipt of Acps is essential for progression beyond the first few stages of morphological change. Furthermore, females that received small quantities of Acps reached slightly later stages than females that received no Acps. Our results suggest that timely morphological changes in the female reproductive tract, possibly muscular in nature, may be needed for successful sperm storage, and that Acps from the male are needed in order for these changes to occur.