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.     

Spermatogenesis, changes in reproductive structures, and time constraint associated with insemination in Podisus nigrispinus

Males of the predatory stinkbug, Podisus nigrispinus (Dallas) (Hemiptera: Pentatomidae), accomplish long and multiple matings. We hypothesize that this behavior is due to time requirement for spermatozoa production and their transference to the females. Thus, this work investigated the effect of mating status of males and mating duration on spermatozoa transference to the females and the location of spermatozoa in the male reproductive tract during mating. On females, morphological alterations on female spermatheca and associated structures during a mating were investigated. Analyses of male reproductive tract showed presence of spermatozoa in the lumen of vas deferens was independent of mating status (ca. virgin, 0, 12 and 24 h after having a full mating), indicating continuous spermatogenesis which is supported by the absence of a seminal vesicle for spermatozoa storage. Female spermatheca had no changes associated with the duration of mating. However, females exhibited spermathecal elastic duct swelling by 30 min of mating duration. The success of males in filling the female spermatheca with spermatozoa depends on duration of mating. Thus, the results indicate that multiple mating is a requirement for reproductive success in the species by transference of spermatozoa and accessory substances stored in the female spermathecal duct. Likewise, the long mating is a male requirement to transfer materials in appropriate amount to the female but it is not dependent on spermatozoa alone.     

Male housefly (Musca domestica L.) genital system as a source of mating pheromone

Male volatile mating pheromone is released by male houseflies placed in close proximity to females or in contact with female hydrocarbon extract. The pheromone induces the aggregation of virgin females in the vicinity of the male. Preparations of macerated testicular and ejaculatory duct tissue exert a similar effect, and these organs are, apparently, the source of the pheromone. Such preparations are not attractive to males or mated females. The pheromone also appears to induce female receptivity, since females that are exposed to emanations from male system preparations show a quicker rate of pairing with males. The pheromone effect results from the testes and ejaculatory ducts acting together, as preparations of each of these alone are inactive. Pheromone activity is age dependent and the greatest female reaction is observed when tissues of 6–10-day old males are used.     

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.     

Morphology of the reproductive system and antennal lobes of gynandromorphic and normal black cutworm moths,agrotis ipsilon (Hufnagel) (Lepidoptera : Noctuidae)

Sexually dimorphic characteristics of a bilaterally asymmetric gynandromorphic black cutworm moth, Agrotis ipsilon (Hufnagel), were compared with those of normal males and females. On one side of the body, the gynandromorphs wings were larger and darker than on the other side, and the antenna was filiform. On the other side of the body, the wings were smaller and lighter in color, and the antenna was pectinate. Females were usually larger and more pigmented than males. Female antenna were filiform and those of males pectinate. At the tip of the abdomen, the gynandromorph had 2 valves, as normal males do, but the one on the female side was smaller. The antennal lobes of the gynandromorphs brain included only ordinary glomeruli on the female side, and ordinary glomeruli plus a partially developed macroglomerular complex (MGC) at the base of the antenna on the male side. Normal female antennal lobes contained only ordinary glomeruli. Normal male antennal lobes contained ordinary glomeruli and a fully developed MGC, consisting of one large and 3 smaller glomeruli. In the gynandromorph, female reproductive organs were partially developed or absent. A portion of the oviduct was missing together with several ovarioles, and no spermatheca or seminal duct were found. The male reproductive track was complete, except for the paired structures, which in the gynandromorph were single. Normal females had paired accessory glands and ovaries, and single oviduct, bursa copulatrix, and spermatheca. Normal males had fused testes, paired accessory glands, and a single ejaculatory duct and aedeagus. The gynandromorph assumed a calling posture and attracted one male, but it did not respond to the pheromone from females.     

Sperm stock and mating of males in a parasitoid wasp

Hymenoptera are haplodiploid insects, consequently sex ratio depends on female's sperm management which itself arises from the reproductive capacity of neighbouring males. To study the influence of ageing on male reproductive potential, laboratory experiments were conducted on Dinarmus basalis (Hymenoptera, Pteromalidae) males, a tropical wasp in which sperm counts are known to constrain sex ratio. Two groups of virgin males were compared: 1-day and 30-days old. Parameters recorded were sperm quantity and viability in seminal vesicles, shape of testis, mating ability in both individual and competitive situations and sperm stored by females after male multiple mating. Older males had twice as much sperm as young males, but their reproductive capacities did not differ. They were able to copulate with 20 successive virgin females in a short period. Sperm stored in spermathecae decreased with female mating order. In competition, old and young males had the same access to females. The difference between old and young males was visible at the level of reproductive tract: young males have functional testis and old males have empty non-functional testis. Spermatozoa are kept viable in male seminal vesicles for long periods. In this species, the reproductive potential of males is not altered by ageing. At the population level this may represent an adaptation for maintaining continuous reserves of sperm at the disposal of females.     

Copulation, the dynamics of sperm transfer and female refractoriness in the leafhopper Balclutha incisa (Hemiptera: Cicadellidae: Deltocephalinae)

Abstract.  Mature sperm of the leafhopper Balclutha incisa (Matsumara) (Cicadellidae: Auchenorrhyncha: Hemiptera) are stored as a series of sperm bundles within seminal vesicles prior to ejaculation. During transfer, sperm are pumped from the vesicles into the ejaculatory duct to the complex aedeagus. Sperm transfer is marked by a c . 30-fold expansion of the spermatheca to accommodate both sperm and seminal fluid. Sperm number increases exponentially with male age, reaching a maximum of 700 000 after 14 days, while the number of sperm available on days 2–5 is between 70 000 and 100 000. During mating, maximum sperm transfer occurs after 7 min and mating is complete after about 10 min. Ejaculate size, defined by both sperm and associated accessory gland fluid, is influenced by male mating status and the interval since the previous mating. There is a positive correlation between duration of copulation and both ejaculate and the time to subsequent mating. Sperm are more likely to be retained in the testes during mating by males of 2–5 days post-emergence than older males. The number of sperm received by the female can be manipulated experimentally by mating males once (medium ejaculate) or twice (small ejaculate) immediately after their first mating. Females that receive small ejaculates from sperm-depleted males have a far shorter refractory period than females receiving medium to large ejaculates. Both ejaculate size and the time after males have mated influence the female post-mating refractory period as measured by the female's responsiveness to male sexual signalling.     

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.     

Female ovarian cycle phase affects the timing of male sexual activity in free-ranging Barbary macaques (Macaca sylvanus) of Gibraltar

Although all macaques have a multimale multifemale mating system, the degree of promiscuity shown by the Barbary macaque is considered to be extreme in terms of both mating frequency and number of mating partners. How mating activity is distributed throughout the female menstrual cycle and whether or not copulations are concentrated around the fertile phase as in other members of the genus is, however, not known. To examine this, we collected data on rates of copulation throughout 29 ovarian cycles from 13 free-ranging females of the Gibraltar Barbary macaque population and related them to the time of ovulation and the female fertile phase as determined from fecal hormone analysis. In addition, patterns of male inspection of females and time spent in consortship, both indicators of female attractivity, were also analyzed. The results indicate that both mating behavior and female attractivity vary predictably with ovarian cycle stage. Rates of copulation were found to increase toward the time of ovulation, with a distinct peak of ejaculatory (but not non-ejaculatory) copulations occurring in the fertile phase. Additionally, we show that frequency of inspection of females by males and time spent in consortship were also highest during the fertile phase and that ejaculatory copulations and male pericopulatory behaviors were significantly correlated with levels of female sex hormones. Our findings indicate that the Barbary macaque shows a mating pattern during the cycle similar to that described for other members of the genus. More importantly, however, our study provides clear evidence that despite an extreme degree of promiscuity Barbary macaque males concentrate their reproductive effort to the fertile phase, implying that they are able to discern this period and that thus timing of ovulation is not concealed from them. Estrogen-related cues appear to be involved in the process of recognition of female reproductive status by males, but the exact nature of these cues and how male Barbary macaques use them remains to be clarified.     

Effects of age and repeated mating on male sperm supply and paternity in a parasitoid wasp

Post-copulatory paternity biases after female multiple mating are major constraints on both male and female reproductive systems. The outcome of paternity in certain situations is only controlled directly by male sperm stock. This was tested experimentally in the parasitoid wasp Anisopteromalus calandrae (Howard) (Hymenoptera: Pteromalidae), in which sperm stocks are small (several hundred) and the fertilizing efficiency of stored sperm is high (the ratio of sperm stored/fertilized eggs is about 0.75). Sperm in seminal vesicles and paternity of males of different status (virgin young, virgin old, or young previously mated) were measured after female single and double mating. The amount of sperm in the seminal vesicle differed according to male status (increasing from previously mated males to old males), but there was no difference in sperm stored by females after a single mating. In double mating experiments with two males of different status, paternity increased linearly with the relative amount of sperm in seminal vesicles. Paternity distribution conforms to 'a fair raffle' of sperm from both donors following complete mixing of sperm prior to fertilization. Thus, in a female multiple mating context, male fitness depends principally on their sperm stock, which in turn depends on life history parameters, such as age and previous mating.     

Female mating receptivity after injection of male-derived extracts in Callosobruchus maculatus

The effects of male-derived extracts on female receptivity were investigated in Callosobruchus maculatus (Coleoptera: Bruchidae). Injection of aqueous extracts of the male reproductive tract into the abdomen of females reduced receptivity. Aqueous extracts of male reproductive tracts were divided to three molecular weight (MW) fractions by ultrafiltration: Fractions: (I) MW<3 kDa, (II) 3-14 kDa, and (III)>14 kDa. Fraction II reduced female receptivity from 3h after injection, and Fraction III reduced female receptivity from 2 days after injection. On the other hand, no effect on receptivity was found for Fraction I. Furthermore, male reproductive tract organs were divided into accessory gland, testis, and seminal vesicle including the ejaculatory duct. Aqueous extracts of the seminal vesicle reduced receptivity of females immediately following injection, while aqueous extracts of the accessory gland reduced receptivity at the second day. The results suggest that the components of Fraction II existed in the seminal vesicle, and those of Fraction III in the accessory gland. The results of the present and the previous studies in Callosobruchus chinensis, a species closely related to C. maculatus, were compared and are discussed from the viewpoint of the significance of ejaculation in the two species.     

FEMALES RECEIVE A LIFE-SPAN BENEFIT FROM MALE EJACULATES IN A FIELD CRICKET

Abstract.— Mating has been found to be costly for females of some species because of toxic products that males transfer to females in their seminal fluid. Such mating costs seem paradoxical, particularly for species in which females mate more frequently than is necessary to fertilize their eggs. Indeed, some studies suggest that females may benefit from mating more frequently. The effect of male ejaculates on female life span and lifetime fecundity was experimentally tested in the variable field cricket, Gryllus lineaticeps. In field crickets, females will mate repeatedly with a given male and mate with multiple males. Females that were experimentally mated either repeatedly or multiply lived more than 32% longer than singly mated females. In addition, multiply mated females produced 98% more eggs than singly mated females. Because females received only sperm and seminal fluid from males in the experimental matings, these life‐span and fecundity benefits may result from beneficial seminal fluid products that males transfer to females during mating. Mating benefits rather than mating costs may be common in many animals, particularly in species where female mate choice has a larger effect on male reproductive success than does the outcome of sperm competition.     

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.     

Female mate preference in an invasive phytopathogen vector: how learning may influence mate choice and fecundity in Diaphorina citri

In some arthropods, females learn in the context of reproduction to refine their mate choices and avoid males displaying traits associated with impotency. Previous studies have shown that males of the Asian citrus psyllid, Diaphorina citriKuwayama (Hemiptera: Psyllidae), associate female odor with copulatory rewards. However, it is unclear whether females similarly learn about male traits. We compared mate choice in females previously mated to either blue or orange males and found that females may associate male color with reproductive success, and avoided blue males after previous experience. Orange males mated more frequently than blue males and appeared to be more sexually aggressive in their mating attempts. In addition, females mated to orange males laid approximately twice as many eggs as those mated to blue males. We dissected male psyllids and measured the size of their reproductive organs to determine whether abdominal color was associated with reproductive development. Our morphometric analysis indicated that blue males may not be physiologically immature compared with orange males. Rather, blue males displayed larger testes and seminal vesicles than orange males, suggesting that differences in reproductive output may be behavioral in nature. Based on our data, we suggest that females learn about the appearance and mating behaviors of blue and orange males from early mating experiences, and subsequently avoid blue males in future matings. Although the roles of blue males within a population are unclear, it is possible that blue morphs may specialize in other functions such as dispersal.     

Relationship between sexual interactions and the timing of the fertile phase in captive female Japanese macaques (Macaca fuscata)

Japanese macaques live in multi‐male/multi‐female social groups in which competition between males, female mate choice, and alternative male mating strategies are important determinants of mating and reproductive success. However, the extent to which adult males rely on female behavior to make their mating decisions as well as the effect of social rank on mating success are not clear as results are inconclusive, varying from study to study. In this study, we combined behavioral and endocrine data of 14 female Japanese macaques to examine the relationship between ovarian cycle phase and frequency of sexual behaviors, and to investigate how social rank influences sexual behavior in this species. We found that there was no increase in female proceptive behaviors during the fertile phase of the ovarian cycle, suggesting that female behaviors did not clearly signal the probability of conception. In spite of that, the frequencies of ejaculatory copulations were highest during this phase, indicating that the attractivity of females increased significantly during the period with higher probability of conception. Males, and especially the highest ranking male, were able to discriminate females nearing ovulation and to concentrate their mating effort, implying that the timing of ovulation was not concealed from them. The α male seemed able to monopolize most female matings, which is probably due in part to the low number of females simultaneously ovulating and to the limited number of inconspicuous places that the lower ranking males have to mate with females and avoid α male aggression. All together, these results suggest that different males may have access to different signals of ovulation and/or are differentially restrained as to how they can act on that information. The exact nature of the estrogen‐related cues males use to recognize female reproductive status, and to what extent males use them warrants further investigation. Am. J. Primatol. 71:868–879, 2009. © 2009 Wiley‐Liss, Inc.