Female control of sperm ejection and retention in the cornsilk fly Euxesta eluta (Diptera: Ulidiidae).

Promiscuous mating systems provide the opportunity for females to bias fertilization toward particular males. However, distinguishing between male sperm competition and active female sperm choice is difficult for species with internal fertilization. Nevertheless, species that store and use sperm of different males in different storing structures and species where females are able to expel all or part of the ejaculates after copulation may be able to bias fertilization. We report a series of experiments aimed at providing evidence of female sperm choice in Euxesta eluta (Hendel), a species of ulidiid fly that expels and consumes ejaculates after copulation. We found no evidence of greater reproductive success for females mated singly, multiply with the same male, or mated multiply with different males. Female E. eluta possesses two spherical spermathecae and a bursa copulatrix for sperm storage, with a ventral receptacle. There was no significant difference in storing more sperm in spermathecae 24 h after copulation than immediately after copulation. Females mated with protein-fed males had greater reproductive success than similar females mated to protein-deprived males. Protein-fed females prevented to consume the ejaculate, retained more sperm when mated to protein-fed males than when mated to protein-deprived males. Our results suggest that female E. eluta can exert control of sperm retention of higher quality males through ejaculate ejection.

Postcopulatory Behavior of Tephritid Flies.

Mating produces profound changes in the behavior of female flies, such as an increase in oviposition, reduction in sexual receptivity, increase in feeding, and even excretion. Many of these changes are produced by copulation, sperm, and accessory gland products that males transfer to females during mating. Our knowledge on the function of the male ejaculate and its effect on female insects is still incipient. In this article, we review peri- and postcopulatory behaviors in tephritid flies. We address the effects of male copulatory behavior; copula duration; and the male ejaculate, sperm, and accessory gland products on female remating behavior. Many species from these families are pests of economic importance; thus, understanding male mating effects on female behavior contributes to both developing more effective environmentally friendly control methods and furthering our understanding of evolutionary implications of intersexual competition and sexual conflict.

Characterization of reproductive proteins in the Mexican fruit fly points towards the evolution of novel functions.

Seminal fluid proteins (Sfps) modify female phenotypes and have wide-ranging evolutionary implications on fitness in many insects. However, in the Mexican fruit fly, Anastrepha ludens, a highly destructive agricultural pest, the functions of Sfps are still largely unknown. To gain insights into female phenotypes regulated by Sfps, we used nano-liquid chromatography mass spectrometry to conduct a proteomic analysis of the soluble proteins from reproductive organs of A. ludens. The proteins predicted to be transferred from males to females during copulation were 100 proteins from the accessory glands, 69 from the testes and 20 from the ejaculatory bulb, resulting in 141 unique proteins after accounting for redundancies from multiple tissues. These 141 included orthologues to Drosophila melanogaster proteins involved mainly in oogenesis, spermatogenesis, immune response, lifespan and fecundity. In particular, we found one protein associated with female olfactory response to repellent stimuli (Scribble), and two related to memory formation (aPKC and Shibire). Together, these results raise the possibility that A. ludens Sfps could play a role in regulating female olfactory responses and memory formation and could be indicative of novel evolutionary functions in this important agricultural pest.

Mating, but Not Male Accessory Gland Products, Changes Female Response to Olfactory Cues in Anastrepha Fruit Flies.

Copulation and/or ejaculate components can alter female physiological state and female post-mating behavior. The objective of the present study was to determine if copulation and male reproductive accessory gland products (MAGs) modify the behavior of female Anastrepha ludens (Loew) and Anastrepha obliqua (Macquart; Diptera: Tephritidae) in response to two stimuli: male-emitted pheromone and oviposition host volatiles. Olfactometry studies revealed that mated females of both A. ludens and A. obliqua have a stronger response for host volatiles compared to unmated females, which have a stronger response for male pheromone. We also examined olfactory responses of females mated to testectomized males who could transfer MAGs but not sperm. In both species, MAGs alone did not cause the change in the olfactory response observed after copulation, unlike what has been found in Ceratitis capitata (Wiedemann). Females mated to testectomized males responded equally to the male sex pheromone or to host volatiles, thus suggesting that the whole ejaculate is needed to elicit the complete behavioral switch in olfactory response. The function of MAGs is still unknown in these two pests of economic importance. The response for host volatiles by mated females has implications for the development of baits and traps that should preferably attract and target this population.

Promiscous female Euxesta eluta derive nutrients for ovarian development by expelling and consuming ejaculates.

Seminal gifts range from important material donations to items that provide little direct benefit to females. Promiscuous, female silk corn flies Euxesta eluta expel and consume male ejaculates immediately after mating. The evolution and function of this peculiar behavior are currently unknown. We performed a series of experiments aimed to: determine if females under different dietary regimes derive nutrients or water for survival and/or reproduction from ejaculate consumption, if males suffer a fitness cost from supplying females with ejaculates, and if females prefer to mate and/or are more likely to store sperm from well fed than nutritionally stressed presumably inferior males. Experiments revealed that protein deprived E. eluta females derive nutrients for ovarian development through consumption of ejaculates of protein fed males. No seminal products affecting survival appear to be transferred in the consumed ejaculate. However, ovarian development, in contrast to testes growth, occurs in detriment of longevity. Females preferred to mate with protein fed males, yet sperm retention in spermathecae was extremely rare after a single mating. This finding suggests that females could be exerting post copulatory control. A key question that remained to be addressed for the understanding of this puzzling and promiscuous mating system is what ecological factors or male traits drive females to retain sperm from one or several males in order to achieve and/or maximize fertilization potential.

Male age and strain affect ejaculate quality in the Mexican fruit fly.

Aging in all organisms is inevitable. Male age can have profound effects on mating success and female reproduction, yet relatively little is known on the effects of male age on different components of the ejaculate. Furthermore, in mass-reared insects used for the Sterile Insect Technique, there are often behavioral differences between mass-reared and wild males, while differences in the ejaculate have been less studied. The ejaculate in insects is composed mainly of sperm and accessory gland proteins. Here, we studied how male age and strain affected (i) protein quantity of testes and accessory glands, (ii) the biological activity of accessory gland products injected into females, (iii) sperm viability, and (iv) sperm quantity stored by females in wild and mass-reared Anastrepha ludens (Diptera: Tephritidae). We found lower protein content in testes of old wild males and lower sperm viability in females mated with old wild males. Females stored more sperm when mated to young wild males than with young mass-reared males. Accessory gland injections of old or young males did not inhibit female remating. Knowledge of how male age affects different ejaculate components will aid our understanding on investment of the ejaculate and possible postcopulatory consequences on female behavior.

The male ejaculate as inhibitor of female remating in two tephritid flies.

The inhibition of female receptivity after copulation is usually related to the quality of the first mating. Males are able to modulate female receptivity through various mechanisms. Among these is the transfer of the ejaculate composed mainly by sperm and accessory gland proteins (AGPs). Here we used the South American fruit fly Anastrepha fraterculus (where AGP injections inhibit female receptivity) and the Mexican fruit fly Anastrepha ludens (where injection of AGPs failed to inhibit receptivity) as study organisms to test which mechanisms are used by males to prevent remating. In both species, neither the act of copulation without ejaculate transfer nor sperm stored inhibited female receptivity. Moreover, using multiply mated sterile and wild males in Mex flies we showed that the number of sperm stored by females varied according to male fertility status and number of previous matings, while female remating did not. We suggest female receptivity in both flies is inhibited by the mechanical and/or physiological effect of the full ejaculate. This finding brings us closer to understanding the mechanisms through which female receptivity can be modulated.

Female receptivity in Anastrepha ludens (Diptera: Tephritidae) is not modulated by male accessory gland products.

In numerous insects, accessory gland products (AGPs) transferred from males to females during mating are responsible for female sexual inhibition, but these products can be affected by male condition. Here, we investigated the effect of AGPs on female receptivity of the Mexican fruit fly Anastrepha ludens (Loew), and the effect of male and female strain, male irradiation, AGP dose and sexual activity period on the effectiveness of these AGPs in inhibiting female remating. Injections of aqueous extracts of male accessory glands into the abdomen of females did not reduce their receptivity either at 0.2 or 0.8 male equivalent. Females injected with AGPs behaved like virgin females and not as mated females. Neither male origin, female origin (wild versus mass-reared), nor male irradiation (sterile versus fertile males) had an effect in inhibiting female remating. Also, injections of glands obtained during the sexual calling period of males, or obtained during the morning when males are not sexually active had no effect on female remating behavior. Mated mass-reared females were more likely to remate than wild females. We conclude that inhibition of female sexual receptivity of A. ludens is mediated by factors other than AGPs, such as the number of sperm stored by females, the stimulus of copulation per se or more probably, mediated by a combination of factors. More research is needed to elucidate the role of AGPs in this species.

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.

Effects of male nutrition on sperm storage and remating behavior in wild and laboratory Anastrepha fraterculus (Diptera: Tephritidae) females.

Male physiological condition can affect his ability to modulate female sexual receptivity. Thus, studying this aspect can have biological and practical implications. Here, we examine how male nutritional status affected the amount of sperm stored, remating rate and refractory period of the tephritid fruit fly Anastrepha fraterculus (Wiedemann) females. Both wild and laboratory flies were evaluated. We also examine female sperm storage patterns. Experiments were carried out by manipulating male adult diet and exposing these males to virgin females. Females mated with differently treated males were either dissected to count the amount of sperm stored or exposed to virgin males to determine remating rate and the length of the refractory period. We found that male nutritional status affected the amount of the sperm stored and the renewal of sexual receptivity in wild flies. For laboratory flies, male nutritional status affected the length of the refractory period but not the amount of sperm stored by females. In addition, we report that the ventral receptacle is not an important organ of sperm storage in this species. We conclude that male nutritional condition influences the ability to modulate female sexual receptivity, possibly through a combination of the quantity and quality of the ejaculate. From an applied perspective, providing males with an enriched diet will likely result in increased efficacy of the sterile insect technique.