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.     

Evolutionary Consequences of Desiccation Resistance in the Male Ejaculate

Avoiding water loss for insects is critical for survival. Selection for reduced water loss will depend on trade-offs between resources allocated for reproduction and those allocated for resisting desiccation. However, we lack knowledge on how selection for desiccation resistance can affect the male ejaculate. Furthermore, as male ejaculate composition is complex, desiccation resistant females could evolve traits that enable them to derive longevity benefits from mating. Here, we assessed how selection for desiccation resistance impacts male testes and accessory gland size, protein content of these organs, female sperm storage and male ability to inhibit female remating behavior, in the Mexican fruit fly Anastrepha ludens. Additionally, we tested if mating increased longevity and fecundity in desiccation resistant females. Males selected for resistance to desiccation stress had smaller accessory glands and seminal vesicles and females mating with these males stored less sperm compared to control males. Females mating with resistant males had lower fecundity compared to females mating with control males. Desiccation resistant females lived longer than control females, yet this was irrespective of mating. Rapid evolutionary responses to hydric stress can have correlated effects in reproductive capabilities, which are not restricted to pre-copulatory traits. Trade-offs between resistance to desiccation stress are reflected in decreased allocation of resources to reproductive organs. Thus, production of the ejaculate may be costly for A. ludens males. Knowledge on the evolution of ejaculate traits and reproductive organ size in response to directional selection for desiccation resistance, will aid our understanding of differential sex-specific responses to environmental stress.     

Disparity in sexual behaviour between wild and mass‐reared Mexican fruit flies

The sterile insect technique (SIT) is currently used to control Mexican fruit fly Anastrepha ludens Loew (Diptera: Tephritidae). However, mass‐rearing can alter the quality of released males. If males that are mass‐reared have behaviours different from those of their wild counterparts, then this may diminish the effectiveness of SIT. Questions remain as to whether wild females may be able to detect the male condition before, during and/or after copulation with a mass‐reared male. In the present study, copula duration, female remating, female fecundity and fertility of both mass‐reared and wild A. ludens are evaluated. Marked differences are found between mass‐reared and wild females. Specifically, mating latency is longer and copula duration is shorter for wild females compared with mass‐reared females. Importantly, there are no significant differences in mating latency, copula duration or remating probability between wild females paired with either mass‐reared or wild males. All mass‐reared females remate, whereas only approximately half of the wild females remate after first mating with either a wild or mass‐reared male. Fecundity of wild females mated to either wild or mass‐reared males is approximately one‐third lower than that of mass‐reared females, confirming that mass‐reared females may have been selected for high fecundity and are adapted to laboratory conditions. Fertility of females that mate with a wild male for only 10 min is not significantly different from that achieved via a full‐length copulation. By contrast, females mating with mass‐reared males need copulation durations of at least 40 min to achieve fertility comparable with that achieved via a full‐length copulation. The findings of the present study have important implications for A. ludens controlled through SIT and broaden our understanding on the copulatory and post‐copulatory behaviours between wild females and mass‐reared males.     

Female Age Determines Remating Behavior in Wild Mexican Fruit Flies

Male and female age are important factors that can influence mating and remating behavior. Females can discriminate against or prefer older males, but there have been relatively fewer studies on how female and male age influence female remating. Here we showed in wild flies of the Mexican fruit fly Anastrepha ludens (Loew), that when females were given a choice between males of different ages, younger females preferred to mate with younger males over older males, while older females were less selective. Also, when given a choice between males of different ages, older females had longer copulation durations than younger females. On the other hand, older males and females had lower mating success, compared with young and middle-aged flies under no choice conditions. However, middle-aged females mated faster compared to young females and young males mated faster compared to middle-aged males. Male age did not influence female remating, while female age strongly determined female remating, with no females remating when they were old. It is unclear if female receptivity mechanisms are switched off at older ages, or if females are reluctant to remate due to possible costs of mating. We discuss our results in terms of how male and female age can influence mating decisions.     

Remating by female Mediterranean fruit flies (Ceratitis capitata, Diptera: Tephritidae): Temporal patterns and modulation by male condition

We determined the temporal pattern of female remating in the Mediterranean fruit fly, Ceratitis capitata, and how mating with sterile males affects remating. In addition, we examined the hypotheses that sterile male nutrition and age affect the subsequent receptivity of their mates. Temporally, female receptivity varied significantly throughout the experimental period. Relatively high levels of remating (14%) on the days following the first copulation were followed by a decline, with a significantly low point (4.1%) 2 weeks after mating. Subsequently, receptivity is gradually restored (18%) 3 and 4 weeks after the initial copulation. When females were first mated to sterile males, significantly higher remating percentages were recorded. The ability of sterile males to inhibit receptivity of both wild and laboratory reared females on the day of first mating was significantly improved when they were fed a nutrient rich diet. Male age at first mating also affected female receptivity: sterile males of intermediate age (11 days old) inhibited female remating significantly more than younger or older flies. Although further studies are needed to determine the relative roles of natural and sexual selection in modulating patterns of female sexual receptivity, the Sterile Insect Technique may be improved by releasing well nourished, older sterile males.     

Male and female condition influence mating performance and sexual receptivity in two tropical fruit flies (Diptera: Tephritidae) with contrasting life histories

Recent recognition of widespread polyandry in insects has generated considerable interest in understanding why females mate multiple times and in identifying factors that affect mating rate and inhibit female remating. However, little attention has been paid to understanding the question from both a female and male perspective, particularly with respect to factors that may simultaneously influence female remating rates. Here, we report on a study aimed at ascertaining the possible interactive effects that male and female size and diet, and female access to a host could have on mating latency, probability, and duration and female refractory period using two tropical fruit fly species with contrasting life histories. Of all factors tested, adult diet played the most significant role. Both Anastrepha ludens and Anastrepha obliqua males which had constant access to protein and sucrose mated more often, had shorter copulations and induced longer refractory periods in females than males fed a low quality diet (sucrose offered every third day). Female size and the interaction with male diet determined how quickly female A. ludens mated for the first time. Smaller females mated sooner with low quality fed males than with high quality fed males while there was no difference for large females, suggesting that male choice may be at play if high quality fed males discriminate against smaller females. Copulation duration also depended on both male and female nutritional condition, and the interaction between male diet and female size and diet. Large and high quality fed females had shorter copulations regardless of male condition. Importantly, for A. ludens, female refractory period depended on male size and the nutritional condition of both males and females, which could indicate that for this species, female receptivity does not depend only on the condition of the male ejaculate. For A. obliqua refractory period was associated with the interaction between male size and diet and male diet and host presence. We discuss our results in terms of male ability to inhibit female remating and the relative contribution of female condition to this behavior. We also address the importance of studying effects simultaneously on species with contrasting life histories.     

Precocious Mexican fruit fly methoprene‐fed males inhibit female receptivity and perform sexually as mature males

The sterile insect technique (SIT) has been used successfully for the control of fruit flies. The efficiency of this technique can be significantly reduced when sterile released insects are exposed to adverse conditions and predators, as a great number of sterile insects die before reaching sexual maturity and thus fail to mate with wild females. Treatments with juvenile hormone (JH) analogues such as methoprene (M) significantly reduce the time to reach sexual maturity by sterile Anastrepha ludens (Loew) (Diptera: Tephritidae) males. In this study, we compared the sexual performance of non‐treated sexually mature males with young males that had been sexually accelerated with M. Furthermore, we compared the ability of M‐fed males in inhibiting female remating compared with sexually mature males. Results showed that at 5 days M‐fed males had lower mating success than mature males; however, 6‐day‐old (0.1%) M‐fed males had the same amount of matings as mature 13‐day‐old males. Young 5‐ to 10‐day‐old M‐fed males also had similar number of matings as mature non‐treated 12‐ to 17‐day‐old males. There were no differences in copula duration between treatments. Moreover, there were no differences between the fertility, fecundity or refractory period of females mated with either young male fed M or normal sexually mature males. These results indicated that young males that were sexually accelerated with M have the same sexual performance as non‐treated sexually mature males. Implications of using M as a pre‐release treatment for A. ludens controlled through SIT are discussed.     

Remating Behavior of Cnephasia jactatana Walker Females (Lepidoptera: Tortricidae)

Cnephasia jactatana Walker is an important pest of kiwifruit in New Zealand. We investigated, under laboratory conditions, the effects of multiple mating on the reproductive performance of C. jactatana females and how such effects varied with male virginity and larval nutrition. We found that in permanent pairs, remating increased female fecundity and fertility but suboptimally fed females benefited more from remating. Regardless of this benefit, mass-reared pairs had a lower remating frequency. Females remating with a virgin male or a male that had delivered a spermatophore presented similar fecundity and fertility; however, females receiving a second ejaculate from a virgin male had increased daily fecundity. Female weight clearly affected remating behavior since those that received a second ejaculate were significantly heavier. Neither mating length nor size of the first spermatophore influenced female remating. Further, mass-reared and individually reared males produced spermatophores of similar size.     

Effects of delayed mating and male mating history on the reproductive potential of Leucoptera coffeella (Lepidoptera: Lyonetiidae)

Abstract 1 Despite the importance of Leucoptera coffeella (Guérin‐Mèneville) in coffee production worldwide, there is a lack of information on its reproduction. This knowledge will help in mass rearing, and support the development of behavioural control techniques for this insect. The present study determined the effects of delayed mating and previous matings of male L. coffeella on fecundity, egg viability and frequency of female remating. 2 The highest levels of fecundity and egg viability were obtained from matings of 1–3‐day‐old females. When females mated at 5 days of age, there were reductions of 40% in oviposition and of 43% in egg viability. 3 Females mated with 2‐day‐old virgin males were more fecund than those mated with older males; egg viability was also low (18%) from females mated with older males. 4 Virgin females that mated with virgin males laid a greater number of eggs than those mated with previously copulated males. Egg viability decreased with the increase in the number of previous male matings. 5 Five‐day‐old females remated in greater proportion than 2–3‐day‐old females. Females that copulated with males that had previously mated three times had higher rates of remating than those that copulated with virgin males. 6 The results obtained indicate that 1–3 days after emergence is the optimum age for mating. The implications of these findings for the control of L. coffeella by synthetic sex pheromone are discussed.     

Male larval nutrition in Choristoneura rosaceana (Lepidoptera: Tortricidae): an important factor in reproductive success

This study examines how Choristoneura rosaceana male quality, as determined by larval diet, age and mating history, affects the reproductive success of both sexes. While the size of the spermatophore produced at first mating increased linearly with male age, the frequency of mating was significantly higher for middle-aged males (2–4 days old) than younger (0–2 days old) or older (6–8 days old) individuals, when both sexes were fed on artificial diet. However, the duration of copulation was longer in couples with older than younger males. The observed age-related changes in spermatophore size had no significant effect on female longevity, fecundity or fertility, suggesting no direct relationship between male investment and spermatophore size under these experimental conditions. Different larval food sources (artificial diet, maple and hazelnut) did not affect the proportion of 2-day-old virgin males that mated; however, the proportion that remated was significantly higher for males reared on high-quality food (maple and artificial diet) than those on hazelnut, a poorer food source. There was a 5-fold decline in spermatophore size between the first and second matings on all diets, but female reproductive output was reduced by only 25%. In contrast, while the first spermatophore produced by males on hazelnut was 1.5 times smaller than those produced on maple and artificial diet, the fecundity of their mates was 40% less than those mated with high-quality virgin males. These results provide additional support to the idea that spermatophore size is not a valuable indicator of male quality. Most tethered females placed in the field during the first flight period mated with virgin males (based on the size of the spermatophore), suggesting that female choice exists in this species. These results are discussed in relation to the incidence of polyandry in naturally occurring populations of Choristoneura and the potential use of size and/or chemical cues by females to assess male quality.     

Male diet and age influence to inhibit female remating in Ceratitis capitata (Diptera: Tephritidae)

This study evaluated the influence of age and adult nutritional status of Ceratitis capitata males on their ability to inhibit female remating. Their roles and that of copula duration on the amount of sperm transferred to female spermathecae were also analysed. After emergence, adults were kept in separate groups according to their diets (either high protein – 6.5 g of brewer’s yeast, or low protein – 3.5 g of brewer’s yeast) and their age at the time of use in experiments (4, 8, 12, 16 and 20 days old). The results demonstrated that: (i) male age was not a factor that influenced remating when females mated with well‐nourished males; however, the youngest males (4 days old) in the low‐protein group were less efficient in preventing female remating; (ii) 12‐ and 16‐day‐old males fed on a high‐protein diet transferred and produced more sperm than males of other groups; (iii) there was no correlation between copula duration and the amount of sperm transferred to the female; the longest copula durations were observed among low‐protein‐diet/20‐day‐old males. These results suggest that age and nutrition influence the ability of the males to inhibit female remating. The sterile insect technique is most effective when females do not remate after coupling with sterile males, and therefore, the capacity to inhibit female remating is an important characteristic of males released in the field.     

Methoprene application and diet protein supplementation to male melon fly,Bactrocera cucurbitae,modifies female remating behavior

Methoprene (an analogue of juvenile hormone) application and feeding on a protein diet is known to enhance male melon fly, Bactrocera cucurbitae Coquillett (Diptera: Tephritidae), mating success. In this study, we investigated the effect of these treatments on male B. cucurbitae's ability to inhibit female remating. While 14‐d‐old females were fed on protein diet, 6‐d‐old males were exposed to one of the following treatments: (i) topical application of methoprene and fed on a protein diet; (ii) no methoprene but fed on a protein diet; (iii) methoprene and sugar‐fed only; and (iv) sugar‐fed, 14‐d‐old males acted as controls. Treatments had no effect on a male's ability to depress the female remating receptivity in comparison to the control. Females mated with protein‐deprived males showed higher remating receptivity than females first mated with protein‐fed males. Methoprene and protein diet interaction had a positive effect on male mating success during the first and second mating of females. Significantly more females first mated with sugar‐fed males remated with protein‐fed males and females first mated with methoprene treated and protein‐fed males were more likely to remate with similarly treated males. Females mating latency (time to start mating) was significantly shorter with protein‐fed males, and mating duration was significantly longer with protein‐fed males compared with protein‐deprived males. These results are discussed in the context of methoprene and/or dietary protein as prerelease treatment of sterile males in area‐wide control of melon fly integrating the sterile insect technique (SIT).     

Differential investment in pre‐ vs. post‐copulatory sexual selection reinforces a cross‐continental reversal of sexual size dimorphism in Sepsis punctum (Diptera: Sepsidae)

Theory predicts that males have a limited amount of resources to invest in reproduction, suggesting a trade‐off between traits that enhance mate acquisition and those that enhance fertilization success. Here, we investigate the relationship between pre‐ and post‐copulatory investment by comparing the mating behaviour and reproductive morphology of four European and five North American populations of the dung fly Sepsis punctum (Diptera) that display a reversal of sexual size dimorphism (SSD). We show that the geographic reversal in SSD between the continents (male biased in Europe, female biased in North America) is accompanied by differential investment in pre‐ vs. post‐copulatory traits. We find higher remating rates in European populations, where larger males acquire more matings and consequently have evolved relatively larger testes and steeper hyper‐allometry with body size. American populations, in sharp contrast, display much reduced, if any, effect of body size on those traits. Instead, North American males demonstrate an increased investment in mate acquisition prior to copulation, with more mounting attempts and a distinctive abdominal courtship display that is completely absent in Europe. When controlling for body size, relative female spermathecal size is similar on both continents, so we find no direct evidence for the co‐evolution of male and female internal reproductive morphology. By comparing allopatric populations of the same species that apparently have evolved different mating systems and consequently SSD, we thus indirectly demonstrate differential investment in pre‐ vs. post‐copulatory mechanisms increasing reproductive success.     

Influence of mating histories and age on female remating behaviour in a few closely related species of Drosophila nasuta subgroup

Female remating with more than one male leads to coexistence of sperm from different males in the same female, thus creating a selection pressure on sperm. To understand the extent of divergence in the reproductive behaviour among closely related species, in the present study, the influence of first mating histories like mating latency, duration of copulation and age of flies have been analysed on female remating behaviour in closely related Drosophila nasuta subgroup species with varying levels of reproductive isolation. The time taken for the once mated females to remate varied from 7 days in D. s. sulfurigaster to 19 days in D. s. neonasuta after first mating. The female remating frequency varied from a minimum of 29% in D. s. neonasuta to a maximum of 95% in D. s. sulfurigaster. The younger flies, which had remating latency of three times less than aged flies, show 100% remating frequency. In addition, it was observed that the duration of copulation in the first mating influences the remating behaviour among the nasuta subgroup members. The results revealed that D. nasuta subgroup members despite being closely related differ in their reproductive behaviour.     

The Effects of Experimentally Induced Polyandry on Female Reproduction in a Monandrous Mating System

Females of most insect species maximize their fitness by mating more than once. Yet, some taxa are monandrous and there are two distinct scenarios for the maintenance of monandry. While males should always benefit from inducing permanent non‐receptivity to further mating in their mate, this is not necessarily true for females. Since females benefit from remating in many species, cases of monandry may reflect successful male manipulation of female remating (i.e. sexual conflict). Alternatively, monandry may favor both mates, if females maximize their fitness by mating only once in their life. These two hypotheses for the maintenance of monandry make contrasting predictions with regards to the effects of remating on female fitness. Here, we present an experimental test of the above hypotheses, using the monandrous housefly (Musca domestica) as a model system. Our results showed that accessory seminal fluid substances that males transfer to females during copulation have a dual effect: they trigger female non‐receptivity but also seem to have a nutritional effect that could potentially enhance female fitness. These results suggest that monandry is maintained in house flies despite potential benefits that females would gain by mating multiply.     

Age‐Related Male Reproductive Investment in Courtship Display and Nuptial Gifts in a Moth,Ostrinia scapulalis

Due to a trade‐off between current and future reproduction, costly reproductive investments should be increased towards the end of a lifespan when the probability of reproduction becomes low (terminal investment hypothesis). We investigated age‐related changes in male reproductive investment towards courtship display and the spermatophore in three age classes (young, middle‐aged and old) of a monandrous moth, Ostrinia scapulalis. As predicted, old males had higher mating success than young and middle‐aged males in no‐choice tests. Moreover, two‐choice tests revealed that middle‐aged males had a higher success rate than young males because of their higher courtship frequency rather than any female preference for them. It was found that old males produced a larger spermatophore than young and middle‐aged males, suggesting greater reproductive effort. The protein content of spermatophores also tended to increase with male age. Despite the age‐related variation in spermatophore size and protein content, age did not affect female fecundity or longevity. A decrease in the number of sperm in the older males might counteract the nutritional benefit of larger spermatophores. Alternatively, fitness components other than longevity and fecundity may be influenced by male age.     

Age-related variation in primary sexual characters in a passerine with male age-related fertilization success, the bluethroat Luscinia svecica

In passerine species with frequent extrapair mating, young (second calendar year) males often have a lower fertilization success than older (after second calendar year) males. This pattern might be explained by male- or female-driven mechanisms, such as female preference for older males or higher competitive ability of older males. In this study we measured the size of the testes, the seminal glomera and the cloacal protuberance as well as the size and motility of the sperm, in individual bluethroats Luscinia svecica . In this species, nearly all extrapair fertilizations (EPFs) are obtained by older males. We found that the mass of the testes and the seminal glomera were highly positively correlated and that older males had significantly larger testes (38%), seminal glomera (15%) and cloacal protuberance (23%) than young males. In contrast, there was no difference between age groups in average sperm size or sperm motility. Our results are consistent with the idea that higher fertilization success by older males in this species is due to their higher rate of sperm production, allowing larger ejaculates and/or more frequent copulations. Unequal sperm production capacities by young and older males have important implications for the interpretation of paternity patterns in extrapair mating systems.     

Female Remating Behavior in a Lekking Moth

In polyandrous species, male reproductive success will at least partly be determined by males' success in sperm competition. To understand the potential for post‐mating sexual selection, it is therefore important to assess the extent of female remating. In the lekking moth Achroia grisella, male mating success is strongly determined by female choice based on the attractiveness of male ultrasonic songs. Although observations have indicated that some females will remate, only little is known about the level of sperm competition. In many species, females are more likely to remate if their first mating involved an already mated male than if the first male was virgin. Potentially, this is because mated males are less well able to provide an adequate sperm supply, nutrients, or substances inhibiting female remating. This phenomenon will effectively reduce the strength of pre‐copulatory sexual selection because attractive males with high mating success will be more susceptible to sperm competition. We therefore performed an experiment designed both to provide a more precise estimate of female remating probability and simultaneously to test the hypothesis that female remating is influenced by male mating history. Overall, approximately one of five females remated with a second male. Yet, although females mated to non‐virgin males were somewhat more prone to remate, the effect of male mating history was not significant. The results revealed, however, that heavier females were more likely to remate. Furthermore, we found that females' second copulations were longer, suggesting that, in accordance with theory, males may invest more sperm in situations with an elevated risk of sperm competition.     

Interpopulation variation in female remating is attributable to female and male effects in <Emphasis Type="Italic">Callosobruchus chinensis</Emphasis>

The evolution of female multiple mating is best understood by consideration of male and female reproductive perspectives. Females should usually be selected to remate at their optimum frequencies whereas males should be selected to manipulate female remating to their advantage. Female remating behavior may therefore be changed by variation of male and female traits. In this study, our objective was to separate the effects of female and male strains on female remating for the adzuki bean beetle, Callosobruchus chinensis, for which there is interstrain variation in female remating frequency. We found that interstrain variation in female remating is primarily attributable to female traits, suggesting genetic variation in female receptivity to remating in C. chinensis. Some interstrain variation in female remating propensity was attributable to an interaction between female and male strains, however, with the males of some strains being good at inducing nonreceptivity in females from one high-remating strain whereas others were good at inducing copulation in nonvirgin females from the high-remating strain. There is, therefore, interstrain variation in male ability to deter females from remating and in male ability to mate successfully with nonvirgin females. These results suggest that mating traits have evolved along different trajectories in different strains of C. chinensis.     

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.