Multiple mating reduces male survivorship but not ejaculate size in the polygamous insect Stenomacra marginella (Heteroptera: Largidae)

In polygamous species, successful males should be able to inseminate multiple females, to defeat sperm from previous males, to avoid sperm displacement by other males, and to induce females to use his sperm during fertilization. Since resources are limited, adaptations to perform any of these functions may conflict with each other (and with other life-history traits) and trade-offs are expected to evolve. We studied if males of the polygamous true bug Stenomacra marginella face a trade-off between multiple mating and survivorship, by comparing the survivorship of virgin and multiply mated males. We also looked for physiological costs of ejaculate production by examining ejaculate production in consecutive matings in multiple mated males. Multiply mated males were able to produce ejaculates of similar size in up to six consecutive copulations but they had decreased survivorship in comparison with virgin males. There was no difference in survivorship between males mated three and six consecutive times, suggesting that the negative relation between survivorship and number of copulations is not linear. The decrease in survivorship seems to be a cost of mating and ejaculate production. This cost could favor the evolution of prudence in the allocation of resources to ejaculate production (e.g., cryptic male choice).     

Reduction in the attraction of males to females after mating in the rice leaf bug Trigonotylus caelestialium

The influence of mating on the extent to which males are attracted to females in Trigonotylus caelestialium (Heteroptera: Miridae) was examined. No differences in attraction of males to mated and virgin females were observed within 3–5 h of mating, but males became less attracted to females 1 to 2 days after the first mating. The difference in male attraction to mated vs virgin females disappeared at 4 days after mating. These results indicate that reduced attraction of males to mated females occurs after a certain time interval, and persists for a few days. Furthermore, males were less attracted to females that had mated with virgin vs recently mated males, i.e. males that had just mated with another female at 1 and 2 days after mating. The ejaculate expenditure of recently mated males was less than that of virgin males. Hence, the amount of male ejaculate transferred to females during mating, rather than the act of mating, might influence the attraction of males to females. The results demonstrate that mating reduces the attraction of males to females in T. caelestialium on the basis of direct observation of male behavior.     

Male age and sexual experience affect male mating behavior in the hawthorn spider mite,Amphitetranychus viennensis

As is reported, in species with first-male sperm precedence, male age and previous sexual experience play crucial roles in male mating behavior. In the hawthorn spider mite, Amphitetranychus viennensis Zacher, previous studies showed that only females that copulated for the first time could achieve fertilization. Based on this, the effects of male age and mating history on male mate choice and male mate competition were investigated. It was confirmed that males could distinguish virgins from fertilized females but they were unable to discriminate between virgins and unfertilized females. Interestingly, the copulation duration of males mated with fertilized females was much shorter than that of males mated with virgins or unfertilized females. Additionally, for male mating choice, males of all ages and more experienced males preferred 5-day-old virgin females, whereas only less experienced males preferred 1-day-old virgin females. In male-male competition, 3-day-old males were more competitive and obtained more copulations compared with others. Copula duration was closely related to male age. Though no significant differences were observed in mating competition between virgin and mated males, copula duration of males in first copulation was the longest and gradually shortened in subsequent copulations. In all, this investigation demonstrated that male age and sexual experience affected male mate choice and male-male competition, leading to further insight into the influences of male age and sexual experience on the reproductive fitness of both sexes.

     

Mating status regulates post‐mating refractory period in Striacosta albicosta females

In polyandrous insect species, males may transfer substances to reduce sperm competition by affecting female sexual receptivity. In this study, we determined the incidence of polyandry in females of Western bean cutworm (WBC), Striacosta albicosta (Smith) (Lepidoptera: Noctuidae), and investigated the influence of both previous female and male mating history on the duration of mating, the female refractory period, and subsequent calling behavior of females under controlled laboratory conditions. The mating status of WBC males influenced mating duration, with copulations involving previously mated males taking longer, possibly related to the time required to produce an ejaculate. The duration of the female refractory period and the onset time of recalling during the scotophase were both affected by female mating history, but not by that of the males. Females had a shorter refractory period and resumed calling activity earlier after their second and third matings than after their first mating. The earlier onset of calling by previously mated females could reduce competition with virgin females and their shorter refractory period could explain the high incidence of polyandry observed in nature.     

Remating in Ceratitis capitata sterile males: Implications in sterile insect technique programmes

Sterile insect technique (SIT) is used, among other biological control tools, as a sustainable measure for the management of Ceratitis capitata Wiedemann (Diptera: Tephritidae) in many agricultural regions where this pest can trigger severe economic impacts. The tendency of wild females to remate multiple times has been deeply studied; it has been a common point of controversy when evaluating SIT programmes. Nevertheless, the remating potential of the released sterile males remains unknown. Here, under laboratory conditions, the remating capability of mass-reared sterile males was determined. Wild-type virgin females were offered to sterile males (Vienna-8 strain), which had the opportunity to mate up to four consecutive times. The remating assays were carried out at 24 hr, 48 hr, 4 days and 7 days after the first mating. At the end of each tested time period, males were divided according to their mating response, mated or unmated, and subsequently reused for the next round of mating assays. The frequency of successful remating in each tested time period was obtained. Insemination was confirmed by determining the sperm transfer in mated female spermathecae by quantitative real-time PCR. Our results demonstrate that 73% of the mass-reared sterile males were able to remate 24 hr after the first mating, 55% of which remated again the day after. Close to 25% of the V8 sterile males tended to copulate in all of the four mating opportunities. The qPCR analysis of the spermathecae contents verified an effective transfer of V8 sperm to wild females with every mating; 99% of copulations resulted in sperm transfer. These findings shed light on the remating potential of V8 sterile males, an aspect until now underestimated in many SIT programmes.     

Costly sexual harassment in a beetle

Abstract The optimal number of mating partners for females rarely coincides with that for males, leading to sexual conflict over mating frequency. In the bruchid beetle Callosobruchus maculatus, the fitness consequences to females of engaging in multiple copulations are complex, with studies demonstrating both costs and benefits to multiple mating. However, females kept continuously with males have a lower lifetime egg production compared with females mated only once and then isolated from males. This reduction in fitness may be a result of damage caused by male genitalia, which bear spines that puncture the female’s reproductive tract, and/or toxic elements in the ejaculate. However, male harassment rather than costs of matings themselves could also explain the results. In the present study, the fitness costs of male harassment for female C. maculatus are estimated. The natural refractory period of females immediately after their first mating is used to separate the cost of harassment from the cost of mating. Male harassment results in females laying fewer eggs and this results in a tendency to produce fewer offspring. The results are discussed in the context of mate choice and sexual selection.     

Mating strategy in Aleochara bilineata: sperm storage and allocation

Abstract By contrast to females that can maximize reproductive success with only one or a few copulations, males generally increase their fitness with frequency of mating. Sperm storage and allocation is therefore crucial for both male and female fitness. Sperm storage in Aleochara bilineata (Coleoptera; Staphylinidae) is investigated by measuring the number of spermatozoa stored in the female spermatheca after single, double or triple successive copulations with different males. The potential advantages of polyandry are studied in terms of the number of sperm stored by females mated twice with the same male (i.e. repeated copulation), compared with females mated twice with two different virgin males (i.e. polyandry). Level of polygyny is also estimated by measuring sperm allocation when ten successive mates are offered to a virgin male. Aleochara bilineata females store the sperm of the same or different males additively, suggesting no advantage for polyandry in terms of the number of sperm stored. A virgin male is able to inseminate ten different females but the number of sperm transferred decreases linearly. Finally, the latencies and durations of copulations are measured in all experiments to estimate changes according to the male or female status (i.e. virgin or mated). The latency before mating is higher when females are virgin than when females have already mated.     

Mating with sperm-depleted males does not increase female mating frequency in the parasitoid Lariophagus distinguendus

Sexual conflicts due to divergent male and female interests in reproduction are common in parasitic Hymenoptera. The majority of parasitoid females are monandrous, whereas males are able to mate repeatedly. Thus, accepting only a single mate might be costly when females mate with a sperm‐depleted male, which may not transfer a sufficient amount of sperm. In the present study, we investigated the reproductive performance in the parasitoid Lariophagus distinguendus Först. (Hymenoptera: Pteromalidae) and studied whether mating with experimentally sperm‐depleted males increases the tendency of females to remate. Males were able to mate with up to 17 females offered in rapid succession within a 10‐h test period. The resulting female offspring, as an indirect measure of sperm transfer, remained constant during the first six matings and then decreased successively with increasing number of copulations by the males. Experimentally sperm‐depleted males continued to mate even if they transferred only small amounts or no sperm at all. Unlike males, the majority of females mated only once during a 192‐h test period. A second copulation was observed only in a few cases (maximum 16%). The frequency of remating was not influenced by the mating status of the first male the females had copulated with, suggesting that these events are not controlled by sperm deficiency of the females. Furthermore, we investigated male courtship behaviour towards mated females. Male courtship intensity towards mated females decreased with increasing time. However, females that had mated with an experimentally sperm‐depleted male did not elicit stronger or longer‐lasting behavioural responses in courting males than those that had mated with a virgin male. As the observed behaviours in L. distinguendus are known to be elicited by a courtship pheromone, these results suggest that females no longer invest in pheromone biosynthesis after mating (as indicated by ceasing behavioural responses of courting males), irrespective of whether they have received a sufficient amount of sperm or not. We discuss the results with respect to a possible mating strategy of sperm‐depleted males.     

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.     

Factors affecting female sexual receptivity in the planthopper, Prokelisia dolus

Abstract. The mating system of Prokelisia dolus Wilson (Homoptera: Delphacidae) was characterized by determining: if males and females multiply mate; when transitions occur in female sexual receptivity, what triggers sexual refractoriness; and what behaviours characterize unreceptive virgins, receptive virgins, and unreceptive mated females. Males copulated with up to six females in less than 1 h, but completely inseminate, on average, only the first four females. Females rarely mated more than once, unless males were depleted of sperm due to previous copulations or if copulation was interrupted (if duration was<2 min). Male and female calling was associated (100% and 91%, respectively) with sexual receptivity and resultant matings. The transition from unreceptive virgin to receptive (calling) mature virgin occurred 48 h posteclosion, and all were mated by day 4. Females that were sexually immature and those completely inseminated did not call. Rejection of males by females included walking away from approaching males (65%), female kicking (7%), and abdominal lifting (5%). Rejection of males was observed by immature, mature and calling, and mated females. Sexual refractoriness was not triggered by acoustic and visual stimuli or mechanical stimulation of genitalia. Refractoriness was also not triggered by reception of small quantities of sperm because some females laid a few viable eggs yet calling was not terminated. Sexual refractoriness was activated by a substance in the ejaculate as demonstrated by injection into the haemocoel of male accessory glands or testes and homogenates of seminal vesicles. This is the first study that documents the role of male ejaculate in inhibiting female sexual receptivity in Hemiptera (Homoptera).     

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.     

Female Reproductive Status and Mate Choice in the Hide Beetle, Dermestes maculatus

Models of mutual mate choice predict that when the costs of mating indiscriminately increase, so may the choosiness of the conventionally non-choosy sex. In a series of behavioral trials, we tested both male and female chemo-reception and mate choice in the hide beetle, Dermestes maculatus. Using olfactometer bioassays we found that males detected and responded more rapidly to individual mated females but had a limited ability to detect virgin females or discriminate between virgin and mated females when presented simultaneously. Both mated and virgin females were unable to detect the presence of virgin males. Copulation behaviors were comparable when males were presented with a virgin or mated female either individually or simultaneously. However, when presented simultaneously, virgin females had a higher probability of receiving a copulation than their mated counterparts. Our data show that males respond to variation in female reproductive status, but that female propensity to mate does not change. We suggest that mutual mate choice may be an important force, even in systems in which there is apparently strong selection for female mate discrimination.     

The effect of male mating history on paternal investment, fecundity and female remating in the seed beetle Callosobruchus maculatus

1. In many organisms, males provide nutrients to females via ejaculates that can influence female fecundity, longevity and mating behaviour. The effect of male mating history on male ejaculate size, female fecundity, female longevity and female remating behaviour in the seed beetle Callosobruchus maculatus was determined.
2. The quantity of ejaculate passed to females declined dramatically with successive matings. Despite the decline, a male's ability to fertilize a female fully did not appear to decline substantially until his fourth mating.
3. When females multiply mated with males of a particular mated status, the pattern of egg production was cyclic, with egg production increasing after mating. Females multiply mated to virgins had higher fecundity than females mated to non-virgins, and females mated to twice-mated males had disproportionately increased egg production late in their life.
4. Females that mated to multiple virgins, and consequently laid more eggs, experienced greater mortality than females mated only once or mated to non-virgins, suggesting that egg production is costly, and rather than ameliorating these costs, male ejaculates may increase them by allowing or stimulating females to lay more eggs.
5. Females mating with non-virgin males remated more readily than did females mated to virgins. Females given food supplements were less likely to remate than females that were nutritionally stressed, suggesting that females remate in part to obtain additional nutrients.     

Male mating costs in a butterfly that produces small ejaculates

In many insects, ejaculate size is positively related to male fitness, although the production of large ejaculates entails physiological costs that can result in decreased male longevity. Ejaculate production costs have been studied in butterfly species because males generally produce large ejaculates that are rich in resources. However, lepidopterans show interspecific variation with respect to relative investment in ejaculates (i.e. percentage of male body weight invested in ejaculates) that could influence the magnitude of costs. The physiological costs of ejaculate production are expected to differ between males with different amounts of resources stored. The effect of male size (an estimate of the amount of resources stored) on ejaculate production and on survivorship costs of multiple mating is studied in males of Leptophobia aripa Boisduval (Pieridae), the butterfly with the smallest relative investment in spermatophores recorded to date (0.31%). ‘Small males’ produce smaller spermatophores, although male size has no effect on ejaculate size (spermatophore + accessory substances), indicating that ‘small males’ make a relatively larger investment in the production of accessory substances. The results indicate that L. aripa males are able to modify the relative amount of resources invested in ejaculate production. Multiple mating has a negative effect on male longevity and this effect depends on male size: in comparison with virgin males, the longevity of ‘small males’ decreases after just one copula, whereas the longevity of ‘large males’ decreases after two copulations. The possible reasons why ‘small males’ invest relatively more resources in ejaculate production, despite the fact that they pay larger costs, are discussed.     

Males mate with females even after sperm depletion in the two-spotted spider mite

Generally, males increase their reproductive success by mating with as many females as possible, whereas females increase their reproductive success by choosing males who provide more direct and indirect benefits. The difference in reproductive strategy between the sexes creates intense competition among males for access to females, therefore males spend much energy and time for competition with rival males for their reproduction. However, if they do not need to engage themselves into male competition and females are in no short supply, how many females can a male mate with and fertilize? We address this question in the two-spotted spider mite, Tetranychus urticae Koch. In this study, we investigated how many females a young, virgin male mated in 3 h, and checked whether the mated females were fertilized. We found that on average males mated with 12–13 females (range: 5–25). As latency to next mating did not change with the number of matings, the males are predicted to engage in even more matings if the mating trial were continued beyond 3 h. Copulation durations decreased with the number of matings and typically after 11 copulations with females any further copulations did not lead to fertilization, suggesting that males continued to mate with females even after sperm depletion. We discuss why spider mite males continue to display mating and copulation behaviour even after their sperm is depleted.

     

Female sexual receptivity and male copula guarding during prolonged copulations in the damselflyIschnura senegalensis (Odonata:Coenagrionidae)

Laboratory experiments were conducted to clarify the relationship between female sexual receptivity and male copula guarding inI. senegalensis, a species that copulates for several hours. In insectaries, most copulations were initiated early in the morning, and terminated relatively synchronously between 11 00 and 13 00. Females refused males with wing-flutter display and oviposited alone in the afternoon regardless of copulation events of that morning. Females could sexually receive males only in the morning. Males copulated for several hours until 12 00 after which females could oviposit. To determine whether copulations that last for hours function as male copula guarding or only of sperm displacement, emerged males were kept at various densities and permitted to copulate with virgin and mated females in insectaries. Both with virgin and mated females, “social” (not solitary; 2–4 males / insectary) males initiated copulations early in the morning and always terminated at around 12 00. However, both with virgin and mated females, solitary (one male / insectary) males terminated copulations in the morning. In both cases, duration of copulations did not significantly differ for virgin females and mated females. Therefore, long (several hour) copulation is more likely to function as male copula guarding than as sperm displacement, and duration of copulations is predicted to be shortened when male density is very low.     

Pattern of sperm storage and migration in the reproductive tract of the swallowtail butterfly Papilio xuthus: cryptic female choice after second mating

Females of the swallowtail butterfly Papilio xuthus L. (Lepidoptera: Papilionidae) mate multiply during their life span and use the spermatophores transferred to increase their longevity as well as fecundity. Sperm from different males may be stored in the sperm storage organs (bursa copulatrix and spermatheca). To clarify the pattern of sperm storage and migration in the reproductive tract, mated females are dissected after various intervals subsequent to the first mating, and the type and activity of sperm in the spermatheca are observed. When virgin females are mated with virgin males, the females store sperm in the spermatheca for more than 10 days. Sperm displacement is found in females that are remated 7 days after the first mating. Immediately after remating, these females flush out the sperm of the first male from the spermatheca before sperm migration of the second male has started. However, females receiving a small spermatophore at the second mating show little sperm displacement, and the sperm derived from the small spermatophore might not be able to enter the spermatheca. Females appear to use spermatophore size to monitor male quality.     

Repeated mating with the same male increases female longevity and fecundity in a polyandrous leaf beetle Galerucella birmanica (Coleoptera: Chrysomelidae)

Female multiple mating (polyandry) is widespread across Insecta, even if mating can be costly to females. To explain the evolution and maintenance of polyandry, several hypotheses, mainly focusing on the material (direct) and/or the genetic (indirect) benefits, have been proposed and empirically tested in many species. Considering only the direct benefits, repeatedly‐mated females are expected to exhibit the same fitness as multiply‐mated females under the same mating frequency. In the present study, we compare the fitness of females received monandrous repeated mating (MM) and polyandrous multiple mating (PM) in a polyandrous leaf beetle Galerucella birmanica and assess female mate preference with regard to polyandry or monandry. Our data indicate that the longevity and the egg‐laying duration of MM females are significantly longer than that of PM females. MM females produce significantly more hatched eggs than PM females over their lifetime under the same mating frequency, which results from the high hatching rate of eggs produced by MM females. PM females mated with novel virgin males in the second mating suffer decreased longevity and lifetime fecundity compared with PM females mated with novel mated males in the second mating. Once‐mated females are more likely to re‐mate with familiar males than novel males. By contrast to expectations, the results of the present study suggest that repeated mating provides females with more direct benefits than multiple mating in G. birmanica, and females prefer to re‐mate with familiar males. The possible causes of this finding are discussed.     

Silk‐ and volatile‐based male mate choice in the genital plug‐producing spider

Female mating history can have a strong effect on male fertilization success. Although males often prefer to mate with virgin females, they often also engage with mated females. As the intensity of sperm competition can differ among mated females, males are expected to evolve means to identify their status. In spiders, males often use female silk to gather information about female quality. Males of many spider species deposit mating plugs into female genitalia to hinder further copulations. We tested whether males of the foliage‐dwelling, plug‐producing spider Philodromus cespitum, which is an important natural enemy of pests, discriminate between females of different mating status and whether they can determine the extent of genital plugging in mated females solely on the basis of cues gained from deposited female silk. We presented males with draglines of females that varied in either mating status (virgin vs. mated), the extent of plugging (small vs. big plug), or the age of the plug (fresh vs. old plug) and examined their mate preferences. Additionally, we tested whether males were attracted to volatile cues produced by female bodies. Our experiments revealed that males preferred draglines of virgin females to those of mated females, and mated females with small plugs to those with large plugs. They were also attracted to female volatile cues. This study suggests that males are able to extract fine‐scale information on mating status from female draglines.     

Strategic ejaculation in the common dung fly Sepsis cynipsea

Sperm competition has been a major selective force acting on male and female behaviour. Theory predicts that when sperm compete numerically, selection will favour males that vary the number of sperm they transfer with sperm competition risk. This often leads to increased copula duration when sperm competition risk is high, the selective advantage of which is increased paternity. We investigated the copulatory behaviour of the common dung fly Sepsis cynipsea in relation to male and female size, female mating status, age, and presence or absence of dung. This fly is unusual in that males mate-guard before copula while females use the sperm of previous males for their current clutch. Body size had no effect on copula duration, but duration of first copulations depended on female age, with older females having longer copulations. For females that copulated twice, there was an interaction between female age and mating status influencing copula duration: old females had longer copulations than young females, but second copulas were longer for young females. Residual testis size of nonvirgin males was smaller than for virgins, and testis shrinkage was significantly associated with copula duration, which indicates that males transfer more ejaculate with longer copulations. We therefore conclude that copulation duration and ejaculate transfer vary in accordance with sperm competition theory.