Opposite Latitudinal Clines for First Mating and Second Mating (Remating) in Males of Drosophila melanogaster

Although female remating has been studied extensively in insects, few studies have been carried out for male remating (second mating). In this study, we analyzed Drosophila melanogaster males for their remating potential, using iso‐female line culture initiated with wild flies collected from eight Indian geographic localities. We examined the association of latitude and percent melanization with first and second male mating (including mating‐related traits). Our results indicated that second male mating has a negative latitudinal cline opposite to that of first mating. Body melanization is negatively correlated with second mating by male and positively with first mating (measured in terms of percent mated pairs). Mating latency during first (ML1) and second (ML2) male mating has a negative latitudinal cline, but slope values differ significantly as ML2 is great at higher latitudes as compared to ML1. The difference between ML1 and ML2 is non‐significant at lower latitudes. However, copulation period of second mating (CP2) has a negative latitudinal cline, whereas copulation period of first mating (CP1) has positive latitudinal cline. Next, the latency and copulation period differ significantly between first and second male mating treatments in within‐population analyses as well as in melanic strains. Furthermore, male remating ability (number of maximum successful remating attempts continuously by a male in 12 h) also follows negative latitudinal cline. The lower latitudinal lighter males have more remating ability as compared to darker males from higher latitudes.     

Female Remating in Drosophila ananassae: Evidence for the Effect of Density on Female Remating Frequency

Drosophila ananassae, a cosmopolitan and domestic species, is largely circumtropical in distribution and belongs to the ananassae species complex of the ananassae subgroup of the melanogaster species group. In the present study, experiments were conducted to investigate the effect of density on female remating frequency by employing different wild-type and mutant strains of D. ananassae. Two experimental designs, i.e., 2-h daily observation and continuous confinement, were used. The results show that there is significant dependence of remating frequency on density in all strains tested under both experimental designs except in a wild-type strain (Bhutan), which shows no dependence of remating frequency on density under 2-h daily observation design. This finding provides evidence that density may increase the frequency of female remating in D. ananassae.     

Female remating inDrosophila ananassae: shorter duration of copulation during second mating as compared to first mating

Drosophila ananassae, a cosmopolitan and domestic species, belongs to theananassae subgroup of themelanogaster species group. Female remating was observed in ten mass culture stocks of this species, which were initiated from flies collected from different geographic localities. The frequency of female remating ranges from 24% to 56% in different strains. Strains show significant variation in remating latency (days). Significant variation has also been found in all the stocks for duration of copulation between first and second matings. The duration of copulation is shorter in second mating as compared to first mating inD. ananassae.     

Intra-specific variation in female remating in Callosobruchus chinensis and C. maculatus

The effects of mating duration on female remating (exp. 1) and under different male densities (exp. 2) were examined in two strains of the adzuki bean beetle, Callosobruchus chinensis and in one strain of the bruchid beetle, C. maculatus. In experiment 1, the frequency of female remating was markedly different between the two strains of C. chinensis. Females of the jC strain, reared long-term in the laboratory, did not remate after being allowed to mate freely (=monogamy), whereas females of the isC strain, recently established from the field, showed high remating frequencies (=polyandry). In both strains, the frequency of female remating increased after the duration of the first mating was deliberately shortened. The relation between mating duration and remating frequency was significantly different, however, between the two strains. In a closely related species, C. maculatus, which manifests polyandry, this relation was more similar to that of the field-derived (=isC) than to that of the laboratory-derived (=jC) strain of C. chinensis. The reasons for the inter-strain variation observed in the remating frequencies of C. chinensis are also discussed. In experiment 2, the mating duration of the three strains was compared under different male densities. Only the lab-derived strain demonstrated a significantly shorter mating duration when one female was placed together with five males than when paired with one male. The shorter mating duration (approximately 26 s) was similar to that of females allowed to remate in the monogamous strain in experiment 1.     

Behavioral genetics of Drosophila ananassae

Drosophila ananassae has a unique status among Drosophila species because of certain peculiarities in its genetic behavior. The most unusual feature of this species is its relatively high frequency of spontaneous male recombination. The results of studies on non-sexual behavior, such as phototactic responses, eclosion rhythm, and preferences for oviposition and pupation sites, lead us to suggest that this behavior is under polygenic control, with a substantial amount of additive genetic variation. Sexual isolation has been reported in D. ananassae with the degree of such isolation being stronger in isofemale lines than in natural populations. The significant variations seen in the mating propensity of several isofemale strains, inversion karyotypes and wild type strains, the diminishing effects of certain mutations on the sexual activity of males, and the positive responses to selection for high and low mating propensity point to a genetic control of sexual behavior in D. ananassae. Males contribute more to variation and thus are more subject to intrasexual selection than females. There is a positive correlation between sternopleural bristle number, mating propensity and fertility in D. ananassae. This correlation between morphometric traits and mating success suggests that larger flies are more successful in mating than smaller ones. There is also evidence for adaptive plasticity and a trade-off between longevity and productivity in D. ananassae. Rare, specific courtship song parameters that provide males with a mating advantage have also been reported in different geographic strains of D. ananassae. The remating behavior of males and females, sperm displacement, and the bi-directional selection for female remating speed indicate that post-mating behavior in this species may also be under genetic control. The occurrence of size assortative mating further indicates that there is size-dependent sexual selection in D. ananassae.     

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.     

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.     

Remating responses are consistent with male postcopulatory manipulation but not reinforcement in D. pseudoobscura

Reinforcement occurs when hybridization between closely related lineages produces low‐fitness offspring, prompting selection for elevated reproductive isolation specifically in areas of sympatry. Both premating and postmating prezygotic behaviors have been shown to be the target of reinforcing selection, but it remains unclear whether remating behaviors experience reinforcement, although they can also influence offspring identity and limit formation of hybrids. Here, we evaluated evidence for reinforcing selection on remating behaviors in Drosophila pseudoobscura, by comparing remating traits in females from populations historically allopatric and sympatric with Drosophila persimilis. We found that the propensity to remate was not higher in sympatric females, compared to allopatric females, regardless of whether the first mated male was heterospecific or conspecific. Moreover, remating behavior did not contribute to interspecific reproductive isolation among any population; that is, females showed no higher propensity to remate following a heterospecific first mating than following a conspecific first mating. Instead, we found that females are less likely to remate after initial matings with unfamiliar males, regardless of species identity. This is consistent with one scenario of postmating sexual conflict in which females are poorly defended against postcopulatory manipulation by males with whom they have not coevolved. Our results are generally inconsistent with reinforcement on remating traits and suggest that this behavior might be more strongly shaped by the consequences of local antagonistic male–female interactions than interactions with heterospecifics.     

Remating, sperm transfer, and sperm displacement in the cactophilic species Drosophila buzzatii Patterson & Wheeler (Diptera: Drosophilidae)

The mating system of Drosophila buzzatii is characterized by short copulation duration, frequent remating in both males and females, and male ejaculate partitioning. Additional features of the system are strong sperm displacement and a high frequency of sterile matings. Remating frequencies and the effects of remating on various mating parameters were studied. In order to characterize variation, five isofemale lines from geographically distant localities in Australia (three localities), Brazil and the Canary Islands were used. Mating parameters studied were: premating time, copulation duration, interval between successive matings, and progeny number as a measure of sperm transfer. Variation for sperm displacement was studied in crosses between laboratory stocks and a number of isofemale lines from Australia. There were significant between‐line differences in female remating frequencies, premating time, copulation duration, interval between successive matings, and progeny numbers, indicating genetic variation for these traits. Females from the five lines mated on average 1.6 to 3.1 times in 4 h, with a maximum of eight matings for one female. The males were given a maximum of ten virgin females in sequence and more than one‐third of the males mated all ten females in the 2 h observation period. Copulation duration decreased and interval between matings increased with copulation number in multiply mated males. Mean copulation duration was c. 2 min. Sperm transfer, measured as the average number of progeny from a single mating, was low (c. 25) and multiply mated females gave more progeny than single mated females, although with much lower progeny numbers than observed in wild‐caught non‐virgin females. A surprisingly high proportion of observed matings gave no progeny, i.e. they were sterile matings. Sperm displacement was strong in most crosses and remained strong in multiply mated females. The results are discussed in relation to the evolution of mating patterns in Drosophila.     

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.     

No evidence for minority male mating advantage in wild type strains of Drosophila ananassae tested in multiple-choice experiments

Minority male mating advantage was tested in wild type strains of Drosophila ananassae through multiple-choice experiments. Mating success of two types of flies present in five different ratios was scored by direct observation in an Elens-Wattiaux mating chamber. We found no evidence for minority male mating advantage in wild type strains of D. ananassae. The relative mating success of two types of females was also compared in the multiple-choice experiments at different ratios; there was no evidence for a rare female effect. Further, there was similarity in the results of experiments employing different methods. The total number of homogamic and heterogamic matings was obtained by combining the data (all five ratios) from each experiment. Homogamic matings were significantly more frequent than heterogamic ones, which demonstrates preferential mating between males and females of the same strain; this was also supported by a lower isolation estimate. There was also a significant difference in the degree of mating preference between the two strains.     

Remating inhibition in female Queensland fruit flies: effects and correlates of sperm storage

Reproductive success of male insects commonly hinges both on their ability to secure copulations with many mates and also on their ability to inseminate and inhibit subsequent sexual receptivity of their mates to rival males. We here present the first investigation of sperm storage in Queensland fruit flies (Tephritidae: Bactrocera tryoni; a.k.a. 'Q-flies') and address the question of whether remating inhibition in females is directly influenced by or correlated with number of sperm stored from their first mates. We used irradiation to disrupt spermatogenesis and thereby experimentally reduce the number of sperm stored by some male's mates while leaving other aspects of male sexual performance (mating probability, latency until copulating, copula duration) unaffected. Females that mated with irradiated rather than normal males were less likely to store any sperm at all (50% vs. 89%) and, if some sperm were stored, the number was greatly reduced (median 11 vs. 120). Despite the considerable differences in sperm storage, females mated by normal males and irradiated males were similarly likely to remate at the next opportunity, indicating (1) number of sperm stored does not directly drive female remating inhibition and (2) factors actually responsible for remating inhibition are similarly expressed in normal and irradiated males. While overall levels of remating were similar for mates of normal and irradiated males, factors responsible for female remating inhibition were positively associated with presence and number of sperm stored by mates of normal but not irradiated males. We suggest seminal fluids as the most likely factor responsible for remating inhibition in female Q-flies, as these are likely to be transported in proportion to number of sperm in normal males, be uninfluenced by irradiation, and be transported without systematic relation to sperm number in irradiated 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.     

Radiation effects on Drosophila suzukii (Diptera: Drosophilidae) reproductive behaviour

Female remating is a widespread behaviour, reported in several insect species. This behaviour can affect the efficiency of sterile insect technique (SIT); however, little is known about the postcopulatory behaviour of some pest species considered as candidates to be controlled by this technique, such as Drosophila suzukii (Matsumura, 1931) (Diptera: Drosophilidae). In this study, we investigated the effects of male and female sterilization on mating and remating behaviour of D. suzukii. First, we tested the occurrence of multiple mating in different combinations between sterile and fertile males and females. Then, we tested the effects of male and female sterility on female propensity to mate and remate. We found an overall low remating rate by D. suzukii females. Male sterility did not influence mating and remating likelihood; however, copula duration of sterile males was shorter compared to fertile males. On the other hand, sterile females were less likely to mate. Our findings encourage further research regarding the use of SIT to control D. suzukii.     

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).     

Remating and sperm displacement in a natural population of Drosophila buzzatii inferred from mother-offspring analysis of microsatellite loci

Prospects for estimation of parameters of models of sperm competition from field data have improved recently with the development of methods that employ multilocus genotype data from brood-structured samples. Sperm competition in Drosophila buzzatii is of special interest because it is possible to directly observe the breeding behaviour of this species in its natural habitat of rotting cactus. Previous laboratory experiments showed that this species exhibits an unusual pattern of frequent remating and sperm partitioning. This paper reports the first attempt to estimate the frequency of female remating and sperm competition in natural populations of D. buzzatii. For the Australian population studied, the mean remating frequency was lower (alpha = 2.12-2.20) than previously estimated in laboratory experiments with the same population, whereas mean sperm displacement (beta = 0.69-0.71) fell within the limits of previous laboratory results. The evolution of the D. buzzatii mating system is discussed.     

A mating plug protein reduces early female remating in Drosophila melanogaster

Mating plugs are formed within the female reproductive tract during mating from male ejaculate constituents or even from male genitalia themselves. Across species, mating plugs have roles in sperm storage and the prevention of female remating. In the fruitfly Drosophila melanogaster, accessory gland proteins such as the sex peptide are known to reduce female remating, however this effect can take some time to establish, hence other ejaculate components must also be involved. We hypothesised a role for the PEBII mating plug protein in the prevention of early female remating. Using RNA interference we produced PEBII knockdown males. We found that these males were significantly less able to prevent female remating in the 4 h following mating. The mating plugs produced by PEBII knockdown males also showed lower levels of autofluorescence in the first 10 min after the start of mating, suggesting they differed in composition to those of control males. Reduced levels of PEBII had no effect, however, on fecundity, progeny production or egg-adult viability in the first 24 after mating, suggesting there were no short-term effects of PEB II on sperm transfer, storage or use. Our results show that PEBII has a subtle but significant role in the prevention of early female remating.     

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.     

No Effect of Male Courtship Intensity on Female Remating in the Butterfly <Emphasis Type="Italic">Pieris napi</Emphasis>

In Pieris napi, female fitness increases with number of matings, but wild females mate at an unexpectedly low rate. From a sexual conflict perspective this could be because males manipulate female remating, or alternatively, because wild females experience costs associated with remating which are not applicable under laboratory conditions. To get an indication which sex controls remating and/or the different sexes’ relative costs and benefits of remating, we here test whether female mating frequency is affected by male courtship intensity. We found no effect on female mating frequency or lifespan. This indicates that (i) females control remating and their optimal mating frequency is lower compared to males, or (ii) males can manipulate female remating. We argue that both these alternatives may apply simultaneously to P. napiand that they are inseparable.     

Function and genetics of long versus short copulations in the cactophilic fruit fly,drosophila mojavensis (diptera: drosophilidae)

Variation in copulation duration of Drosophila mojavensisstrains was influenced by both sexes. Males maintained predominant control, as copulation duration of pairs from different strains was more similar to that of the strain from which the male was derived, but female origin also contributed significantly to the duration of copulation. Variation among strains was controlled by genes acting additively in both sexes. The size of both males and females also affected copulation duration. Small males copulated longer on average than large males, while males paired with large females copulated longer than those paired with small females. The importance of copulation duration to fitness was tested by correlation analyses with male size, female size, female remating latency, and number of eggs laid prior to female remating. Longer copulations stimulated earlier oviposition, possibly by increasing accessory gland secretions that are passed by males during copulation.