Male butterfly investment in successive ejaculates in relation to mating system

In Lepidoptera, female mating systems range from strict monandry to strong polyandry. Males transfer an ejaculate during copulation that contains both sperm and accessory gland substances. In butterflies the male ejaculate has at least three effects: it (1) contains sperm that can fertilize the eggs of the male’s mating partner, (2) influences the refractory period of the mated female, and (3) contains nutrients that can be used by the female to increase her reproductive output. A number of recent studies have shown that males in polyandrous mating systems are endowed with adaptations to increase mating capabilities. Relative to males in more monandrous species they transfer larger first ejaculates which contain relatively more protein, and allocate proportionally more resources to reproduction. The objective of this study was to compare male reproductive investment in successive ejaculates, in terms of both mass and quality, to determine if males of polyandrous species are capable of maintaining the production of larger and more nutritious ejaculates than males of more monandrous species. We used three species of pierid butterfly, with mating systems ranging from relative monandry to polyandry. The degree of polyandry had a substantial effect on the reproductive performance of males. The cumulative protein content of ejaculates increased with the degree of polyandry. Only males of the most polyandrous species, Pieris rapae, produced three successive ejaculates of similar mass and protein content. In the relatively monandrous species, Aporia crataegi and P. brassicae, males in subsequent matings never produced another ejaculate as large as that transferred by males mating for the first time, although the protein content (mg) of ejaculates did not differ in second and third ejaculates. Moreover, the ability to remate varied with the degree of polyandry. Given the opportunity to remate, the majority of P. rapae males mated three times, whereas relatively few A. crataegi and P. brassicae males were able to perform three matings. These results suggest that male capacity to produce large, nutritious ejaculates is limited in relatively monandrous species. In this study only males belonging to the most polyandrous species had the ability to recuperate quickly from a mating event and remate. Our results suggest that males in polyandrous systems are better adapted to mating more than once. Received: 3 November 1995/Accepted after revision: 13 July 1996     

Polyandrous female butterflies forage for matings

Butterfly mating systems exhibit great variation and range from strict monandry to strong polyandry. During mating males transfer ejaculates containing both sperm and accessory substances to females. In the polyandrous green-veined white butterfly, Pieris napi (Lepidoptera, Pieridae) these ejaculates average 15% of male body mass, but can represent up to 23% of body mass for individual males. Hence, mating is costly to males, and recently mated males increase copula duration but decrease ejaculate mass transferred to females. Substances transferred to females during mating are later incorporated into female soma and reproductive tissues, and multiply mated female butterflies have higher lifetime fecundity, lay proportionately larger eggs, and live longer compared to once mated females. Here we report that females of P. napi allowed to mate at liberty with recently mated males only (i.e. males that delivered a small ejaculate) increased their lifetime number of matings compared to females allowed to mate with virgin males only (i.e. males that delivered large ejaculates), the former group mating on average 5.1 times (range 2–10) and the latter group mating on average 2.8 times (range 1–4). The lifetime fecundity of the two groups of females did not differ significantly. Because nutrient donation from males is essential for females to realize their potential fecundity, we conclude that females of the polyandrous green-veined white actively forage for matings.     

Protein content of spermatophores in relation to monandry/polyandry in butterflies

The evolution of a mating system, and specifically mating frequency, is dependent on the costs and benefits to both sexes of mating once or several times. In butterflies, males transfer a spermatophore that contains both sperm and accessory gland products. Accessory gland substances contain nutrients which, in some species, females use to increase their reproductive output and longevity. Nutrients contained in these packaged ejaculates represent investment by males in reproduction. Consequently, the nutritional composition of spermatophores may vary depending on the mating system. There are two lines of arguments concerning the evolution of the nutrient content of ejaculates. One hypothesis argues that male nuptial gifts evolved in the context of certainty of paternity and ease of finding mates; thus spermatophores of polyandrous males (with lower certainty of paternity and greater ease of finding mates) should contain less protein than those of monandrous males, since more spermatophores are produced on average. The other hypothesis argues that polyandry evolved in the context of maximization of male transfer of nutrients to females, and hence spermatophores of polyandrous males should contain more protein than those of monandrous males. In an attempt to distinguish between these two hypotheses, we determined how protein content of ejaculates varied with the degree of polyandry in nine species of pierid and two species of satyrid butterflies. We found that both relative ejaculate mass and protein content increased with the degree of polyandry. Hence our results are consistent with the view that polyandry has evolved in the context of male transfer of nutrients to females, and provides another example of a male adaptation to multiple mating in butterflies.     

Different ejaculate delivery strategies in first versus subsequent matings in the swallowtail butterfly Papilio machaon L.

Summary Evidence suggests that males incur costs in producing ejaculates. Therefore some discrimination should be expected with respect to their pattern of allocating ejaculates. In a laboratory study, males of the European swallowtail butterfly Papilio machaon were mated with conspecific virgin females up to six times. The weight of the spermatophore and of the accessory substances delivered by males, the duration of mating, and the number of sperm in ejaculates were measured. The weight of the first spermatophore was related to male weight but that of accessory substances was not. The weight of the spermatophore delivered by males upon remating was always less than that of the first spermatophore. Second spermatophores Produced by males were smallest when delivered on the day after the first mating and increased asymptotically to about half the initial size with time elapsed since the first mating. Copula duration was highest for males remating on the day after the first mating but decreased to the duration of the first mating for males that were remated on the third day after the first mating or later. The weight of accessory substances delivered by males and the number of sperm in ejaculates were also higher in first matings compared to all subsequent matings. Both of two males that were handpaired on three consecutive days after the first mating produced a cumulative ejaculate mass equalling that transferred at the first mating. Thus the fact that males delivered smaller ejaculates upon remating on the third day after the first mating or later requires explanation, especially in view of the fact that copulations were not prolonged. We argue that the male ejaculate delivery strategy in mildly polyandrous species should be to mate for the first time with virgin females, when a maximum size ejaculate should be delivered, and then to maximize the number of matings. The rationale behind the male shift towards smaller ejaculate sizes delivered at subsequent matings is that: (1) the size of the ejaculate that can be produced is dependent on the time elapsed since the last mating; (2) the abundance of virgin females decreases as the flight season proceeds; and (3) there is an inverse relationship between the size and number of ejaculates that a male can produce in his life-time.     

Effects of size and nuptial gifts on butterfly reproduction: can females compensate for a smaller size through male-derived nutrients?

In butterflies and other insects, fecundity generally increases with female adult weight. Hence, most butterflies are essentially "capital breeders", because nutrients acquired during the larval stage are stored and subsequently used for egg production during the adult stage. However, in some species, males transfer a large nutritious ejaculate to the female at mating. These females can partly be characterized as "income breeders", and female mass can potentially be decoupled from fecundity to some extent. In the gift-giving green-veined white butterfly Pieris napi, it has been shown that female fecundity and longevity increase with number of matings and also that females mature at smaller size under poor food conditions compared to males. So it has been suggested that females can compensate for their smaller size through nuptial feeding. Here we test this hypothesis in P. napi by assessing female fecundity and longevity in relation to female mass and polyandry. The results showed no support for the hypothesis. Smaller females were not capable of increasing their mating rate to compensate for a low weight at eclosion. Instead, larger females remated sooner. Also, smaller females suffered from both a reduced daily and total fecundity compared to larger females and this decrease in fecundity was independent of female mating status, i.e. females allowed to mate only once and multiply mated females suffered to the same extent from their smaller size.     

Fallow deer polyandry is related to fertilization insurance

Polyandry is widespread, but its adaptive significance is not fully understood. The hypotheses used to explain its persistence have rarely been tested in the wild and particularly for large, long-lived mammals. We investigated polyandry in fallow deer, using female mating and reproduction data gathered over 10 years. Females of this species produce a single offspring (monotocous) and can live to 23 years old. Overall, polyandry was evident in 12 % of females and the long-term, consistent proportion of polyandrous females observed, suggests that monandry and polyandry represent alternative mating strategies. Females were more likely to be polyandrous when their first mate had previously achieved high numbers of matings during the rut or was relatively old. However, polyandry was not related to the following factors: female age, the stage of the rut, the dominance ranks of mates, or the number of daily matings achieved by males. Polyandrous and monandrous multiple-mating females were not more likely than single-mating females to be observed with an offspring during the following year, and there were no significant differences in offspring size between these females. These results provide support for a fertility insurance hypothesis, with females remating if fertilization from the first mating was uncertain due to possible sperm depletion. The potential for different female mating strategies among large, polygynous mammals has generally been overlooked. Our findings highlight the complexity of female reproductive strategies and the possible trade-offs between fertilization success, preferences for high-quality males, and potential costs of polyandry, particularly for monotocous species.     

Female benefit,male risk: Polyandry in the true armyworm Pseudaletia unipuncta

In Lepidoptera polyandry is common and females may increase their lifetime reproductive output through repeated matings if they acquire essential resources from male ejaculates. However, the paternity of males mating with previously-mated females is far from assured unless sperm precedence is absolute. In this study on the polyandrous armyworm, Pseudaletia unipuncta, we used two strains of male (the black-eyed wild type and a red-eyed homozygous, recessive mutant), mated with red-eyed females, to determine (i) whether male investment has any impact on female reproductive output, and (ii) if females do benefit from multiple matings, to what extent males fertilize the eggs to which they contributed. Multiple mating resulted in a significant increase in both the fecundity and longevity of females. However, the degree of sperm precedence (those eggs fertilized by the second male) varied from 0–100%, but was not affected by either male size or age, or by the duration of copulation. In cases where sperm precedence was <50% (x = 12%) females produced significantly more eggs (1384 versus 940) prior to the second mating than females where sperm precedence was >50% (x = 89%), indicating that the quality of the first mating influenced the fertilization success of the female's second mate.     

A female preference for experienced males in the almond moth, Cadra cautella

Male mating status can affect female reproductive output if male ejaculate investment declines over consecutive matings. Accordingly, females are predicted to mate preferentially with virgin males. In mildly polyandrous lepidopterans, female fitness is less affected by reduced male investment than in more polyandrous species, and so the predictions for female mating preferences are less clear. We examined female mating preferences in the mildly polyandrous almond moth, Cadra cautella, in which ejaculate size does not affect female reproductive output. First, we allowed females to mate with virgin or once-mated males, in which the males were presented individually or simultaneously. We recorded the latency to mating and, in the case of the simultaneously presented trials, the identity of the successful, copulating male. We found that females mated more frequently with mated males (when simultaneously presented with both males), yet females did not differ in the time taken to initiate copulation with any male. We further examined if this mated male advantage was due to differential mate detection or locomotory behaviour of the male treatments. We tested the ability of virgin and mated males to locate a receptive female within a wind tunnel using long-distance pheromone cues and recorded their activity budget. We found no difference in the ability of mated or virgin males to locate or approach a receptive female, or in their activity levels. These data suggest a female preference for mated males in this species, a preference that may minimise other potential costs of mating.     

Sperm competition and the level of polyandry in a bushcricket with large nuptial gifts

Variation in paternity due to sperm competition or post-copulatory female choice has a major influence on animal mating system evolution and on the levels of genetic variability in natural populations. However, there are relatively few studies comparing the outcome of sperm-competition experiments in the laboratory with natural variation in polyandry among families from the field. In the bushcricket Ephippiger ephippiger, females mate multiply, and the males provide them with a large, nutritious, and probably expensive, donation at mating. We examined paternity in a series of laboratory matings, where females mated with two males, and amongst a series of families collected from a natural population. In the laboratory, paternity was highly bimodally distributed: 24% of families had offspring fathered by the first male to mate, 68% by the second male (in only 8% was paternity shared). In the field, paternity was more mixed: only 27% of families had a single father, 14% had more than two fathers, whilst 59% had two fathers. While unsuccessful matings may contribute to the highly biased paternity in the laboratory, they cannot fully explain the high incidence of complete P2 families. Nonrandom sperm utilisation is therefore likely. Greater sperm mixing in the field probably results from females mating with more males, but the distribution of paternity also reflects an active process of nonrandom sperm utilisation. Confidence of paternity due to last male advantage may be relatively high in this species, and therefore may have facilitated the evolution of the large spermatophore in E. ephippiger.Communicated by D. Gwynne     

Polyandry and enhanced reproductive success in the harlequin-beetle-riding pseudoscorpion

The growing molecular evidence that females of many species mate with several males calls for a critical reassessment of the selective forces which act to shape female mating tactics. In natural populations of the harlequin-beetle-riding pseudoscorpion, Cordylochernes scorpioides, females are polyandrous and typically produce mixed-paternity broods. Laboratory behavioral analyses and breeding experiments indicate that polyandry in this pseudoscorpion is an active strategy which increases female reproductive success. Females restricted to mating with a single male experienced a higher rate of embryo failure and produced significantly fewer offspring than either females mated to more than one male in the laboratory or females naturally inseminated in the field. Forced copulation, insufficient sperm from a single mating, male nutrient donations and variation in inherent male genetic quality cannot explain the greater number of nymphs hatched by polyandrous females in this study. Evidence suggests that, by mating with several males, C. scorpioides females may exploit postcopulatory mechanisms for reducing the risk and/or cost of embryo failure resulting from fertilization by genetically incompatible sperm. Received: 5 May 1996 / Accepted after revision: 16 October 1996     

Sperm traits in the quacking frog (<Emphasis Type="Italic">Crinia georgiana</Emphasis>), a species with plastic alternative mating tactics

In species where males use alternative reproductive tactics and male phenotypes are confronted with different risks of sperm competition, theory predicts that between-male-type differences in sperm expenditure may evolve. In the frog Crinia georgiana big males can monopolize females, whereas small males often engage in polyandrous matings. Consequently, big males may experience a lower risk of sperm competition than do small males. We tested if the predictions from theoretical models can be applied to the mating system of C. georgiana. Our results showed that small males do not have larger testes relative to their body size compared to their larger counterparts and that the efficiency with which sperm number, size, motility, and longevity are produced by the testes does not differ between small and large males in the predicted way. These results are not in alignment with predictions from a loaded raffle model of sperm competition on sperm expenditure in males with alternative phenotypes. The plasticity in mating tactics used by C. georgiana males and a high intraseasonal variation in male densities may have prevented the evolution of enhanced sperm performance in smaller males. A fair raffle in the sperm competition game played by C. georgiana males could also explain the observed patterns in sperm traits. Future investigations determining the parameters responsible for the deviation from theoretical predictions in this system will test the degree to which current theoretical models can indeed be applied to species with plastic reproductive tactics.     

Facultative polyandry and the role of infant-carrying in wild saddle-back tamarins (Saguinus fuscicollis)

Summary Wild saddle-back tamarins (Saguinus fuscicollis) in southeastern Peru have a variable mating system that can differ both between territories at any one time and within territories over time. Groups are usually monogamous or cooperatively polyandrous, but are occasionally even polygynous. This study addressed the following questions: why does this population contain both monogamous and polyandrous groups simultaneously? What factors determine whether specific groups are monogamous or polyandrous? The data from this study population tentatively support the hypothesis that adults should mate monogramously only if they have nonreproductive helpers (usually older offspring) to help rear infants. Without helpers, the reproductive success of both males and females is hypothesized to be higher, on average, if they mate polyandrously than if they mate monogamously. The proposed benefits of polyandry to males and females differ quantitatively, but in both cases benefits stem from the help that males provide in rearing young. The following results support this hypothesis. (1) Lone pairs were never seen to attempt breeding, and calculations suggest that the costs of lactation and infant-carrying are too great for lone pairs to have a high probability of being able to raise twin offspring (the normal litter size). (2) Polyandrous males and nonreproductive offspring contributed substantially to infant care, particularly infant-carrying (fig. 2). (3) Adult males carried infants approximately twice as often as did lactating females, presumably because of the combined costs of (a) lactation (Fig. 3) and (b) infant-carrying (Fig. 4). The proximate causes of cooperative polyandry inS. fuscicollis appear to be different from those responsible in several bird species, showing that cooperative polyandry is a complex phenomenon.     

Sperm competition as a mechanism of female choice in the field cricket,Gryllus bimaculatus

Summary While traditionally viewed as an extension of intermale competition, mechanisms of sperm competition may be used by multiply mating females for mate choice. In the field cricket G. bimaculatus sperm were shown to mix in the spermatheca. The proportion of offspring sired by the second male increased with spermatophore attachment duration and, therefore, the number of sperm transferred. There was no second male advantage for single matings after an initial double mating. However, the proportion ofoffspring sired by the second male increased in proportion to the number of times he mated such that second males mating three times after an initial double mating had the advantage at fertilization. The data suggested that sperm were utilized in proportion to their numerical representation in the spermatheca. The mechanism of sperm precedence may, therefore, be one of sperm dilution. Female G. bimaculatus may control the degree of sperm competition as a mechanism of mate choice. By accepting large quantities of sperm from chosen males they may determine the paternity of their offspring by diluting out the sperm stored from previous matings.     

Effects of risks of sperm competition on the numbers of eupyrene and apyrene sperm ejaculated by the moth Plodia interpunctella (Lepidoptera: Pyralidae)

This study examines the effects of different risks of sperm competition upon ejaculate characteristics in the moth Plodia interpunctella. In this short-lived species, females will remate and thus generate sperm competition, while males have a limited sperm supply. We therefore predict males to have evolved prudence in ejaculate allocation and investigate the effects of (1) rival male presence, (2) female mating history and (3) female age, upon the ejaculation of eupyrene (fertilizing) and apyrene (non-fertile) sperm numbers. We found no effect of the presence of rival males upon ejaculate characteristics, and conclude, due to the mating system of P. interpunctella, that rival males do not represent a proximate risk of sperm competition. We controlled female mating history by allowing females to receive different and predictable numbers of sperm which they then store for at least 7 days. In subsequent matings (7 days later) we found that new males ejaculated significantly more eupyrene sperm to females that had previously received larger numbers of sperm. We conclude that males increase numbers of eupyrene sperm to maintain success in sperm competition with rival sperm already in storage in the female. We found no effect of female mating history upon the ejaculation of apyrene sperm. Female age, however, had a significant negative effect upon both sperm types. We discuss these results in relation to sperm competition theory and apyrene sperm function.     

High degree of multiple paternity in the viviparous Shiner Perch, <Emphasis Type="Italic">Cymatogaster aggregata</Emphasis>, a fish with long-term female sperm storage

The Shiner Perch (Cymatogaster aggregata Gibbons) exhibits a viviparous reproductive mode and long-term female sperm storage, two biological features that may predispose this fish species for both intense sperm competition and frequent multiple paternity within broods. To test these hypotheses, we used polymorphic microsatellite markers to identify sires and quantify paternal contributions to the progeny arrays of 27 pregnant females from a natural population of C. aggregata. The number of sires per brood ranged from one to eight (mean 4.6), typically with skewed distributions of fertilization success by the fathers but no correlation between sire number and brood size. The extraordinarily high incidences of multiple paternity in this species probably are due in part to high rates of mate encounter, but selection pressures related to the avoidance of maternal–fetal incompatibility may further have promoted the evolution of polyandrous mating behaviors in this female-pregnant species. Our genetic data are consistent with the hypothesis that viviparity, long-term sperm storage, and extreme polyandry are interrelated reproductive phenomena that should promote the evolution of post-copulatory sperm competition and/or cryptic female choice in these fishes.     

The benefits of multiple mating to female seaweed flies, Coelopa frigida (Diptera: Coelpidae)

Female seaweed flies, Coelopa frigida, have the potential to benefit from mating more than once. Single matings result in low fertility so females may benefit directly from multiple copulations by sperm replenishment. A chromosomal inversion associated with larval fitness, with heterokaryotypic larvae having higher viability than homokaryotypes, means that polyandrous homokaryotypic females have a higher probability of producing genetically fit offspring than monandrous homokaryotypic females. We allowed females to mate only once, repeatedly four times to the same male, or polyandrously four times to four different males. Multiply mated and polyandrous females laid more eggs and produced more offspring than singly mated and monandrous females, respectively. Polyandrous females laid more eggs, had higher egg-to-adult survival rates and produced more offspring than repeatedly mated females. Fertility rates did not differ between treatments. The observed fitness patterns therefore resulted from increased oviposition through multiple mating per se, and a further increase in oviposition coupled with higher egg-to-adult offspring survival benefits to polyandry. Daily monitoring of individual females over their entire life spans showed that multiple copulations induced early oviposition, with polyandrous females ovipositing earlier than repeatedly mated females. Singly mated and polyandrous females incurred a longevity cost independent of egg production, whereas repeatedly mated females did not. This suggests that repeatedly mating with the same male may counteract a general cost of mating. Longevity, however, was not correlated with overall female fitness. Our data are discussed in the overall context of the seaweed fly mating system.Communicated by G. Wilkinson     

Multiple mating in the ant Acromyrmex versicolor: a case of female control

In eusocial insects, polyandrous mating has the potential to reduce genetic relatedness of individuals within a colony, which may have a profound effect on the stability and social structure of the colony. Here we present evidence that multiple mating is common in both males and females of the desert leaf-cutter ant Acromyrmex versicolor. Females seem to have complete control over the number of matings, and mate on average with three males, even though the sperm transferred in a single copulation is sufficient to fill the spermatheca. To determine whether there is a bias in the representation of sperm from different mates in the spermatheca, females were mated to three or four males in controlled mating experiments and were subsequently allowed to found colonies in the laboratory. Paternity analysis of the offspring by random amplified polymorphic DNA analysis showed that all males that have been mated to a female successfully contributed sperm to the production of her offspring. No significant asymmetry in sperm use was detected, suggesting complete sperm mixing. Different hypotheses to explain polyandrous mating are discussed, and it is argued that the best hypothesis to explain polyandrous mating and complete sperm mixing in A. versicolor is that utilizing genetically diverse sperm confers a selective advantage on females. Received: 14 June 1995/Accepted after revision: 26 November 1995     

Sperm removal and ejaculate size correlate with chelae asymmetry in a freshwater crayfish species

Asymmetry in traits of sexual relevance may impair copulation behaviour and sexual performance of males, ultimately resulting in a fitness cost. Freshwater crayfish males use chelae, a sexually selected trait, to secure and position the female prior to and during mating. Thus, a relatively large chelae asymmetry, resulting from accidental loss and regeneration of one cheliped after autotomy, could have great consequences for male sexual behaviour. We studied copulatory behaviour and sperm expenditure of males paired to a mated female in Austropotamobius italicus, a freshwater crayfish species where both male and female mate multiply and where last-mating males are able to actively remove previously deposited sperm. We aimed at assessing whether male sperm removal and expenditure varied according to sperm allocated by first-mating males, and according to copulation behaviour and phenotypic traits (carapace length, chelae length and relative chelae asymmetry) of second-mating males. Second-mating males did not adjust their ejaculate size in relation to first-mating male ejaculate, nor to the first-mating male’s sperm removed. Moreover, the amount of sperm removed by second-mating males increased with increasing first-mating males ejaculate size, and first-mating male sperm remaining after removal did not correlate with the original first-mating male ejaculate size. Interestingly, the amount of sperm removed by second-mating males decreased with increasing relative chelae asymmetry, while increasing with male body size. However, second-mating (but not first-mating) asymmetric-clawed males produced larger ejaculates than symmetric-clawed ones. Importantly, the proportion of second-mating male sperm remaining after the two matings did not vary with relative chelae asymmetry nor with body size of second-mating males. Thus, small, asymmetric-clawed crayfish males appear to adopt sperm allocation tactics that allow them to fully compensate for their inferior sperm removal ability.     

Polyandrous mating in treetops: how male competition and female choice interact to determine an unusual carnivore mating system

The diversity of mammalian mating systems is primarily shaped by sex-specific reproductive strategies. In the present study, we explored determinants and consequences of a unique mating system exhibited by fossas (Cryptoprocta ferox), the largest Malagasy carnivore, where females mate polyandrously on traditional mating trees, and males exhibit intrasexual size dimorphism. Males face both contest and scramble competition, and inter-sexual size dimorphism can be pronounced, but its magnitude depends on the male morph. Using a continuous behavioral observation of six estrous females over 4 years, we investigated correlates of male contest competition and female choice based on 316 copulations. Furthermore, we assessed correlates of male scramble competition based on testes size and movement data obtained from GPS tracking. We found that females dominated males regardless of their smaller size and that females actively solicited copulations. Heavy males had highest mating success during the female’s peak mating activity, but were discriminated against afterwards. Female choice and male–male competition thus converged to generate a mating advantage for heavier males. Our results suggest that females actively seek polyandrous matings, presumably for indirect genetic benefits. Since body mass is the major determinant of male mating success and is at the same time dependent on the degree of sociality and associated hunting mode of the respective male morph, a male’s feeding ecology is likely to influence its reproductive tactic. A combination of benefits from female polyandry and the consequences of different subsistence strategies may thus ultimately explain this unusual mating system.