Spermatophore and Sperm Allocation in Males of the Monandrous Butterfly Pararge aegeria: the Female’s Perspective

In insects, spermatophore production represents a non‐trivial cost to a male. Non‐virgin males have been shown to produce small spermatophores at subsequent matings. Particularly in monandrous species, it may be an issue to receive a sufficiently large spermatophore at the first and typically only mating. Females of the monandrous Speckled wood butterfly Pararge aegeria (L.) produce fewer offspring after mating with a non‐virgin male. After mating, females spend all their active time selecting oviposition sites and typically ignore other males. Here, we show that females did not discriminate between a virgin male and a recently mated male in our laboratory experiments. We demonstrate that the number of eupyrene sperm bundles relative to spermatophore mass differed with subsequent male matings. Males transferred a significantly smaller spermatophore after the first copulation, but the spermatophore mass did not decrease further with subsequent matings. However, the number of eupyrene sperm bundles decreased linearly. Therefore, there was proportionally more eupyrene sperm in the male’s second spermatophore compared with the first and the later spermatophores. Such a pattern has been shown in polyandrous species. Hence, it suggests that differences in sperm allocation strategy between polyandrous and monandrous butterflies may be quantitative rather than qualitative. There was also a tendency for females that had mated with a recently mated male to have higher propensity to remate than did females that had mated with a virgin male. We discuss the results relative to the mating system in P. aegeria, including female remating opportunities in the field and male mate‐locating behaviour.     

Sperm Numbers and Female Fertility in the Moth Plodia interpunctella (Hübner) (Lepidoptera; Pyralidae)

I investigated two possible reasons for remating in female Plodia interpunctella: i) females remate to obtain sufficient sperm to maintain fertility; and ii) male investment in non-sperm components increases female fecundity and longevity. The number of sperm and the mass of the spermatophore transferred by males decreases on successive matings. Sperm numbers and potential male investment were varied by allowing females to mate either once or twice with males either on their first or second mating. Females receiving a single small spermatophore containing few sperm (from a male on his second mating) had sufficient sperm to fertilize all their eggs. Females did not show increased fecundity or longevity as a result of obtaining more spermatophore material. I discuss why females remate when they already have sufficient sperm to fertilize all their eggs.     

Sperm transfer during mating, movement of sperm in the female reproductive tract, and sperm precedence in the common cutworm Spodoptera litura

Abstract. Mating behaviour, sperm transfer and sperm precedence were studied in the moth Spodoptera litura (Fabr.) (Lepidoptera: Noctuidae). There existed a rhythmic, diel pattern of mating behaviour of this moth during the scotophase, presumably set with respect to an endogenous activity rhythm. Approximately 30 min after copulation had started, the formation of the corpus of the spermatophore began in the bursa copulatrix of the female moth, but full inflation of the corpus was not completed until 45–60 min after mating had started. The mature spermatophore contained about 350 eupyrene sperm bundles and a large number of individual (loose) apyrene spermatozoa. The mating status and the age of the male insect influenced the number of sperm transferred to the female within the spermatophore, and also affected the consequent fertility. There was no evidence of sperm reflux within the male tract. Within the female, dissociation of eupyrene sperm bundles was evident within the spermatophore less than 15 min after the completion of mating. Spermatozoa began to move from the bursa (in which the spermatophore is lodged) into the spermatheca 30–45 min after the end of the copulation, and the quantity of sperm in the spermatheca reached a plateau at 90 min after mating. Apyrene sperm reached the spermatheca first, followed by eupyrene sperm. Examination of total (apyrene plus eupyrene) sperm in the female tract showed that 86% of mated females received an apparently normal amount of total sperm from the male. Examination of eupyrene sperm alone showed that 81% of matings resulted in an apparently normal transfer of eupyrene sperm. A small proportion (approximately 8%) of the matings, however, were identified as transferring a clearly subnormal quantity of eupyrene sperm to the spermatheca. The eggs produced as a result of such pairings displayed much reduced fertility (about 43%) compared to those from matings confirmed to have transferred normal quantities of sperm, which showed about 92% fertility. This shows that the availability of eupyrene sperm in the spermatheca may be an important constraint on fertility in normal populations of insects. In the laboratory, S. litura females exhibited multiple matings. Of the females, 93% mated, and the mean frequency of mating was 1.69. Mating with a fertile male led to the oviposition of an increased number of eggs. This effect continued even when the female subsequently mated with an infertile male. Displacement of sperm from previous matings is known to be an important factor in the evolution of multiple mating strategies. Our results on sperm utilization by S. litura indicated that after a second mating, the sperm utilized for subsequent fertilization were almost exclusively from the last mating with little mixing. The proportion of eggs fertilized by sperm from the second mating (P₂) was calculated as 0.95, indicating almost complete sperm precedence from the last mating.     

Determinants of paternity in a butterfly

Success in sperm competition is of fundamental importance to males, yet little is known about what factors determine paternity. Theory predicts that males producing high sperm numbers have an advantage in sperm competition. Large spermatophore size (the sperm containing package) also correlates with paternity in some species, but the relative importance of spermatophore size and sperm numbers has remained unexplored. Males of the small white butterfly, Pieris rapae (Lepidoptera: Pieridae), produce large nutritious spermatophores on their first mating. On their second mating, spermatophores are only about half the size of the first, but with almost twice the sperm number. We manipulated male mating history to examine the effect of spermatophore size and sperm numbers on male fertilization success. Overall, paternity shows either first male or, more frequently, second male sperm precedence. Previously mated males have significantly higher fertilization success in competition with males mating for the first time, strongly suggesting that high sperm number is advantageous in sperm competition. Male size also affects paternity with relatively larger males having higher fertilization success. This may indicate that spermatophore size influences paternity, because in virgin males spermatophore size correlates with male size. The paternity of an individual male is also inversely correlated with the mass of his spermatophore remains dissected out of the female. This suggests that females may influence paternity by affecting the rate of spermatophore drainage. Although the possibility of female postcopulatory choice remains to be explored, these results clearly show that males maximize their fertilization success by increasing the number of sperm in their second mating.     

Delayed mating and its cost to female reproduction in the butterfly,Eurema hecabe

The timing of mating of females under semi-natural condition, male ejaculate production and their effects on female fecundity were examined inEurema hecabe. Age of the first mating of females varied, and the number of matings increased with age. Male spermatophore production depended on age and body mass. The spermatophore mass at the second mating depended only on the interval between the first and second matings. The timing of the first mating and the spermatophore mass did not affect female fecundity. The timing of mating of females relative to the role of male spermatophores in female fecundity and male mating strategy are discussed.     

Strategic sperm allocation in the Small White butterfly Pieris rapae (Lepidoptera: Pieridae)

1. In species where females mate multiply, it is important for males to recuperate quickly in order to maximize their fertilization success. Butterflies produce a spermatophore at mating containing accessory secretions and sperm of two types: a large number of non-fertile 'apyrene' sperm and fewer fertile 'eupyrene' sperm. Many butterfly species eclose with most nutrients for reproduction already present. Males must therefore decide how to allocate resources to the various spermatophore components at any given mating.
2. Recovery rates of apyrene and eupyrene sperm number and spermatophore size was studied in the polyandrous Small White butterfly Pieris rapae . The mass of the first spermatophore increases with time since eclosion, as does the number of both types of sperm. Similarly, on a male's second mating, both the mass of the spermatophore and the number of sperm increases with time since the first mating.
3. However, the rate of increase in eupyrene sperm numbers is higher after the first mating. The difference in rate of increase may be the result of different probabilities of virgin and non-virgin males obtaining future matings.
4. Males have a sperm storage organ, the duplex, in which they retain sperm after their first mating. This ensures that high sperm numbers are available for their second mating, even when remating only 1 h later. Thus, males do not ejaculate all available sperm on any given mating, and seem to have different strategies on their first and second matings.
5. It can be argued that Small White butterfly males allocate sperm strategically according to the probability of obtaining subsequent matings, and the level of sperm competition.     

The Effect of Repeated Matings on Sperm Numbers in Successive Ejaculates of the Cabbage White Butterfly Pieris rapae (Lepidoptera: Pieridae)

The effect of repeated matings on sperm numbers in successive ejaculates of the cabbage white butterfly, Pieris rapae, was examined. First ejaculates were larger than successive ones, which did not differ among themselves. Moreover, the cumulative mass of previous spermatophores was not correlated with that of the last mating. The number of eupyrene sperm bundles in the ejaculate did not differ between first and successive matings. Multiplying by 256, a male transfers about 11,000 eupyrene sperm at every mating. First ejaculates contained about 46,000 apyrene sperm, whereas successive ejaculates contained higher numbers. The sperm density increased after the first mating, though the spermatophore mass decreased. The significance of change in sperm quantity with mating number is discussed from the viewpoint of male investment.     

Effects of male mating interval on spermatophore formation,transfer, and subsequent female receptivity and fecundity in the sorghum plant bug,Stenotus rubrovittatus

Males of the sorghum plant bug, Stenotus rubrovittatus (Matsumura) (Heteroptera: Miridae), transfer a spermatophore to females during copulation. After a 1‐day interval between the first and second copulation, males transferred both sperm and a spermatophore to females during the second copulation. However, when male mating interval was <1 h, they transferred sperm but no spermatophores to females during the second copulation. Therefore, the male mating interval probably produces two types of mated females, those with and those without a spermatophore. Mated females of S. rubrovittatus do not remate for at least 3 days after mating, even when courted, and lay more eggs than virgin females at the beginning of the oviposition period. The effects of spermatophores on female sexual receptivity and fecundity were examined using mated females with or without a spermatophore. Only one of the 40 (2.5%) mated females with a spermatophore remated, whereas 10 of the 26 (38.5%) without a spermatophore remated. Furthermore, mated females with a spermatophore laid more eggs than those without a spermatophore. These results suggest that spermatophores participate in reducing female sexual receptivity and enhancing female fecundity in S. rubrovittatus.     

When do the Costs of Spermatogenesis Constrain Sperm Expenditure? Remarks on the Pattern of the Spermatogenic Cycle

The costs of spermatogenesis constrain sperm expenditure when sperm production per day is limited. Thus, males are challenged to allocate available resources to sperm production and other life history functions. However, this prevailing assumption is not applicable to species in which spermatogenesis becomes quiescent during the breeding season. Males of these species prepare large quantities of sperm before the breeding season. Among these species, constraints on ejaculates have been intensively investigated in salamanders that deposit spermatophores. Although it is predicted that sperm expenditure should not be limited because of abundantly prepared sperm, spermatophore deposition is often limited during the breeding season when vas deferens are full of sperm. We tested a hypothesis regarding limited spermatophore deposition by measuring sperm quantity and volume of spermatophores sequentially deposited by male eastern newts Notophthalmus viridescens. A male newt rarely deposits more than three spermatophores per mating. If depletion of non‐sperm components of spermatophores limits spermatophore deposition, we predicted that spermatophore volume decreases while sperm quantity remains constant as a male deposits more spermatophores. Alternatively, some regulative mechanisms allow a limited portion of available sperm to be expended per mating, in which sperm quantity is predicted to decrease while the spermatophore volume remains constant. Finally, depletion of non‐sperm components may regulate sperm expenditure, which predicted that both spermatophore volume and sperm quantity decrease. We found that both sperm quantity and the spermatophore volume decreased as a male deposited more spermatophores during a single mating. Sperm expenditure was constrained without the costs involved in active spermatogenesis, and depletion of non‐sperm components likely regulate sperm quantity loaded in spermatophores. In dissociated spermatogenesis, constrained sperm expenditure do not mean that costly spermatogenesis is directly limiting male mating capacity but rather suggest that the evolution of physiological mechanisms regulating sperm expenditure per mating maximizes male reproductive success.     

Last–male sperm precedence in the bushcricket Poecilimon veluchianus (Orthoptera, Tettigonioidea) demonstrated by DNA fingerprinting

Males of the bushcricket Poecilimon veluchianus pass a large spermatophore to the female during mating. The spermatophore is eaten by the female after copulation. Because females mate with several males during their reproductive life, the competition between spermatozoa of different males affects a male's reproductive success. In order to determine the outcome of sperm competition, the paternity of the progeny of double–mated females was established by DNA fingerprinting with the oligonucleotide (GATA)₄. Typical P. veluchianus DNA fingerprints consisted of 15 scoreable fragments per individual. The proportion of bands shared between presumably unrelated bushcrickets was 17%. After the second copulation the second mating male clearly predominated at fertilization. The mean proportion of eggs fertilized by the second male was 90.1%. There was no significant relationship between the level of sperm precedence and the time of ovipositions after the second mating. If female P. veluchianus increase the fitness of their offspring by the incorporation of spermatophore–derived substances in developing eggs, there is little chance for the feeding male to fertilize eggs containing his nutrients, because of the very short mating intervals of females and the observed high level of last–male sperm precedence in this species. Under such conditions the last mating male would fertilize many eggs containing nutrients from a prior male. Because nuptial gifts, like the tettigoniid spermatophore, function only as paternal investment if the donating male's progeny benefit from the gift, a paternal investment function of the P. veluchianus spermatophore seems to be unlikely.     

Multiple mating and lifetime reproductive output in female dobsonflies that receive nuptial gifts

In mating of the dobsonfly, Protohermes grandis (Thunberg), the male attaches the spermatophore externally to the female genitalia. The spermatophore includes a large gelatinous mass which the female detaches and feeds on after mating. While the female consumes this nuptial food gift, sperm is evacuated from the remaining portion of the spermatophore (sperm package) into her reproductive tract. Under laboratory conditions, mated females maintained receptivity throughout their lifetime, and they remated even on the day following copulation. A single insemination may supply enough sperm, as females mated only once deposited fertile eggs throughout life and, when dissected after death, all females had sperm in the spermatheca. There was a positive correlation between longevity and the number of matings. Lifetime fecundity also increased as mating multiplied. However, the size of eggs and hatchlings was not influenced by the number of matings. It seems that large spermatophore consumption by female P. grandis provides nutrients that increase fitness not in offspring quality, but in their quantity.     

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.     

Limits to Nuptial Gift Production by Male Fireflies, Photinus ignitus

Males of diverse insect species provide females with nuptial gifts, and limits on males' ability to produce these gifts may influence courtship behavior and mating systems. In the firefly Photinus ignitus, males transfer a complex spermatophore to females during mating. We provided firefly males unlimited access to responsive females to examine whether spermatophore production limits male mating success. Male spermatophore mass decreased significantly across sequential matings, and the percentage of successful matings declined during the second half of each male's life span. Male body mass explained a significant proportion of variation in size of the first spermatophore produced by P. ignitus males, but this relationship disappeared with second spermatophores. This study indicates that males' ability to produce spermatophores declines over their lifetime and that limits on nuptial giftproduction can constrain male mating success in Photinus fireflies.     

Resolving mechanisms of short-term competitive fertilization success in the red flour beetle

Postcopulatory sexual selection occurs when sperm from multiple males occupy a female’s reproductive tract at the same time and is expected to generate strong selection pressures on traits related to competitive fertilization success. However, knowledge of competitive fertilization success mechanisms and characters targeted by resulting selection is limited, partially due to the difficulty of discriminating among sperm from different males within the female reproductive tract. Here, we resolved mechanisms of competitive fertilization success in the promiscuous flour beetle Tribolium castaneum. Through creation of transgenic lines with fluorescent-tagged sperm heads, we followed the fate of focal male sperm in female reproductive tracts while tracking paternity across numerous rematings. Our results indicate that a given male’s sperm persist and fertilize eggs through at least seven rematings. Additionally, the proportion of a male’s sperm in the bursa (the site of spermatophore deposition), which is influenced by both timing of female’s ejecting excess sperm and male size, significantly predicted paternity share in the 24 h following a mating. Contrary to expectation, proportional representation of sperm within the female’s specialized sperm-storage organ did not significantly predict paternity, though spermathecal sperm may play a role in fertilization when females do not have access to mates for longer time periods. We address the adaptive significance of the identified reproductive mechanisms in the context of T. castaneum’s unique mating system and ecology.     

Impact of male condition on his spermatophore and consequences for female reproductive performance in the Glanville fritillary butterfly

In butterflies, male reproductive success is highly related to the quality and the size of the spermatophore transferred to the female. The spermatophore is a capsule produced by the male during copulation, which in many species contains sperm in addition to a nuptial gift, and which is digested by the female after copulation. The nuptial gift may contribute to egg production and offspring quality, and in some cases also to female body maintenance. The production of the spermatophore, however, represents a cost for the male and, in polyandrous species, ejaculates are sometimes allocated adaptively across matings. Nonetheless, although the ecological factors affecting the reproductive success of female butterflies have been the topic of numerous studies, little information exists on the factors affecting males’ contribution to reproduction, and the indirect impacts on female fecundity and fitness. We used the Glanville fritillary butterfly, Melitaea cinxia (Linnaeus, 1758) (Nymphalidae), in order to assess variation in male allocation to matings. In this species, smaller males produce smaller spermatophores, but variation in spermatophore size is not correlated with female reproductive success. We show that spermatophore size increases with male age at first mating, decreases with mating frequency and adult food‐deprivation, and is not influenced by developmental food‐limitation. The length of copulation period does not influence the spermatophore size nor influences the polyandrous mating behavior in this species. Male contribution to his spermatophore size is clearly influenced by his condition and adult‐resource at the time of mating. Despite this variation, spermatophore size does not seem to have a direct impact on female reproductive output or mating behavior.     

Eupyrene sperm migrates to spermatheca after apyrene sperm in the swallowtail butterfly, Papilio xuthus L. (Lepidoptera: Papilionidae)

When swallowtail butterflies, Papilio xuthus, are mated by the hand-pairing method, both types of sperm, eupyrene and apyrene sperm, are transferred from the male to the spermatheca via the spermatophore in the bursa copulatrix. This mechanism is demonstrated by two different kinds of experiments. The first set of experiments employed interrupted copulation, and the second set was examination of the sperm in the spermatophore and spermatheca after the termination of copulation. The sperm was transferred 30 min after the start of copulation. The eupyrene sperm was still in the bundle; the number of the bundles ranged from 9 to 108 (mean, 42.7; n = 27). The bundles were gradually released after the completion of copulation, and the free eupyrene spermatozoa then remained in the spermatophore at least 2 h before migrating to the spermatheca. On the other hand, about 160 000 apyrene spermatozoa were transferred to the spermatophore and remained there for more than 1 h. We observed 11 000 apyrene spermatozoa in the spermatheca 12 h after the completion of copulation, but most of this type of sperm disappeared shortly thereafter. In contrast, the eupyrene sperm arrived in the spermatheca more than 1 day after the completion of copulation and remained there at least 1 week. Therefore, these findings suggest that apyrene sperm migrate from the spermatophore to the spermatheca earlier than eupyrene sperm. Accordingly, if females mated multiply, the time difference might avoid the mixing of sperm. In addition, the predominance of sperm from the last mating session may occur not in the bursa copulatrix but in the spermatheca. Received: January 7, 2000 / Accepted: May 24, 2000     

The outcome of sperm competition is affected by behavioural and anatomical reproductive traits in a simultaneously hermaphroditic land snail

Sperm competition is important in species with reproductive strategies that involve multiple mating and prolonged sperm storage such as the simultaneously hermaphroditic land snail Cornu aspersum. Double mating trials in this species have revealed that mating order and courtship behaviour affect paternity success. We investigated the effect of behavioural and anatomical reproductive traits on paternity success from triple mating trials. Triple mating resulted in triple fertilization in 58% of the cases whereas zero paternity was observed in 16% of sperm donors. Third sperm donors achieved higher paternity followed by first and second sperm donors. Snails with a longer epiphallus, the spermatophore forming organ, sired more offspring regardless of their mating order. Genetic compatibility between sperm donor and recipient did not influence paternity success. The results of the present study identified mating order and epiphallus length, as traits affecting the outcome of sperm competition in this species.     

Variations in sperm number in relation to larval crowding and spermatophore size in the armyworm, Pseudaletia separata

1. Number of sperm and its relationship with larval rearing density were investigated in the armyworm Pseudaletia separata . Males that emerged from crowded larvae produced significantly more apyrene sperm than those from solitary larvae (375 700 ± 116 600 and 290 300 ± 99 600 at a mating with a 3-day old virgin, respectively), with no significant difference in number of eupyrene sperm between the two types being observed.
2. For both solitary- and crowded-type, the amount of fertile sperm the males produced at a mating exceeded the number needed to fertilize all of a female's eggs, suggesting that sperm competition may be a major selective force for keeping sperm numerous. The production of more apyrene sperm by crowded-type males may be an adaptation to cope with the increased sperm competition from rival males at high density.
3. The relationship between number of sperm and spermatophore size was also studied using solitary-type moths. Large spermatophores were found to have more eupyrene and apyrene sperm than small ones.     

Multiple matings increase the fecundity of the yellow swallowtail butterfly,Papilio xuthus L., in summer generations

Females of the yellow swallowtail butterfly, Papilio xuthus,were reared in the laboratory. They were divided into four groups held under different mating conditions: nonmating (virgin) and mated once, twice, and three times. The number of eggs in the ovaries was counted by dissection. Virgin females produced increasing numbers of mature eggs, up to about 30, in the week following emergence. When the female had mated once, the number of mature eggs was significantly higher than that of virgin females by the second day after emergence. However, the double- and triplemated females did not increase the number of eggs in each state further than the singlemated females. The double-mated females deposited significantly more eggs than the singlemated females in the laboratory. The triplemated females also deposited more eggs on the day after the third mating than the doublemated females. Thus, multiple matings increased the number of eggs deposited. The change in the hatchability and the morphology of the spermatophore in the bursa copulatrix suggested that the sperm from the last mating had precedence.     

Motility and dynamics of eupyrene and apyrene sperm in the spermatheca of the monandrous swallowtail butterfly Byasa alcinous

Lepidopteran males produce two sperm types: nucleated eupyrene sperm and non‐nucleated apyrene sperm. Although apyrene sperm are infertile, both sperm types migrate from the spermatophore to the spermathecal after copulation. As a dominant adaptive explanation for migration of apyrene sperm in polyandrous species, the cheap filler hypothesis suggests that the presence of a large number of motile apyrene sperm in the spermatheca reduces female receptivity to re‐mating. However, apyrene sperm are also produced in males of the monandrous swallowtail butterfly Byasa alcinous Klug. To identify the role of apyrene sperm in these males, the present study examines the number of spermatozoa produced and transferred and the dynamics and motility of spermatozoa in the spermatheca for each type of sperm. Apyrene sperm represents approximatey 89% of the sperm produced and transferred, which is comparable to polyandrous species. Two‐day‐old males transfer approximately 17 000 eupyrene and 230 000 apyrene spermatozoa to a spermatophore; approximately 5000 eupyrene and 47 000 apyrene spermatozoa arrive at the spermatheca. Eight days after copulation, most eupyrene spermatozoa remain in the spermatheca and a quarter of them are still active. However, the number of apyrene spermatozoa decreases and those remaining lose their motility after the arriving at the spermatheca. Consequently, 8 days after copulation, no motile apyrene sperm are found. The high proportion of apyrene sperm in the spermatophore, as well as in sperm migration, suggests that the production and migration of apyrene sperm is not simply an evolutionary vestigial trait. The possible functions of apyrene sperm in monandrous species are discussed.