Size-assortative mating and non-reciprocal copulation in a hermaphroditic intertidal limpet: test of the mate availability hypothesis

Mating behaviour and mating patterns are affected to a large extent by body size in both hermaphrodites and gonochorists. Detailed research on mating patterns, mate choice, pre- and post-copulatory sexual selection in marine pulmonates is wanting, thus warranting more attention as a study system in the future. The simultaneous hermaphroditic limpet Siphonaria capensis (Pulmonata: Basommatophora) shows size-dependent fecundity, and acts as a suitable organism to test the effects of body size on mating patterns, mating success, gender expression and reciprocity in hermaphroditic marine gastropods. We mainly used bootstrap resampling techniques to estimate the effects of different factors on mating patterns. In the populations studied, a strong size-assortative mating pattern was observed, where small-scale spatial distribution of potential mating partners (the mate availability hypothesis) could explain 65% of this pattern, while mate choice and mating constraints explaining the rest. No significant difference in mating success between limpets with different body size was found, even though in one population, the sperm recipients were larger than the non-copulating limpets. Interestingly, we found that intromission was non-reciprocal during copulations. This may mean that this species does not conform to the common rule of reciprocity predicted for hermaphrodites, unless there is sex-role alternation between individuals in a mating pair. The mating partners consisted of similar sized, acting males and females, thus without any indication of the body size determining the gender expression. The matings took place in early mornings only at spring tides and the animals were observed laying egg masses only during neap tides. Since siphonariid limpets possess both a spermatheca and a seminal vesicle, which may have either a sperm storing and/or digestive function, post-copulatory sexual selection (e.g., sperm competition) cannot be dismissed.     

Body size preferences in the pot-bellied seahorse <Emphasis Type="Italic">Hippocampus abdominalis:</Emphasis> choosy males and indiscriminate females

Male seahorses (genus Hippocampus) provide all post-fertilization parental care, yet despite high levels of paternal investment, these species have long been thought to have conventional sex roles, with female mate choice and male–male competition. Recent studies of the pot-bellied seahorse (Hippocampus abdominalis) have shown that sex-role reversal occurs in high-density female-biased populations, indicating that male mating preferences may lead to sexual selection on females in this species. Egg size, egg number, and offspring size all correlate positively with female body size in Hippocampus, and by choosing large mating partners, male seahorses may increase their reproductive success. While male brood size is also positively correlated with body size, small H. abdominalis males can carry exceptionally large broods, suggesting that the fecundity benefits of female preference for large partners may be limited. We investigated the importance of body size in reproductive decisions of H. abdominalis, presenting focal individuals of both sexes with potential mating partners of different sizes. Mating preferences were quantified in terms of time spent courting each potential partner. Male seahorses were highly active throughout the mate-choice trials and showed a clear behavioral preference for large partners, while females showed significantly lower levels of activity and equivocal mating preferences. The strong male preferences for large females demonstrated here suggest that sexual selection may act strongly on female body size in wild populations of H. abdominalis, consistent with predictions on the importance of female body size for reproductive output in this species. An erratum to this article can be found at     

Multiple mating affects offspring size in the opisthobranch Chelidonura sandrana

Offspring size can have pervasive effects throughout the life history stages of many marine invertebrates. Although maternal offspring investment is largely determined by the environmental conditions experienced by the mother, egg size might additionally vary in response to the number and quality of previous mating partners. Positive effects of mating multiply with several different males (polyandry) have been confirmed for a variety of species, whereas such investigations are lacking for marine invertebrates. Here we differentiated between the effects of ejaculate amount (repeatedly mated) and ejaculate diversity (polyandry) on maternal offspring investment in the simultaneously hermaphroditic sea slug Chelidonura sandrana. We found that focal “females” mated with four different “males” produced significantly larger egg capsules and larger veligers, while focal “females” mated four times with the same “male” suffered from reduced mid-term fecundity. We found no effect of veliger size on veliger survival. Our results show that female mating patterns are an important addition to understanding the variation in offspring size in internally fertilizing marine invertebrates.     

Hermaphrodite sex role preferences: the role of partner body size, mating history and female fitness in the sea slug Chelidonura sandrana

Costs and benefits associated with matings and the effects of mating frequency on fitness commonly differ between the sexes. As a result, outcrossing simultaneous hermaphrodites may prefer to copulate in the more rewarding sex role, generating conflicts over sperm donation and sperm receipt between mates. Because recent sex role preference models remain controversial, we contrast here some of their assumptions and predictions in the sea slug Chelidonura sandrana. For this hermaphrodite with sperm storage and internal fertilisation, risk-averse models assume that fitness pay-offs are constantly higher in the female than in the male function in any single mating. Moreover, excluding mutual partner assessment, these models predict male mating behaviour to be independent of receiver traits. The competing gender ratio hypothesis assumes that relative fitness pay-offs, and thus the preferred mating roles, vary and may reverse between matings and predicts that ejaculation strategies co-vary with receiver quality. We found that field mating rates of C. sandrana substantially exceeded what is required to maintain female fertility and fecundity, indicating large variation in direct female benefits between matings. We further demonstrate that male copulation duration adaptively increased with partner body size (i.e. fecundity) but decreased with recent partner promiscuity. These findings are compatible with the gender ratio hypothesis but contradict risk-averse models.     

Sperm exchange in a simultaneous hermaphrodite

Many simultaneously hermaphroditic animals cross-inseminate when copulating, even though unilateral sperm exchange is functionally possible. The question is why reciprocity is the rule. This study addresses sperm exchange in a hermaphroditic planarian flatworm, Dugesia polychroa, in which self-fertilisation does not occur. We investigated the availability of self sperm for inseminating a partner, how self sperm affects the likelihood of sperm donation, and whether sperm donation depends on reciprocation by the partner. The amount of self sperm depended on body size, duration of isolation and experimental date, and animals that had more self sperm were more likely to mate. Depletion of allosperm (sperm received from partners) played only an indirect role, suggesting that animals copulate more to donate sperm rather than to replenish allosperm reserves. Among 60 copulating pairs, reciprocal insemination was more common (63.3%) than unilateral transfer (18.3%). A surprising 18.3% did not exchange sperm in either direction. Individuals were more likely to behave like their partners, resulting in more symmetrical matings (either reciprocity or no sperm exchange) than asymmetrical matings (unilateral sperm donation). The amount of self sperm donated during a copulation depended mainly on the amount available before the copulation and not on the amount received from the partner. The results suggest that conditional sperm exchange, or sperm trading, takes place in D. polychroa. Received: 31 May 1997 / Accepted after revision: 1 December 1997     

The role of body size in early mating behavior in a simultaneous hermaphrodite, <Emphasis Type="Italic">Chelidonura sandrana</Emphasis>

Intraspecific variation in mating behavior is widespread among simultaneous hermaphrodites but its underlying sources remain largely unexplored. In the sea slug Chelidonura sandrana, most matings are reciprocal. However, despite non-conditional sperm exchange and potential polygamy-mediated benefits, 30% of matings end after unilateral insemination. To resolve this apparent inconsistency, we here investigated the effect of body size on the frequency of reciprocal matings by testing the following two hypotheses. First, sex-allocation theory predicts that the likelihood of reciprocity depends on the size difference between mating partners. Second, if both sex functions temporally differ in reaching maturity, reciprocal matings should be more frequent with increasing absolute body size of the smaller partner. The likelihood of reciprocity increased with body size of the smaller partner. Moreover, smaller individuals acted more often as males among unilateral matings. These findings suggest that the ability to donate sperm develops prior to female functionality in C. sandrana.     

Sperm allocation in relation to male traits, female size, and copulation behaviour in freshwater crayfish species

Sperm competition is a well-recognised agent in the evolution of sperm and ejaculate structure, as well as variation in female quality. Models of the evolution of ejaculate expenditure predict that male body condition, female fecundity and the risk and intensity of sperm competition may be the ultimate factors shaping optimal ejaculate size. We investigated sperm allocation in Austropotamobius italicus, a freshwater crayfish exhibiting a coercive mating system and external fertilisation, in relation to male and female traits and copulation behaviour under laboratory conditions. We found that mating males were sensitive to female size and produced larger ejaculates when mating with larger females, which were more fecund in terms of number of eggs produced. We found no evidence for female egg production being sperm-limited, as the number of eggs was not dependent on male sperm expenditure. Copulation duration and number of ejaculations reliably predicted the amount of sperm transferred, and both these behavioural measures positively covaried with female body size. These results indicate that male freshwater crayfish can modulate their sperm expenditure in accordance with cues that indicate female fecundity. In addition, a novel finding that emerged from this study is the decrease in sperm expenditure with male body size, which may either suggest that large, old male crayfish are better able than small males to economise sperm at a given mating to perform multiple matings during a reproductive season, or that they experience senescence of their reproductive performance.     

Male lagoon gobies, <Emphasis Type="Italic">Knipowitschia panizzae</Emphasis>, prefer more ornamented to larger females

Female ornamentation may be directly sexually selected, by male choice or female competition, or occurs as the result of a genetic correlation, arising from sexual selection on males. However, increasing evidence supports the former hypothesis, suggesting that males actively choose their partner preferring traits indicative of female quality. In the lagoon goby, Knipowitschia panizzae, a polygynous species whose males perform parental care to eggs, body length and the size of a sex-specific yellow patch on the belly are known to be reliable indicators of female fecundity. In this paper, we tested, using dummies, the male’s mating preferences for female body and yellow belly patch sizes. The two experimental trials in which a single female trait was variable showed that males prefer a larger belly patch and a larger body size, indicating that both these characters are selected by male mate choice. However, when faced with dummies exhibiting an inverse combination of body and belly patch sizes (experiment 3), males significantly preferred the smaller ones with larger yellow belly patches. A calculation of dummy theoretical fecundity reveals that in the first two experiments, males would have received an immediate benefit from their choice in terms of egg number, whereas in the third one, males chose partners that would have provided them with fewer eggs. The male lagoon goby preference for females with larger belly patches, regardless of their size, suggests that this trait, in addition to indicating fecundity, conveys information about other aspects of female and/or egg quality.     

Large donors transfer more sperm,but depletion is faster in a promiscuous hermaphrodite

Males are limited in sperm and seminal fluid, which can lead to prudent sperm allocation across consecutive matings. While sperm depletion is usually considered a population characteristic, we investigated an individual size-related variation in sperm depletion in the promiscuous sea slug Chelidonura sandrana. We found that sperm counts substantially declined across four successive copulations, indicating sperm depletion. Sperm depletion occurred at a disproportionally fast rate in larger sperm donors. Based on our finding of body size-dependent sperm depletion, we further investigated whether this translates into prudent sperm allocation across ejaculates. We found no evidence that slugs adjusted the number of delivered sperm in response to partner body weight, a tight proxy of fecundity. Instead, sperm counts increased with sperm donor body weight. Sperm donation independent of partner body size and—as found in previous work—partner novelty leaves scope for currently unidentified traits like the sperm competition environment to affect male sperm allocation. Alternatively, we propose that, in C. sandrana, a low optimal male mating rate close to the female optimum as indicated by our data may ultimately render indiscriminate sperm allocation the most beneficial male mating strategy.     

Influence of male harassment and female competition on female feeding behaviour in a sexual–asexual mating complex of mollies (<Emphasis Type="Italic">Poecilia mexicana</Emphasis>, <Emphasis Type="Italic">P. formosa</Emphasis>)

Differential interests between the sexes regarding the number of copulations can result in sexual harassment. Hence, females may have less time available for foraging. Male sexual harassment often leads to fitness reduction in females. We used the mating complex of the bisexual fish Poecilia mexicana and the co-occurring all-female Poecilia formosa to study sexual harassment and its incurred cost on female feeding efficiency. P. formosa is a sperm-dependent parthenogen that requires mating with host males to induce embryogenesis, but the male genes are not used. We therefore predicted P. mexicana males to prefer conspecific females. Hence, costs of male sexual harassment should not occur in unisexuals. While P. formosa are at a disadvantage compared to P. mexicana females due to male mate choice (leading to sperm limitation), this could be traded-off by suffering less from sexual harassment. In our experiment, we found males to direct significantly more pre-copulatory mating behaviour towards conspecific females, whereas actual mating attempts did not differ between species. Contrary to our prediction, both types of females started feeding later and spent less time feeding in the presence of a male partner compared to the time spent feeding with another female, suggesting that females of both species suffer from male harassment. The focal females' feeding time declined with increasing body size of the female competitor, and the same pattern was found when a male was present. We discuss that—besides sexual harassment—other factors such as food competition and female mate choice may affect female feeding efficiency.     

The ultimate function of nuptial feeding in the bushcricket Poecilimon veluchianus (Orthoptera : Tettigoniidae : Phaneropterinae)

Summary During mating the males of the bushcricket Poecilimon veluchianus transfer a large spermatophore of about a quarter of their body weight to the female. Such nuptial feeding is often thought to function as paternal investment by increasing the fitness of the male's offspring. According to an alternative, though not mutually exclusive, hypothesis, the size of the spermatophore is maintained because of its function as a sperm protection device. In this case the cost to the male should be classified as mating effort. To discriminate between these two hypotheses we measured the duration of sperm transfer into the female spermatheca and the time taken for spermatophore consumption. A comparison of durations revealed that spermatophore consumption interferes with the process of sperm transfer (Fig. 4). There was no significant effect of spermatophore consumption on number of eggs laid, weight of eggs or absolute weight of hatched larvae. The relative dry weight of hatched larvae, however, was increased as a result of spermatophore consumption (Table 1). Thus spermatophylax size is adjusted in accordance with a sperm protection function and the spermatophylax therefore represents mating effort. The increase in relative dry weight indicates that there may also be a paternal investment effect of the spermatophylax, if the offspring that benefit from spermatophylax materials are fathered by the donating male. Correspondence to: K. Reinhold     

Fitness benefits of multiple mating versus female mate choice in the cellar spider (Pholcus phalangioides)

Recent studies have demonstrated that mating with multiple males can be beneficial for females and her offspring even if males contribute nothing but sperm. This was mainly established for species in which sperm from several males mix in the reproductive tract of the female, thus allowing sperm competition and/or female sperm choice. However, in species with last male sperm precedence, female re-mating decides against the previous male by strongly limiting his reproductive success. We tested the effect of female re-mating behaviour using the cellar spider Pholcus phalangioides, which shows strong last males sperm precedence and moderate levels of polyandry under natural situations. We predicted that females prevented from remating even though they are receptive would show reduced reproductive success compared to females that accept two copulations and females that reject a second male, since the latter two treatments were allowed to behave according to their decisions. However, if the number of matings per se had an effect on oviposition or on offspring performance, double-mated females should perform better compared to both treatments of once-mated females. We measured female fecundity and fertility over a period of 140 days, comparable to the species' natural reproductive peak season. Two thousand one hundred and fifty-two offspring from 67 first egg sacs were reared under two feeding levels. We registered development time and survival, and measured offspring adult size and mass. We found a positive effect of double mating, as in this treatment, oviposition probability was higher compared to the other treatments. Interestingly, adult female offspring of the DM treatment that were raised under low food level had a higher condition index compared to those from FS and RM, but development time, size and mass at adulthood were not affected by mating treatment. Female choice only seemed to affect hatching latency of the offspring. Overall, the main predictor of female reproductive output and success was female body size.     

Mating effort or paternal investment? Incorporation rate and cost of male donations in the wartbiter

Summary Provision of nuptial gifts and incorporation of male-derived substances occurs in several insects. There has been some discussion as to whether these gifts should be regarded as a form of paternal investment or part of mating effort. One assumption of the paternal investment hypothesis is that the donating male is expected to father all, or the majority of, the offspring. The rate of incorporation into developing eggs is therefore assumed to be rapid enough to assure the donating male of paternity of the offspring in which he is investing. Spermatophore production in a paternal investment system is also expected to be costly, since males are providing females with highly nutritious gifts during copulation, thereby increasing female fecundity. Therefore, under conditions of food limitation males are expected to alter either mating frequency or spermatophore size. Experiments on the wartbiter (Decticus verrucivorus), using radioactive isotopes, show that the incorporation rate of male-derived substances is so slow that females become sexually receptive before any material received from the last male have been incorporated into eggs laid by the female. Since sperm mixing occurs in this species and since the label is present in eggs laid up to more than 30 days after mating, males might potentially benefit another male's offspring. The nuptial gift (the spermatophylax) was found, regardless of mating frequency and diet, to have a low protein content. These findings coincide with previous results showing that consumption of the gift had no effect on female fecundity. Instead, wartbiter males keep mating frequency high, are capable of mating every day regardless of food availability, invest the same proportion of the body weight in spermatophore production regardless of size, and produce a spermatophylax that correlates with ampulla size. These results indicate that the nuptial gift mainly functions to increase fertilization success in the wartbiter.     

Size-dependent mating and gender choice in a simultaneous hermaphrodite, Bulla gouldiana

Simultaneous hermaphrodites are predicted to optimally divide resources between male and female function, which can result in both size-dependent mating behaviors and conflict between potential mates. Predicted strategies include size-assortative mating, conditional exchange of gametes, and mating patterns where relative size affects investment in each sexual role. This study investigated the effect of body size on the mating strategies of a hermaphroditic opisthobranch, Bulla gouldiana. Although individuals were spatially aggregated in the field with high levels of movement and size variation, there was little evidence for predictions. Laboratory experiments, however, revealed complicated effects of mass on the probability and duration of mating, as well as gender choice. Pairs were more likely to mate if they included at least one large animal, with the larger animal typically inseminating the smaller. When both individuals were large, they were more likely to each mate in both sexual roles by switching roles once. Although B. gouldiana did not usually alternate between sexual roles multiple times within mating events, paired individuals behaved similarly (neither or both mating as sperm donors) more often than expected by chance. This suggests some level of reciprocity, which is unlikely to be conditional given rates of unilateral mating. When the larger member of the mating pair inseminated the smaller, the duration of insemination increased with the size of the smaller sperm recipient. Copulations lasted longer in pairs that switched sexual roles than in those that did not switch roles. This study suggests that variation in body size can lead to size-dependent mating patterns, but only some of the patterns in B. gouldiana support theoretical predictions. We review other studies that have addressed similar issues, providing inconsistent mating patterns in sperm-storing hermaphrodites.     

No cost of male mating experience on female reproductive success in the almond moth, <Emphasis Type="BoldItalic">Cadra cautella</Emphasis> (Lepidoptera; Pyralidae)

Male copulation experience may have a profound impact on female reproductive success if male reproductive investment declines over consecutive copulations and if females are unlikely to re-mate. Male reproductive investment is particularly interesting in lepidopterans because males produce dimorphic sperm: a fertilizing (eupyrene) and a non-fertilising (apyrene) sperm. In two experiments, we explored the lifetime reproductive investment of male almond moths, Cadra cautella (also known as Ephestia cautella) and examined its influence on female reproductive success. In the almond moth, females re-mate infrequently and males transfer sperm in a spermatophore. Attached to the spermatophore is a large chitinous process, the function of which is unknown. One group of males were permitted consecutive copulations with virgin females and the amount of sperm and size of the spermatophore transferred were compared for all females. We found that the number of both eupyrene and apyrene sperm per ejaculate decreased with his increased mating frequency, while the size of the spermatophore process decreased dramatically after the male’s first copulation. In a second experiment, we allowed males to mate with females throughout their lives and then compared female fecundity and fertilisation success. We found no obvious decrease in female fecundity and fertilisation success with increased male copulation experience, despite the likely reduction in male gametic investment. We discuss potential explanations for the development of this enlarged and elaborate first spermatophore of male almond moths given that it confers no clear fitness advantage to females.     

Independence, not conflict, characterizes dart-shooting and sperm exchange in a hermaphroditic snail

Although the sexes are united in hermaphrodites, conflict can still occur because the male and female functions have separate interests. We examined the evidence for conflict in the mating system of the terrestrial snail Cantareus aspersus (formerly Helix aspersa) where sharp, calcareous darts are ‘shot’ during courtship. We predicted that the use of the dart would either reflect or create conflict and this would be evident in either the courtship behavior or the transference of sperm. Previous studies demonstrated that the dart functions after sperm transfer to increase sperm survival. Using detailed observations of mating snails, we examined the factors that determine dart shooting order, the behavioral responses after being hit by a dart, the accuracy of dart shooting, and the allocation of sperm resources. We found that each dart was shot independently, and each animal appeared to be interested only in getting off the best possible shot, probably one that penetrates deeply near the genital pore. There is no evidence of mating conflict. Every snail transfers sperm to its partner, and the size of the donation does not depend on the success or failure of either snail’s dart shot. Although the receipt of a dart does not appear to cause harm, it may produce indirect costs due to the partial loss of control over fertilization. We conclude that mating in C. aspersus is a partnership in which independent actors demonstrate unconditional reciprocity during courtship and sperm transfer.     

The cost of mating with a non-virgin male in a monandrous butterfly: experimental evidence from the speckled wood, Pararge aegeria

Contrary to vertebrates, sperm production in insects may bear considerable costs for males. This is especially true in species that donate spermatophores containing sperm and nutrient-rich accessory gland products like in butterflies. Hence, spermatophores at first and subsequent copulations can differ in a quantitative and qualitative way. Such effects have particularly been shown in polyandrous species providing large spermatophores. Here we experimentally tested the effect of male mating status (virgin male vs recently mated male) on copulation duration, spermatophore size and females’ fitness components in a monandrous butterfly Pararge aegeria that typically donates small spermatophores. Copulations with non-virgin males lasted on average five times longer than that with virgin males and resulted in a spermatophore which was on average three times smaller. Number of eggs laid and female life span were not affected by the mating status treatment, but there was a significant effect on the number of living caterpillars a female produced, as copulations with virgin males resulted in higher numbers of larval offspring. Interestingly, the difference in spermatophore mass at the first and the second copulation increased with male body size. This suggests differential spermatophore allocation decisions among males of different size. Consequences for females and potential mechanisms influencing female fitness components are discussed. Given the small absolute size of spermatophores in P. aegeria, components other than consumable nutrients (perhaps hormones) should cause the observed effects.     

Determinants of ejaculate size in a grasshopper (Chorthippus parallelus)

Models for the evolution of ejaculate expenditure predict that ejaculate size (ES) depends on male body condition, on female fecundity, or on the risk of sperm competition. These ideas were tested in the grasshopper Chorthippus parallelus. ES ranged over four orders of magnitude, around a mean of 442,000 sperm. ES was independent of male body size and female fecundity. None of the male condition parameters - age, degree of parasitism, macropterism score (a measurement of competitive energy allocation to other sources), and the mass-femur length residuals - explained variation in ES. As expected in species where females are cyclically non-receptive after mating, male-male encounters before copulation were a more reliable indicator of the risk of sperm competition than the number of males present during copulation. In particular, the summative number of male-male encounters rather than the male-male encounter frequency was a significant predictor of ES variation (24.9%).     

Effects of female mating status on copulation behaviour and sperm expenditure in the freshwater crayfish <Emphasis Type="Italic">Austropotamobius italicus</Emphasis>

Sperm competition models predict that males should adjust their sperm expenditure according to the risk and/or intensity of sperm competition. In this paper, we analysed copulatory behaviour of both sexes and sperm expenditure in relation to female mating status (virgin or mated) in the freshwater crayfish Austropotamobius italicus, a species where males have been reported to feed on and remove sperm laid by other males. The same females were allowed to be inseminated sequentially by two males, and we compared the sexual behaviours of partners between the first (virgin females) and the second mating (mated females). We found that female resistance did not differ between the first and the second mating, nor males refused or took more time to mount a mated female. However, when mating with a mated female, males reached an effective copulation position significantly later. This occurred because second-mating males removed, by eating, all or most spermatophores previously deposited by first males. As removal was often incomplete, this resulted in a larger amount of sperm being deposited on female ventral parts after the second mating, although second males did not allocate more sperm to mated females than first males did. Thus, the peculiar mode of sperm competition, where males remove previously deposited sperm, and the consequent predictable strong last male prevalence in paternity likely led to the observed lack of adjustment of sperm expenditure to female mating status in this species.     

Multiple paternity and individual variation in sperm precedence in the simultaneously hermaphroditic land snail Arianta arbustorum

Intraspecific variation in the proportion of offspring sired by the second mate with a female (P₂) is an aspect of sperm competition that has received little attention. I examined the effects of delay between copulations (range 9–380 days) and size of sperm donor on sperm precedence in double-mated individuals of the simultaneously hermaphroditic land snail Arianta arbustorum. Using shell colour as a genetic marker, paternity was analysed in 132 broods produced by 35 snails that had mated with two partners of different genotype. Sperm precedence (P₂) was influenced by the time between the two matings when the mating delay exceeded 70 days (one reproductive season). P2 averaged 0.34 in the first brood of snails that mated twice within 70 days indicating first mate sperm precedence. In contrast, P₂ averaged 0.76 in broods of snails that remated in the following season, indicating a decreased viability of sperm from the first mate. The size of sperm-donating individuals had no effect on the fertilization success of their sperm in the first brood produced after the second copulation. Analysis of long-term sperm utilization in 23 snails that laid three to nine egg batches over 2 years revealed striking differences among individuals. Five snails (21.7%) exhibited first-mate sperm precedence throughout, eight snails (34.8%) showed second-mate sperm precedence throughout, whereas ten snails (43.5%) exhibited sperm mixing in successive batches. It is suggested that the individual variation in sperm precedence in A. arbustorum may partly be due to differences in the amount of sperm transferred. Paternity analysis in 34 batches laid by 19 wild-caught individuals of A. arbustorum indicated that at least 12 snails (63.2%) used sperm from two or more mates for the fertilization of their eggs. This suggests a high incidence of multiple paternity in broods of A. arbustorum under field conditions.