Determinants of paternity in the garden snail Helix aspersa

Despite the likely importance of post-copulatory sexual selection in simultaneous hermaphrodites, the factors influencing sperm competition in these organisms are generally unknown. We have investigated the effects of dart-shooting, mating order, and several other predictors on the proportion of offspring fathered by penultimate (P_n-1) and ultimate (P_n) sperm donors in multiply mated garden snails, Helix aspersa. While paternity ratios were biased towards the penultimate donor (mean P_n-1=0.61), the magnitude of this advantage was dependent upon which of the two donors successfully darted the recipient. Mean P_n-values increased from 0.17 when the recipient was hit by the penultimate donor to 0.39 when it was hit by the ultimate donor. Furthermore, the effect of the dart was more pronounced in the clutches of smaller recipients. From these results, and observations of live sperm in the storage organs, we propose a novel mechanism to explain the detected pattern of sperm utilization in helicid snails. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00265-002-0519-6.     

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.     

Intertidal slope of coral sand beach as a unique habitat for fish: meiobenthic diet of the transparent sand dart, <Emphasis Type="Italic">Kraemeria cunicularia</Emphasis> (Gobiidae)

Coral sand beaches harbor gobiid sand darts (genus Kraemeria), the only fish known to live in the sand throughout their adult life. Despite the uniqueness of sand-dwelling habitat as a vertebrate, the biology of this fish remains unclear. To explore how this unique fish utilize an unusual habitat, we investigated diurnal patterns of microhabitat use and prey consumption by the transparent sand dart, Kraemeria cunicularia, at a sandy beach on Iriomote Island, the Ryukyu Archipelago, Japan. Sand darts were found in sediment in the lower intertidal zone throughout the daytime regardless of changes in tidal level, whereas at nighttime these fish were found swimming. Gut content analyses revealed that the sand dart diet was dominated by harpacticoid copepods throughout the day. Analyses of meiobenthic distribution indicated that these copepods were most abundant at lower intertidal zones where highest numbers of sand darts were found during the daytime; thus, it is possible that microhabitat use of the fish is largely determined by food availability. An extensive distributional survey throughout the Ryukyu Archipelago further indicated that sand darts prefer sandy beaches with well-sorted, coarse sand. These results provide novel insights into how sand darts respond to the tidal rhythm and highlight putative key environmental factors that determine their distribution at both regional and microhabitat scales. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Competition,fluctuating asymmetry and sperm transfer in male gryllid crickets (<Emphasis Type="Italic">Gryllus bimaculatus</Emphasis> and <Emphasis Type="Italic">Gryllodes sigillatus</Emphasis>)

Sperm competition theory predicts that ejaculate expenditure by males should increase with the risk of competition from rivals. The tendency to modulate expenditure, however, may reflect various constraints acting on different individuals. We test this idea here by looking at the effects of competition and fluctuating asymmetry (FA) in two species of gryllid cricket. Male Gryllus bimaculatus and Gryllodes sigillatus modulated the sperm content of their spermatophores in relation to both apparent competition and FA in limb size. Males of both species increased the amount of sperm transferred when an apparent male competitor was present, sperm number increasing regardless of the species of apparent competitor in G. bimaculatus, but significantly more with a conspecific competitor in G. sigillatus. In addition, the relative size of the spermatophylax in G. sigillatus increased in the presence of apparent competition, while spermatophore transfer time decreased in G. bimaculatus. Sperm number showed opposite relationships with limb asymmetry in the two species, decreasing with increasing asymmetry in males in G. sigillatus, but increasing with asymmetry in both males and females in G. bimaculatus. G. bimaculatus, but not G. sigillatus, also transferred more sperm when paired with a larger female. Relationships between reproductive measures and limb asymmetry were significant only when competitors were present in G. bimaculatus, but were significant regardless of competition in G. sigillatus. The differences may reflect the additional burden of producing a spermatophylax in G. sigillatus.     

Among- and within-population variation in sperm quality in the simultaneously hermaphroditic land snail Arianta arbustorum

Sperm competition models on the evolution of sperm size assume associations with another sperm quality trait, sperm longevity. Sperm length can also provide an indication of possible mechanisms affecting motility and thus fertilization success. Despite their importance, however, detailed mechanisms of sperm competition at the gamete level are poorly understood. In simultaneously hermaphroditic land snails, sperm traits and cryptic female choice are assumed to be crucial in determining fertilization success. We examined the variation in sperm length and number among individuals from four natural populations of the land snail Arianta arbustorum, a species with multiple mating and long-term sperm storage. We also assessed variation in velocity, motility and longevity of sperm in snails from two of the four populations. Independent of shell size, sperm length differed among populations and, to a minor extent, even among individuals within populations. Mean sperm length of a snail was not correlated with the number of sperm delivered in a spermatophore. The mean sperm velocity (=VCL) did not differ between snails from two populations. However, VCL varied among snails. Percentage motility and longevity of sperm differed between snails from the two populations. No correlations were found between length, velocity, percentage motility and longevity of sperm. To conclude, individual snails differed in sperm quality, and this variation may partly explain the differential fertilization success between A. arbustorum snails. Moreover, our findings did not support the positive association between sperm length and longevity assumed by sperm competition models for internally fertilizing species.     

Intranest relatedness and nestmate recognition in the meadow ant Formica pratensis (R.)

The impact of intranest relatedness on nestmate recognition was tested in a population of polydomous and monodomous nests of the mound-building ant Formica pratensis. Nestmate recognition was evaluated by testing aggression levels between 37 pairs of nests (n=206 tests). Workers from donor colonies were placed on the mounds of recipient nests to score aggressive interactions among workers. A total of 555 workers from 27 nests were genotyped using four DNA microsatellites. The genetic and spatial distances of nests were positively correlated, indicating budding and/or fissioning as spread mechanisms. Monodomous and polydomous nests did not show different aggression levels. Aggression behavior between nests was positively correlated with both spatial distance and intranest relatedness of recipient colonies, but not with genetic distance or intranest relatedness of donor colonies. Multiple regression analysis revealed a stronger effect of spatial distance than of genetics on aggression behavior in this study, indicating that the relative importance of environment and genetics can be variable in F. pratensis. Nevertheless, the positive regression between intranest relatedness of recipient colonies and aggression in the multiple analysis supports earlier results that nestmate recognition is genetically influenced in F. pratensis and further indicates that foreign label rejection most likely explains our data.     

Estimation of age in the sex-changing, coral-inhabiting snail Coralliophila violacea from the growth striae on opercula and a mark–recapture experiment

Change of sex in the coral-inhabiting snail Coralliophila violacea (Lamarck) may occur in a wide range of sizes in the field. One proposed explanation for this is that the snails change their sex at a certain age and that individuals have different growth rates caused by microhabitat differences. In this study, we attempt to establish a method to determine the age of this snail and age at sex change. The growth striae on the operculum were studied and compared to the age estimated by the Gompertz growth function based on growth data obtained from mark-recapture experiments in southern Taiwan. There is a significant correlation between the number of striae on the operculum and the age estimated from the Gompertz growth function, and the relationship is 1:1. These results suggest that the number of striae on the operculum can be used as an age index, with each stria representing 1 year of age. The age of sex change of this snail, according to our estimates by both stria number and aperture-length inferences, occurs between 4 and 6 years old. Growth rates of the snails are negatively correlated to size. Furthermore, individuals undergoing sex-change grow faster than males and females.     

Females influence sperm storage and use in the yellow dung fly Scathophaga stercoraria (L.)

Summary The influence of the female on the process of sperm storage and use was examined. Copula duration, the condition of the female and whether or not a copula terminated naturally influenced the number of spermathecae (of three) in which once-mated females stored sperm. Females stored more sperm the larger their mate and the sperm from larger males were stored more unevenly amongst the spermathecae than were those from smaller males. Double-mated females had sperm in fewer spermathecae the larger the second of their mates and these spermathecae tended to be the ones which lay together within the female. The P2 values over three successive clutches were constant and sperm precedence was complete when the larger male was second to mate but began low and increased over subsequent clutches when the smaller male mated second. These results suggest females prefer, and are able, to use the sperm of larger males to fertilise their eggs. It is proposed that multiple spermathecae in Diptera have evolved to give females better control over offspring paternity.     

Male sperm reserves and copulation frequency in birds

Summary Male passerine birds store sperm in a cloacal protuberance during the breeding season. We consider three hypotheses to explain interspecific variation in relative cloacal protuberance size. The copulation efficiency hypothesis states that a relatively large cloacal protuberance facilitates sperm transfer and predicts more rapid copulation in species with larger protuberances. There is insufficient data to test this idea rigorously, but we found no evidence for such an effect. The spermatozoa size hypothesis is a non-functional hypothesis which states that cloacal protuberance size is merely a consequence of sperm size, and predicts that species with larger spermatozoa have relatively larger cloacal protuberances. Sperm length was positively correlated with protuberance size, providing support for this hypothesis, although it seems unlikely that variation in sperm size alone is sufficient to account for variation in protuberance size because the numbers of sperm stored in the cloacal protuberance were also positively correlated with its size. The sperm competition hypothesis states that the cloacal protuberance is a sperm store and predicts that when sperm competition is intense, as measured by male copulation frequency and or mating system, males will have relatively large protuberances and testes mass. The sperm competition hypothesis was supported: in a comparative study in which we controlled for phylogenetic effects, relative cloacal protuberance size was significantly and positively correlated with copulation frequency. Across all mating systems protuberance size was also positively correlated with the mass of seminal glomera tissue, the number of sperm stored in the seminal glomera, and with relative testes size. These results suggest that where sperm competition is intense, a large cloacal protuberance is required to maintain a large sperm reserve for a high copulation rate.Correspondence to: T.R. Birkhead     

Biased sperm use by polyandrous queens of the ant<Emphasis Type="Italic"> Proformica longiseta</Emphasis>

The occurrence and genetic effects of polyandry were studied in the ant Proformica longiseta using three microsatellite markers. The average queen mating frequency (QMF) estimated from the sperm dissected from the spermathecae of 61 queens was 2.4 with 69% of the queens being multiply mated. QMF estimated from worker offspring in a subsample of eight monogynous colonies was 3.5, but the effective paternity (m_e,p) was only 1.23. The difference between these values reflected unequal sperm use by the queens. Most colonies of P. longiseta were polygynous and the average relatedness among workers was 0.35. Polyandry thus added only marginally to the genetic diversity of colonies, and our results gave little support to the genetic-variability hypothesis for explaining polyandry. Diploid male load was low, as only 1% of males were diploid. A large majority (92%) of nests produced one sex only, with males produced in colonies that had higher than average worker relatedness. This contradicted the predictions derived from worker control of sex ratios. Males produced enough sperm to fill the spermathecae of several queens. Thus, the results indicated that diploid male load, sperm limitation and sex ratio conflict are also unlikely explanations of polyandry. Plausible hypotheses for polyandry include mating by convenience, as the sex ratio is male biased and the mating costs to a female can be low because the females are wingless and have no mating flight. The observed unequal sperm use furthermore points to sperm choice and sperm competition as important factors in the evolution of polyandry.     

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.     

Cascading effects of hydrodynamics on an epiphyte–grazer system in intertidal seagrass beds of the Wadden Sea

This study examines experimentally how water movement may alter epiphyte-grazer systems in intertidal seagrass beds. Field observations in the Sylt-Rømø Bay (German Wadden Sea, SE North Sea) showed that the biomass of seagrass epiphytes was highest on seagrasses exposed to water movement, whereas at sheltered sites the epiphyte cover was negligible. In contrast, the seagrass shoot density and aboveground biomass was comparably sparse and the abundance of Hydrobia ulvae was extremely low at exposed areas, but showed maximum values at sheltered seagrass beds. Cross transplantation experiments and enclosure experiments between sheltered and exposed seagrass beds showed that adhering snails were washed off from seagrasses soon after transplantation into an exposed seagrass bed, and epiphytes started to grow. After 4 weeks the epiphyte biomass was similar to the that of the adjacent exposed seagrass bed. When heavily epiphytised seagrasses were transplanted from exposed into sheltered areas, the epiphytes were completely grazed down by immigrating snails within a week. Experiments carried out by means of an in situ "three-current-flume", modifying the entire current velocity, showed that snail density was significantly negatively correlated with increasing current velocity, whereas epiphyte biomass showed a significant positive correlation with current speed. These results suggest a cascading impact of hydrodynamics on an epiphyte-grazer system in intertidal seagrass beds, by directly affecting the density of grazers and indirectly leading to enhanced epiphyte growth, thereby inhibiting seagrass development. Additionally it shows that cascading effects within the trophic web cannot only be triggered by biotic interdependencies, but can also be caused by physical factors.     

Sexual conflict,sexual selection and sperm competition in the spawning decisions of bitterling, <Emphasis Type="Italic">Rhodeus</Emphasis><Emphasis Type="Italic">sericeus</Emphasis>

Sexual selection may operate through either direct selection on preference genes or indirect genetic benefits. However, in many species both direct and indirect selection may operate and can influence female mate and oviposition choice. Adaptations by males for sperm competition can also affect female mate and oviposition choice and can lead to sexual conflict. We investigated the role of direct and indirect benefits and male adaptation to sperm competition for female oviposition decisions in the bitterling, Rhodeus sericeus, a freshwater fish that spawns on the gills of living unionid mussels. Field experiments showed that females chose mussels for spawning on the basis of the number of embryos already in mussels and the body size and/or extent of red coloration of the eyes of territorial males. There was also a significant interaction between these direct and indirect benefits of oviposition choice. Territorial male bitterling avoided leading females to mussels into which rivals had already released sperm. Males also increased their rate of sperm release into mussels in response to sperm release by sneaking males. We discuss the possibility that sexual conflict in oviposition choice occurs in this species.     

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.     

Male dimorphism, sperm traits and immunology in the corkwing wrasse (Symphodus melops L.)

The evolution of alternative male mating phenotypes inevitably involves variation in risk or intensity of sperm competition. Males that typically mate in disfavoured roles (i.e. sneakers/parasitic males) are predicted to experience higher magnitudes of sperm competition than males mating in favoured roles (preferred by females/exhibiting mate monopolisation). Here, we investigated whether two distinct male phenotypes in corkwing wrasse (Symphodus melops) differ with respect to adaptations for sperm competition in terms of sperm quantity, sperm motility and fertilising capacity. We found that males exhibiting female mimicry (disfavoured role) had both absolutely and relatively larger gonads than males with typical secondary sexual characters (favoured role). Moreover, a higher proportion of sperm from female mimics was motile 5 min after activation compared to sperm of territorial males. There were, however, no differences in sperm concentration or in in vitro fertilisation ability between the two male morphs. We also examined whether the male phenotypes differed in some immune parameters and if any such variation is associated with sperm traits. Current theories predict that ejaculate quality should be negatively correlated with the level of immunocompetence. Territorial males had relatively heavier spleens than female mimics, but whether this reflects differing immunocompetence is uncertain, since the spleen is also an important organ for erythrocyte storage. Furthermore, there were no differences in the densities of circulating lymphocytes or granulocytes, and the immune parameters did not covary with the sperm traits. Differences in immunocompetence are therefore unlikely to provide a major proximate explanation for variations in sperm traits in corkwing wrasse.     

Strategic mating effort in a simultaneous hermaphrodite

Sexual selection theory for simultaneously hermaphroditic animals predicts an overall preference for inseminating partners that have a relatively higher female fecundity. Previous work on the link between male mating decisions and female fecundity has primarily focused on the effect of the partners’ body size using existing variation in this trait within a study population. On the assumption that the body size is positively correlated with female fecundity, sperm donors should preferentially inseminate relatively larger individuals to obtain a higher fitness gain through their male sex function. However, empirical evidence for such size-dependent mate choice in simultaneous hermaphrodites is equivocal, possibly because of confounding variables. We studied the mating behavior of the simultaneously hermaphroditic flatworm Macrostomum lignano and tested for a strategic mating effort in response to the feeding status of the partner. We experimentally manipulated the feeding status of potential mating partners in order to generate variation in female fecundity among them and tested whether this affected the copulation number and the number of sperm that the focal worm managed to store in the partner’s sperm storage organ. We found that the manipulation of the feeding status had a strong effect on the body size of the potential mating partners and that focal worms copulated more frequently with, and stored more sperm in well-fed partners compared to unfed partners. Our results suggest that M. lignano adjusts its mating effort in response to the feeding status of the mating partner.     

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%).     

Reproductive cycle and maternal effects on offspring size and number in the neogastropod Buccinum undatum (L.)

This paper examines relationships between maternal phenotype and offspring phenotype in the commercially exploited marine neogastropod Buccinum undatum (L.). The study also aimed to estimate the effects of female size on subsequent recruitment, and to determine the reproductive cycle of B. undatum in Swedish waters. The evaluation of gonad development in field-sampled animals from August 1998 to February 2000 revealed a definite annual reproductive cycle with a single major egg-laying period in the autumn. In addition, adult whelks of varying sizes from two locations and in two consecutive years were kept in aquaria to study the influence of female size on fecundity. Twenty-seven females deposited eggs in the laboratory. Offspring quality, measured as egg and hatchling size, was independent of female size, although larger females produced more eggs and hatchlings than smaller females, and thus probably account for a disproportionately large share of potential recruitment; the relative fecundity (number of eggs or hatchlings per gram of body weight) was constant with female size. I found no evidence for a longer time interval between successive egg-layings in larger females, i.e. mature females of all sizes probably spawn equally often. The present results therefore indicate that spawning-stock biomass is probably a better measure of spawning-stock size than the number of mature individuals in the population. Effects of an individual female's characteristics on stock productivity have largely been overlooked by fishery managers but could help to explain the weakness often found in stock-recruitment relationships.     

Egg production in the arrow squid Nototodarus gouldi (Cephalopoda: Ommastrephidae), fast and furious or slow and steady?

The process of reproductive maturation and egg release was examined in the temperate shelf squid Nototodarus gouldi. The energy allocation between somatic and reproductive growth from juvenile to mature adult was investigated throughout the life span to determine the underlying energetic strategy adopted by individuals. The relative weight of the mantle, fin and digestive gland remained unchanged during ovarian development, with no significant correlations found between the mantle length (ML)-gonad residuals and the ML-mantle (r=0.01, P>0.05), ML-fin (r=0.07, P>0.05) and ML-digestive gland (r=0.07, P>0.05) residuals. This suggested that energy was not being diverted away from somatic growth during sexual development, and consequently neither muscle nor digestive gland was being utilised as an energy store. Since squid in all maturity stages contained some food in their stomachs (e.g. 66.7% of mature animals), it is likely that the cost of maturation in this species is largely being met by food intake. The energy investment in reproductive tissues was relatively low (mean gonado-somatic index for mature individuals was 9.29% - 0.40%), indicating that only small amounts of energy were being allocated to reproduction at anyone point in time, which is characteristic of a multiple-spawning strategy. Furthermore, oviduct weight was not correlated with body size (r=0.256, P>0.05), suggesting that eggs are not stored for a single release. In all except one individual, ovary weight was consistently heavier than oviduct weight, suggesting that the ovary is not being depleted of oocytes as mature ova move into the oviducts. Additionally, the ovaries of mature females contained a range of oocyte sizes with discrete peaks, indicating a continued production and development of oocyte cohorts. The presence of some individuals with stretched empty oviducts is further evidence that the reproductive strategy of N. gouldi is slow and steady, with eggs possibly being released in discrete batches over a period of time.     

Reproductive biology of a small isopod symbiont living on a large isopod host: from the maternal marsupium to the protective grip of guarding males

Mating systems of many symbiotic crustaceans are characterised by a high degree of mate guarding. A peculiar case of mate guarding has been reported for small symbiotic janirid isopods where males mate with immature females. Field samples of individual hosts and laboratory experiments were conducted to reveal the mating behaviour of the symbiont in a natural environment, that is, on their hosts. Along the coast of the Magellan Strait, Chile, the janirid isopod Iais pubescens was frequently found on the shore-living isopod Exosphaeroma gigas. Symbiont prevalence (percent hosts occupied) was high at eight of the nine sampling sites. Mean symbiont intensity was very low at one site (<<1 individual host^-1), intermediate at two sites (1-10 individuals host^-1) and high at the other sites (10-40 individuals host^-1). The mean sex ratio (males:females) was male biased at most sampling sites (n=7). Females of I. pubescens reached substantially larger sizes (1.5-3.0 mm body length, BL) than males (1.1-1.9 mm BL). The majority of males were carrying small juveniles (66.15%), and males with juveniles were significantly larger than males without juveniles - this suggests that males prefer virgin juveniles to adult females and that they compete for small juveniles. In laboratory observations, males were seen to manipulate the marsupium of adult females that were about to release small juveniles. Males obtained virgin juveniles in this manner. Juveniles were carried for ~7 days, and they moulted shortly before being fertilised and released by males. The high proportion of juveniles carried by males in the field (68.2%) supports previous observations that males initially are not able to distinguish male and female juveniles. It is suggested that the mating system of symbiotic janirid isopods with long-term sperm storage and continuous receptivity in females and male mating with virgin females has evolved in response to highly unpredictable encounter probabilities between the sexes. Mate guarding and manipulation of small virgin juveniles may be favoured on the highly mobile hosts of symbiotic janirid isopods. Furthermore, adult females may gain by leaving their emerging offspring in the protective grip of guarding males, thereby reinforcing the maintenance of this peculiar mating system.