Male genital traits and mating interval affect male fertilization success in the water strider Gerris lacustris

Parker's seminal work brought attention to the possibility of postmating sexual selection by non-random fertilization success. Mechanisms for these processes are still only partly understood and there is clearly a need for more studies of intraspecific variation in sperm precedence. Here, we report results from an experimental study of the variation in fertilization success between males of the water strider Gerris lacustris. Genital morphology, male body size, and copulation duration were examined as possible correlates of paternity. The significance of guarding duration was also analysed. Only male genital morphology was correlated to fertilization success. This is one of the first studies showing a relationship between male genital traits and fertilization success, supporting the view that sexual selection may be responsible for the rapid and divergent evolution of genital structures in animals with internal fertilization. The fertilization success of last males varied considerably after double matings with a short mating interval (10 min). Last-male priority ranged from 0 to 100% and usually one of the males involved fertilized almost all the eggs. After double matings with a short mating interval, the proportion of eggs fertilized by the last male averaged 0.68 and was greater than 0.5. In contrast, the average fertilization success was biased towards the first male when the matings were more spread out over time (24 h). These results do not support earlier suggestions of a widespread last-male sperm priority in water striders. Received: 28 July 1998 / Received in revised form: 15 March 1999 / Accepted: 28 March 1999     

Patterns of sperm use in the scorpionfly Panorpa germanica L. (Mecoptera: Panorpidae)

Sperm competition can be a powerful selective force in the evolution of reproductive strategies and mating systems. In studies on sperm competition, patterns of sperm use are typically reported as the mean species value of P ₂, determined as the proportion of offspring sired by the second male to copulate with a doubly mated female. However, the within-species variance in P ₂ has mostly been ignored, although taking this variance into account may be crucial for understanding the underlying mechanisms of sperm competition. Paternity analysis among the offspring of doubly mated females of Panorpa germanica (Mecoptera, Panorpidae) revealed a relationship between relative copulation durations of both males and the proportion of offspring each male will sire. This correlation between proportional copulation durations and paternity suggests mixing of sperm from different males inside the female’s spermatheca. Yet, sperm mixing appears to be incomplete, as paternity was overall slightly shifted towards the second male on average fathering a higher proportion of the offspring than its relative copulation duration would predict in case of complete sperm mixing. For individual males, however, the outcome of sperm competition is rather unpredictable as the intraspecific variance in P ₂ was found to be very high, irrespective of copulation durations. Possible causes of the observed variance in P ₂ and the partial last male sperm precedence are discussed.     

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

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

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

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

Paternity analysis of two male mating tactics in the fiddler crab, Uca mjoebergi

The fiddler crab Uca mjoebergi mates both underground in male-defended burrows and on the surface near female-defended burrows. The reproductive tract of Uca species facilitates last-male precedence, suggesting that males that do not guard-mated females are likely to gain very little paternity if the female re-mates with another male. Here, we test the reproductive success of burrow and surface matings using paternity analysis. We found that 100?% of the females that mated in burrows extruded a clutch of eggs. Furthermore, we show conclusively, for the first time in a fiddler crab species, that last-male sperm precedence results in the majority of the female’s eggs being fertilised by the burrow-mated male. In contrast, surface matings resulted in significantly fewer females extruding eggs (5.6?%). Paternity analysis also revealed that more than half of the clutches from burrow-mated females showed low levels of extra-pair paternity from previous matings. Although multiple matings appear common in U. mjoebergi, burrow-mated males that guard females are guaranteed a successful mating with extremely high rates of assured paternity. Surface matings therefore appear to be an opportunistic tactic that may increase male reproductive success in a highly competitive environment.     

Sperm transfer and storage in relation to sperm competition in Callosobruchus maculatus

This paper examines the underlying mechanisms of sperm competition in the beetle Callosobruchus maculatus (F.) (Coleoptera: Bruchidae). Recently developed mathematical models of sperm competition are combined with an empirical investigation of the processes of sperm transfer and storage. During a single insemination virgin males transfer approximately 46000 sperm, 85% more sperm than females can effectively store in their spermathecae. Many of these sperm remain in the bursa copulatrix where they are apparently rapidly degraded and can therefore play no role in fertilization. The spermatheca (primary site of sperm storage) is filled by a single insemination and sperm are lost from this organ at a constant rate. This rate of sperm loss from the spermatheca is insufficient for sperm mixing (without displacement) or sperm stratification to account for the degree of last male sperm precedence measured as P ₂; the proportion of offspring fathered by the second male to mate reported for this species (P ₂ = 0.83, when two inseminations are separated by 24 h). Models of sperm displacement correctly predict high levels of sperm precedence although the precision of these predictions is limited because the proportion of sperm entering the spermatheca cannot be accurately determined. The results suggested that last male sperm precedence in C. maculatus the result of sperm displacement, although the exact mechanism of displacement (sperm-for-sperm or fluid displacement) remains unknown. Possible constraints imposed by female genital anatomy on sperm displacement are discussed.     

Sperm precedence, mating interval, and a novel mechanism of paternity bias in a beetle (Tenebrio molitor L.)

When females mate with more than one male, the ensuing sperm competition leads to the evolution of male mechanisms that skew paternity. Males of the yellow mealworm beetle (Tenebrio molitor) transfer a spermatophore to females during copulation, but sperm release and storage occur later. We investigated how the interval between two matings with different males affects sperm precedence by varying the interval between the copulations so that the second mating was either: (1) before sperm release from the first spermatophore (<5 min); (2) after sperm release but before spermatophore ejection (15–20 min); (3) after spermatophore ejection but before sperm storage (4 h), or (4) after complete sperm storage (24 h). We collected offspring over a period of 2 weeks and determined paternity by protein electrophoresis. There was second-male sperm precedence in all treatments, but when the interval was <5 min, the second male usually (86% of cases) had complete sperm precedence (i.e., P ₂=1). Investigations into the mechanism of second-male sperm precedence during <5-min mating intervals indicate that sperm release from the first spermatophore is inhibited, a phenomenon which has not been previously documented. Received: 31 January 2000 / Revised: 9 June 2000 / Accepted: 26 August 2000     

Mate replacement in experimentally widowed collared flycatchers (Ficedula albicollis): determinants and outcomes

There is increasing evidence that sexual selection may be intense even in socially monogamous birds, resulting from both mate choice and sperm competition. We studied these two modes of sexual selection experimentally by removing paired male collared flycatchers, Ficedula albicollis, from their mates for 2 days and investigating the factors that influenced the likelihood of a replacement male appearing and how the removals influenced paternity. Replacement males (usually neighbouring males) appeared at 81% (n = 37) of nests where males were removed. The likelihood of this appearance was unaffected by the probable reproductive value of the female's clutch to the replacing male. A replacement was, however, less likely when the original male had a large forehead patch, a trait previously shown to be subject to sexual selection in this population. Experimental removal of males increased the level of sperm competition: 74% of experimental broods were multiply sired, compared to 29% of unmanipulated broods in a previous study. Only two factors predicted how paternity was shared between males: removed males fathered more young if removed closer to laying, and if they had larger forehead patches. The former result is consistent with last-male sperm precedence determining paternity, whereas the latter adds to other evidence that forehead patch size is the target of female preference in this species. Our results suggest that females exert some control over male replacement, and also that they may influence the fertilisation success of males by behavioural means. Received: 15 July 1998 / Received in revised form: 16 March 1999 / Accepted: 28 March 1999     

Factors influencing paternity in multiply mated female red-sided garter snakes and the persistent use of sperm stored over winter

In some species, sperm is stored within the female reproductive tract for months to years, and yet remains viable to fertilize eggs and produce offspring. Female red-sided garter snakes store sperm for over 7 months of winter dormancy. In previous work, we demonstrated that these stored sperm account for an average of 25 % paternity of a litter when the female mates with a male at spring emergence. Here, we tested whether last-male sperm precedence was prevalent when a female mates with two males during the spring. On average, paternity was shared equally among the first (P₁ proportion of paternity of the first male to mate) and second males (P₂) to mate in the spring, and stored sperm (P_ss), but the variance in paternity was high. Thus, last male sperm precedence may diminish when a female has more than two mates. Male size did not affect paternity, but, as the interval between matings increased, P₁ increased at the expense of P_ss. Interestingly, as the second spring male’s copulation duration increased, P₁ also increased at the expense of P₂. This result suggests that female influence over sperm and/or copulatory plug transfer during matings may also affect which male fathers her offspring in response to coercive matings as we assisted females to mate for their second mating. Finally, all females were spring “virgins”; consequently, sperm stored from autumn matings (and/or previous spring matings) remain competitive even when faced with two rivals in sperm competition and is likely the driver of the evolution of sperm longevity.     

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

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

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.     

Patterns of paternity skew in Formica ants

Reproductive skew among cooperatively breeding animals has recently attracted considerable interest. In social insects reproductive skew has been studied in females but not in males. However, cooperative breeding of males occurs when two males mate with the same queen and father offspring. Here we present the first analysis of comparative data on paternity skew in ants. We show that, across seven species of Formica ants, the average skew in paternity among worker offspring of doubly mated queens is negatively correlated with the population-wide frequency of multiple (mostly double) mating. We also demonstrate that this trend is relatively robust in additional analyses taking phylogenetic relationships between species into account. The observed trend is opposite to the one normally found in non-social insects with second-male precedence through sperm displacement, but agrees with predictions based on queen-male conflict over sperm allocation as a consequence of facultative, worker controlled, sex allocation – an interpretation which assumes first-male precedence. However, alternative (but not mutually exclusive) explanations are possible and further studies will be needed to discriminate between these alternatives. Received: 16 May 1997 / Accepted after revision: 26 September 1997     

Female mediation of sperm competition in the millipede Alloporus uncinatus (Diplopoda: Spirostreptidae)

The tropical spirostreptid millipede Alloporus uncinatus has a polygynandrous mating pattern that is apparently shaped by sperm competition (Telford and Dangerfield 1990, 1993a). In the present study radioisotopic labelling of ejaculates was used to quantify the temporal effects of double mating sequences on sperm precedence patterns. Ejaculates of successive males mix completely within the sperm storage organs and are concentrated close to the site of fertilisation, the spermathecal-oviduct junction. When matings follow each other immediately (experiment 1), equal volumes of the ejaculates of each male are stored within the spermathecae, and both males have equal probability of paternity. Where matings are separated by a 24-h delay (experiment 2), the volumetric contribution of the first male is reduced by 54.8 % and last male precedence operates. Although the distal ends of the gonopods play a primary role in the redistribution of ejaculates (Barnett and Telford 1994), by using a single mating sequence we show that they are not responsible for the reduction in ejaculate volume. The most likely explanation is absorption and/or ejection of the first male's ejaculate by the female. Genital functional morphology is used to reconstruct the mechanism of ejaculate distribution.     

Mating behaviour of Macrophthalmus hirtipes (Brachyura: Ocypodidae)

 We examined the mating behaviour of the New Zealand ocypodid crab Macrophthalmus hirtipes in the laboratory between February and June 1998. This species has a discrete breeding season. Mating and moulting were not linked and only intermoult females with mobile gonopore opercula were attractive to males. Allometry and compatibility of gonopods and gonopores of different-sized crabs was investigated. Under laboratory conditions, the opercula of intermoult females remained mobile on average for 11.4 d, but the duration of receptivity did not appear to be under female control. The operational sex ratio in the laboratory fluctuated greatly, but was always male-dominated. During the period of opercular mobility, females mated many times with several different males. Matings in the absence of burrows were relatively short (mean duration = 23 min, max. = 122 min) and the mating behaviour of M. hirtipes lacked courtship and mate-guarding. Males used a search-intercept method to acquire mates, with very low levels of intrasexual competition. There was no evidence of mate preference in M. hirtipes, and males spent just as long mating with ovigerous females as with non-ovigerous ones. Although M. hirtipes has ventral-type spermathecae, as do several other ocypodid crabs, it is unclear whether this promotes last-male sperm precedence. The role of burrows in modifying the mating behaviour of M. hirtipes in the field remains to be established. Received: 7 January 2000 / Accepted: 5 June 2000     

Sperm usage in honey bees

Sperm usage was investigated in a naturally mated honey bee queen. We collected worker progeny arising from eggs that were laid sequentially during three sampling periods. Paternity was determined by analysis of three polymorphic microsatellite loci, leading to the conclusion that the queen had mated with seven males. Direct analysis of the sperm from the spermatheca revealed no evidence that sperm from additional males was present inside the spermatheca. Frequencies of different subfamilies differed significantly and ranged from 3.8% to 27.3%. In the short term, the frequencies of subfamilies among the eggs laid did not change over time. The frequency of eggs of a particular subfamily was statistically independent of the previous egg's subfamily. Thus, there is no evidence for non-random fine-scale sperm usage, and we estimate the effect of sperm clumping to be less than 6%. We conclude that the sperm is mixed completely inside the queen's spermatheca. Our results suggest that taking brood samples from comb cells next to each other is a statistically correct way of independent sampling of subfamilies at a given time in honey bee colonies. Furthermore, any bias in subfamily frequencies in offspring queens due to sperm usage can be excluded. However, the analyses of progeny samples taken 12 months apart do not allow us to exclude moderate fluctuations of subfamily frequencies in the long-term. Received: 11 August 1997 / Accepted after revision: 14 November 1997     

Multiple insemination increases reproductive success of female Montandon’s newt (<Emphasis Type="Italic">Triturus montandoni</Emphasis>, Caudata,Salamandridae)

The adaptive significance of multiple matings for females is a matter of much controversy. In insects, supplying the female’s sperm reserves with portions of fresh spermatozoa may be the main function of multiple matings. This simple explanation may also be applied to other animals which produce large numbers of eggs over prolonged periods of time. We tested the fertility insurance hypothesis in Montandon’s newt (Triturus montandoni, Amphibia, Salamandridae). T. montandoni females are inseminated internally by spermatophores they have picked up, and subsequently lay eggs fertilized by spermatozoa released from the spermatheca. We compared the reproductive success of singly and multiply inseminated females of Montandon’s newt in the laboratory. Multiply inseminated females laid more eggs and had a lower percentage of non-developing eggs than females who mated only once. Our data suggest that remating increases the reproductive success of multiply inseminated females by replenishing sperm reserves in the spermatheca or by supplying females with fresh portions of spermatozoa with high fertilizing capacity. Received: 7 January 2000 / Revised: 13 September 2000 / Accepted: 7 October 2000     

Sperm competition and evidence of sperm fertilization patterns in the carrion ball-roller beetle Canthon cyanellus cyanellus LeConte (Scarabaeidae: Scarabaeinae)

Field observation and laboratory experiments were conducted to investigate sperm competition and patterns of sperm fertilization under different experimental conditions in the carrion ball-roller beetle Canthon cyanellus cyanellus. Males in nature can mate with females whose spermathecae contains fertile sperm from other males. Sperm precedence was investigated using a visible genetic marker. The progeny of red (homozygous recessive) virgin females mated once with a red male and afterward, once with a green beetle (homozygous dominant) and vice versa, revealed that regardless of its color, the last male to mate fertilized c.a. 50% of the eggs, suggesting strong sperm competition. Males were able to achieve higher levels of paternity (more than 80%) when mated ad libitum with previously mated females, although large amount of variance in paternity does not exclude the possibility of first male sperm precedence or female cryptic choice. These results suggest that repeated mating and sperm replacement are the mechanisms by which last males achieve sperm precedence.     

Paternal investment in Poecilimon veluchianus bushcrickets: beneficial effects of nuptial feeding on offspring viability

In most bushcrickets, males transfer a large spermatophore during copulation that is afterwards consumed by the female. In some species this nuptial gift enhances offspring fitness and is therefore believed to function as paternal investment. To determine whether this is the case, I examined whether a male's own offspring benefit from spermatophore consumption in the bushcricket Poecilimon veluchianus. Females that consumed a spermatophore produced offspring with increased residuals of dry weight compared to females that were prevented from feeding on the spermatophore. This beneficial effect of spermatophore consumption occurred within the first 4 days after copulation. An increased dry weight indicates higher energy reserves because offspring dry weight correlates significantly with the lifespan of starved larvae and because spermatophore consumption increased the lifespan of starved offspring. During egg-laying, females apply a liquid substance to the soil that hardens and probably serves as protection for the egg clutch. The amount of this substance correlated with the number of eggs laid but did not differ between spermatophore treatments. In P. veluchianus, females mate frequently and there is last-male sperm precedence. The spermatophore thus only constitutes paternal investment when offspring produced before female remating benefit from spermatophore consumption. The dry weight of offspring increased during the first 4 days after spermatophore consumption and thus within the natural remating interval. This shows that the spermatophore has a paternal investment function in addition to its already known sperm protection function. Received: 15 May 1998 / Accepted after revision: 11 October 1998     

Why do male Callosobruchus maculatus beetles inseminate so many sperm?

Male Callosobruchus maculatus F. (Coleoptera: Bruchidae) inseminate more sperm than females can effectively store in their spermathecae. This study examines the adaptive significance of excess sperm transfer by measuring components of male and female reproductive success in response to manipulating the number of sperm inseminated. The number of sperm transferred during copulation was reduced from 56,000 ±4,462 to 8,700±1,194 by sequentially mating males to virgin females. Reducing the number of sperm inseminated by the first male to mate had no effect on the extent of sperm precedence, but reducing the number of sperm inseminated by the second male resulted in a significant reduction in the extent of sperm precedence. When large numbers of sperm are inseminated the remating refractory period of females is increased. These results indicate that males transferring large numbers of sperm during copulation have a two-fold advantage at fertilization; they are more effective at preempting previously stored sperm and they are likely to father more offspring by delaying the time of female remating. The transfer of excess sperm does not appear to serve as nonpromiscuous male mating effort; the number of eggs laid, their fertility and the subsequent survival of zygotes were unaffected by manipulating the number of sperm inseminated. The underlying mechanisms of sperm precedence were also examined. Simple models of sperm displacement failed to accurately predict the patterns of sperm precedence observed in this species. However, the results do not provide conclusive evidence against the models but rather serve to highlight our limited understanding of the movement of sperm within the female's reproductive tract.     

Sperm allocation in an uncertain world

Models of optimal sperm allocation are constructed using stochastic dynamic programming techniques, yielding predictions about sperm allocation when males mate sequentially, breeding time is finite, sperm stores are limited and females vary in quality. The models suggest that uncertainty of future reproductive opportunities should favour withholding of sperm, so that males tend to allocate less (for a given level of stored sperm) earlier in the breeding season. This effect is more pronounced the greater the variance in female quality. We also show that while allocation will be influenced by mate value, it is not necessarily optimal to allocate preferentially to high-quality females, since the benefits of a higher-quality mate may be offset by increased risk of rejection of sperm or higher sperm competition. The relationship between mate quality and level of allocation will depend strongly on the amount of remaining stored sperm, with males whose supplies are depleted being more likely to favour lower-quality partners. Received: 12 September 1997 / Accepted after revision: 28 June 1998