Determinants of sperm precedence in a noctuid moth Heliothis virescens: a role for male age

1. Females of the noctuid moth Heliothis virescens F. mate more than once. Thus, sperm from two or more males normally compete for fertilisations within the female reproductive tract. The eggs are typically fertilised by sperm from only one male, either the female's last mate or an earlier mate. Twice‐mated females store only one ejaculate's worth of fertilising sperm (eupyrene) but nearly two ejaculates' worth of a nonfertilising sperm morph (apyrene), which is thought to play a role in sperm competition. 2. The mechanism of sperm use in H. virescens was investigated by examining factors that vary with paternity, which was assigned based on allozyme variation. The factors included male and female body masses and ages, male genital characters, the size of the sperm package, and the number of sperm stored by the female. 3. One male typically gained sperm precedence; this was nearly twice as likely to be the second male as it was to be the first. Two factors were found to vary significantly with paternity: female mass and male age. The second male to mate was more likely to gain sperm precedence if the female was larger and if the male was older than the female's first mate. 4. The significance of male age and female mass to several hypothetical models of the mechanism of sperm use is discussed.     

Factors influencing paternity success in Antechinus agilis: last‐male sperm precedence,timing of mating and genetic compatibility

We describe the patterns of paternity success from laboratory mating experiments conducted in Antechinus agilis, a small size dimorphic carnivorous marsupial (males are larger than females). A previous study found last‐male sperm precedence in this species, but they were unable to sample complete litters, and did not take male size and relatedness into account. We tested whether last‐male sperm precedence regardless of male size still holds for complete litters. We explored the relationship between male mating order, male size, timing of mating and relatedness on paternity success. Females were mated with two males of different size with either the large or the small male first, with 1 day rest between the matings. Matings continued for 6 h. In these controlled conditions male size did not have a strong effect on paternity success, but mating order did. Males mating second sired 69.5% of the offspring. Within first mated males, males that mated closer to ovulation sired more offspring. To a lesser degree, variation appeared also to be caused by differences in genetic compatibility of the female and the male, where high levels of allele‐sharing resulted in lower paternity success.     

Impact of cryptic female choice on insemination success: Larger sized and longer copulating male squid ejaculate more,but females influence insemination success by removing spermatangia

In polyandrous mating systems, sperm competition and cryptic female choice (CFC) are well recognized as postcopulatory evolutionary forces. However, it remains challenging to separate CFC from sperm competition and to estimate how much CFC influences insemination success because those processes usually occur inside the female's body. The Japanese pygmy squid, Idiosepius paradoxus, is an ideal species in which to separate CFC from sperm competition because sperm transfer by the male and sperm displacement by the female can be observed directly at an external location on the female's body. Here, we counted the number of spermatangia transferred to, removed from, and remaining on the female body during single copulation episodes. We measured behavioral and morphological characteristics of the male, such as duration of copulation and body size. Although males with larger body size and longer copulation time were capable of transferring larger amounts of sperm, females preferentially eliminated sperm from males with larger body size and shorter copulation time by spermatangia removal; thus, CFC could attenuate sperm precedence by larger males, whereas it reinforces sperm precedence by males with longer copulation time. Genetic paternity analysis revealed that fertilisation success for each male was correlated with remaining sperm volume that is adjusted by females after copulation.     

Discriminating Males and Unpredictable Females: Males Bias Sperm Allocation in Favor of Virgin Females

When both sexes mate with multiple partners, theory predicts that males should adjust their investment in ejaculates in response to the risk and/or intensity of sperm competition. Here, we demonstrate that, in the harlequin beetle riding pseudoscorpion, Cordylochernes scorpioides, males use cues deposited on females by previous males to distinguish between virgin, once‐mated, and multiply‐mated females and adjust sperm allocation accordingly. Sperm number declined in direct proportion to the number of previous males, with virgin females receiving nearly three times more sperm than females exposed to three previous males. Given the lack of first‐male sperm precedence in C. scorpioides, this pattern is not consistent with current sperm competition models and appears best explained by a significant risk of wasting ejaculates on deceitful, mated females. In C. scorpioides, males transfer sperm indirectly to females via a stalked spermatophore deposited on the substrate. Mated females often feign sexual receptivity and cooperate throughout mating, only to reject the sperm packet produced by the male. While indirect sperm transfer facilitates a high level of female deceit and control, females of many species are able to influence the number and fate of sperm transferred during copulation and are likely to conceal their sexual unreceptivity to minimize male retaliation. If males cannot accurately assess female receptivity, increased risk of sperm rejection by mated females could outweigh the risk of sperm competition and favor greater sperm allocation to virgin females.     

FUNCTIONAL INCOMPATIBILITY BETWEEN THE FERTILIZATION SYSTEMS OF TWO ALLOPATRIC POPULATIONS OF CALLOSOBRUCHUS MACULATUS (COLEOPTERA: BRUCHIDAE)

Abstract Recent studies indicate that postcopulatory sexual selection may represent an important component of the speciation process by initiating reproductive isolation via the evolutionary divergence of fertilization systems. Using two geographically isolated populations of the polyandrous beetle Callosobruchus maculatus, we investigated divergence in fertilization systems by determining the extent of postcopulatory functional incompatibility. Through reciprocal, cross‐population matings we were able to separately estimate the effects of male and female population origin and their interaction on the extent of last‐male sperm precedence, female receptivity to further copulation and female oviposition. Our results indicate partial incompatibility between the fertilization systems of the two populations at all three functional levels. Males derived from the same population as females outcompete rival, allopatric males with respect to sperm preemption, sperm protection, and ability to stimulate female oviposition. This pattern is reciprocated in both populations indicating that postcopulatory, prezygotic events represent important mechanisms by which between‐population gene flow is reduced. We suggest the partial gametic isolation observed is a by‐product of the coevolution of male and female fertilization systems by a process of cryptic female choice. Our results are consistent with a mechanism akin to conventional mate choice models although they do not allow us to reject antagonistic sexual coevolution as the mechanism of cryptic female choice.     

Males Prefer Small Females in a Dichotomous Choice Test in the Poeciliid Fish Heterandria formosa

Male choice is expected to evolve when females differ in quality, even if male investment in each mating is low. The family Poeciliidae is an example of fishes in which males show little parental investment as they only provide sperm. Up until now, a preference for large females has been found in all species studied. Here we show that unexpectedly, males of the least killifish (Heterandria formosa) prefer to interact with small instead of large females in a dichotomous male choice test, even though large females are more fecund. During a free‐swimming choice experiment, males did not discriminate between females based on their size. We suggest that this unique preference for small females, or the lack of preference for large females, results from strong first male sperm precedence in this species. Smaller females are younger and therefore more likely to be virgin, which probably makes them more profitable mates for males. When presented with a virgin and a mated female of similar size, males showed no preference for either type. This suggests that males do not use pheromone cues to assess female mating status but that they are likely to use female size as a proxy for it.     

Copula duration and sperm storage in Mediterranean fruit flies from a wild population

In the Mediterranean fruit fly (Ceratitis capitata Weidemann, ‘medfly’), a lekking tephritid, evidence from laboratory studies of flies from laboratory strains suggests that copulation is shorter, and sperm storage more abundant, if males are large or protein‐fed, and that copulation is longer when females are large. In addition, sperm tend to be stored asymmetrically between the female’s two spermathecae and this asymmetry declines with abundance of stored sperm. The primary objective of this study was to investigate whether these trends persist in other experimental contexts that bear closer resemblance to nature. Accordingly, we carried out experiments in a field‐cage using males derived as adults from a wild population and virgin females reared from naturally infested fruit. The results of this study were consistent with laboratory studies in that copula duration increased with female size, that sperm were stored asymmetrically between the females’ spermathecae, and that this asymmetry declined with number of sperm stored. However, we also found some previously unreported effects of female size; large females stored more sperm and stored sperm more asymmetrically between their two spermathecae than did small females. Unlike the laboratory studies, copula duration and sperm storage patterns were unaffected by male size and diet. This may be due to overwhelming variation from other sources in the wild‐collected males used, as well as environmental variability in the semi‐natural setting.     

Size, operational sex ratio, and mate-guarding success of the carrion beetle, Necrophila americana

When male insects guard females until oviposition, the benefitsfrom last-male sperm precedence must outweigh the costs of relinquishingadditional fertilizations. The profitability of guarding isincreased when males guard large, fecund females and when femalesare scarce because fewer fertilizations are sacrificed. However,the male reproductive success is not only determined by theprofitability of guarding but also by his ability to maintainguarding. In this study, we used male carrion beetles (Necrophilaamericana) to examine the effects of sex ratio, male relativesize, and female quality on the ability to guard. First, wepresent a model of mate guarding that explores factors, suchas sperm precedence, sex ratio, male size, and female quality,that influence the profitability of postcopulatory riding. Ourmodel predicts that large N. americana males should preferentiallyguard the largest female only when the sex ratio is male biasedand sperm precedence is above 80%. In contrast, small malesgain little from guarding because they are not likely to maintainit and be the last male to mate. Then, we tested these predictionsby manipulating sex ratio, relative male size, and female quality.All males in equal sex ratio and large males in male-biasedsex ratio guarded females significantly longer than did malesin female-biased sex ratio. In male-biased sex ratio, largemales guarded significantly longer and achieved more takeoversthan small males. Large females were guarded longer. The successof guarding males in this beetle depends on their size relativeto other males and the operational sex ratio.     

Male and female effects on sperm precedence in the giant sperm species <Emphasis Type="Italic">Drosophila bifurca</Emphasis>

Sperm competition is expected to be a driving force in sexual selection. In internally fertilized organisms, it occurs when ejaculates from more than one male are present simultaneously within the female’s reproductive tract. It has been suggested that greater sperm size may improve the competitive ability of sperm, but studies provide contradictory results depending on the species. More recently, the role of females in the evolution of sperm morphology has been pointed out. We investigate here the male and female effects that influence sperm precedence in the giant sperm species, Drosophila bifurca Patterson & Wheeler. Females were mated with two successive males, and the paternity outcomes for both males were analyzed after determining sperm transfer and storage. We found very high values of last male sperm precedence, suggesting a strong interaction between rival sperm. However, the data also indicate high frequencies of removal of the sperm of the first male from the female reproductive tract prior to any interaction with the second male. This implies that successful paternity depends mainly on successful sperm storage. Knowing what happens to the sperm within females appears to be a prerequisite for disentangling post-copulatory sexual interactions between males and females.     

Intraspecific variation in sperm precedence in the bruchid beetle Callosobruchus maculatus

Abstract.

• 1 By examining potential sources of intraspecific variation in sperm precedence, the underlying mechanisms of sperm competition in Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) were investigated.
• 2 The extent of sperm precedence was not related to either copulatory behaviour or body size (male and female).
• 3 The extent of sperm precedence increased during the egg-laying period, suggesting that the stratification of sperm within the spermatheca is not the mechanism of sperm precedence.
• 4 Direct removal of sperm from the female's reproductive tract was not observed.
• 5 Four other mechanisms (not mutually exclusive) are proposed to account for last-male sperm precedence in this species.

     

The effect of sperm storage and timing of mating on offspring sex ratios in the yellow dung fly Scatophaga stercoraria

1. Offspring sex ratios in the yellow dung fly Scatophaga stercoraria were examined in the laboratory. 2. Previous work indicated that females using previously stored sperm to fertilise their eggs produced male‐biased sex ratios. This result may have been due to female influences or the effects of sperm storage per se. 3. This pattern was not reproduced in the study presented here. Females that were allowed to mate just prior to oviposition produced similarly male‐biased sex ratios to those females that used previously stored sperm to fertilise their clutch. 4. Captive‐reared females may have perceived a lack of males in the population and thus produced a male‐biased offspring sex ratio. Alternatively, gamete ageing or extra‐chromosomal sex ratio distorters may have produced the male bias.     

Male Competition over Access to Females in a Spider with Last-Male Sperm Precedence

Agonistic behaviour between male cellar spiders (Pholcus phalangioides) was investigated to test whether (1) size difference determines which male achieves access to the female, (2) males are able to monopolize access to the female until egg laying and whether (3) female resource value increases before egg laying because of last‐male sperm precedence. We further investigated whether (4) there is variation in time and energy spent on courtship and copulation depending on the degree of sperm competition, i.e. with or without rival present. In three experimental settings we introduced two males of either different or similar sizes, or a single male to a female. The mating units were constantly video‐observed until the females produced their first egg sac. Experience, ownership and female resource value in terms of body size was controlled. Our results show that larger males achieve almost exclusive access to females. Size symmetrical settings resulted in increased fighting activity and duration but dominance did not influence mating success. If copulations were disturbed by the rival male, copulations were terminated earlier in symmetrical settings compared with asymmetrical settings. In 94.8% of trials only one copulation took place, suggesting that the copulating male successfully monopolized access to the female. Males confronted with a rival copulated longer but courted significantly shorter than lone males. Although the last male to copulate sires 88% of the offspring in P. phalangioides, neither fighting nor courtship activity increased before the female laid a batch of eggs. This suggests that males have no indication of the timing of oviposition.     

SPERM COMPETITION AND THE EVOLUTION OF NUPTIAL FEEDING BEHAVIOR IN THE CRICKET,GRYLLODES SUPPLICANS (WALKER)

The pattern of sperm predominance in doubly mated female crickets, Gryllodes supplicans, was investigated using a radiation-sterility technique. Female G. supplicans made significant use of sperm from both males in fertilizing eggs; overall, first males to mate enjoyed a small advantage, fertilizing about 60% of the offspring produced subsequent to the second mating. The combined use of the sperm of both males in fertilizing eggs occurred soon after the second mating; evidently, mixing of ejaculates within a female's spermatheca does occur. Male G. supplicans provide females with a nuptial gift, the spermatophylax, which influences the time at which a female removes the externally attached sperm-ampulla; this in turn determines the quantity of sperm that is transferred. Moreover, the degree of sperm precedence achieved by a male may be positively related to the time at which the female removes his sperm ampulla. Thus males, by feeding females, ensure not only that a sufficient number of sperm are transferred to fertilize all of a female's eggs, but also may increase the certainty of their paternity. In mating systems in which females control sperm transfer and paternity is influenced by numbers of sperm (i.e., numerical sperm competition), an increase in prezygotic investment in females may be an adaptive male response.     

Female polyandry and size‐assortative mating in isolated local populations of the Japanese common toad Bufo japonicus

In anurans, female polyandry under male harassment is distributed across taxa because of external aquatic fertilization. According to the sexual selection theory, male–male competition for access to females is affected by the operational sex ratio (OSR) and population density. The Japanese common toad, Bufo japonicus, is widespread in mainland Japan, and like the European common toad, B. bufo, it engages in explosive breeding. In this study, we observed the breeding behaviour of B. japonicus in isolated local populations for over four years in two breeding ponds with different population sizes and densities: large‐low (L‐pond) and small‐high (S‐pond). We analysed the relative polyandry ratio in egg clutches laid by females and estimated the size‐assortative mating pattern to be an indicator of male–male competition in the two ponds. Both ponds tended to exhibit a size‐assortative mating pattern; however, the frequency of polyandry was different in the two ponds (L‐pond = 20% and S‐pond = 90%). Our results showed that polyandry could occur without multiple amplexus with a high population density, i.e. eggs were often fertilized by free‐swimming sperm in the small shallow pond. We propose that high female polyandry ratios without continuous male harassment are generated because of a male‐biased OSR and a high population density in the small pond. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 236–242.     

Sperm storage and use in polyandrous females of the globally invasive fruitfly, Ceratitis capitata

The medfly, Ceratitis capitata, is an invasive species in which polyandry, associated with sperm precedence, is a common behaviour in the wild. In this species, characterized by internal fertilization, we disclose how the sperm from two males are stored in the female storage organs and how they are used in terms of paternity outcome. The experiments were designed to furnish comparable and unbiased estimates of sperm numbers and progeny in twice-mated females. Results are incorporated in a model through which it is possible to relate the amount of stored sperm with the progeny of twice-mated females. The results show that polyandrous medfly females conserve equal amounts of sperm from the two males to fertilize their eggs. However, we observed a clear advantage of the second male's sperm in siring progeny, which interestingly decreases in favor of the first male as ovipositions progress. The results enable us to exclude differential sperm mortality and suggest that it is the mechanics governing the storage organs which causes the initial, but decreasing second male sperm precedence during the female reproductive life. These outcomes allow us to correlate sperm use in polyandrous females with the mating strategies and invasiveness of this fly.     

Short-and long-term sperm precedence in the beetle Tenebrio molitor: a test of the 'adaptive sperm removal' hypothesis

Abstract. Sperm removal in Tenebrio molitor L. (Coleoptera: Tenebrionidae) has been proposed as an adaptation to sperm competition and has been documented when the remating interval between successive copulations is short, but not when it is long (Gage, 1992). If sperm removal is adaptive, it follows that there should be different fertilization outcomes from double matings with different remating intervals.
Sperm precedence patterns were assessed using reciprocal double matings of normal and γ-irradiated (sterile) virgin males of controlled size and age with virgin females of controlled size and age.
Immediate last male sperm precedence was high whether the remating interval was short (<10 min) (P_2,= 0.89) or long (24h) (P₂= 0.92).
Sperm precedence in eggs laid in a 16-day period after the last copulation showed no difference in the pattern of change between females with short and long remating intervals.
By examining the aedeagus of males we show that sperm are removed at the end of copulation by the first and the second male to mate with a virgin female regardless of whether the remating interval is short or long.
We conclude that sperm removal is unlikely to be the primary mechanism by which males gain such high levels of last male sperm precedence.     

Growth and survivorship as proximate causes of sexual size dimorphism in peninsula dragon lizards Ctenophorus fionni

Males and females differ in body size in many animals, but the direction and extent of this sexual size dimorphism (SSD) varies widely. Males are larger than females in most lizards of the iguanian clade, which includes dragon lizards (Agamidae). I tested whether the male larger pattern of SSD in the peninsula dragon lizard, Ctenophorus fionni, is a result of sexual selection for large male size or relatively higher mortality among females. Data on growth and survivorship were collected from wild lizards during 1991–1994. The likelihood of differential predation between males and females was assessed by exposing pairs of male and female lizards to a predator in captivity, and by comparing the frequency of tail damage in wild‐caught males and females. Male and female C. fionni grew at the same rate, but males grew for longer than females and reached a larger asymptotic size (87 mm vs. 78 mm). Large males were under‐represented in the population because they suffered higher mortality than females. Predation may account for some of this male‐biased mortality. The male‐biased SSD in C. fionni resulted from differences in growth pattern between the sexes. The male‐biased SSD was not the result of proximate factors reducing female body size. Indeed SSD in this species remained male‐biased despite high mortality among large males. SSD in C. fionni is consistent with the ultimate explanation of sexual selection for large body size in males.     

FEMALE CONTROL OF SPERM TRANSFER AND INTRASPECIFIC VARIATION IN SPERM PRECEDENCE: ANTECEDENTS TO THE EVOLUTION OF A COURTSHIP FOOD GIFT

Manipulation of ejaculates is believed to be an important avenue of female choice throughout the animal kingdom, but evidence of its importance to sexual selection remains scarce. In crickets, such manipulation is manifest in the premature removal of the externally attached spermatophore, which may afford females an important means of postcopulatory mate choice. We tested the hypothesis that premature spermatophore removal contributes significantly to intraspecific variation in sperm precedence by (1) experimentally manipulating spermatophore attachment durations of competing male Gryllodes sigillatus and (2) employing protein electrophoresis to determine the paternity of doubly mated females. The relative spermatophore attachment durations of competing males had a significant influence on male paternity, but the pattern of sperm precedence deviated significantly from the predictions of an ideal lottery. Instead, paternity data and morphological evidence accorded best with a model of partial sperm displacement derived here. Our model is similar to a displacement model of Parker et al. in that sperm of the second male mixes instantaneously with that of the first throughout the displacement process, but the novel feature of our model is that the number of sperm displaced is only a fraction of the number of sperm transferred by the second male. Regardless of the underlying mechanism, female G. sigillatus can clearly alter the paternity of their offspring through their spermatophore-removal behavior, and employ such cryptic choice in favoring larger males and those providing larger courtship food gifts. We discuss how female control of sperm transfer and intraspecific variation in sperm precedence may be important precursors to the evolution of gift giving in insects.     

A paternity advantage for speedy males? Sperm precedence patterns and female re-mating frequencies in a sexually cannibalistic praying mantid

Scramble competition polygyny is expected when females and/or resources are widely dispersed and not easily monopolized by males, or when there is an abundance of mates during an extremely restricted reproductive period. Additional factors such as first male sperm precedence or low female re-mating rate might further explain the propensity of males to engage in scramble competition. The sexually cannibalistic praying mantid Pseudomantis albofimbriata exhibits a polygynous mating system, where females exist in low-density populations and male competition manifests as the race to find females rather than as direct physical fighting. Here, we aim to determine whether there is a paternity advantage for the first-male to mate and/or a low frequency of female re-mating. First, we determined sperm precedence patterns in P. albofimbriata using the sterile male technique. Second, we tested the likelihood of female re-mating in P. albofimbriata by comparing the close-range approach behaviour and frequency of successful mating attempts for males when paired with virgin as opposed to recently mated females, and by comparing the frequency of long-distance male attraction between virgin and mated females. We found no paternity advantage for the first male to mate, rather a second male advantage. Although mated females were not rejected by males when approached from close-range, they were chemically unattractive to males searching from a distance. Since initial mate attraction in many praying mantids, including P. albofimbriata, is mediated via long-distance chemical communication, we believe the latter result is more ecologically relevant and therefore more important. These results suggest that the relatively low frequency of female re-mating observed in P. albofimbriata may be an additional factor driving scramble competition in this system.     

Polyandry in the medfly - shifts in paternity mediated by sperm stratification and mixing

Background

In the Mediterranean fruit fly (medfly), Ceratitis capitata, a highly invasive agricultural pest species, polyandry, associated with sperm precedence, is a recurrent behaviour in the wild. The absence of tools for the unambiguous discrimination between competing sperm from different males in the complex female reproductive tract has strongly limited the understanding of mechanisms controlling sperm dynamics and use.

Results

Here we use transgenic medfly lines expressing green or red fluorescent proteins in the spermatozoa, which can be easily observed and unambiguously differentiated within the female fertilization chamber. In twice-mated females, one day after the second mating, sperm from the first male appeared to be homogenously distributed all over the distal portion of each alveolus within the fertilization chamber, whereas sperm from the second male were clearly concentrated in the central portion of each alveolus. This distinct stratified sperm distribution was not maintained over time, as green and red sperm appeared homogeneously mixed seven days after the second mating. This dynamic sperm storage pattern is mirrored by the paternal contribution in the progeny of twice-mated females.

Conclusions

Polyandrous medfly females, unlike Drosophila, conserve sperm from two different mates to fertilize their eggs. From an evolutionary point of view, the storage of sperm in a stratified pattern by medfly females may initially favour the fresher ejaculate from the second male. However, as the second male's sperm gradually becomes depleted, the sperm from the first male becomes increasingly available for fertilization. The accumulation of sperm from different males will increase the overall genetic variability of the offspring and will ultimately affect the effective population size. From an applicative point of view, the dynamics of sperm storage and their temporal use by a polyandrous female may have an impact on the Sterile Insect Technique (SIT). Indeed, even if the female's last mate is sterile, an increasing proportion of sperm from a previous mating with a fertile male may contribute to sire viable progeny.