Male genital length and mating status differentially affect mating behaviour in an earwig

Phenotypic variation in male genitalia may affect copulation behaviour, which can have important fitness consequences for males. Male genitalia commonly possess traits that increase male control over copulation, but in species where females control mating, a poor functional understanding often prevents insight into the processes responsible for such effects. Here, I investigate the effect of male genital length on copulation behaviour in the earwig Euborellia brunneri, where both sexes exhibit extremely elongated genitalia that correspond in shape. This model system is particularly suitable because pairs mate repeatedly and females can limit both the number and duration of copulations. I used both virgin and mated males and females in a double-mating design because longer male genitalia confer benefits in sperm competition. Consistent with a greater predicted male mating effort in mated females, the duration of individual copulations increased, but this traded off against mating frequency as cumulative mating duration remained unchanged. In contrast, male genital length increased both individual and cumulative mating duration, regardless of mating status. This difference suggests that, while males may modify copulation duration in response to mating status, females facultatively adjust mating frequency to prevent mating excessively or express preferences for increased male genital length. Notably, this study demonstrates that male genital phenotypes that are successful in sperm competition also enjoy female-mediated mating benefits.     

Choice in Varroa jacobsoni Oud. between honey bee drone or workerbrood cells for reproduction

Summary The effect of variation in copulation duration on sperm precedence (P₂) of the last male was investigated in a non-territorial libellulid using irradiated-male techniques and observations in a large field enclosure. Copulation duration varied between 6 min and more than 1 h (X = 23.19 min ± 12.74). In the clutch deposited immediately after the copulation, most copulation durations resulted in high sperm precedence for the last male. Several experimentally interrupted short matings still resulted in 100% precedence. However, most interrupted copulations of 10 min or less resulted in a low P₂. After long, uninterrupted copulations, P₂ remained high in clutches that were collected up to 9 days after the mating. After interrupted short copulations, P₂ fluctuated or remained at a low level from the first clutch onwards. This indicates that sperm mixing probably starts within 24 h and that after a long copulation virtually no rival sperm is left in the female's sperm storage organs. As most natural copulations are long, complete removal of rival sperm seems to be common in S. danae. Only 3 f 17 variables investigated explained a significant amount of the variation in copulation duration. Copulations shortened with increasing temperature and for each additional mating of the male. The strongest effect however, was due to time of day: early morning copulations lasted longer than midday copulations. Adjustment of the copulation duration to the decreasing chance of obtaining additional matings was found to be the most likely of five explanatory hypotheses that were evaluated.     

Sperm transfer,displacement and precedence in Ischnura graellsii (Odonata : Coenagrionidae)

Summary Copulation in Ischnura graellsii may be divided into three stages, according to the movements and position of the male's abdomen. We measured sperm volumes in males and females interrupted at different phases of copulation in laboratory-reared and field specimens. The results showed that males remove sperm from the female during stage I, and do not transfer sperm until stage Il of the copulation. In the field females interrupted during stage I of copulation had less sperm than postcopula females, and the volume of sperm in laboratory females mated once or twice was similar. These results suggest that males can remove most of the sperm during stage I of copulation. Preparations of in-copula specimens showed the horns of the penis (used to remove sperm) inside the bursa copulatoox and the spermatheca. Therefore males can remove sperm from both organs, in contrast to the other Ischnura species so far studied, where males can empty only the bursa. The length of these horns is positively correlated with male body length, and there are significant differences in length between the left and right horns of individual males. This suggests great variability in the male's ability to remove sperm. On the other hand, ejaculate volume is positively related to male and female size, and negatively to male age. Males are likely to be able to detect the presence of sperm in females: if the effect of population density and time of start of copulation are taken into account, copulations are longer with mated than with virgin females. Using genetic markers, sperm precedence was studied by rearing the female offspring of 6 females mated with two males of different genotype. In 5 out of 6 crosses, the second male fertilized all the eggs laid by the female in her first clutch. On average, the following clutches were progressively more fertilized by the first male, but there were striking differences between crosses. These differences are probably due to the variability in the amount of sperm transferred and/or removed. Offprint requests to: A. Cordero     

Prolonged copulation: A male ‘postcopulatory’ strategy in a promiscuous species,Lygaeus equestris (Heteroptera: Lygaeidae)

Summary Copulation in Lygaeus equestris L. (Heteroptera, Lygaeidae) is known to last 0.5–24 h. Variations in copula duration of field-collected insects were studied in the laboratory, and different hypotheses concerning the significance of prolonged copulations were tested.Through reciprocal matings with normal and sterile (irradiated) males, sperm displacement was estimated at about 90%. A male could thus increase his fitness by preventing subsequent matings of an inseminated female.Copulations with virgin insects, observed over 15 h, were classified in two categories: short (0.5–8.0 h) and long (> 15 h) duration (Fig. 1). No difference in the number of fertilized eggs was found between long and short copulations, and the insemination rate was highest during the first hour of a couplation (Fig. 2). Long-lasting couplations did not give rise to increased sperm displacement (Table 2). These results indicate that there is no difference in the amount of sperm transferred during short and long copulations and that no insemination takes place during the latter part of a prolonged copulation.Longer lasting copulations occurred when the sex ratio was male-biased than when it was female-biased (Fig. 1). The frequency of prolonged copulations was higher when the female was gravid than when she contained no eggs (virgin) (Figs. 3 and 4). Support is thus given to the hypothesis that prolonged copulation is a male postinsemination strategy to prevent subsequent matings of a female.Females subjected to male competition over a longer period, laid fewer eggs and died earlier than females that were mated but subsequently isolated from males (Fig. 5). Egg batches from females living with males were larger than batches from mated, isolated females (Table 3). This is probably due to a combination of many matings with short intermissions and prolonged copulations, both of which postpone oviposition.Comparisons between copulations of males with either gravid or virgin females during these experiments, led to the conclusion that short copulations with virgin females result from a male decision to terminate copulation after insemination is completed, whereas copula duration with gravid females is more likely to depend on a combination of male and female behavior.     

Copulation duration and sperm competition in white-faced dragonflies (Leucorrhinia intacta; Odonata: Libellulidae)

Summary In many odonates, females mate with more than one male while laying a single clutch of eggs. We studied paternity of eggs laid by remated females of Leucorrhinia intacta, a small libellulid dragonfly, at a pond near Syracuse, NY, USA. The probability of a female remating is a function of male density on the pond. The length of copulations differs considerably among males active on the study pond at the same time. Much of this variation was correlated with differences in mating tactics of the males; copulations by males that stayed on their territories during copulation were shorter than those by other males (Fig. 2). Eggs collected from females mated to irradiated, sterile males and to free-living, fertile males indicated that the average paternity expectation was higher for long than for short copulations, and that the variance in paternity expectation was lower for long than for short copulations. Some possible causes of the high variation in paternity at low copulation durations and possible reasons for differences in copulation duration between male mating tactics are discussed.     

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.     

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     

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.     

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

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

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.     

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

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

Male remating and female fitness in the wolf spider Pardosa astrigera: the role of male mating history

Although the effects of male mating history on female reproductive output and longevity have been studied in insects, few such studies have been carried out in spiders. In a mating system in which females are monandrous while males are polygynous, females may incur the risk by mating with successful males that have experienced consecutive matings and suffer from the possible depletion of sperm and/or associated ejaculates. Here, we examine the effects of male mating history on male courtship and copulation duration, female reproductive fitness, and female adult longevity of the wolf spider, Pardosa astrigera. Results indicated that male mating frequency had little effect on their subsequent copulation success, and of 35 males tested, about half of the males were able to copulate with five virgin females successively at an interval of 24 h. Male mating history had little effect on their courtship duration. However, male mating history significantly affected male copulation duration, female adult longevity, and reproductive output. Males that mated more frequently copulated longer and more likely failed to cause their mates to produce a clutch, although there was no significant difference in the number of eggs laid and the number of eggs hatched regardless of the first clutch or the second one. Multiple mating of male P. astrigera resulted in significant reduction in female adult longevity. Our results indicate that monandrous females mating with multiple-mated males may incur substantial fitness costs.     

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.     

Mating system,optimal number of matings,and sperm transfer in the Argentine ant Iridomyrmex humilis

Summary In ants, because males have a finite sperm supply and females mate only at the beginning of their reproductive lives, it is possible to infer which is the limiting sex from a few parameters: the amount of sperm produced by males, the amount of sperm stored by females, and the numerical sex ratio. In the Argentine ant Iridomyrmex humilis mating takes place in the nest. Laboratory experiments and field data showed that the numerical sex ratio is heavily male-biased (10.1:1) and that the maximum number of sperm a female can store is similar to the number of sperm a male possesses. Thus females are the limiting sex in this species. In a set of mating experiments, one queen was presented with 1–20 males. The highest proportion of successful matings occurred when females were presented with two males. There was a significant negative correlation between the amount of sperm queens stored and the number of males present in the mating arena. This relationship most likely resulted from male interference during the copulation process. When several males were present in the arena, the mating pair was frequently disturbed by other males trying to copulate. Newly mated queens collected from the field stored 172,000 ± 76,000 sperm, a quantity most similar to that measured in laboratory mating experiments with a ratio of 5 or 10 males per queen. Because the operational sex ratio in I. humilis is highly male-biased, male interference may also decrease the amount of sperm queens store in the field. In many ants, fewer sperm stored by queens should decrease their reproductive success because they would run out of sperm earlier in their reproductive lifetimes. However, comparison of the amount of sperm present in young and old I. humilis queens collected in the field suggests that most use only a small proportion of their sperm supply during their lifetimes. Males mate once and discharge all their sperm during a single mating. Females may mate with several males but dissection of these males indicated that in most cases only one of them had empty seminal vesicles thus suggesting that a single male is responsible for most of the sperm transfer. Thus caution should be exercised in inferring multiple inseminations, as is frequently done in eusocial insects, from the observation of multiple copulations. Correspondence to: L. Keller     

A comparative analysis of sperm storage and mating strategies in five species of brachyuran crabs

Variation in female sperm storage is explained, in part, by the amount of sperm transferred at mating. Laboratory mating experiments were conducted on Eurypanopeus depressus and Rhithropanopeus harrisii from the Chesapeake Bay and Pachygrapsus transversus from Florida, while mated pairs of Uca beebei and U. terpsichores were collected from mudflats in Panama. All experiments and collections were conducted during the summer of 2006 and 2007. More sperm was transferred to larger than smaller females, and by species with long copulation durations (R. harrisii and E. depressus). These two species live in cryptic habitats, have high sperm/egg ratios, and likely store sperm across multiple broods. In contrast, P. transversus and U. beebei mate conspicuously, have short copulations, transfer fewer sperm, and have low sperm/egg ratios. Comparisons of sperm transfer across different mating strategies and habitats provide a better understanding of female sperm storage in the Brachyura.     

Prolonged mating in the milkweed leaf beetle Labidomera clivicollis clivicollis (Coleoptera: Chrysomelidae): a test of the “sperm-loading” hypothesis

Summary Female milkweed leaf beetles (Labidomera clivicollis clivicollis) frequently mate with more than one male, and pairs form mating associations which last for up to 42 h in the field. I tested the hypothesis that males remaining with females for long periods of time benefit by numerically overwhelming the sperm of their competitors. Male L.c.clivicollis copulated intermittently with females throughout an 11 hour period in the laboratory. When virgin females were allowed a single copulation, 94.3% of the sperm they received were located in the spermatheca immediately afterward. Males were not sperm-depleted, for they had large numbers of sperm available after one copulation (mean=230,000±43,200); the maximal number of sperm a male transferred to a female in 24 h was 30,500. There was a positive linear relationship between the number of sperm transferred and time up to 24 h after mounting (r ²=0.178, P<0.003). These data suggest that males transfer increasing numbers of sperm throughout a 24-h-period. Mating duration was the most important determinant of paternity when females were placed with one male for 24 h and another male for 6 hours. Females whose first matings were longer showed first male sperm predominance (as determined by starch-gel electrophoresis), while females whose second matings were longer showed last male sperm predominance. In view of these data, it is puzzling that males do not inseminate with large numbers of sperm immediately after mounting the female. It is possible that female refractory behaviors make insemination difficult and favor prolonged mating by male milkweed leaf beetles.     

Variation in copulation duration and the resultant degree of sperm removal inOrthetrum cancellatum (L.) (Libellulidae: Odonata)

Summary Orthetrum cancellatum showed two distinct copulation durations at a study site in the south of France. These different durations are correlated with different degrees of sperm displacement from the female sperm stores; long copulations (894±142 s) resulting in almost 100% sperm removal, and short copulations (21.0±13.5 s) resulting in 10–15% removal. The difference in copulation duration is also related to the site of copulation and to the relative age of the copulating males. At oviposition sites males who gained copulations were relatively young and copulated for long periods. The difference in the duration of copulation and the degree of sperm removal is discussed with reference to the female habit of ovipositing before remating during oviposition episodes, and the possibility of differences in the rate at which males encounter receptive females at the two sites.     

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

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

Polyandry and enhanced reproductive success in the harlequin-beetle-riding pseudoscorpion

The growing molecular evidence that females of many species mate with several males calls for a critical reassessment of the selective forces which act to shape female mating tactics. In natural populations of the harlequin-beetle-riding pseudoscorpion, Cordylochernes scorpioides, females are polyandrous and typically produce mixed-paternity broods. Laboratory behavioral analyses and breeding experiments indicate that polyandry in this pseudoscorpion is an active strategy which increases female reproductive success. Females restricted to mating with a single male experienced a higher rate of embryo failure and produced significantly fewer offspring than either females mated to more than one male in the laboratory or females naturally inseminated in the field. Forced copulation, insufficient sperm from a single mating, male nutrient donations and variation in inherent male genetic quality cannot explain the greater number of nymphs hatched by polyandrous females in this study. Evidence suggests that, by mating with several males, C. scorpioides females may exploit postcopulatory mechanisms for reducing the risk and/or cost of embryo failure resulting from fertilization by genetically incompatible sperm. Received: 5 May 1996 / Accepted after revision: 16 October 1996     

Potential direct fitness consequences of ornament-based mate choice in a butterfly

Female mate choice has been shown to provide direct mating benefits in several animal groups. In butterflies, for which there are increasing reports of fine-scale color-based mate choice, the evolutionary benefits that accrue from such mating biases, if any, are largely unknown. We addressed this issue in the butterfly Colias eurytheme, a species in which females choose mates on the basis of iridescent ultraviolet (UV) wing ornamentation and in which males donate reproductively beneficial nuptial gifts. In the first experiment, we assessed the mass of gifts donated to 77 virgin females by males sampled directly from a field encounter site. Despite large variance in the male adult phenotype and ejaculate, no single aspect of dorsal wing coloration, including UV brightness, chroma, or hue, was related to ejaculate mass. There was, however, an interesting interaction between the effects of male body size and copula duration upon ejaculate mass, with size scaling positively with ejaculate mass among males involved in shorter copulations (those lasting <70 min) but negatively among males in longer copulations. In the second experiment, we assessed the lifetime fecundity, fertility, and longevity of 85 females mated under similar circumstances to free-flying wild males. Although several wing color parameters proved subtly informative in more sophisticated multivariable models, no model predicted more than about 20% of the variation in any single female fitness parameter. The duration of copulation, which ranged from 35 min to over 16 h and which carries putative costs for females, was, again, only very weakly predicted by male wing color parameters (i.e., R ² = 0.089). Given the overall minor predictive power of male wing coloration in general and of UV brightness in particular, our results do not strongly support the hypothesis that female C. eurytheme prefer bright UV males to obtain direct benefits or to minimize the costs associated with lengthy copulations.