Microsatellite analysis of sperm-use patterns in the bushcricket Requena verticalis

Abstract.— The proportion of offspring sired by the second male to mate with a doubly mated female, P2, is a ubiquitously measured statistic in the study of insect sperm competition. Nevertheless, the underlying mechanisms of sperm transfer, storage, and use that determine P2 are poorly understood. Typically the second male to mate gains moderate to high paternity. More rarely, the first male to mate gains the majority of fertilizations. Here we examine the transfer, storage, and use of sperm in the bushcricket Requena verticalis , a species with male parental investment and almost complete first male paternity. Sperm drain from an externally attached spermatophore into the female's reproductive tract, where they are transported to the sperm store or spermatheca. We find that only sperm from the first male to mate are transported to the spermatheca. We provide some data that address a number of different mechanisms that might account for the lack of storage of second-male sperm. DNA microsatellite markers are developed to assign paternity. By manipulating the numbers of sperm transferred by first and second males, we show that the size of the ejaculate transferred by the first male has a major impact on paternity; increasing ejaculate size of the first male assures his paternity. Paternity assurance in R. verticalis holds significant implications for the evolution of paternal investment via the male's nuptial gift.     

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.     

Sperm competition and small size advantage for males of the golden orb‐web spider Nephila edulis

Sexual selection, through female choice and/or male–male competition, has influenced the nature and direction of sexual size dimorphism in numerous species. However, few studies have examined the influence of sperm competition on size dimorphism. The orb‐web spider Nephila edulis has a polygamous mating system and extreme size dimorphism. Additionally, the frequency distribution of male body size is extremely skewed with most males being small and few large. The duration of copulation, male size and sexual cannibalism have been identified as the significant factors determining patterns of sperm precedence in spiders. In double mating trials, females were assigned to three treatments: either they mated once with both males or the first or the second male was allowed to mate twice. Paternity was strongly associated with the duration of copulation, independent of mating order. Males that were allowed to mate twice not only doubled the duration of copulation but also their paternity. Small males had a clear mating advantage, they copulated longer than large males and fertilized more eggs. Males of different sizes used different tactics to mate. Large males were more likely to mate through a hole they cut into the web, whereas small males approached the female directly. Furthermore, small males usually mated at their first attempt but large males required several attempts before mating took place. There was no obvious female reaction towards males of different sizes.     

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.     

Ejaculate size, second male size, and moderate polyandry increase female fecundity in a seed beetle

Several hypotheses have been proposed to explain the evolutionof polyandry in species that provide nuptial gifts. When nuptialgifts are in the form of nutritional elements in the ejaculateand ejaculate size is correlated with male body size, femalescan accrue both direct (nutritional) and indirect (genetic)benefits from multiple mating. We examined remating decisionsin females of the seed beetle Stator limbatus and, using pathanalysis, examined the effects of male body size on the sizeof his ejaculate, the amount of ejaculate that was successfullytransferred to females, and the overall effect of these variableson female fecundity. Larger males produced larger ejaculatesand consequently transferred a larger ejaculate to females,but the effects on female fecundity differed between the females'first and second mates. Both larger first and second males wereable to transfer more of their ejaculate to females than weresmaller males. Both the total amount of ejaculate transferredby these males and polyandry (number of matings) were positivelycorrelated to female fecundity independently of each other.However, larger second males were more successful at stimulatingfemale fecundity independently of how much ejaculate they transferred.We also provide evidence that females are choosy during theirsecond mating opportunity. Both female choosiness and higherfemale investment after mating with larger second males suggestthat females may benefit from both direct and indirect effectsfrom multiple mating. We also conclude that male body size isunder both directional fecundity selection and directional sexualselection.     

Old‐male paternity advantage is a function of accumulating sperm and last‐male precedence in a butterfly

Old‐male mating advantage has been convincingly demonstrated in Bicyclus anynana butterflies. This intriguing pattern may be explained by two alternative hypotheses: (i) an increased aggressiveness and persistence of older males during courtship, being caused by the older males' low residual reproductive value; and (ii) an active preference of females towards older males what reflects a good genes hypothesis. Against this background, we here investigate postcopulatory sexual selection by double‐mating Bicyclus anynana females to older and younger males, thus allowing for sperm competition and cryptic mate choice, and by genotyping the resulting offspring. Virgin females were mated with a younger virgin (2–3 days old) and afterwards an older virgin male (12–13 days old) or vice versa. Older males had a higher paternity success than younger ones, but only when being the second (=last) mating partner, while paternity success was equal among older and younger males when older males were the first mating partner. Older males produced larger spermatophores with much higher numbers of fertile sperm than younger males. Thus, we found no evidence for cryptic female mate choice. Rather, the findings reported here seem to result from a combination of last‐male precedence and the number of sperm transferred upon mating, both increasing paternity success.     

Genetic benefits of mate choice separated from differential maternal investment in red junglefowl (Gallus gallus)

Abstract Females may choose more attractive mates to obtain better viability or attractiveness genes for their offspring. A number of studies have demonstrated a positive relationship between paternal attractiveness and offspring quality. However, this pattern could be due to inheritance of paternal genes and/or it could be due to increased maternal investment in the offspring of more attractive males. To isolate female responses to male appearance from paternal genetic effects, I housed female red junglefowl ( Gallus gallus ) with vasectomized (sterile) males and artificially inseminated them. Male junglefowl with larger combs are more attractive to females. Females laid more eggs when housed with a large-combed, as opposed to a small-combed, vasectomized mate. Neither egg volume nor offspring body condition was associated with comb size of the mother's vasectomized mate. Paternal genetics appeared important. Body condition and comb size were greater for the sons of large-combed sperm donor males. This is consistent with the hypothesis that genetic benefits to offspring maintain female preference for the most ornate males. It is possible that greater body condition and comb size in sons of large-combed sires was not caused by genetic differences, but instead was due to compounds in the ejaculate of large-combed sperm donors inducing greater reproductive investment from females. However, females artificially inseminated by large-combed males did not produce more or larger eggs than females artificially inseminated by small-combed males, and thus there is no other evidence consistent with ejaculate-induced differential investment. Furthermore, only in older chicks was body condition significantly related to sire comb size, suggesting genetic rather than differential investment mechanisms.     

Male insemination decisions and sperm quality influence paternity in the golden orb-weaving spider

In polyandrous species, paternity may be influenced by the timingand frequency of mating. Female spiders possess 2 genital openingsthat lead to separate sperm-storage structures. Thus, even whenmating with a previously mated female, a male may reduce directsperm competition by inseminating the opposite opening to herfirst mate. Such morphology may provide females with greatercontrol over paternity. We examined simultaneously whether malesavoided already inseminated female genital openings and whetherthis behavior varied with the time between successive matings.To explore these questions, we mated female golden orb weaverspiders, Nephila edulis, each to 2 males and manipulated thetiming of their second mating. We documented male inseminationpatterns and explored the influence of male mating decisionson paternity success using the irradiated male technique. Wefound that 60% of males avoided sperm competition by discriminatingagainst inseminated genital openings. Moreover, male matingbehavior had a dramatic impact on the paternity success of irradiatedmales. When males inseminated the same genital opening, thecompetitive ability of the irradiated male's sperm was dramaticallyreduced resulting in lower paternity success. In contrast, whenthe 2 males inseminated opposite genital openings both malessired equal proportions of offspring regardless of their radiationstatus. There was no evidence that the timing of the secondmating affected patterns of paternity. Our data suggest thatdifferences in sperm quality may influence paternity successof N. edulis males under a sperm-competitive scenario. In contrast,females appear to have limited postmating control over paternity.     

Postmating sexual selection: the effects of male body size and recovery period on paternity and egg production rate in a water strider

The role of male body size in postmating sexual selection wasexplored in a semiaquatic insect, the water strider Gerris lateralis.To separate effects of male size per se from those due to numericsperm competition, male recovery period (shown here to be proportionalto ejaculate size) was manipulated independently of body sizein a factorial experiment where virgin females were mated firstwith sterile males and then with focal males. Both relativemale fertilization success and female reproductive rate were measured.The number of sperm transferred increased with male recoveryperiod, an effect that was mediated by longer copulation duration,but there were no effects of body size on ejaculate size. Neithermale size nor recovery period had any significant direct effectson male fertilization success. However, copulation durationinfluenced relative fertilization success, suggesting that malesable to transfer more sperm also achieved higher fertilizationsuccess. Females exercised cryptic female choice by modulatingtheir reproductive rate in a manner favoring large males andmales that were successful in terms of achieving high relativefertilization success. Thus, successful males gained a twofoldadvantage in postmating sexual selection. This study has important implicationsfor previous estimates of sexual selection in this group of insectsbecause pre- and postmating sexual selection will be antagonisticdue to limitations in male sperm production: males mating frequently(high mating success) will on average transfer fewer sperm ineach mating and will hence tend to fertilize fewer eggs permating (low fertilization success).     

Influences of sex, size, and symmetry on ejaculate expenditure in a moth

Although sperm fundamentally function to fertilize eggs, forcesarising from both sexes select for optimal ejaculate composition.Sperm competition is one recognized agent in the evolution ofsperm and ejaculate structure. Few studies, however, have examinedhow female factors influence ejaculate structure, despite somebehavioral evidence for male mate choice. Male Plodia interpunctella(Lepidoptera, Pyralidae) accrue all resources for reproductionas larvae. Adults emerge with a limited sperm complement andare therefore under intense selection to optimize gamete allocation.I detected no effect of male body weight on ejaculate size.However, female reproductive potential (ovary masses) was dictatedby body weight In addition, heavier females had greater spermathecalvolumes, but there was no such relationship with bursal size.Finally, heavier females showed a higher mating frequency. Ifound that mating males were sensitive to female size and producedlarger ejaculates when mating with heavier females. Males mayejaculate more sperm into larger females either because it paysthem to "spend" more reproductive resources on matings thatprovide greater reproductive potential, or because heavier (longerlived and more attractive) females mate more frequently andhave larger spermathecal volumes. Alternatively, females maycontrol spermatophore formation and "accept" an appropriateejaculate to maximize fertility. Males may therefore be alsoselected to ejaculate more sperm into larger females to counteractgreater risks of sperm competition associated with heavier females.There was no association between male or female femur asymmetryand ejaculate size. P.interpunctella may be selected to exercisemodulation of ejaculate size because males invest paternally,sperm for the single reproductive episode are limited, and femalefecundity and mating pattern vary between individuals and areassociated with body weight. More obvious variability in malereproductive behavior and choice may therefore be paralleledat the cryptic gametic level by plasticity in ejaculate allocation.     

Patterns of Sperm Transfer in the Golden Orb‐Weaver Nephila edulis

Sperm competition studies typically identify copulation duration as an important predictor of paternity as it may determine the quantity of sperm transferred and thus paternity success. This study explores the relationship between copulation duration, male body size, male age and sperm transfer in the golden orb‐weaving spider, Nephila edulis. Paternity in this species is strongly associated with the relative frequency and duration of copulation, which is also influenced by male size. We determined the number of sperm transferred during copulation, by performing sperm counts in both the male copulatory organs (palps) and female sperm receptacle (spermatheca) of recently mated pairs. The total number of sperm recorded (the sum in the male palps and female spermathecae) was greater for younger males than older males, but did not vary with male body size. In general, younger males transferred more sperm and a greater proportion of their sperm supplies than older males and, among these younger males, larger individuals transferred more sperm. However, there were no significant size effects for older males. More sperm was transferred with longer copulations, but in contrast with previous studies, we found that larger males copulated for longer. The rate of sperm transferred was negatively correlated with the duration of copulation, suggesting that the variation in copulation duration in N. edulis may represent strategic investment by males to alter patterns of paternity, in addition to transferring additional sperm.     

Determinants of paternity in a butterfly

Success in sperm competition is of fundamental importance to males, yet little is known about what factors determine paternity. Theory predicts that males producing high sperm numbers have an advantage in sperm competition. Large spermatophore size (the sperm containing package) also correlates with paternity in some species, but the relative importance of spermatophore size and sperm numbers has remained unexplored. Males of the small white butterfly, Pieris rapae (Lepidoptera: Pieridae), produce large nutritious spermatophores on their first mating. On their second mating, spermatophores are only about half the size of the first, but with almost twice the sperm number. We manipulated male mating history to examine the effect of spermatophore size and sperm numbers on male fertilization success. Overall, paternity shows either first male or, more frequently, second male sperm precedence. Previously mated males have significantly higher fertilization success in competition with males mating for the first time, strongly suggesting that high sperm number is advantageous in sperm competition. Male size also affects paternity with relatively larger males having higher fertilization success. This may indicate that spermatophore size influences paternity, because in virgin males spermatophore size correlates with male size. The paternity of an individual male is also inversely correlated with the mass of his spermatophore remains dissected out of the female. This suggests that females may influence paternity by affecting the rate of spermatophore drainage. Although the possibility of female postcopulatory choice remains to be explored, these results clearly show that males maximize their fertilization success by increasing the number of sperm in their second mating.     

Revisiting telegony: offspring inherit an acquired characteristic of their mother's previous mate

Newly discovered non‐genetic mechanisms break the link between genes and inheritance, thereby also raising the possibility that previous mating partners could influence traits in offspring sired by subsequent males that mate with the same female (‘telegony’). In the fly Telostylinus angusticollis, males transmit their environmentally acquired condition via paternal effects on offspring body size. We manipulated male condition, and mated females to two males in high or low condition in a fully crossed design. Although the second male sired a large majority of offspring, offspring body size was influenced by the condition of the first male. This effect was not observed when females were exposed to the first male without mating, implicating semen‐mediated effects rather than female differential allocation based on pre‐mating assessment of male quality. Our results reveal a novel type of transgenerational effect with potential implications for the evolution of reproductive strategies.     

Last–male sperm precedence in the bushcricket Poecilimon veluchianus (Orthoptera, Tettigonioidea) demonstrated by DNA fingerprinting

Males of the bushcricket Poecilimon veluchianus pass a large spermatophore to the female during mating. The spermatophore is eaten by the female after copulation. Because females mate with several males during their reproductive life, the competition between spermatozoa of different males affects a male's reproductive success. In order to determine the outcome of sperm competition, the paternity of the progeny of double–mated females was established by DNA fingerprinting with the oligonucleotide (GATA)₄. Typical P. veluchianus DNA fingerprints consisted of 15 scoreable fragments per individual. The proportion of bands shared between presumably unrelated bushcrickets was 17%. After the second copulation the second mating male clearly predominated at fertilization. The mean proportion of eggs fertilized by the second male was 90.1%. There was no significant relationship between the level of sperm precedence and the time of ovipositions after the second mating. If female P. veluchianus increase the fitness of their offspring by the incorporation of spermatophore–derived substances in developing eggs, there is little chance for the feeding male to fertilize eggs containing his nutrients, because of the very short mating intervals of females and the observed high level of last–male sperm precedence in this species. Under such conditions the last mating male would fertilize many eggs containing nutrients from a prior male. Because nuptial gifts, like the tettigoniid spermatophore, function only as paternal investment if the donating male's progeny benefit from the gift, a paternal investment function of the P. veluchianus spermatophore seems to be unlikely.     

Investment in Mate Guarding May Compensate for Constraints on Ejaculate Production in the Cricket Gryllodes sigillatus

Although there is a corpus of evidence that females of many taxa are choosy about males, there is less information on how males may react to females of different 'quality' (i.e. potential fecundity). The cricket Gryllodes sigillatus shows distinct mate guarding behaviour. We examined how long males mate guard females of different sizes (reflecting egg load and potential fecundity). We also examined the sperm number in ampullae donated to females of different sizes to see if males make a concomitant difference in investment in ejaculate. We also examined mate-guarding behaviour and ejaculate size of males mated to virgin and nonvirgin females of the same size to see if males equate size with increased age and increased likelihood of mating (increased sperm competition). The results showed that males mate guard larger females for longer but make no difference in ejaculate investment between sizes of female. Males make no significant difference in mate guarding investment or ejaculate investment between virgins and nonvirgins of the same size. There is evidence that other species of crickets do alter their ejaculate depending on the female size and mating history, but have less distinct guarding behaviour. We suggest that mate-guarding investment in G. sigillatus may serve a similar function to that of ejaculate investment in other crickets.     

Sequential mate encounters: female but not male body size influences female remating behavior

Whether and how individuals choose sequentially among matesis an important but largely neglected aspect in sexual selectionstudies. Here, we explore female remating behavior in the cellarspider Pholcus phalangioides. We focus on body size as one ofthe most important traits involved in mate choice. Large andsmall females (n = 216) were double mated with large or smallmales in all eight possible combinations. All females copulatedwhen virgin, but only 82% accepted a second male. The chanceof a female remating was not significantly predicted by thebody size of the second or first male or by the size differencebetween the two. In contrast, a previous study demonstrateda male size effect in that larger males monopolized femalesuntil egg laying when two males of different sizes were present.We suggest that sequential encounters are more common undernatural conditions than male monopolization of females becauseestimates of concurrent multiple paternity together with observationsin a natural population do not favor mate guarding as the predominantmating strategy in this species. It follows from our study thatthe intensity of sexual selection on male size may be greatlyoverestimated when using a competitive laboratory setting fora species in which females generally encounter mates in a sequentialfashion. Female remating probability was significantly predictedby female size, with large females remating with higher probabilitythan small females. Thus, when mating with large females, malesmay gain higher fertilization success through increased femalefecundity but also face a higher sperm competition risk.     

Male phenotype predicts insemination success in guppies

Theory predicts that mate choice can lead to an increase in female fecundity if the secondary sexual traits used by females to assess male quality covary with the number of sperm transferred during copulation. Where females mate multiply, such a relationship between male attractiveness and ejaculate size may, additionally (or alternatively), serve to augment the effect of indirect selection by biasing paternity in favour of preferred males. In either case, a positive correlation between male attractiveness and the size of ejaculates delivered at copulation is predicted. To date, some of the most convincing (indirect) evidence for this prediction comes from the guppy, a species of fish exhibiting a resource-free mating system in which attractive males tend to have larger sperm reserves. We show that, during solicited copulations, male guppies with preferred phenotypes actually transfer more sperm to females than their less-ornamented counterparts, irrespective of the size of their initial sperm stores. Our results also reveal that, during coercive copulations, the relationship between ejaculate size and the male's phenotype breaks down. This latter result, in conjunction with our finding that mating speed--a factor under the female's control-is a significant predictor of ejaculate size, leads us to speculate that females may exert at least partial control over the number of sperm inseminated during cooperative matings.     

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.     

Sperm Competition in a Viviparous Fish

We investigated multiple paternity and sperm precedence in the Amarillo fish, Girardinichthys multiradiatus (Goodeidae). We allowed females to mate with two different-sized males consecutively and assessed the paternity of the ensuing broods using allozyme electrophoresis. We presented one-half of the females the larger, and the other half the smaller, male first. Allozyme variation among individuals was low, yielding conservative estimates of multiple paternity. Half the broods were of mixed paternity, but one male always sired more than 70% of the embryos in each brood. The proportion of the brood sired was not related to mating sequence, but when we classified males by relative size, the larger male of each pair usually fathered greater proportions of offspring than the smaller male. This association disappeared when we used the actual size of the males in the analysis. Instead, for any pair of males, the difference in number of offspring sired was correlated to differences in the rate of courtship displays, rather than size differences, suggesting that courtship intensity is a better predictor of paternity than male size. Within a pair, the larger male usually displayed more than the smaller one, but there was no correlation between male size and display rate across all males. Parsimony suggests a correlation between courtship rate and sperm production, but we cannot rule out the possibility that females allocate paternity according to the relative merits of the males.     

Male Mate Choice in the Guppy (Poecilia reticulata): Do Males Prefer Larger Females as Mates?

Although females are the choosier sex in most species, male mate choice is expected to occur under certain conditions. Theoretically, males should prefer larger females as mates in species where female fecundity increases with body size. However, any fecundity‐related benefits accruing to a male that has mated with a large female may be offset by an associated fitness cost of shared paternity if large females are more likely to be multiply mated than smaller females in nature. We tested the above hypothesis and assumption using the Trinidadian guppy (Poecilia reticulata) by behaviourally testing for male mate choice in the laboratory and by ascertaining (with the use of microsatellite DNA genotyping) patterns of male paternity in wild‐caught females. We observed significant positive relationships between female body length and fecundity (brood size) and between body length and level of multiple paternity in the broods of females collected in the Quaré River, Trinidad. In laboratory tests, a preference for the larger of two simultaneously‐presented virgin females was clearly expressed only when males were exposed to the full range of natural stimuli from the females, but not when they were limited to visual stimuli alone. However, as suggested by our multiple paternity data, males that choose to mate with large females may incur a larger potential cost of sperm competition and shared paternity compared with males that mate with smaller females on average. Our results thus suggest that male guppies originating from the Quaré River possess mating preferences for relatively large females, but that such preferences are expressed only when males can accurately assess the mating status of encountered females that differ in body size.