Sperm competition in Callosobruchus maculatus (Coleoptera: Bruchidae): a comparison of two methods used to estimate paternity

Abstract.

• 1 This study investigates sperm competition in Cullosobruchus maculatus (F.) (Coleoptera: Bruchidae), and is the first study to make a direct, controlled comparison of the sterile male and genetic marker techniques to estimate sperm precedence.
• 2 P₂ values (the proportion of offspring fathered by the second male) obtained from the two methods were similar: P₂ (sterile male) = 0.82, P₂ (genetic marker) = 0.85. Both methods are therefore suitable for studying sperm precedence if the appropriate correction factors are applied.
• 3 The importance of general fertilization ability, differential fertilizing capacity and differential zygote mortality are examined and discussed.

     

Sperm competition in the melon fly,Bactrocera cucurbitae (Diptera: Tephritidae): Effects of sperm “longevity” on sperm precedence

Sperm competition inBactrocera cucurbitae was studied by double matings of one female with normal and sterile males, with different intervals between the first and the second matings and with or without allowing oviposition after the first or the second mating. When the interval was less than 4 days, the last-male sperm precedence,P ₂ , was not different from 0.5, but as the interval was prolonged,P ₂ was higher than 0.5. There was no significant difference between treatments in which females were allowed to oviposit after the first mating and only after the second mating. The reason for the higherP ₂ when the interval was long was therefore attributed not to sperm usage for egg fertilization during the two matings but, possibly, to sperm mortality. ThatP ₂ was 0.5 for shorter intervals suggests that particular sperm replacement mechanisms such as removal and inactivation are absent in B. cucurbitae. Our study is the first to demonstrate a significant effect of short sperm longevity on the last-male sperm precedence.     

Sperm stratification and paternity success in red flour beetles

Abstract. When females are inseminated by multiple males, male paternity success (sperm precedence) is determined by the underlying processes of sperm storage and sperm utilization. Although informative for many questions, two-male sperm competition experiments may offer limited insight into natural mating scenarios when females are likely to mate with several males. In this study, genetic markers in Tribolium castaneum are used to trace paternity for multiple sires, and to determine whether displacement of stored sperm that occurs after a third mating equally affects both previous mates, or if fertilizations are disproportionately lost by the female's most recent mate. For 20 days after triple-matings, first males retain significantly higher paternity success (relative to first male paternity in double-matings) compared with second males. These results demonstrate that when females remate before sperm mixing occurs, sperm stratification results in differential loss of sperm from the most recent mate. This study provides insight into the mechanisms underlying sperm precedence in a promiscuous mating system, and suggests that T. castaneum females could limit paternity success of particular mates by remating with more highly preferred males.     

Ejaculate size varies with socio-sexual situation in an insect

Abstract.

• 1 Males operate within a finite energy budget and cannot produce limitless supplies of sperm. On the other hand, when a female mates with a second male while still containing fertile sperm from a rival male, selection should favour the male that inseminates more sperm. Optimal strategy should thus be for males to exercise discretion in the allocation of sperm to individual females. Assuming the outcome of sperm competition to be based on either the raffle or kamikaze principles, the sperm competition hypothesis predicts a positive association between the probability that the sperm will find themselves in competition with sperm from rival males and the number of sperm inseminated.
• 2 The beetle, Tenebrio molitor L., behaves in accordance with this hypothesis. Males accompanied by a rival male before and during mating inseminate more sperm per ejaculate than unaccompanied males. Accompanied males are also faster to initiate mating and more likely to show post-copulatory guarding. Adjustment of number of sperm inseminated was shown by males subjected to both long-term (5 days) and short-term (5 min) exposure to potential intrasexual competitors. Individual males exposed to both levels also demonstrated the ability to adjust their ejaculate according to socio-sexual situation.
• 3 We conclude that male T.molitor adjust the number of sperm they inseminate according to some perception of the risk of sperm competition.

     

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.     

Variation in sperm precedence in Chorthippus grasshoppers (Caelifera: Gomphocerinae)

Variation in the proportions of offspring fathered by a second male to mate (the P₂ value) has been studied in two species of grasshoppers, Chorthippus parallelus (Zetterstedt) and Ch. biguttulus (Linnaeus), by means of the sterile‐male technique. In both species the P₂‐values of the first egg pods laid were in the range of 50–100%, and the temporal variation of P₂‐values appeared to follow two modes. In one, the P₂‐value steadily declined with time, in the other it remained constant at a high level. It is concluded that sperm is passively lost between two matings. The remaining sperm then either mixes within the spermatheca or is stratified. Further variability in sperm precedence can possibly be explained by spermatophore ejection or differential use of stored sperm.     

RAPID DIVERSIFICATION OF SPERM PRECEDENCE TRAITS AND PROCESSES AMONG THREE SIBLING DROSOPHILA SPECIES

Postcopulatory sexual selection is credited with driving rapid evolutionary diversification of reproductive traits and the formation of reproductive isolating barriers between species. This judgment, however, has largely been inferred rather than demonstrated due to general lack of knowledge about processes and traits underlying variation in competitive fertilization success. Here, we resolved processes determining sperm fate in twice‐mated females, using transgenic Drosophila simulans and Drosophila mauritiana populations with fluorescently labeled sperm heads. Comparisons among these two species and Drosophila melanogaster revealed a shared motif in the mechanisms of sperm precedence, with postcopulatory sexual selection potentially occurring during any of the three discrete stages: (1) insemination; (2) sperm storage; and (3) sperm use for fertilization, and involving four distinct phenomena: (1) sperm transfer; (2) sperm displacement; (3) sperm ejection; and (4) sperm selection for fertilizations. Yet, underlying the qualitative similarities were significant quantitative differences in nearly every relevant character and process. We evaluate these species differences in light of concurrent investigations of within‐population variation in competitive fertilization success and postmating/prezygotic reproductive isolation in hybrid matings between species to forge an understanding of the relationship between microevolutionary processes and macroevolutionary patterns as pertains to postcopulatory sexual selection in this group.     

Sperm transfer during mating, movement of sperm in the female reproductive tract, and sperm precedence in the common cutworm Spodoptera litura

Abstract. Mating behaviour, sperm transfer and sperm precedence were studied in the moth Spodoptera litura (Fabr.) (Lepidoptera: Noctuidae). There existed a rhythmic, diel pattern of mating behaviour of this moth during the scotophase, presumably set with respect to an endogenous activity rhythm. Approximately 30 min after copulation had started, the formation of the corpus of the spermatophore began in the bursa copulatrix of the female moth, but full inflation of the corpus was not completed until 45–60 min after mating had started. The mature spermatophore contained about 350 eupyrene sperm bundles and a large number of individual (loose) apyrene spermatozoa. The mating status and the age of the male insect influenced the number of sperm transferred to the female within the spermatophore, and also affected the consequent fertility. There was no evidence of sperm reflux within the male tract. Within the female, dissociation of eupyrene sperm bundles was evident within the spermatophore less than 15 min after the completion of mating. Spermatozoa began to move from the bursa (in which the spermatophore is lodged) into the spermatheca 30–45 min after the end of the copulation, and the quantity of sperm in the spermatheca reached a plateau at 90 min after mating. Apyrene sperm reached the spermatheca first, followed by eupyrene sperm. Examination of total (apyrene plus eupyrene) sperm in the female tract showed that 86% of mated females received an apparently normal amount of total sperm from the male. Examination of eupyrene sperm alone showed that 81% of matings resulted in an apparently normal transfer of eupyrene sperm. A small proportion (approximately 8%) of the matings, however, were identified as transferring a clearly subnormal quantity of eupyrene sperm to the spermatheca. The eggs produced as a result of such pairings displayed much reduced fertility (about 43%) compared to those from matings confirmed to have transferred normal quantities of sperm, which showed about 92% fertility. This shows that the availability of eupyrene sperm in the spermatheca may be an important constraint on fertility in normal populations of insects. In the laboratory, S. litura females exhibited multiple matings. Of the females, 93% mated, and the mean frequency of mating was 1.69. Mating with a fertile male led to the oviposition of an increased number of eggs. This effect continued even when the female subsequently mated with an infertile male. Displacement of sperm from previous matings is known to be an important factor in the evolution of multiple mating strategies. Our results on sperm utilization by S. litura indicated that after a second mating, the sperm utilized for subsequent fertilization were almost exclusively from the last mating with little mixing. The proportion of eggs fertilized by sperm from the second mating (P₂) was calculated as 0.95, indicating almost complete sperm precedence from the last mating.     

Effective population size,reproductive success and sperm precedence in the butterfly,Bicyclus anynana,in captivity

A pedigree approach is used to estimate the effective population size yn two population cages of the butterfly, Bicyclus anynana. Each cage was founded with 54 individually marked adults of each sex. Matings were recorded over a 3‐day period. Eggs were then collected from each female over a similar period before the numbers of hatching larvae were counted to assess progeny number. The males showed a higher variance in reproductive success than the females. Since about one‐quarter of all females mated more than once, we also examined the pattern of sperm precedence using molecular markers or, in separate crossing experiments, wing pattern mutants. Both instances of complete first and last male sperm precedence, as well as of sperm mixing, were found. In some crosses a ‘leakiness’ was found in which some of the early eggs laid by a female were fertilized by a male partner which was subsequently completely unsuccessful. However, the estimates of effective population size were largely unaffected by the pattern of sperm precedence. Estimates for N_e : N in each cage were close to 0.60. The possibility of obtaining comparable estimates in selected natural populations of butterflies is discussed.     

Adaptive control of copulation duration by males under sperm competition in the mite,Macrocheles muscaedomesticae

In a manure-inhabiting predatory mite, Macrocheles muscaedomesticae (Gamasida, Macrochelidae), when the female mates with two males, the first male takes nearly perfect fertilization priority (Yasui, 1988). The present study examined whether the first-male's sperm precedence is influenced by the copula-duration of the first and second males mating with the same female, and whether males control their copulation duration by assessing the probability that the mate has been inseminated by other males. Results of the artificial interruption of copulation showed that sperm precedence value, P₂ (the proportion of the offspring fathered by the second male), was negatively correlated with the copulation duration of the first male but positively correlated with that of the second male. There was a threshold (ca. 180–300 seconds) in the first-male's copulation duration beyond which P₂ decreased drastically; when length of the first copulation exceeded this threshold, the second males did not fertilize eggs, whereas they fertilized more than half of the eggs when the first-copulation duration was shorter than the threshold. Almost all males copulated for a longer period (average 509.8 seconds) than this threshold if the copulation duration of the previous male had not exceeded the threshold, but if it was longer than the threshold, second males had shortened their copulation (67.6 seconds). These results suggest that males are able to assess the insemination status of their mates and to adjust their copulation duration depending on the probability of fertilizing eggs by their own sperm. A mechanistic explanation for sperm precedence (i.e., plug-formation within sperm receptive organ of the females) is proposed.     

CONSPECIFIC SPERM PRECEDENCE IS AN EFFECTIVE BARRIER TO HYBRIDIZATION BETWEEN CLOSELY RELATED SPECIES

Conspecific sperm precedence is widespread in animals, appears to evolve rapidly, and is thought to have the potential to prevent hybridization between closely related species. However, to date no study has tested the isolating potential of such a barrier in mixed populations of two taxa under conditions in which other potential barriers to gene flow are controlled for or are prevented from operating. We tested the isolating potential of conspecific sperm precedence in the ground crickets Allonemobius fasciatus and A. socius in population cage experiments in which the frequency of the two species was varied. Despite the observation of abundant interspecific matings, the proportions of hybrid progeny were low and differed statistically from the proportions expected in the absence of conspecific sperm precedence. The results demonstrate that conspecific sperm precedence can severely limit gene flow between closely related species, even when one species is less abundant than the other.     

Nontransitivity of sperm precedence in Drosophila

Abstract.— Sperm competition is an important component of fitness in Drosophila , but we still do not have a clear understanding of the unit of selection that is relevant to sperm competition. Here we demonstrate that sperm competitive ability is not a property of the sperm haplotype, but rather of the diploid male's genotype. Then we test whether the relative sperm competitive ability of males can be ranked on a linear array or whether competitive ability instead depends on particular pairwise contests among males. Sperm precedence of six chromosome-extracted lines was tested against three different visible marker lines ( cn bw, bw^D , and Cy ), and the rank order of the six lines differed markedly among the mutant lines. Population genetic theory has shown that departures from transitivity of sperm precedence may be important to the maintenance of polymorphism for genes that influence sperm competitive ability. The non-transitivity seen in sperm precedence should theoretically increase the opportunity for polymorphism in genes that influence this phenotype.     

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.     

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.     

Linear models for assessing mechanisms of sperm competition: the trouble with transformations

Abstract.— Although sperm competition is a pervasive selective force shaping the reproductive tactics of males, the mechanisms underlying different patterns of sperm precedence remain obscure. Parker et al. (1990) developed a series of linear models designed to identify two of the more basic mechanisms: sperm lotteries and sperm displacement; the models can be tested experimentally by manipulating the relative numbers of sperm transferred by rival males and determining the paternity of offspring. Here we show that tests of the model derived for sperm lotteries can result in misleading inferences about the underlying mechanism of sperm precedence because the required inverse transformations may lead to a violation of fundamental assumptions of linear regression. We show that this problem can be remedied by reformulating the model using the actual numbers of offspring sired by each male, and log-transforming both sides of the resultant equation. Reassessment of data from a previous study (Sakaluk and Eggert 1996) using the corrected version of the model revealed that we should not have excluded a simple sperm lottery as a possible mechanism of sperm competition in decorated crickets, Gryllodes sigillatus .     

Sexual selection in flour beetles: the relationship between sperm precedence and male olfactory attractiveness

Sperm precedence, defined as nonrandom differential fertilizationsuccess among mating males, is an important postmating componentof sexual selection. This study examined the relationship betweenpremating and postmating components of sexual selection in malesof the flour beetle (Tribolium castanewn). Male olfactory attractivenessto females was positively correlated with a male's subsequentfertilization success: more attractive males achieved highersecond-male sperm precedence when allowed to mate with previouslyinseminated females. Attractive males may achieve compoundedgains in their reproductive success through enhanced matingopportunities as well as through greater fertilization success.Thus, the relationship between these reproductive fitness componentsmay augment differences in reproductive success among males.Female fecundity, estimated as the number of adult progeny produced,increased significantly with multiple malings. This result supportsincreased female reproductive success as a direct benefit ofmultiple mating in T. caslaneum and suggests that progeny productionis partially limited by sperm availability. Total progeny productionby doubly mated females remained constant at all levels of second-malesperm precedence. However, higher sperm precedence was associatedwith a decline in firstmale progeny and a concomitant increasein second-male progeny. This pattern of progeny production suggeststhat more attractive males may achieve higher fertilizationsuccess through a combination of displacement of previouslystored sperm, transfer of greater sperm quantities, or females'preferential use of sperm of attractive males for fertilizations.     

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.     

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.     

Possible glimpses into early speciation: the effect of ovarian fluid on sperm velocity accords with post‐copulatory isolation between two guppy populations

Identifying mechanisms of reproductive isolation is key to understanding speciation. Among the putative mechanisms underlying reproductive isolation, sperm–female interactions (post‐mating–prezygotic barriers) are arguably the hardest to identify, not least because these are likely to operate at the cellular or molecular level. Yet sperm–female interactions offer great potential to prevent the transfer of genetic information between different populations at the initial stages of speciation. Here, we provide a preliminary test for the presence of a putative post‐mating–prezygotic barrier operating between three populations of Trinidadian guppies (Poecilia reticulata), an internally fertilizing fish that inhabits streams with different levels of connectivity across Trinidad. We experimentally evaluate the effect of female ovarian fluid on sperm velocity (a predictor of competitive fertilization success) according to whether males and females were from the same (native) or different (foreign) populations. Our results reveal the potential for ovarian fluid to act as a post‐mating–prezygotic barrier between two populations from different drainages, but also that the strength of this barrier is different among populations. This result may explain the previous finding that, in some populations, sperm from native males have precedence over foreign sperm, which could eventually lead to reproductive isolation between these populations.