Double Mating Research Articles

Meiotic drive does not impede success in sperm competition in the stalk-eyed fly, Teleopsis dalmanni.

Male X-linked meiotic drive systems, which cause the degeneration of Y-bearing sperm, are common in the Diptera. Sperm killing is typically associated with fitness costs that arise from the destruction of wildtype sperm and collateral damage to maturing drive sperm, resulting in poor success under sperm competition. We investigate X-linked meiotic drive fertility in the stalk-eyed fly, Teleopsis dalmanni. Drive male paternity was measured in double mating trials under sperm competition against a wildtype male. Drive males sired the same number of offspring as wildtype males, both when mated first or second. This is the first evidence that drive males can compete equally with non-drive males in double matings, challenging the assumption that drive males inevitably suffer reduced fertility. The finding is in accord with previous work showing that the number of sperm per ejaculate transferred to females during non-competitive single matings does not differ between drive and wildtype males, which is likely due to the adaptive evolution of enlarged testes in drive males. Future experiments will determine whether the competitive ability of drive males is maintained under higher rates of female remating likely to be experienced in nature.

Sperm competition intensity affects sperm precedence patterns in a polyandrous gift-giving spider.

Sperm competition drives traits that enhance fertilization success. The amount of sperm transferred relative to competitors is key for attaining paternity. Female reproductive morphology and male mating order may also influence fertilization, however the outcome for sperm precedence under intense sperm competition remains poorly understood. In the polyandrous spider Pisaura mirabilis, males offer nuptial gifts which prolong copulation and increase sperm transfer, factors proposed to alter sperm precedence patterns under strong sperm competition. First, we assessed the degree of female polyandry by genotyping wild broods. A conservative analysis identified up to four sires, with a mean of two sires per brood, consistent with an optimal mating female rate. Then we asked whether intense sperm competition shifts sperm precedence patterns from first male priority, as expected from female morphology, to last male advantage. We varied sexual selection intensity experimentally and determined competitive fertilization outcome by genotyping broods. In double matings, one male monopolised paternity regardless of mating order. A mating order effect with first male priority was revealed when females were mated to four males, however this effect disappeared when females were mated to six males, probably due to increased sperm mixing. The proportion of males that successfully sired offspring drastically decreased with the number of competitors. Longer copulations translated into higher paternity shares independently of mating order, reinforcing the advantage of traits that prolong copulation duration under intense competition, such as the nuptial gift. Sperm competition intensity enhances the impact of competitive sexual traits and imposes multiple effects on paternity.

The handsome liar: Male spiders offering worthless gifts can benefit increasing mating duration

AbstractFemales of nuptial gift‐giving species are known to mate with several males, so that they secure multiple ejaculates and food via nuptial gifts. However, gifts may decrease females’ reproductive success, for instance, if they received non‐nutritive gifts (worthless). The gift‐giving mating system of the spider Paratrechalea ornata creates a scenario in which females may receive either a nutritive prey or a worthless inedible item wrapped in silk. The worthless gift tactic is expected to be less successful as females should prefer to mate and to mate for longer with males offering nutritive gifts. However, at the moment, the scarce information suggests that both mating tactics may have similar reproductive success. By exposing females to a double mating experimental design, we examined whether mating access and duration correlate with the gift type (nutritive‐worthless), mating order (first‐second), and male size, and additionally explored possible effects on female fitness (e.g., fecundity). We found that the females accepted to mate with courting males independently of gift type, mating order, or size. But, mating duration was positively correlated with male size, and longer when males offered worthless gifts. Furthermore, females’ fecundity increased with mating duration and female size. This demonstrated that males offering worthless gifts might enhance their reproductive success by transferring more sperm than those offering nutritive gifts. Thus, either due to female choice or male effort, male size might be a more relevant feature for reproductive success than gift type, which can explain the high frequencies of worthless gifts in the field.

Reproductive interference between Aedes albopictus and Aedes flavopictus at a place of their origin.

Aedes (Stegomyia) albopictus and Aedes (Stegomyia) flavopictus are related species that have overlapping distributions from southern to central Japan. To understand how they interact, we studied reproductive interference between them, particularly focusing on the body size difference between the mating pair. Here, we examined the effects of conspecific, heterospecific and double mating (i.e. heterospecific mating followed by conspecific mating) on copulation duration, egg production and hatchability of eggs using mosquitoes that varied in body size. Females mated only with heterospecific males produced few viable eggs, indicating that post-mating isolation is almost complete. When mated with heterospecific males before conspecific mating, the production of viable eggs was lower than when mated only with conspecific males, revealing the occurrence of reproductive interference. The degree of reproductive interference was larger in Ae. flavopictus than in Ae. albopictus when the female size was small but did not differ between them when the female size was large. Aedes albopictus females appear to be able to distinguish Ae. flavopictus males from conspecific males and larger females are more successful in the rejection of heterospecific males. On the other hand, Ae. flavopictus were not able to discriminate between conspecific and heterospecific males.

Queen Execution, Diploid Males, and Selection For and Against Polyandry in the Brazilian Stingless Bee Scaptotrigona depilis.

Female mating frequency varies. Determining the causes of this variation is an active research area. We tested the hypothesis that in stingless bees, Meliponini, single mating is due to the execution of queens that make a matched mating at the complementary sex determination locus and have diploid male offspring. We studied the Brazilian species Scaptotrigona depilis. We made up 70 test colonies so that 50% (single matched mating), 25% (double mating), 12.5% (quadruple mating), or 0% (single nonmatched mating) of the emerging brood were diploid males. Queen execution following diploid male emergence was equal and high in colonies producing 50% (77% executed) and 25% (75%) diploid males versus equal and low in colonies producing 12.5% (7%) and 0% (0%) diploid males. These results show that queens that mate with two males with similar paternity suffer an increased chance of being executed, which selects against double mating. However, double mating with unequal paternity (e.g., 25∶75), which occasionally occurs in S. depilis, is selectively neutral. Single mating and double mating with unequal paternity form one adaptive peak. The results show a second adaptive peak at quadruple mating. However, this is inaccessible via gradual evolutionary change in a selective landscape with reduced fitness at double mating.

Sperm competition tactics shape paternity: adaptive role of extremely long copulations in a wolf spider

Polyandry may occur in many species. In spiders when multiple males mate sequentially, conservative anatomical female traits largely, although not entirely, determine whether the first or last male gains precedence in siring the offspring. More flexible behavioural traits show remarkable variation among spiders, including the occurrence of extremely long copulation times. In the wolf spider Pardosa agrestis copulations can last several hours, even though complete fertilization of the female's eggs requires a small fraction of this time. We tested the hypothesis that long copulations in P. agrestis may be a way to overcome a strict first-male precedence, expected from the genital anatomy of female entelegyne spiders. In a large number of mating tests we observed complete and interrupted copulations in both positions of a double mating set-up. Using the sterile male technique we found that paternity was not exclusively determined by the mating order of males; interruption of mating had a dramatic effect on paternity, but did not affect offspring number in a single mating scenario (i.e. the copulation duration was sufficient for the fertilization of all eggs). Females that had an uninterrupted mating were less willing to mate again and became choosy, while females whose first mating was interrupted behaved similarly to virgin females. Also, males seemed to adjust copulation duration to female reproductive status, mating for less time with nonvirgin females, irrespective of whether the female's first mating was interrupted or not. We conclude that extremely long copulations are likely to be an adaptive behaviour manipulating female reproductive behaviour and enabling males to overcome phylogenetically determined limitations in paternity.

When is the change in sperm precedence in the millipede <em>Centrobolus inscriptus</em> (Attems, 1928) (Diplopoda, Pachybolidae)?

There were three scenarios of sperm precedence; first-male, last-male and no order effect. I replicated the H3+ radiolabel technique and showed sperm precedence patterns in the sexually dimorphic millipede Centrobolus inscriptus (Attems, 1928). Disintegration rates (D.P.M.) from single labelled mating were significantly lower than those of unlabelled-labelled mating (U=-2.6, n=13, p=0.005) but not to labelled-unlabelled double mating (U=0.45, n=14, p=0.65). In the initial experiment without the interval V2=43.66% and with the interval V2=74.59%. D.P.M. from the immediate dissections of twice mated females were higher (Z = 3.81, n = 12, 12, P = 0.0001) than females with a remating interval of up to 24 hours. When the experiment was replicated significant differences (H = 30.67, d.f. = 5, P = 0.00001) between total D.P.M values were again found. D.P.M. values were higher when second mating followed immediately after the first (V2= 21%). D.P.M. from labelled-unlabelled mating were significantly lower than the D.P.M. of unlabelled-labelled mating after 10 hours (600 minutes) resulting in a reduction of up to 57% in favour of the second male when second-male ejaculate volume precedence arises to 77%.

Underlying sperm precedence pattern in the millipede Centrobolus inscriptus (Attems, 1928) (Diplopoda, Pachybolidae)

Three scenarios exist for sperm precedence; first-male, last-male and no order effect. I replicated the H3+ radiolabel technique to show sperm precedence patterns in the arthropod Centrobolus inscriptus (Attems, 1928). Male and female morphometric parameters illustrated a clear sexual dimorphism. Disintegration rates (d.p.m.) from immediate dissections of once-mated females were higher than females dissected after 24 hours (L (0) » L (24)). A significant difference between mean d.p.m. values occurred in double mating (L-UL vs. UL- L). D.p.m. values were higher when second mating followed immediately after the first (UL-L (O) >UL-L (24) and L-UL (0) » L-UL (24)). D.p.m. from L mating were significantly lower than those of UL-L mating but not then L-UL double mating. There was a reduction of up to 57% in favour of the second male over 24 hours in C. inscriptus.

How Efficient Is Fertilization by Traumatic Insemination in Orius insidiosus (Hemiptera: Anthocoridae)?

Traumatic insemination (TI) can be injurious to females, and females have evolved various paragenital structures to mitigate these impacts. We examined the mating behavior of Orius insidiosus (Say) and the consequences of single and double matings for female fitness. A total of 100 virgin females (4-6-d old) were directly observed while they mated with virgin males. Some of these females were mated a second time with a different, nonvirgin male 3-5 d later, after they oviposited in sunflower stems. Females were held in isolation, fed eggs of Ephestia kuehniella Zeller, and reproductive success was tracked for 30 d. Six females died during their first copulation (6%), and another within 48 h, without laying eggs. Four percent of the females died during their second copulations. Copulations lasting less than 90 s usually did not result in successful fertilization, and duration of copula was positively correlated with egg fertility in singly-mated females. Duration of copula was more than halved in second matings, twice as variable, and negatively correlated with 30 d fecundity. Thirty-seven percent of singly-mated females and 31% of twice-mated females were infertile, with fewer than half of all females producing 88% of all eggs. We conclude that O. insidiosus females are likely monandrous in the wild, and that TI in this species is inefficient, contributing to high variation in female fitness. Thus, mating involves a significant mortality risk for females, despite their possession of complex paragenital structures that ostensibly mitigate copulatory injury.

No support for the sexy-sperm hypothesis in the seed beetle: Sons of monandrous females fare better in post-copulatory competition.

The sexy‐sperm hypothesis posits that polyandrous females derive an indirect fitness benefit from multi‐male mating because they increase the probability their eggs are fertilized by males whose sperm have high fertilizing efficiency, which is assumed to be heritable and conferred on their sons. However, whether this process occurs is contentious because father‐to‐son heritability may be constrained by the genetic architecture underlying traits important in sperm competition within certain species. Previous empirical work has revealed such genetic constraints in the seed beetle, Callosobruchus maculatus, a model system in sperm competition studies in which female multi‐male mating is ubiquitous. Using the seed beetle, I tested a critical prediction of the sexy‐sperm hypothesis that polyandrous females produce sons that are on average more successful under sperm competition than sons from monandrous females. Contrary to the prediction of the sexy‐sperm hypothesis, I found that sons from monandrous females had significantly higher relative paternity in competitive double matings. Moreover, post hoc analyses revealed that these sons produced significantly larger ejaculates when second to mate, despite being smaller. This study is the first to provide empirical evidence for post‐copulatory processes favoring monandrous sons and discusses potential explanations for the unexpected bias in paternity.

Female control over multiple matings increases the opportunity for postcopulatory sexual selection.

It is widely acknowledged that in most species sexual selection continues after mating. Although it is generally accepted that females play an important role in generating paternity biases (i.e. cryptic female choice, CFC), we lack a quantitative understanding of the relative importance of female-controlled processes in influencing variance in male reproductive fitness. Here, we address this question experimentally using the guppy Poecilia reticulata, a polyandrous fish in which pre- and postcopulatory sexual selection jointly determine male reproductive fitness. We used a paired design to quantify patterns of paternity for pairs of rival males across two mating contexts, one in which the female retained full control over double (natural) matings and one where sperm from the same two males were artificially inseminated into the female. We then compared the relative paternity share for a given pair of males across both contexts, enabling us to test the key prediction that patterns of paternity will depend on the extent to which females retain behavioural control over matings. As predicted, we found stronger paternity biases when females retained full control over mating compared with when artificial insemination (AI) was used. Concomitantly, we show that the opportunity for postcopulatory sexual selection (standardized variance in male reproductive success) was greater when females retained control over double matings compared with when AI was used. Finally, we show that the paternity success of individual males exhibited higher repeatability across successive brood cycles when females retained behavioural control of matings compared with when AI was used. Collectively, these findings underscore the critical role that females play in determining the outcome of sexual selection and to our knowledge provide the first experimental evidence that behaviourally moderated components of CFC increase the opportunity for sexual selection.

Paternity success in ladybirds: function of mating interval and order

Multiple matings result in varying paternity share based on mating interval and order. Thus, assessing the effect of mating interval and order on patterns of sperm usage and paternity is crucial. We designed consecutive and delayed double-mating experiments to investigate paternity variation in ladybird, Menochilus sexmaculatus (Fabricius) (Coleoptera: Coccinellidae), using two distinct morphs of the species as phenotypic markers of paternity. The time to commence mating, copulation duration and reproductive output were recorded. The morphs of the offspring from the two setups were taken as a measure of paternity accumulated by the males. The time to commence mating decreased for the second mating in the consecutive mating treatment, while the reverse was observed in the delayed mating treatment. Consecutive double matings reduced the mating duration. Fecundity increased when second mating occurred after a few days, though percent egg viability remained unaffected. The second male accrued higher paternity (P2 = 0.61) than the first male (P1 = 0.39) in the consecutive mating treatment, while in the delayed mating treatment, the overall paternity share of the first 0.49 (P1) and last male was equal 0.51 (P2). Thus, our study revealed that both mating order and the time interval between successive matings regulate the male paternity share. This finding is reported for the first time in this ladybird species.

Sexual conflict over the duration of copulation in Centrobolus inscriptus (Attems)

Centrobolus inscriptus provides evidence of a conflict of interests over control of copulation duration. Copulation in C. inscriptus lasts for 173 ± 41 min. The re-mating interval between double matings was not impacted by the first copulation duration but was negatively related to the second copulation duration, so that when a female endured a long interval the following copulation was shorter. Female girth was positively correlated to the second copulation duration. The first copulation duration was not significantly dependent on the sexual size dimorphism within copulating pairs, but the second copulation durations was. In these matters of physical combat, the relative sizes of the combatants are thought to be of primary importance.

Precopulatory but not postcopulatory male reproductive traits diverge in response to mating system manipulation in Drosophila melanogaster.

Competition between males creates potential for pre‐ and postcopulatory sexual selection and conflict. Theory predicts that males facing risk of sperm competition should evolve traits to secure their reproductive success. If those traits are costly to females, the evolution of such traits may also increase conflict between the sexes. Conversely, under the absence of sperm competition, one expectation is for selection on male competitive traits to relax thereby also relaxing sexual conflict. Experimental evolution studies are a powerful tool to test this expectation. Studies in multiple insect species have yielded mixed and partially conflicting results. In this study, we evaluated male competitive traits and male effects on female costs of mating in Drosophila melanogaster after replicate lines evolved for more than 50 generations either under enforced monogamy or sustained polygamy, thus manipulating the extent of intrasexual competition between males. We found that in a setting where males competed directly with a rival male for access to a female and fertilization of her ova polygamous males had superior reproductive success compared to monogamous males. When comparing reproductive success solely in double mating standard sperm competition assays, however, we found no difference in male sperm defense competitiveness between the different selection regimes. Instead, we found monogamous males to be inferior in precopulatory competition, which indicates that in our system, enforced monogamy relaxed selection on traits important in precopulatory rather than postcopulatory competition. We discuss our findings in the context of findings from previous experimental evolution studies in Drosophila ssp. and other invertebrate species.

Field observations of simultaneous double mating in the wolf spider Rabidosa punctulata (Araneae: Lycosidae)

Males of many species of spider engage in alternative mating tactics that do not involve pre-mating courtship. Here I report field observations of a novel opportunistic mating tactic of the wolf spider Rabidosa punctulata (Hentz, 1844): simultaneous double mating, whereby males that encounter copulating pairs also mount and achieve inseminations concurrently with the first male. On three separate occasions, female R. punctulata were observed mating with two males simultaneously. Males that mate with already copulating females likely receive multiple fitness benefits. It may allow courtship parasitism of other males while also reducing male agonistic interactions, eliminate the need to court or subdue the female, and reduce pre-mating cannibalism risk. If such behavior is common, it may limit sexual selection acting on male courtship displays by reducing the effectiveness of pre-mating female choice while also increasing sperm competition.

Female remating decisions and a shorter inter-mating interval diminish last-male sperm precedence

Highly variable within and across species, patterns of sperm use not only are often driven by post-copulatory sexual selection but can also be impacted by experimental design. In investigations of paternity bias using competitive double matings, the inter-mating interval is a temporal factor that can affect sperm use patterns if the first male’s sperm is used or lost at an appreciable rate between matings or if its viability or relative competitiveness is influenced by the time since ejaculation. Rapid loss of first-male sperm within the female after mating has been established in the seed beetle (Callosobruchus maculatus), a model system in sperm competition studies. However, our understanding of sperm precedence in this species, which disproportionately favors the last (second) male to mate, is based on long inter-mating intervals. Here, I determine the effect of a shortened inter-mating interval on second-male paternity (P 2) and, importantly, the extent to which females in this species are willing to remate immediately. I find that P 2 is significantly reduced when females remate immediately than when they remate 24 or 48 h after the first mating and that immediate remating is common, indicating that there is a substantial potential for female remating decisions to influence the intensity of sperm competition within species. To understand the variation in inter-mating intervals from a female perspective, I further identify key differences between females that did and did not remate at three inter-mating intervals (0, 24, and 48 h after the initial mating) and discuss potential mechanisms for the observed variation in female refractoriness. Females often mate with multiple males, and in insects it is common for the last male to mate to obtain the greatest share of paternity. One possible explanation for this bias towards last-male sperm use is the passive loss of sperm from the first male with time since mating. Here, I examine the effect of different intervals between matings in a seed beetle species to determine if first-male paternity is increased when females remate sooner. My results show that first males do achieve higher paternity when females remate sooner, which is more common than expected in this species. I also show the differences between females that do and do not remate, which can help explain potential causes for variation in female remating behavior. Together, these results show the significant impact that female remating decisions can have on male competition and, ultimately, their reproductive success.

Size matters in myriapod copulation

Size correlates with mating in Centrobolus. I tested for the presence of relationships between male length and copulation duration in C. inscriptus by performing double mating. Males differed from females in mass, length, and dorsal tergite width. Male length was positively related to the duration of the first mating but negatively related to the duration of the second mating which related to sexual size dimorphism (SSD).

Last male wins the egg fertilization fight: A case study in ladybird, Menochilus sexmaculatus

Sexual selection and the mechanisms involved in sperm competition have not been greatly explored in ladybird beetles. The present study was conducted to investigate the processes of sperm competition and the role of mate guarding behaviour in its regulation in ladybird beetles. We investigated these questions in polyandrous females of the ladybird, Menochilus sexmaculatus (Coleoptera: Coccinellidae) using a phenotypic marker (typical and intermediate morph) to assess paternity of offspring; to determine sperm competition. We conducted two double mating experiments: (i) complete first and second matings, and (ii) disrupted first and complete second matings each using homomorphic and heteromorphic pairing in alternation. Males which mated last were found to sire up to 72% of the offspring produced, indicating last male sperm precedence. Morph itself, independent of mating order, did not have a significant effect on proportion of offspring sired. Paternity share of the last male was negatively associated with mating duration of the first male; mating duration of the first male being indicative of mate guarding. This therefore indicates that prolonged matings by first males are essentially examples of post-copulatory mate guarding to prevent last male sperm precedence.

Sperm dumping in <em>Centrobolus inscriptus</em> Attems (Spirobolida: Trigoniulidae)

Differential ejaculate usage was tested in the diplopod Centrobolus inscriptus. Copulation duration, post-mating interval, and ejaculate volume (disintegrations per minute of H3+) were analysed for single and double mating sequences. During the 24h post-mating, ejaculate volumes decline was identified. The significant drop in ejaculate volumes (dpm) post-mating (Z = 3.00, n = 6, 7, P = 0.003) indicates a female process thought to be sperm dumping.

Sperm storage in <em>Centrobolus inscriptus</em> Attems (Spirobolida: Trigoniulidae)

Differential ejaculate usage was tested in the diplopod Centrobolus inscriptus. Copulation duration, post-mating interval, and ejaculate volume (disintegrations per minute of H3+) were analysed for single and double mating sequences. During the 24h post-mating, ejaculate volumes decline was identified. The significant drop in ejaculate volumes (dpm) post-mating (Z = 3.00, n = 6, 7, P = 0.003) indicates a female process thought to be sperm dumping.