Mating competition among females: testing the distinction between natural and sexual selection in an insect.

In species where females compete for mates, the male often provides the female with resources in addition to gametes. A recently suggested definition of sexual selection proposed that if females only benefit from additional resources that come with each mating and not additional gametes, female intrasexual competition for mating opportunities would result in natural selection rather than sexual selection. The nuptial gift-giving bushcricket Kawanaphila nartee has dynamic sex roles and has been a textbook example of sexual selection acting on females via mating competition. We investigated whether females of this species gain fitness benefits from nuptial gifts, additional ejaculates or both by controlling the number of matings and whether the female was allowed to consume the nutritious gift (spermatophylax) at mating. We found that egg production per day of life increased with the number of additional matings, both with and without spermatophylax consumption, but consuming the spermatophylax had an additional positive effect on the number of eggs. These effects were particularly strong in females with shorter lifespans. We discuss how the recently suggested definition of sexual selection applies to nuptial-feeding insects and conclude that both natural and sexual selections influence mating competition in K. nartee females.

Experience matters: genetic variation affects male reproductive success across sequential mating events in Drosophila melanogaster.

The cost of reproduction is well studied in females but only recently have the costs of mating been investigated in males. Research suggests that males allocate resources between subsequent mating events, resulting in differential success across mating bouts. Selection should favor allocation strategies that match the likelihood of successive matings. The complexity of the system, however, suggests that one fixed strategy is unlikely to be universally favored and thus I predict that genetic variation for different allocation strategies will be segregating in natural populations. To test this, I measured several components of reproductive performance in eight inbred genotypes of Drosophila melanogaster across three sequential mating events. As predicted, there was genetic variation for how previous experience affected a male's reproductive performance for both the proportion of matings that produced offspring and the proportion of offspring sired (P1). Some genotypes had the highest success in their first matings and declined in successive matings while other genotypes did best in later matings. Mating experience had consistent effects across genotypes on fertility and induced refractoriness to remating. On average, virgin matings produced the highest fertility and third matings most effectively induced refractoriness. Genotype also had a significant effect on fertility. These results have important implications for understanding how selection may be acting on males when there is variation in the likelihood of multiple mating events and could affect the evolution of male allocation strategies in the face of perceived competitors.

A new dataset of pheromone and pheromone-gene structures from the ciliate, Euplotes crassus.

Like many other organisms, ciliates communicate and interact socially via diffusible chemical signals, named pheromones, that are functionally associated with a genetic mating-type mechanism of cell self/not-self recognition. In Euplotes species, pheromones form species-specific families of small, globular, and disulfide-rich proteins folding into exclusively helical secondary structures. Each is specified by one of a series of high-multiple alleles that are inherited in Mendelian fashion with relationships of co-dominance at the so-called mat genetic locus of the cell transcriptionally inert micronuclear genome, and expressed in the transcriptionally active macronuclear genome as individual DNA molecules in which the central coding region is flanked by 5'-leader and 3'-trailer noncoding regions ending with C4A4/T4G4 telomeric repeats. In E. crassus, a cosmopolitan marine species with a long tradition in the study of ciliate mating systems and breeding patterns, oligonucleotides specific to amino acid sequences of pheromones Ec-1 and Ec-α were previously used to clone and sequence a first set of four structurally distinct macronuclear (mac) pheromone coding genes, mac-ec-α, mac-ec-1, mac-ec-2 and mac-ec-3, from two interbreeding strains, L-2D and POR-73. The use of these oligonucleotides in PCR amplifications of macronuclear DNA preparations from three other E. crassus interbreeding strains, ES10, Fava4 and MN4, has now resulted in the characterization of a second set of eight new pheromone coding genes, mac-ec-ß, mac-ec-γ, mac-ec-δ, mac-ec-ε, mac-ec-µ, mac-ec-4, mac-ec-5 and mac-ec-6. Multiple alignment between previously and newly determined pheromone-gene sequences reinforces the concept that the E. crassus pheromone-gene family includes two sub-families, which likely reflect a duplication of the micronuclear mat gene locus and represent an apomorphic trait of the E. crassus clade. Members of one sub-family (each identified with a Greek letter) show a 500-bp 5'-leader noncoding region rich in AGGA/AGGGA repetitions, and encode 56-amino acid pheromones with eight conserved Cys residues. Members of the other sub-family (each identified with an Arabic numeral) show an 800-bp 5'-leader noncoding region without AGGA/AGGGA repetitions, and encode 45-amino acid pheromones with ten conserved Cys residues.

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.

A Limiting Factor of Sex Attractants of Bactrocera dorsalis (Diptera: Tephritidae), Verified under Laboratory Conditions.

At present, sexual attractants mainly control insect populations by killing males. However, the effect of sex attractants may be limited by the mating ability of the attracted insects. The Oriental fruit fly, Bactrocera dorsalis (Hendel), has a strong reproductive capacity; it brings great losses to agricultural production, which can be controlled by methods using sex attractant methyl eugenol that mainly attracts males. Therefore, we studied the multiple and continuous (as well as consecutive) mating ability of B. dorsalis through behavioral experiments. The results show that male B. dorsalis can mate 11 times on average, with females mating only 1.93 times, and that 10.81% of males mate more than 20 times. The reproductive capacity of male B. dorsalis decreased significantly after four to five instances of continuous mating. In different mating patterns, the reproductive fitness of polyandry is not the highest, rather, interval mating is the best. A limiting factor of the sex attractant effect was revealed in B. dorsalis through behavioral evidence.

Asymmetry in the reproductive interference between two closely related species of spider mites, Panonychus citri and Panonychus osmanthi (Prostigmata: Tetranychidae).

An incomplete species recognition system may cause reproductive interference. Interspecific mating between Panonychus citri and Panonychus osmanthi produced apparently normal F1 hybrid females. However, the F1 females laid few eggs, and the produced eggs did not hatch (suggesting hybrid breakdown). Several common host plants of P. citri and P. osmanthi have been determined, including Japanese pear, peach, and citrus. However, P. osmanthi has been found only on its particular host plants in the genus Osmanthus. To assess the potential for reproductive interference between P. citri and P. osmanthi, we evaluated the competitiveness (1 ♀ × 2 ♂♂) and preference (2 ♀♀ × 1 ♂) of males in mating and guarding behavior and assessed the effect of second mating on offspring paternity using Japanese pear leaf disks. We found that P. citri males were superior competitors. Moreover, the species recognition ability of P. citri males was lower than that of P. osmanthi males, and frequent occurrence of reproductive interference should therefore be expected. In a mixed population (2 ♀♀ × 2 ♂♂), 41% of females copulated with heterospecific males, although the mating superiority of P. citri was not proven. The ratio of effective second mating with conspecific males after heterospecific mating was higher in the crosses to P. citri females (77.8%) than P. osmanthi females (14.3%). Therefore, P. citri females had more chance than P. osmanthi females to recover from the fitness cost imposed by heterospecific mating through subsequent conspecific mating. To date, P. osmanthi has not been detected on host plants other than Osmanthus species. Reproductive interference by P. citri males may therefore narrow the range of host plants available for P. osmanthi.

X chromosome drive is constrained by sexual selection and influences ornament evolution.

Experimental evolution provides an integrative method for revealing complex interactions among evolutionary processes. One such interaction involves sex-linked selfish genetic elements and sexual selection. X-linked segregation distorters, a type of selfish genetic element, influence sperm transmission to increase in frequency and consequently alter the population sex ratio and the opportunity for sexual selection, while sexual selection may impact the spread of X-linked distorters. Here we manipulated sexual selection by controlling female mating opportunities and the presence of a distorting X chromosome in experimental lines of the stalk-eyed fly, Teleopsis dalmanni, over 11 generations. We find that removal of sexual selection leads to an increase in the frequency of the X-linked distorter and sex ratio across generations and that post-copulatory sexual selection alone is sufficient to limit the frequency of distorters. In addition, we find that male eyestalk length, a trait under pre-copulatory sexual selection, evolves in response to changes in the strength of sexual selection with the magnitude of the response dependent on X chromosome type and the frequency of distorting X chromosomes. These results reveal how a selfish X can interact with sexual selection to influence the evolution of sexually selected traits in multiple ways.

Transgenerational effects of multiple mating in Spodoptera litura Fabricius (Lepidoptera: Noctuidae).

Polyandrous mating can result in sexual conflict and/or promote the evolution of mating patterns. Does multiple mating by females support the genetic benefits hypothesis and can it be validated as an evolutionary strategy? If we are to decipher the consequences of sexual interactions and understand the interplay of sexual conflict and multiple generational benefits, the transgenerational effects need to be followed over multiple generations. We investigated the effects of three mating patterns, single mating, repeated mating, and multiple mating, on parental Spodoptera litura copulation behavior, and then identified the impact on the development, survival, and fecundity of the F1 and F2 generations. Fecundity was not significantly affected in the F1 generation but was substantially enhanced in the F2 generation. There was a reversal of offspring fitness across the F2 generations from the F1 generations in progeny produced by multiple mating. In addition, the intrinsic rate of increase, finite rate of increase and net reproductive rate in the F1 generation the multiple mating treatment was significantly lower than in the single mating treatment, but there was no apparent effect on the F2 generation. Repeated mating had no significant effects on progeny fitness. We postulate that multiple mating imposes cross-transgenerational effects and may ultimately influence multigenerational fitness in S. litura.

The carboxypeptidase B and carbonic anhydrase genes play a reproductive regulatory role during multiple matings in Ophraella communa.

Seminal fluid proteins (SFPs) are key factors in sexual reproduction and are transferred to females during mating with sperm. SFPs have a nutritional value because they protect and activate sperm storage and release to optimize fecundity. Multiple matings promote ovipositioning in several insect species. Therefore, insects may obtain more SFP through multiple matings to maximize reproduction, but this process has not yet been clearly confirmed. Here, the relationship between multiple matings and the SFPs in Ophraella communa (Coleoptera: Chrysomelidae), a biological control agent of the common ragweed Ambrosia artemisiifolia (Asterales: Asteraceae), was studied. Multiple matings significantly increased female fecundity and ovary egg deposition. Carboxypeptidase B (OcCpb) and carbonic anhydrase (OcCa) genes were identified as putative SFP genes in O. communa and they showed strong male-biased expression. Additionally, OcCpb and OcCa expression was upregulated in the bursa copulatrix of mating females compared to that in virgin females, but their expression gradually declined after copulation. Furthermore, OcCpb and OcCa knockdown in males led to a decrease in insect fecundity compared to that in the control. The reproductive tract of females mated with dsRNA-treated males was dissected and observed and, notably, the ovaries produced significantly fewer eggs. These data suggest that OcCpb and OcCa play regulatory roles during multiple matings in O. communa.

Multiple paternity is related to adult sex ratio and sex determination system in reptiles.

The adult sex ratio (ASR, the proportion of males in the adult population) is an emerging predictor of reproductive behaviour, and recent studies in birds and humans suggest it is a major driver of social mating systems and parental care. ASR may also influence genetic mating systems. For instance male-skewed ASRs are expected to increase the frequency of multiple paternity (defined here as a clutch or litter sired by two or more males) due to higher rates of coercive copulations by males, and/or due to females exploiting the opportunity of copulation with multiple males to increase genetic diversity of their offspring. Here, we evaluate this hypothesis in reptiles that often exhibit high frequency of multiple paternity although its ecological and life-history predictors have remained controversial. Using a comprehensive dataset of 81 species representing all four non-avian reptile orders, we show that increased frequency of multiple paternity is predicted by more male-skewed ASR, and this relationship is robust to simultaneous effects of several life-history predictors. Additionally, we show that the frequency of multiple paternity varies with the sex determination system: species with female heterogamety (ZZ/ZW sex chromosomes) exhibit higher levels of multiple paternity than species with male heterogamety (XY/XX) or temperature-dependent sex determination. Thus, our across-species comparative study provides the first evidence that genetic mating system depends on ASR in reptiles. We call for further investigations to uncover the complex evolutionary associations between mating systems, sex determination systems and ASR.

Female-limited responses in remating rate and mating duration in the experimental evolution of a beetle Callosobruchus chinensis.

Mating rate optima often differ between the sexes: males may increase their fitness by multiple mating, but for females multiple mating confers little benefit and can often be costly (especially in taxa without nuptial gifts or mala parental care). Sexually antagonistic evolution is thus expected in traits related to mating rates under sexual selection. This prediction has been tested by multiple studies that applied experimental evolution technique, which is a powerful tool to directly examine the evolutionary consequences of selection. Yet, the results so far only partly support the prediction. Here, we provide another example of experimental evolution of sexual selection, by applying it for the first time to the mating behaviour of a seed beetle Callsorobruchus chinensis. We found a lower remating rate in polygamy-line females than in monogamy-line (i.e. no sexual selection) females after 21 generations of selection. Polygamy-line females also showed a longer duration of first mating than monogamy-line females. We found no effect of male evolutionary lines on the remating rate or first mating duration. Though not consistent with the original prediction, the current and previous studies collectively suggest that the observed female-limited responses may be a norm, which is also consistent with the conceptual advances in the last two decades of the advantages and limitations of experimental evolution technique.

Effect of Parental Age and Mating Status on Reproductive Performance of Orius laevigatus (Hemiptera: Anthocoridae).

The reproductive potential of biological control agents (BCAs) is crucial for efficient mass-rearing and field performance, and it all begins with mating. Fecundity can be strongly influenced by intrinsic conditions, such as female age and, often neglected, male age and mating status. However, little is known about the impact of parental status at mating on female reproductive outcomes in BCAs. Orius laevigatus (Fieber) (Hemiptera: Anthocoridae) is widely used to control thrips in protected crops. We evaluated how many consecutive females a male could successfully mate and the effect on a female's reproductive output. In addition, we studied the effects of male and female age on mating. In the multiple mating experiment, the males showed a high capacity to fertilize females successively, not reducing fecundity until the sixth mated female. In the delayed mating experiment, copulation duration and fecundity increased with male age but decreased with female age. In contrast, fertility followed an opposite pattern, increasing with female age but decreasing with male age. However, fecundity gains outweighed fertility declines in both sexes. Therefore, reproductive capacity is increased when mating newly emerged females with males a few days old. The implications of our results for mass rearing and field performance are discussed.

The ontogeny of selection on genetic diversity in harvester ants.

Selection may favour traits throughout an individual's lifetime or at a particular life stage. In many species of social insects, established colonies that are more genetically diverse outperform less diverse colonies with respect to a variety of traits that contribute to fitness, but whether selection favours high diversity in small colonies is unknown. We tested the hypothesis that selection favours genetically diverse colonies during the juvenile period using a multi-year field experiment with the harvester ant, Pogonomyrmex occidentalis. We used controlled matings to generate colonies that varied in genetic diversity and transplanted them into the field. We monitored their survival for seven (the 2015 cohort, n = 149) and six (the 2016 cohort, n = 157) years. Genetically more diverse colonies had greater survival, resulting in significant viability selection. However, in both cohorts survival was not influenced by genetic diversity until colonies were three years old. We suggest that changes in their internal organization enabled colonies to use the benefits of multiple genotypes, and discuss possible mechanisms that can generate this pattern.

Mitonuclear discordance and patterns of reproductive isolation in a complex of simultaneously hermaphroditic species, the Allolobophora chlorotica case study.

Historical events of population fragmentation, expansion and admixture over geological time may result in complex patterns of reproductive isolation and may explain why, for some taxa, the study of mitochondrial (mt) and nuclear (nu) genetic data results in discordant evolutionary patterns. Complex patterns of taxonomic diversity were recently revealed in earthworms for which distribution is largely the result of paleogeographical events. Here, we investigated reproductive isolation patterns in a complex of cryptic species of earthworms in which discordant patterns between mt and nu genetic lineages were previously revealed, the Allolobophora chlorotica aggregate. Using four nu microsatellite markers and a fragment of the cytochrome c oxidase subunit I mt gene, we carried out a parentage analysis to investigate the mating patterns (i) between individuals belonging to two divergent mt lineages that cannot be distinguished with nu markers and (ii) between individuals belonging to lineages that are differentiated both at the mt and nu levels. Amongst the 157 field-collected individuals, 66 adults were used in cross-breeding experiments to form 22 trios based on their assignment to a mt lineage, and 453 obtained juveniles were genotyped. We showed that adults that mated with both their potential mates in the trio produced significantly more juveniles. In crosses between lineages that diverged exclusively at the mt level, a sex-specific pattern of reproduction characteristic to each lineage was observed, suggesting a possible conflict of interest concerning the use of male/female function between mating partners. In crosses between lineages that diverged both at the mt and nu levels, a high production of cocoons was counterbalanced by a low hatching rate, suggesting a post-zygotic reproductive isolation. Different degrees of reproductive isolation, from differential sex allocation to post-zygotic isolation, were thus revealed. Lineages appear to be at different stages in the speciation process, which likely explain the observed opposite patterns of mitonuclear congruence.

Mating status affects female choice when females are signalers.

Sexual selection in animals has been mostly studied in species in which males are signalers and females are choosers. However, in many species, females are (also) signalers. In species with non-signaling females, virgin females are hypothesized to be less choosy than mated females, as virgins must mate to realize fitness and the number of available males is generally limited. Yet, when females signal to attract males, mate limitation can be overcome. We tested how virgin and mated females differ in their calling behavior, mating latency, and in mate choice, using the tobacco budworm Chloridea (Heliothis) virescens as an example for a species in which females are not only choosers but also signalers. We found that virgin females signaled longer than mated females, but virgin and mated signaling females were equally ready to mate, in contrast to non-signaling females. However, we found that virgin signaling females showed weaker mate preference than mated females, which can be explained by the fact that females increase their fitness with multiple matings. Mated females may thus further increase their fitness by more stringent mate selection. We conclude that signaling is a crucial aspect to consider when studying female mate choice because signaling may affect the number of available mates to choose from.

Males mate with females even after sperm depletion in the two-spotted spider mite.

Generally, males increase their reproductive success by mating with as many females as possible, whereas females increase their reproductive success by choosing males who provide more direct and indirect benefits. The difference in reproductive strategy between the sexes creates intense competition among males for access to females, therefore males spend much energy and time for competition with rival males for their reproduction. However, if they do not need to engage themselves into male competition and females are in no short supply, how many females can a male mate with and fertilize? We address this question in the two-spotted spider mite, Tetranychus urticae Koch. In this study, we investigated how many females a young, virgin male mated in 3 h, and checked whether the mated females were fertilized. We found that on average males mated with 12-13 females (range: 5-25). As latency to next mating did not change with the number of matings, the males are predicted to engage in even more matings if the mating trial were continued beyond 3 h. Copulation durations decreased with the number of matings and typically after 11 copulations with females any further copulations did not lead to fertilization, suggesting that males continued to mate with females even after sperm depletion. We discuss why spider mite males continue to display mating and copulation behaviour even after their sperm is depleted.

Female mating status affects male mating tactic expression in the wolf spider Rabidosa punctulata.

Males and females have conflicting interests on the frequency and outcomes of mating interactions. Males maximize their fitness by mating with as many females as possible, whereas choosy females often reduce receptivity following copulation. Alternative male mating tactics can be adaptive in their expression to a variety of mating contexts, including interactions with a relatively unreceptive mated female. Male Rabidosa punctulata wolf spiders can adopt distinctive mating tactics when interacting with a female, a complex courtship display, and/or a more coercive direct mount tactic that often involves grappling with females for copulation. In this study, we set up female mating treatments with initial trials and then paired mated and unmated females with males to observe both female remating frequencies and the male mating tactics used during the interactions. Males adopted different mating tactics depending on the mating status of the female they were paired with. Males were more likely to adopt a direct mount tactic with already-mated females and courtship with unmated females. Already-mated females were considerably less receptive to males during experimental trials, although they did remate 34% of the time, the majority of which were with males using a direct mount tactic. Whereas males adjusting to these contextual cues were able to gain more copulations, the observation of multiple mating in female R. punctulata introduces the potential for sperm competition. We discuss this sexual conflict in terms of the fitness consequences of these mating outcomes for both males and females.

Patterns of sperm use by twice-mated female Queensland fruit flies.

Multiple mating by females, polyandry, is common in insects, including in tephritid fruit flies. Female insects that remate commonly store sperm of multiple males. How the sperm of different males contribute to paternity is an important element of sexual selection. Sexual behavior and reproduction of the Queensland fruit fly (Qfly), Bactrocera tryoni, has been extensively investigated both in relation to understanding this economically important species' reproductive biology and in relation to implications for Sterile Insect Technique (SIT), whereby sterile flies are released to constrain reproduction of pest populations. Despite numerous studies of pre- and postcopulatory sexual selection in Qfly, there have been no direct studies of paternity patterns in polyandrous female Qflies. We used two morphologically distinguishable lines to investigate patterns of sperm use in Qfly. The two lines showed comparable mating performance evidenced by similar mating and remating frequency, copula duration, and proportion of second mate paternity (P2) between reciprocal crosses. The mechanism of sperm usage, with P2 close to 0.5 immediately after the second mating followed by gradual decrease of P2 as females aged, is most consistent with stratification or repositioning of sperm. Patterns observed in the present study are compared with the available information from other tephritid fruit flies, and are discussed in relation to this species' reproductive biology, known patterns of sperm storage, and SIT.

The benefits of being big and diverse: early colony survival in harvester ants.

In sessile organisms such as plants and benthic invertebrates, founding propagules typically suffer extremely high rates of mortality due to both extrinsic and intrinsic factors. Many social insect species share similarities with these groups, but factors influencing early colony survival are relatively unstudied. We used a field experiment to measure the importance of environmental quality relative to intrinsic colony properties in the harvester ant, Pogonomyrmex occidentalis, by monitoring the survival of 584 experimental colonies. We measured survival of transplanted colonies over four months in each of three years (2014-2016) at a site in western Colorado. Colony survival was primarily determined by colony features. Multiple mating by the queen and larger colony size at the time of transplant increased survival, but queen size, maternal lineage and the composition of plant species in the vicinity of the colony did not. Food supplementation increased survival significantly when natural food was scarce, but was not consistently beneficial, in contrast to predictions. Our results emphasize the general importance of rapid growth and early attainment of large size in the survival of sessile species. However, attributes specific to ants that are a consequence of their sociality also strongly affected survival. Colonies with multiply-mated queens were more likely to survive over a wide range of circumstances, highlighting the importance of this trait even at the early stages of colony life.

Evolution of male nuptial gift and female remating: A quantitative genetic model.

In some species of separate sexes, males present a nuptial gift containing nutrition to their mate. Producing a large nuptial gift is a considerable cost to the male, but it may improve his siring success if the female reduces the likelihood to accept another male after receiving a large gift. The female may receive a direct benefit by accepting another male who provides an additional nuptial gift. Additionally, the female may receive an indirect fitness benefit via laying offspring sired by a male who is able to produce a large nuptial gift. We formalized the multivariate quantitative genetics model describing the coevolution of the size of nuptial gift produced by the male (x) and the female's propensity to engage in remating (y). We analyzed the model focusing two cases: [1] remating females receive no indirect fitness benefit, but enjoy direct benefit of nutrition; and [2] remating females receive no direct benefit, but enjoy an indirect fitness benefit due to a positive genetic correlation of x and y, which is possible if random mutations tend to make males produce small nuptial gifts. In both cases, the stable evolutionary equilibrium with neither nuptial gift nor remating (x-=y-=0) always exists. Another stable equilibrium may exist in which male produces nuptial gifts (x->0) and female engage in multiple mating (y->0). We discussed implications to the sexual conflict.