Widespread 'hilltopping' in Acraea butterflies and the origin of sex-role-reversed swarming in Acraea encedon and A. encedana

In some populations of the butterflies Acraea encedon and A. encedana, most females are infected with a bacterium that kills their sons. The resulting shortage of males is associated with females adopting a sex‐role‐reversed mating system, in which females swarm at landmarks such as hilltops and compete for males. We have observed the mating behaviour of Acraea species that are not known to be infected with the male‐killer. In over half of these species, males were found to aggregate on hilltops. It is likely that this behaviour was ancestral to the sex‐role‐reversed swarms of Acraea encedon and A. encedana, and we discuss how the spread of the male‐killing infection may have converted this mating system into sex‐role‐reversed swarming.     

Male-killing Wolbachia and mitochondrial DNA: selective sweeps,hybrid introgression and parasite population dynamics

Mitochondrial DNA (mtDNA) sequences are widely used as neutral genetic markers in insects. However, patterns of mtDNA variability are confounded by the spread of maternally transmitted parasites, which are genetically linked to the mitochondria. We have investigated these effects in the butterflies Acraea encedon (which is host to two strains of male-killing Wolbachia bacteria) and A. encedana (which is host to one strain). Within a population, the mitochondria are in linkage disequilibrium with the different male-killers. Furthermore, there has been a recent selective sweep of the mtDNA, which has led to the loss of mitochondrial variation within populations and erased any geographical structure. We also found that one of the male-killers, together with the associated mtDNA, has introgressed from A. encedana into A. encedon within the last 16,000 years. Interestingly, because butterflies are female heterogametic, this will presumably have also led to the introgression of genes on the W sex chromosome. Finally, in A. encedon the mitochondria in uninfected females are unaltered by the spread of the male-killer and have diverse, geographically structured mtDNA. This means we can reject the hypothesis that the male-killer is at a stable equilibrium maintained by imperfect transmission of the bacterium. Instead, some other form of balancing selection may be maintaining uninfected females in the population and preventing the species from going extinct due to a shortage of males.     

Population biology of tropical African butterflies. Sex ratio and genetic variation in Acraea encedon

In Uganda the butterfly, Acraea encedon (L.), occurs in well-defined populations that are largely genetically and ecologically isolated from each other. During the course of a large-scale capture-recapture programme about 18,000 different individuals were marked and released, and from these and many recaptures aspects of the population structure were investigated. Some populations are composed of almost all females while in other populations the sex ratio is normal. In the predominantly female populations many females remain unmated and produce infertile eggs, laying them on plants that are not foodplants and on each other. In such populations there is intense aggregating behaviour. Males live longer than females and each male may mate with several different females. There is evidence for the existence of a self-regulating mechanism in the predominantly female populations in which population size and male frequency are reciprocally dependent. Acraea encedon exists in a large variety of sympatric colour forms, the frequency of which varies from population to population. Some colour forms survive better than others. The hindwing of Acraea encedon has 20 black spots each of which can be absent in any particular butterfly. The relative frequency of missing spots varies from population to population. The ecological genetics of Acraea encedon is discussed in relation to theories of mimicry and polymorphism.     

HOW CAN SEX RATIO DISTORTERS REACH EXTREME PREVALENCES? MALE‐KILLING WOLBACHIA ARE NOT SUPPRESSED AND HAVE NEAR‐PERFECT VERTICAL TRANSMISSION EFFICIENCY IN ACRAEA ENCEDON

Abstract.— Maternally transmitted bacteria that kill male hosts early in their development are found in many insects. These parasites typically infect 1–30% of wild females, but in a few species of insects, prevalences exceed 95%. We investigated one such case in the butterfly Acraea encedon , which is infected with a male-killing Wolbachia bacterium. We measured three key parameters that affect the prevalence of the parasite: transmission efficiency, rate of survival of infected males, and the direct cost of infection. We observed that all wild females transmit the bacterium to all their offspring and that all infected males die in wild populations. We were unable to detect any physiological cost to infection in lab culture. These observations explain the high prevalence of the A. encedon male killer, as theory predicts that under these conditions the parasite will spread to fixation. This will occur provided the death of males provides some benefit to the surviving infected females. The problem therefore becomes why the bacterium has not reached fixation and driven the butterfly extinct due to the shortage of males. We therefore investigated whether males choose to mate with uninfected rather than infected females, as this would prevent the bacterium from reaching fixation. We tested this hypothesis in the "lekking swarms" of virgin females found in the most female-biased populations, and were unable to detect any evidence of mate choice. In conclusion, this male killer has spread to high prevalence because it has a high transmission efficiency and low cost, but the factors maintaining uninfected females in the population remain unknown.     

Non-random Mating in the Dance Fly Empis borealis: The Importance of Male Choice

In the dance-fly Empis borealis (Diptera, Empididae), females form swarms to which males, carrying a nuptial gift, come for mating. We examined whether males or females were choosy and/or competed for mates. First, measurements of the size relationships between copulating males and females, nuptial gifts and the swarming females from different swarms were assessed. Second, male visiting time in differently sized female swarms was recorded. Larger (wing-length) females participated disproportionately in copulations in each swarm, but not for the population at large. Female mating status (virgin/non-virgin) or proximity to oviposition (egg size) did not influence the likelihood of copulation. No assortative mating pattern was found: male size and size of nuptial gift did not correlate with size of the mating female. The time spent by males in swarms increased with the number of females present and it took longer when males left a swarm without copulation than when doing so. Male visiting time per female was negatively correlated with number of females in swarms. Males more often left smaller than larger swarms without mating. We conclude that E. borealis males discriminate among females but find no evidence for male competition or for female choice. It is still a question to what degree females compete for males.     

Swarm behaviour and mate competition in mayflies (Ephemeroptera)

Although mayfly swarms are frequently cited as an example of lekking by insects, little is known about the behaviour of individuals within a swarm, or how mate-selection takes place. A study of five species of mayfly over a period of 10 consecutive years has revealed species-specific differences in the flight pattern of swarming males and in the ability of males to recognize swarms of their own species. Males of four of the five species jostle other males in the swarm at all times except when mating: mating pairs are not jostled. The pattern of jostling varies with the species. Measurements of the sperm content of the vesicula seminalis and of the wing length of members of individual swarms show that larger wing size is positively correlated with the presence of less sperm. The vesicula seminalis is always filled with sperm at the beginning of the imaginal stage and the testes regress before the beginning of the imaginal stage. If the volume of sperm in the vesicula seminalis is a valid index of mating success then males with larger wings have the highest success. Large wings may bestow an advantage during jostling. The males of Ephemera danica , which do not jostle, glide with outspread wings; these outspread wings may attract females, the largest wings being the most attractive. Females of all five species enter the swarm a few at a time, although many females may be resting beneath the swarm. This phased entry may decrease the attraction of the swarm for predators. The number of females in a swarm is not correlated with swarm size, and the factors which enable females to regulate their entry into a swarm remain obscure.     

Swarming Behavior, Sexual Dimorphism, and Female Reproductive Status in the Sex Role-Reversed Dance Fly Species Rhamphomyia marginata

Dance flies are predaceous insects which often form male mating swarms. In many species males prior to swarming catch an insect prey, which is presented to the female at mating. In Rhamphomyia marginata, females in contrast to males gather to swarm, while males carrying a prey visit swarms for mating. Here I describe the swarming and courtship behavior in R. marginata and provide data on sexual dimorphism and swarming female reproductive status. Females swarm in small clearings in the forests. There was no specific swarm-maker. The swarming period lasted for 2–3 h and peaked around sunset. Identical swarm sites were used each evening and for several years. The mean number of females in swarms (swarm sites with at least one female) was 9.9 ± 9.1 (range, 1–40; n = 107) in 1993 and 7.1 ± 7.0 (range, 1–35; n = 68) in 1994. No obvious competition between females in swarms was observed. The operational sex ratio in swarms was extremely female biased (all swarms, 0.04). Less than one-third of male visits to swarms resulted in mating and males were found more often in larger swarms. Nuptial prey consisted of male midges. Females seem to mate more than once. Swarming females had undeveloped eggs, whereas mated females in swarms had further developed eggs than unmated females. Amount of sperm in the spermatheca was correlated with egg size. Amount of sperm and egg size did not correlate with wet weight, wing length, or wing load, except for egg size and weight. The wing coloration pattern and shape in R. marginata females are unique among dance flies, being greatly enlarged (1.6 times larger than that of males) and bicolored (gray part, 60% of wing area). When females, instead of males, possess extravagant secondary sexual characters, it is predicted from sexual selection theory that females should compete for males and that males should be selective in their choice of partner. A sex-role reversal will evolve when assess to males limit female reproductive success. The dance fly species R. marginata, like Empis borealis, another dance fly species studied earlier and discussed here, seems to fit these predictions.     

Male killing can select for male mate choice: a novel solution to the paradox of the lek

In lekking species, intense directional selection is applied to aspects of the male genotype by female choice. Under conventional quantitative genetics theory, the expectation is that this will lead to a rapid loss in additive genetic variance for the trait in question. However, despite female choice, male variation is maintained and hence it pays females to continue choosing. This has been termed the ''paradox of the lek''. Here we present a theoretical analysis of a putative sex-role-reversed lek in the butterfly Acraea encedon. Sex-role reversal appears to have come about because of infection with a male-killing Wolbachia. The bacterium is highly prevalent in some populations, such that there is a dearth of males. Receptive females form dense aggregations, and it has been suggested that males preferentially select females uninfected with the bacterium. As with more conventional systems, this presents a theoretical problem exactly analogous to the lek paradox, namely what maintains female variation and hence why do males continue to choose? We model the evolution of a male choice gene that allows discrimination between infected and uninfected females, and show that the stable maintenance of both female variation and male choice is likely, so long as males make mistakes when discriminating between females. Furthermore, our model allows the maintenance, in a panmictic population, of a male killer that is perfectly transmitted. This is the first model to allow this result, and may explain the long-term persistence of a male killer in Hypolimnas bolina.     

Reassessment of Sex Role-Reversal Hypothesis in the Tropical Butterfly Acraea encedon (Lepidoptera: Nymphalidae)

Species with extremely female-biased sex ratios are expected to show alteration in the normal sex roles, as a response to male scarcity. The tropical butterfly Acraea encedon is known to be infected with a male-killing bacterium of the genus Wolbachia, which has led to severe sex ratio distortion in some populations where more than 95 % of wild females are infected with the male-killer. Thus, the aggregation of female A. encedon at resource-free landmarks has been interpreted as “female lekking” behaviour, a sex role-reversed form of lekking normally seen in males of many animals. For this paper, sites in Uganda where female-leks have previously been reported (in 1998) were revisited and surveyed for both sex ratio and bacterial prevalence, for 3 years (2005–2007). The hypothesis of sex role-reversal in A. encedon was evaluated in light of the field data obtained. The study concluded that the response of host populations to the gradual spread of the male-killer toward fixation occurs initially at the behavioural level, as sex role-reversal, and finally at the demographic level, by succumbing to extinction.     

Male mate choice, sexual conflict and strategic allocation of copulations in a lekking bird

The males of lekking species are not expected to be choosy about mating because a reduced reproductive rate due to lost mating opportunities should outweigh any benefits of male choice. Females have traditionally not been expected to be competitive in this system since their reproduction has usually been assumed to be unconstrained by male availability. Here we show that, in contrast to these predictions, males are choosy and females may be competitive in the lekking great snipe Gallinago media. Males preferred by many females often refused to copulate with and even chased away females that the male had already copulated with, whereas females seemed to compete for repeated copulations. We conclude that choosiness may sometimes pay for popular males in those lekking species where females copulate repeatedly. Apparently, evolutionary conflicts of interest between individuals may cause a richer repertoire of behavioural adaptations than, to our knowledge, hitherto realized.     

Male body size and mating success in swarms of the mayfly Epeorus longimanus

Epeorus longimanus is a widely distributed mayfly in the western United States that forms relatively large mating swarms. The operational sex ratio of swarms is highly male biased and males are potentially polygynous, suggesting that male-male competition over mates may be intense. We investigated whether body size influenced male mating success in E. longimanus , as evidence of sexual selection. Males collected as mating pairs had significantly greater body lengths compared with males collected randomly from the swarm on each of six sampling dates examined, and had significantly greater head widths than males from random collections on two dates. There was no indication that large males occupied preferred positions within the swarm, and we suspect that the large male advantage may be due to greater success in pursuing females. We found no evidence of size-assortative mating in E. longimanus indicating that males attempt to male with every female encountered, consistent with the brief copulatory period in mayflies and overall low parental investment of males.     

Do males choose their mates in the lekking moth Achroia grisella? Influence of female body mass and male reproductive status on male mate choice

Lekking males aggregate to attract females and contribute solely to egg fertilization, without any further parental care. Evolutionary theory therefore predicts them to be nonchoosy toward their mates, because any lost mating opportunities would outweigh the benefits associated with such preferences. Nevertheless, due to time costs, the production of energetically costly sexual displays, and potential sperm limitation, the mating effort of lekking males is often considerable. These factors, combined with the fact that many females of varying quality are likely to visit leks, could favor the evolution of male mate preferences. Here, we show that males of the lekking lesser wax moth, Achroia grisella, were indeed more likely to mate with heavier females in choice experiments, even at their virgin mating (i.e., when their reproductive resources have not yet been depleted by previous matings). This differential female mating success could not be attributed to female behavior as heavy and light females showed similar motivation to mate (i.e., latency to approach the males) and time to copulate. Males seem to benefit from mating with heavier females, as fecundity positively correlated with female mass. This new empirical evidence shows that male mate choice may have been underestimated in lekking species.     

Swarming behavior in male chironomid midges: a cost-benefit analysis

Aerial mating swarms of nonbiting male midges form at dusk andattract females from the surrounding vegetation. Females flyinto the swarm, and copulation occurs on the wing. Mating andpredation are identified as the major benefit and cost of swarmingand are influenced by swarm size in opposing ways. Swarms varygreatly in size but the individual's probability of mating isgreatest in the smallest swarms. However, the individual predationrisk is also greatest in the smallest swarms. These opposingeffects on swarm size combine in a common currency of matingsuccess per evening to favor males in the smallest swarms. Thereis also an effect of male body size. The smallest males occurpredominantly in the smallest swarms and have the highest matingsuccess. The mechanisms that might maintain the observed swarmsize distribution are discussed.     

Swarming and mating behavior in Ephemera orientalis Mclachlan, 1875 (Ephemeroptera: Ephemeridae) with morphological analyses

Swarming and mating behaviors of a mayfly species, Ephemera orientalis Mclachlan, 1875 were observed in 2015, 2016, and 2018 at a river bank of the Asahi River, Japan. Males started to make swarms between late April and middle May in 2016 and 2018. The numbers of mated pairs in a swarm correlated with the numbers of flying males in a swarm in 2016 and 2018. Swarms were formed during a limited period at dusk most probably because that interval is free from natural enemies. Males competed with each other to copulate with females in swarms. We clarified the function of the forelegs of males, which are significantly longer than those of females. Males used their forelegs to hold up a female from below. Besides forelegs, males have longer tails than females. We will discuss why sexual differences are found in these traits. Our results represent the first observation of swarm mating behavior in E. orientalis.     

An alternative mating system in small male insects

Abstract. 1. Small males of all midge (Diptera: Chironomidae and Chaoboridae) species thus far examined (one chaoborid and six chironomids) are rare in mating swarms. They are found instead in vegetation adjacent to the swarm.
2. We show that it is here that females aggregate prior to embarking on mate acquisition flights. In the vegetation females appear to be accessible to males staying behind.
3. Such behaviour in small males may exploit the mate-attracting activities of large males in the swarm and may, at the same time, reduce competition and conserve energy.
4. The evolution and maintenance of these size-related types of mating behaviour are discussed.     

Swarm Site Fidelity in the Sex Role-Reversed Dance Fly Empis borealis

In the dance fly species Empis borealis (L.), females (1–40) gather to swarm at landmarks (swarm markers, like trees and bushes), and males carrying an insect prey visit these swarms for mating. We noticed earlier that some swarm sites were used for several years and that they appeared to be frequented by a similar number of swarming females in each year, although the numbers of females varied greatly among swarm sites and certain sites attracted more swarming individuals than others. To explore swarm site fidelity in this mating system, in 1993 we monitored the same swarm sites that we studied in 1989, addressing the questions, Would the same swarm sites still attract the same number of females and males after 4 years? and Why do some swarm sites attract more displaying females than others? The number of females swarming at the different markers in 1993 was approximately the same as 4 years earlier. Some of these swarm sites are known to have been used for 18 years. The swarm sites with the largest number of flies had a high sun exposure during the day and were found at coniferous swarm marker trees and in a mixed forest habitat. A swarm site with few females attending and with a low amount of insolation during the day can be predicted to be abandoned as a swarming site soon. Empis borealis swarm sites thus persist over many years and are attended by a similar number of individuals each year. To our knowledge, such site fidelity has not been demonstrated for any swarming insect species earlier.     

The inheritance and population genetics of sex ratio in the butterfly Acraea encedon

In West and East Africa the butterfly, Acraea encedon , occurs in well-defined populations that are often predominantly female. Breeding the butterfly in the laboratory revealed the presence of an all-female strain, which is inherited directly through the female parent. It is probable that the inheritance is controlled by a Y-linked gene, causing meiotic drive in the Y chromosome, but the possibility of cytoplasmic inheritance has not been ruled out. A simple model for the population genetics of a predominantly female population indicates that such a population should rapidly become extinct due to the spreading of the all-female strain, but in most field populations studied extinction does not occur as quickly as predicted, if at all. Three factors which could enable populations to avoid extinction are investigated: suppressing systems, frequency-dependent mating preference and the sequence of emergence of the sexes in normal broods. No positive evidence has yet been found for the existence of a gene or genes capable of suppressing the sex ratio aberration, and no frequency-dependent mating preference was found, but an argument is presented which shows that the sequence of emergence in normal broods could be partly responsible for the maintenance of stable equilibria in predominantly female populations. Attempts to upset the sex ratio in the normal strain by making crosses between widely separated populations were not successful.     

Male Lekking duration and Mating Benefits in a Lek-Forming Hawaiian Drosophila

Variation in lekking duration of males of a Hawaiian Drosophila, D. grimshawi, was examined in laboratory enclosures. The relationship between variation in male lekking activity and number of eggs laid by females, proportion of eggs hatching, and total offspring production was investigated. Females mating highly active males laid fewer eggs and thus had lower offspring production than females mating less active males, even though other studies have shown that D. grimshawi females prefer more active males as mates. These results are discussed in terms of conflict between males and females over mating benefits and in trade-offs faced by males between lekking duration and offspring production per mating. Testis mass was not related to lekking activity levels, but males with heavier testes produced more offspring per mating, suggesting that males may vary in other ways that correlate with possible direct fitness gains for females.     

Absence of male reproductive skew,along with high frequency of polyandry and conspecific brood parasitism in the lekking Houbara bustard Chlamydotis undulata undulata

Behavioural studies have led to the perception that lekking species experience a high male reproductive skew as a consequence of females’ selective mate choice. In addition, observations suggest that females copulate only once and therefore polyandry seems unlikely as females are supposed to choose the best male available. In order to analyse the mating strategy of the Houbara bustard, an endangered lekking species under reinforcement in eastern Morocco, we used microsatellite data to perform paternity analyses. None of our observations followed common expectations under a lek mating system: we found no male reproductive skew suggesting no apparent selective female mate choice and no apparent male benefit from lekking. In contrast, a high level of polyandry (60 % of the nests) was recorded suggesting that sperm competition may operate. In addition, we present another case of conspecific brood parasitism in a lekking species and this was an unexpected alternative strategy for a species presenting high parental cost and low fecundity. The increasing number of studies contradicting common assumptions on lekking species suggests that alternative breeding strategies such as males pursuing an off‐lek mating strategy, female polyandry and even conspecific brood parasitism might be more widespread in lekking species than previously thought.     

Assortative mating in mixed swarms of the mosquito Anopheles gambiae s.s. M and S molecular forms,in Burkina Faso,West Africa

The molecular form composition of Anopheles gambiae Giles s.s. (Diptera: Culicidae) mating swarms and the associated mating pairs (copulae) were investigated during two rainy seasons (July to October, 2005 and July to November, 2006) in the villages of Soumousso and Vallée du Kou (VK7). Although the habitats of these villages differ markedly, sympatric populations of M and S molecular forms of An. gambiae s.s. occur in both places periodically. The main aim was to assess the degree to which these molecular forms mate assortatively. In Soumousso, a wooded savannah habitat, the majority of swarm samples consisted of only S‐form males (21/28), although a few M‐form males were found in mixed M‐ and S‐form swarms. In VK7, a rice growing area, the majority of swarm samples consisted of only M‐form males (38/62), until October and November 2006, when there were nearly as many mixed‐form as single‐form swarms. Overall, ～60% of M‐ and S‐form swarms were temporally or spatially segregated; the two forms were effectively prevented from encountering each other. Of the remaining 40% of swarms, however, only about half were single‐form and the rest were mixed‐form. Of the 33 copulae collected from mixed‐form swarms, only four were mixed‐form pairs, significantly fewer than expected by random pairing between forms (χ² = 10.34, d.f. = 2, P < 0.01). Finally, all specimens of inseminated females were of the same form as the sperm contained within their spermatheca (n = 91), even for the four mixed‐form copulae. These findings indicate that assortative mating occurs within mixed‐form swarms, mediated most probably by close‐range mate recognition cues.