The use of chemical and visual cues in female choice in the butterfly Bicyclus anynana

Investigating the relative importance of multiple cues for mate choice within a species may highlight possible mechanisms that led to the diversification of closely related species in the past. Here, we investigate the importance of close-range pheromones produced by male Bicyclus anynana butterflies and determine the relative importance of these chemical cues versus visual cues in sexual selection by female choice. We first blocked putative androconial organs on the fore- and hindwings of males, while also manipulating the ability of females to perceive chemical signals via their antenna. We found that male chemical signals were emitted by both fore- and hindwing pairs and that they play an important role in female choice. We subsequently tested the relative importance of these chemical cues versus visual cues, previously identified for this species, and found that they play an equally important role in female choice in our laboratory setting. In addition, females will mate with males with only one signal present and blocking both androconial organs on males seems to interfere with male to male recognition. We discuss the possible functions of these signals and how this bimodal system may be used in intra- and interspecific mate evaluation.     

Detection of female mating status using chemical signals and cues

Males of many species choose their mate according to the female's reproductive status, and there is now increasing evidence that male fitness can depend on this discrimination. However, females will also aim to regulate their mating activity so as to maximize their own fitness. As such, both sexes may attempt to dictate the frequency and timing of female mating, reflecting the potentially different costs of female signaling to both sexes. Here, I review evidence that chemical cues and signals are used widely by males to discriminate between mated and unmated females, and explore the mechanisms by which female odour changes post‐mating. There is substantial empirical evidence that mated and unmated females differ in their chemical profile, and that this variation provides males with information on a female's mating status. Although there appears to be large variation among species regarding the mechanisms by which female odour is altered post‐mating, the transfer of male substances to females during or subsequent to copulation appear to play a major role. This transfer of substances by males may be part of their strategy to suppress reproduction by competing males, particularly in species where females mate more than once.     

Male tree weta are attracted to cuticular scent cues but do not discriminate according to sex or among two closely related species

Recognition of conspecifics is an essential precursor of successful mating. Where related species coexist, species discrimination might be important, but because related species are similar, species signal recognition may actually be low. Chemical cues such as cuticular hydrocarbons (CHCs) are frequently used by insects to identify suitable sexual partners. We predicted that New Zealand tree weta (Hemideina spp.), a genus of nocturnal ensiferan Orthoptera that live both allopatrically and sympatrically, use chemical signals from either frass or CHCs to find mates. In a series of six laboratory trials using both H. thoracica and H. crassidens, we found that male tree weta, but not female tree weta, occupied cavities primed with female cuticular cues more often than cavities without. However, males did not discriminate between chemical cues of male and female conspecifics, or between conspecifics and heterospecifics. In field trials, tree weta did not occupy artificial cavities primed with either female frass or female cuticular cues more often than unscented cavities. However, in both trials weta preferentially returned to cavities that had already been occupied earlier in the trials. A final field trial confirmed the presence of mixed species harems during the mating season in one region of sympatry. Our results suggest that selection on sex and species specific chemical cues that could be used to find conspecific mates is weak. Mixed species aggregations suggest that identification of conspecific mating cues has not evolved to be species specific. We infer that for male tree weta, the cost of mating with heterospecifics is likely less than not mating at all.     

Chemosensory cues allow male Tenebrio molitor beetles to assess the reproductive status of potential mates

Males of many insect species, including beetles, choose their mates according to their reproductive status. However, the ways in which male beetles evaluate female reproductive status have received little attention. We tested the existence of male mate choice in the mealworm beetle, Tenebrio molitor, by observing mating and courtship behaviour of males given simultaneous access to pairs of females differing in their reproductive status: (1) mature versus immature; (2) virgin versus previously mated; (3) familiar (mated with the experimental male) versus unfamiliar (mated with a different male). Males courted and mated preferentially with mature and virgin females. To determine whether chemical cues played a role in these discriminations, we exposed males to filter paper squares bearing chemical cues from different types of females: (1) virgin versus mated; (2) mature versus immature. Males were significantly more attracted to those squares bearing chemical cues from virgin and mature females, suggesting that males can assess female reproductive status on the basis of chemical cues alone.     

Beyond magic traits: Multimodal mating cues in Heliconius butterflies

Species coexistence involves the evolution of reproductive barriers opposing gene flow. Heliconius butterflies display colorful patterns affecting mate choice and survival through warning signaling and mimicry. These patterns are called “magic traits” for speciation because divergent natural selection may promote mimicry shifts in pattern whose role as mating cue facilitates reproductive isolation. By contrast, between comimetic species, natural selection promotes pattern convergence. We addressed whether visual convergence interferes with reproductive isolation by testing for sexual isolation between two closely related species with similar patterns, H. timareta thelxinoe and H. melpomene amaryllis. Experiments with models confirmed visual attraction based on wing phenotype, leading to indiscriminate approach. Nevertheless, mate choice experiments showed assortative mating. Monitoring male behavior toward live females revealed asymmetry in male preference, H. melpomene males courting both species equally while H. timareta males strongly preferred conspecifics. Experiments with hybrid males suggested an important genetic component for such asymmetry. Behavioral observations support a key role for short‐distance cues in determining male choice in H. timareta. Scents extracts from wings and genitalia revealed interspecific divergence in chemical signatures, and hybrid female scent composition was significantly associated with courtship intensity by H. timareta males, providing candidate chemical mating cues involved in sexual isolation.     

Simultaneously hermaphroditic shrimp use lipophilic cuticular hydrocarbons as contact sex pheromones

Successful mating is essentially a consequence of making the right choices at the correct time. Animals use specific strategies to gain information about a potential mate, which is then applied to decision-making processes. Amongst the many informative signals, odor cues such as sex pheromones play important ecological roles in coordinating mating behavior, enabling mate and kin recognition, qualifying mate choice, and preventing gene exchange among individuals from different populations and species. Despite overwhelming behavioral evidence, the chemical identity of most cues used in aquatic organisms remains unknown and their impact and omnipresence have not been fully recognized. In many crustaceans, including lobsters and shrimps, reproduction happens through a cascade of events ranging from initial attraction to formation of a mating pair eventually leading to mating. We examined the hypothesis that contact pheromones on the female body surface of the hermaphroditic shrimp Lysmata boggessi are of lipophilic nature, and resemble insect cuticular hydrocarbon contact cues. Via chemical analyses and behavioural assays, we show that newly molted euhermaphrodite-phase shrimp contain a bouquet of odor compounds. Of these, (Z)-9-octadecenamide is the key odor with hexadecanamide and methyl linoleate enhancing the bioactivity of the pheromone blend. Our results show that in aquatic systems lipophilic, cuticular hydrocarbon contact sex pheromones exist; this raises questions on how hydrocarbon contact signals evolved and how widespread these are in the marine environment.     

Experimental evidence for a hierarchy of mate- and host-induced cues in a fish ectoparasite, Argulus coregoni (Crustacea: Branchiura)

Argulus coregoni is an ectoparasite primarily infesting freshwater salmonids. Sexually reproducing parasites such as A. coregoni are confronted with a dilemma between finding a mate and the costs involved in doing so; if mating partners are unavailable on a host, by leaving to search for a mate on a new host, the parasite is exposed to risks such as predation and energy loss. The utilization of chemical cues could enhance the probability of finding a host and/or a suitable mating partner and thus decrease the level of costs associated with detachment from the host. In this study we constructed a Y-maze arena to determine if adult A. coregoni respond to mate- and host-related chemical cues. We also tested the directional response towards light, since it has been suggested that photic cues are the most important cues for juvenile A. coregoni locating a host. Our results showed that both sexes were attracted to light and fish odour. Free-swimming A. coregoni males responded to chemical cues produced by adult females but not vice versa. The hierarchy of these stimuli was analyzed by pitting the cues against one another in the Y-maze, showing that light was the most salient stimulus for both male and female parasites. Moreover, male parasites were more strongly attracted towards light and fish odour than female odour. In another experiment in a semi-natural environment, we examined whether the ability of A. coregoni males to detect female odour influences their host choice. Free-swimming males did not preferentially infest fish infected with female parasites over parasite-free fish. We suggest that a hierarchy of stimulus responses exists, whereby free-swimming parasites first respond to host-related signals and most dominantly to visual cues. However, cues connected to mate finding may become a priority for late adult stages and/or once the parasite has attached to the host.     

RESEARCH PAPER: Multiple Sexual Ornamentation Signals Male Quality and Predicts Female Preference in Minnows

Sexual ornamentation often consists of multiple components. Different sexual signals may indicate different aspects of mate quality or reflect quality in different time scales. On the other hand, same signals can have a dual function and are used both in male–male competition and courtship. Many fish species are capable of rapidly altering their colouration (ephemeral colour changes), but this capability is usually ignored in sexual selection studies. Here, we used experimentally manipulated social environments to study the ephemeral colour changes in multicomponent sexual signals of male minnows (Phoxinus phoxinus) during male–male competition and female choice. We found that the dominant males courted the females more actively and had redder and/or darker skin colouration than the subordinate males. Furthermore, darkness difference between subordinate and dominant males increased in the presence of female, which suggests that the male–male competition may increase the honesty of signalling and thus facilitate female choice. In support of this hypothesis, females had a strong behavioural preference towards the more colourful males, which may indicate female choice. As colourful males often had a higher social status than paler individuals, it is possible that females base their preference on male status, not only the colouration per se. In any case, our results suggest that sexual ornamentation of male minnows may signal status, courting activity and superior quality of the males and that these signals may have a dual function in both male–male competition and female choice. Females preferred different ornamental traits (dark and red colour patterns) relatively equally, indicating that mate choice is based on multiple cues.     

Chemical and visual communication during mate searching in rock shrimp

Mate searching in crustaceans depends on different communicational cues, of which chemical and visual cues are most important. Herein we examined the role of chemical and visual communication during mate searching and assessment in the rock shrimp Rhynchocinetes typus. Adult male rock shrimp experience major ontogenetic changes. The terminal molt stages (named "robustus") are dominant and capable of monopolizing females during the mating process. Previous studies had shown that most females preferably mate with robustus males, but how these dominant males and receptive females find each other is uncertain, and is the question we examined herein. In a Y-maze designed to test for the importance of waterborne chemical cues, we observed that females approached the robustus male significantly more often than the typus male. Robustus males, however, were unable to locate receptive females via chemical signals. Using an experimental set-up that allowed testing for the importance of visual cues, we demonstrated that receptive females do not use visual cues to select robustus males, but robustus males use visual cues to find receptive females. Visual cues used by the robustus males were the tumults created by agitated aggregations of subordinate typus males around the receptive females. These results indicate a strong link between sexual communication and the mating system of rock shrimp in which dominant males monopolize receptive females. We found that females and males use different (sex-specific) communicational cues during mate searching and assessment, and that the sexual communication of rock shrimp is similar to that of the American lobster, where females are first attracted to the dominant males by chemical cues emitted by these males. A brief comparison between these two species shows that female behaviors during sexual communication contribute strongly to the outcome of mate searching and assessment.     

Nonvisual mate choice in the Pyrenean mountain newt (<Emphasis Type="Italic">Euproctus asper</Emphasis>): females prefer small males

In many species, females prefer large males as mates. In this paper, we examined whether female Pyrenean mountain newts (Euproctus asper) would show a preference for large males in a simultaneous choice situation. When we tested for a visual preference relative to male body size using visual cues, females did not prefer large males. When chemical cues were presented, females did show a preference, but contrary to our prediction, they preferred the smaller of two males. E. asper males exhibit coercive mating behavior (amplexus), keeping females from feeding for several hours or even days, which may explain why females avoid large, physically stronger, and thus, more coercive males. The predominantly nocturnal lifestyle may explain why visual cues are less important for mate discrimination in this species.     

Functional coupling of acoustic and chemical signals in the courtship behaviour of the male Drosophila melanogaster

During courtship, the male Drosophila melanogaster sends signals to the female through two major sensory channels: chemical and acoustic. These signals are involved in the stimulation of the female to accept copulation. In order to determine the respective importance in the courtship of these signals, their production was controlled using genetical and surgical techniques. Males deprived of the ability to emit both signals are unable to mate, demonstrating that other (e.g. visual or tactile) signals are not sufficient to stimulate the female. If either acoustic or chemical signals are lacking, the courtship success is strongly reduced, the lack of the former having significantly more drastic effects. However, the accelerated matings of males observed with males bearing wild-type hydrocarbons compared with defective ones, whichever the modality of acoustic performance (wing vibration or playback), strongly support the role of cuticular compounds to stimulate females. We can conclude that among the possible factors involved in communication during courtship, acoustic and chemical signals may act in a synergistic way and not separately in D. melanogaster.     

Sex Recognition via Chemical Cues in the Sex-Role-Reversed Gulf Pipefish (Syngnathus scovelli)

Sexual selection theory predicts that members of the choosy sex, usually females, should employ multiple sensory systems to obtain information about potential mates. Such predictions should also apply to systems in which sexual selection acts most strongly on females (i.e. sex-role-reversed species), and males of these taxa should be the individuals employing multiple cues to assess female attractiveness. Very little work has been directed toward mate choice involving multiple sensory systems in sex-role-reversed taxa, but fishes of the family Syngnathidae (pipefishes, seahorses, and sea dragons) provide an excellent opportunity to contribute to this research enterprise. While much is known about visual communication in pipefish, the role of chemical communication has not been investigated. Using dichotomous choice tests, we found that male, but not female, Gulf pipefish attended to chemical cues of opposite sex conspecifics. Given that males distinguished sex on the basis of chemical cues, we also tested whether males could assess female body size, an important trait with respect to mate choice in pipefish, on the basis of chemical cues alone. When given the choice between chemical cues produced by large vs. small females, males exhibited no preference. Our results suggest that male pipefish can use chemical cues to distinguish between males and females but not to differentiate females of different body size.     

Mutual Mate Assessment in Wolf Spiders: Differences in the Cues Used by Males and Females

When males engage in conspicuous courtship displays, it seems obvious that females would use characteristics of that display in mating decisions. However, males must also have a way to identify and evaluate females prior to engaging in what might be a costly mating ritual. Although it was known that female wolf spiders of the species Pardosa milvina (Araneae; Lycosidae) attract males using volatile chemical cues, the nature of the cues used by males and females in mate selection had not been investigated. Specifically we determined whether males could detect the mating status of the female and if chemotactile cues from the female played a role in that process. In addition, we quantified conspicuous aspects of the male courtship (leg raises and body shakes) to determine if courtship intensity was related to female choice. Although repeated mating occurred in our studies, males were more likely to court and mate with virgin females. Males used substrate‐borne cues deposited by females to discriminate between mated and virgin females. Females used the conspicuous behaviors of males during courtship, body shakes and leg raises, in mate selection. Thus males and females use different kinds of information and different sensory modalities to assess the suitability of a potential mate.     

Allyl‐2,6‐dimethoxyphenol,a female‐biased compound,is robustly attractive to conspecific males of Bactrocera dorsalis at close range

Insect cuticular hydrocarbons (CHCs) play important roles in mate recognition and chemical communication. To explore the cues regulating courtship and mating behaviour in the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), CHCs were extracted from mature virgin female and male oriental fruit flies using n‐hexane. Nine compounds – i.e., 4‐allyl‐2,6‐dimethoxyphenol (designated ‘compound 3’) and eight ester compounds – had significantly greater abundance in female samples than in males. Of these nine compounds, six (1, 2, 4, 5, 6, and 8) elicited electrophysiological responses from the antennae of male flies. Compound 3 did not elicit a detectable male antennal response, but in Y‐tube olfactometer bioassays, it exhibited robust attractiveness to conspecific males at close range. We speculate that compound 3 is a close‐range sex pheromone in B. dorsalis. A mating competition experiment revealed that compound 3 significantly increased the competitiveness of males, which implied that compound 3 might be used in control programs of B. dorsalis.     

Sex recognition by male Andrew's toad Bufo andrewsi in a subtropical montane region

Sex recognition is important for successful reproduction and species usually have efficient systems of signals and responses to find the optimal potential mate. In the present study, we investigated the ability of males to recognize between sexes for two stimulus animals in Andrew's toad Bufo andrewsi, a species widely distributed in western China. When a male was placed with a gravid female and a similar-sized male, the male did not discriminate between them. When two males with distinct size differences were provided with a male, the male chose the larger one. In an experiment in which a gravid female and a different-sized male were offered a test male, males preferred the larger gravid females than smaller males. If a test male clasped a stimulus male, the stimulus male uttered a specific release call that caused the test male to release the stimulus male. These findings suggest that male B. andrewsi can recognize between the sexes probably based on male release calls, and prefer to mate with larger individuals with visual cues.     

Repeatability of Male Mate Choice in the Mosquitofish, Gambusia holbrooki

Male mate choice has evolved in many species in which female fecundity increases with body size. In these species, males are thought to have been selected to favour mating with large females over smaller ones, thereby potentially increasing their reproductive success. While male mate choice is known to occur, it is less well studied than female mate choice and little is known about variation in mating preference among individual males. Here, we presented individual male eastern mosquitofish ( Gambusia holbrooki ) with paired females that differed in body size, and we quantified their mate preference on two consecutive days, allowing us to assess repeatability of preferences expressed. When males were allowed to view paired stimulus females, but not to acquire chemical or tactile cues from them, they exhibited a strong preference for large females over smaller ones. However, individual males were not consistent in the strength of their preference and repeatability was not significant. When individual males were allowed to fully interact with pairs of females, the males again exhibited a preference for large females over smaller ones, as revealed by a greater number of attempted copulations with large females than with smaller ones. In the latter social context, individual male preference was significantly repeatable. These results indicate that male eastern mosquitofish from our Florida study population possess, on average, a mating preference for larger females and that this preference is repeatable when males socially interact freely with females. The significant repeatability for mating preference, based on female body size, obtained for male mosquitofish in the current study is consistent with the presence of additive genetic variation for such preferences in our study population and thus with the opportunity for the further evolution of large body size in female mosquitofish through male mate choice.     

Silk‐ and volatile‐based male mate choice in the genital plug‐producing spider

Female mating history can have a strong effect on male fertilization success. Although males often prefer to mate with virgin females, they often also engage with mated females. As the intensity of sperm competition can differ among mated females, males are expected to evolve means to identify their status. In spiders, males often use female silk to gather information about female quality. Males of many spider species deposit mating plugs into female genitalia to hinder further copulations. We tested whether males of the foliage‐dwelling, plug‐producing spider Philodromus cespitum, which is an important natural enemy of pests, discriminate between females of different mating status and whether they can determine the extent of genital plugging in mated females solely on the basis of cues gained from deposited female silk. We presented males with draglines of females that varied in either mating status (virgin vs. mated), the extent of plugging (small vs. big plug), or the age of the plug (fresh vs. old plug) and examined their mate preferences. Additionally, we tested whether males were attracted to volatile cues produced by female bodies. Our experiments revealed that males preferred draglines of virgin females to those of mated females, and mated females with small plugs to those with large plugs. They were also attracted to female volatile cues. This study suggests that males are able to extract fine‐scale information on mating status from female draglines.     

Female assessment of male functional fertility during mate choice in a promiscuous fish

Sexual selection should favour females that can assess the functional fertility of available sexual partners and avoid mating with recently mated, sperm‐depleted males. Our current understanding of the sensory mechanism(s) underlying female assessment of males based on their functional fertility and avoidance of sperm‐depleted males is incomplete. Female Trinidadian guppies (Poecilia reticulata) are known to avoid mating with males that they had previously observed mating with other females. Here, we investigated experimentally the proximate sensory cues that they use to distinguish between paired size‐ and colour‐matched mated and unmated males in the absence of visual public information on their prior mating histories. When only water‐borne chemical cues from the males were available, females avoided the previously mated male and preferred the unmated one, but they chose randomly when only male visual cues (and no chemical cues) were available. They also preferred unmated over mated males when freely swimming with them in a more sensorially complex environment with multiple male cues (i.e., visual, chemical and mechanical cues) concurrently available. Females exhibited no preference for either stimulus males when both were unmated, irrespectively of the sensory environment. These novel results suggest that, in the absence of prior visual public information on the recent mating histories of males, female guppies use olfactory cues putatively emitted by mated males to avoid mating with them. The source and nature of the implicated olfactory cues and the fitness benefits gained by female guppies in sexually preferring males that have not recently mated remain unknown and warrant further research.     

Lack of species discrimination based on chemical cues by male sailfin mollies, <Emphasis Type="Italic">Poecilia latipinna</Emphasis>

When making mating decisions, individuals may rely on multiple cues from either the same or multiple sensory modalities. Although the use of visual cues in sexual selection is well studied, fewer studies have examined the role of chemical cues in mate choice. In addition, few studies have examined how visual and/or chemical cues affect male mating decisions. Male mate choice is important in systems where males must avoid mating with heterospecific females, as is found in a mating complex of Poecilia. Male sailfin mollies, Poecilia latipinna, are sexually parasitized by gynogenetic Amazon mollies, P. formosa. Little is known about the mechanism by which male sailfin mollies base their mating decisions. Here we tested the hypothesis that male sailfin mollies from an allopatric and a sympatric population with Amazon mollies use multiple cues to distinguish between conspecific and heterospecific females. We found that male sailfin mollies recognized the chemical cues of conspecific females, but we found no support for the hypothesis that chemical cues are by themselves sufficient for species discrimination. Lack of discrimination based on chemical cues alone may be due to the close evolutionary history between P. latipinna and P. formosa. Males from populations sympatric with Amazon mollies did not differentially associate with females of either of the two species when given access to both visual and chemical cues of the females, yet males from the allopatric population did associate more with conspecific females than with heterospecific females in the presence of both chemical and visual cues. The lack of discrimination by males from the sympatric population between conspecific and heterospecific females based on both chemical and visual cues suggests that these males require more complex combinations of cues to distinguish species, possibly due to the close relatedness of these species.     

Male-derived cuticular hydrocarbons signal sperm competition intensity and affect ejaculate expenditure in crickets

Female sexual promiscuity can have significant effects on male mating decisions because it increases the intensity of competition between ejaculates for fertilization. Because sperm production is costly, males that can detect multiple matings by females and allocate sperm strategically will have an obvious fitness advantage. The presence of rival males is widely recognized as a cue used by males to assess sperm competition. However, for species in which males neither congregate around nor guard females, other more cryptic cues might be involved. Here, we demonstrate unprecedented levels of sperm competition assessment by males, which is mediated via the use of chemical cues. Using the cricket Teleogryllus oceanicus, we manipulated male perception of sperm competition by experimentally coating live unmated females with cuticular compounds extracted from males. We found that males adjusted their ejaculate allocation in response to these compounds: the viability of sperm contained within a male's ejaculate decreased as the number of male extracts applied to his virgin female partner was increased. We further show that males do not respond to the relative concentration of male compounds present on females, but rather to the number of distinct signature odours of individual males. Our results conform to sperm competition theory, and show for the first time, to our knowledge, that males can detect different intensities of sperm competition by using distinct chemical cues of individual males present on females.