Conspecific Males Research Articles

Divergent warning patterns influence male and female mating behaviours in a tropical butterfly.

Traits under divergent ecological selection that also function during mating can be important in maintaining species boundaries. Few studies have considered mutual mate choice, where both males and females base mating decisions on the same trait. Wing colouration in Heliconius butterflies evolved as a warning signal but also functions as a mating cue. We investigated the contribution of visual preference to assortative mating in an aposematic butterfly Heliconius cydno in the context of reproductive isolation with its sympatric, visually distinct relative Heliconius melpomene. Heliconius cydno have conspicuous white bands on their forewings, whereas those of H. melpomene are red in colour. We predicted that both sexes of H. cydno contributed to assortative mating by exhibiting visual preference towards conspecific wing colouration. We analysed published and new data from preference experiments, in which males were presented with conspecific and H. melpomene females. We also recorded female responses and mating outcomes in choice experiments, involving conspecific males with either the original white or artificially painted red forewing bands. Both sexes of H. cydno responded more positively towards the conspecific colouration, and males strongly preferred females of its own colours. In contrast, male colouration did not predict mating outcomes in female choice experiments. As courtships are initiated by males in butterflies, our findings suggest that female visual preference might be of secondary importance in H. cydno. Our data also suggest that the contribution of visual preference to reproductive isolation might be unequal between H. cydno and its sympatric relative H. melpomene.

Heterospecific interference modulates the reproductive traits of a ladybird beetle

AbstractReproductive interference between species is vital to understand interspecific interactions along with their ecological and evolutionary patterns in nature. We studied potential reproductive interference between two sympatric species of ladybird beetles Propylea dissecta and Cheilomenes sexmaculata (Coleoptera: Coccinellidae). Our aim was to determine whether heterospecific matings occur between these two species, and if so, how they affect the reproductive output of females of both species. We found that heterospecific mating occurs, with P. dissecta females mating with C. sexmaculata males, but not vice versa. To compare the effect of conspecific and heterospecific males on the reproductive output of P. dissecta females, we used two mixed mating treatments: conspecific mixed (an additional P. dissecta male with conspecific pair of P. dissecta), and heterospecific mixed (a C. sexmaculata male with conspecific pair of P. dissecta). We found that conspecific interference did not affect reproductive parameters. However, the presence of a heterospecific C. sexmaculata male interfered with conspecific matings in P. dissecta through multiple mating attempts, resulting in reduced fitness and reproductive success of P. dissecta. Our results indicate that the presence of heterospecific males significantly affected the reproductive output of P. dissecta, apparently as a result of the greater attempted mating activity of C. sexmaculata males. Our findings may be relevant for effective biocontrol strategies involving these insect species.

Sex-biased phenotypic plasticity affects sexual dimorphism patterns under changing environmental conditions

Sexual dimorphism is almost ubiquitous in animals. A common pattern observed across multiple taxa involves differences in development time (sexual bimaturism) and body size (sexual size dimorphism) between conspecific males and females. Furthermore, a strict association of dimorphism at these traits has been documented in several taxa, where the sex showing shorter development time also has a smaller body size than the other sex. Growth and development are strongly dependent on environmental conditions during individual life-cycle in ectotherms, inducing considerable phenotypic plasticity. However, how phenotypic plasticity affects the association between sexual dimorphism in development time and body size remains unclear. Here, we tracked development time, body size, and body mass throughout the ontogeny of the mosquito Aedes mariae. The larval development of this species is strictly linked to Mediterranean Sea rock-pools, whose highly variable environmental conditions over minimal time frames make this organism-environment system ideal for exploring plasticity-led eco-evolutionary processes. We found differential plasticity between males and females, dissolving the link between dimorphism in development time and body size under increasing temperature and decreasing salinity conditions. These findings contrast with the current hypotheses proposed to explain the origin of the association between sexual bimaturism and sexual size dimorphism, highlighting the condition dependence of sexual dimorphism patterns and the need to consider phenotypic plasticity in future studies on their evolution.

Inter- and intraspecific female behavioral plasticity drive temporal niche segregation in two Tribolium species

Abstract Temporal niche segregation is a way to reduce competition over shared resources. Species with overlapping spatial niches often show plasticity and can use different activity times to minimize competition with and disturbance by other species. In many granivores, especially those living in their food resources, there is low competition over food, but other selective forces can drive distinct temporal activity times. Reproductive interference, that is, the interaction between individuals of different species during mate acquisition, can be such a driving force. Reproductive interference is a strong driving force for niche segregation between the co-occurring flour beetles Tribolium castaneum and Tribolium confusum (Coleoptera: Tenebrionidae). Nevertheless, most studies on Tribolium spp. evaluated other selective forces, such as resource competition, which are potentially weaker. Here, we aimed to characterize the species’ peak activity time and its plasticity when exposed to a possible reproductive interference cue. We used a synthetic aggregative pheromone, shared by both species, as a cue for potential reproductive interference. We compared temporal niche plasticity between the sexes because reproductive interference is known mainly to reduce the female’s fitness. We found that both species’ activity times were hard-wired to an endogenous clock and that the sexes differed in their peak activity time and activity level. However, once exposed to the aggregation pheromone, only females shifted their activity time to match that of conspecific males, hence expressing more plasticity than the males to reduce Reproductive interference. These findings suggest an important role of the aggregation pheromone in the process of niche segregation.

Evidence of a pheromone involved in the behaviour ofDrosophila suzukiiMatsumura (Diptera: Drosophilidae)

AbstractRecent findings have documented the importance of (Z)‐11‐octadecenyl acetate (cVA) in the melanogaster group as a sex pheromone that regulates several sexual and social behaviours. However, this pheromone is missing inDrosophila suzukii, thus posing the question of whether it could have been replaced by another compound similar to cVA. Olfactory responses ofD. suzukiifemales and males to conspecific volatiles in laboratory and field trails suggest the existence of a pheromone. Females attracted more females and males than the control. In field trials, both sexes were successfully captured in traps baited with conspecifics. Females were attracted to conspecific females and males. Gas chromatography coupled with mass spectrometry of volatiles showed that both sexes released 18 compounds, including Z‐9‐tricosene, hexacosane and heneicosane. Our results suggest that these compounds may influence the behaviour ofD. suzukiiand can act as sex or aggregation pheromones.

Light and dark cycles modify the expression of clock genes in the ovaries of Aedes aegypti in a noncircadian manner.

Circadian oscillators (i.e., circadian clocks) are essential to producing the circadian rhythms observed in virtually all multicellular organisms. In arthropods, many rhythmic behaviors are generated by oscillations of the central pacemaker, specific groups of neurons of the protocerebrum in which the circadian oscillator molecular machinery is expressed and works; however, oscillators located in other tissues (i.e., peripheral clocks) could also contribute to certain rhythms, but are not well known in non-model organisms. Here, we investigated whether eight clock genes that likely constitute the Aedes aegypti clock are expressed in a circadian manner in the previtellogenic ovaries of this mosquito. Also, we asked if insemination by conspecific males would alter the expression profiles of these clock genes. We observed that the clock genes do not have a rhythmic expression profile in the ovaries of virgin (VF) or inseminated (IF) females, except for period, which showed a rhythmic expression profile in ovaries of IF kept in light and dark (LD) cycles, but not in constant darkness (DD). The mean expression of seven clock genes was affected by the insemination status (VF or IF) or the light condition (LD 12:12 or DD), among which five were affected solely by the light condition, one solely by the insemination status, and one by both factors. Our results suggest that a functional circadian clock is absent in the ovaries of A. aegypti. Still, their differential mean expression promoted by light conditions or insemination suggests roles other than circadian rhythms in this mosquito's ovaries.

A landmark-free analysis of the pelvic girdle in Sulawesi ricefishes (Adrianichthyidae): How 2D and 3D geometric morphometrics can complement each other in the analysis of a complex structure.

Geometric morphometrics (GM) enable the quantification of morphological variation on various scales. Recent technical advances allow analyzing complex three-dimensional shapes also in cases where landmark-based approaches are not appropriate. Pelvic girdle bones (basipterygia) of Sulawesi ricefishes are 3D structures that challenge traditional morphometrics. We hypothesize that the pelvic girdle of ricefishes experienced sex-biased selection pressures in species where females provide brood care by carrying fertilized eggs supported by elongated pelvic fins ("pelvic brooding"). We test this by comparing pelvic bone shapes of both sexes in species exhibiting pelvic brooding and the more common reproductive strategy "transfer brooding," by using landmark-free 2D and 3D GM, as well as qualitative shape descriptions. Both landmark-free approaches revealed significant interspecific pelvic bone variation in the lateral process, medial facing side of the pelvic bone, and overall external and internal wing shape. Within pelvic brooders, the three analyzed species are clearly distinct, while pelvic bones of the genus Adrianichthys are more similar to transfer brooding Oryzias. Female pelvic brooding Oryzias exhibit prominent, medially pointing tips extending from the internal wing and basipterygial plate that are reduced or absent in conspecific males, Adrianichthys and transfer brooding Oryzias, supporting our hypothesis that selection pressures affecting pelvic girdle shape are sex-biased in Sulawesi ricefishes. Furthermore, both sexes of pelvic brooding Oryzias have overall larger pelvic bones than other investigated ricefishes. Based on these differences, we characterized two reproductive strategy- and sex-dependent pelvic girdle types for Sulawesi ricefishes. Morphological differences between the investigated pelvic brooding genera Adrianichthys and Oryzias provide additional evidence for two independent origins of pelvic brooding. Overall, our findings add to a better understanding on traits related to pelvic brooding in ricefishes and provide a basis for upcoming studies on pelvic girdle function and morphology.

Anuran swingers: misdirected mating attempts occurred early during anuran diversification

Abstract Promiscuity, explosive breeding and male-biased operational sex-ratios can affect the strength of male selectivity and may play major roles in the expression of misdirected matings (with conspecific males, other species, corpses or objects) in anuran amphibians. Yet, misdirected amplexus occur in multiple species diverging from these reproductive traits, suggesting that the expression of such behaviour is widespread, and may have occurred early during anuran diversification. Using two methods of ancestral-trait reconstruction which predict unknown states, we found a very high probability that most anuran genera express misdirected amplexus, suggesting that this by-product of anuran reproductive strategies is likely to have occurred early during anuran diversification. The persistence of misdirected amplexus suggests that its infrequent occurrence may outweigh individual costs of breeding attempts with unfit mates. We found a recent exponential increase in reported observations of misdirected amplexus, which, along with increased research effort and publication rate, may reveal an effect of environmental perturbations known to promote the occurrence of these behaviours.

CRISPR/Cas9 mediated mutagenesis of the major sex pheromone gene, acyl-CoA delta-9 desaturase (DES9) in Fall armyworm Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae)

The Fall armyworm, Spodoptera frugiperda is a significant global pest causing serious yield loss on several staple crops. In this regard, this pest defies several management approaches based on chemicals, Bt transgenics etc., requiring effective alternatives. Recently CRISPR/Cas9 mediated genome editing has opened up newer avenues to establish functions of various target genes before employing them for further application. The virgin female moths of S. frugiperda emit sex pheromones to draw conspecific males. Therefore, we have edited the key pheromone synthesis gene, fatty acyl-CoA Delta-9 desaturase (DES9) of the Indian population of S. frugiperda. In order to achieve a larger deletion of the DES9, we have designed two single guide RNA (sgRNA) in sense and antisense direction targeting the first exon instead of a single guide RNA. The sgRNA caused site-specific knockout with a larger deletion which impacted the mating. Crossing studies between wild male and mutant female resulted in no fecundity, while fecundity was normal when mutant male crossed with the wild female. This indicates that mating disruption is stronger in females where DES9 is mutated. The current work is the first of its kind to show that DES9 gene editing impacted the likelihood of mating in S. frugiperda.

Stored product pest Nemapogon variatella (Clemens): Identification of sex pheromone, putative antagonist and mitotype characterization

AbstractNemapogon variatella (Clemens) (Lepidoptera: Tineidae) is a worldwide widespread stored product pest that damages grain, dried fruits and mushrooms. The most powerful and environmentally friendly tool for insect pest monitoring and control is pheromone application. In our study, we revealed for the first time that the females of N. variatella release (3Z,13Z)‐octadecadien‐1‐ol – a single compound that activates olfactory receptors in the antenna of conspecific males. These data allow to assign this compound, which has been known as an attractant for males only, to the category of sex pheromones of N. variatella moths. Males' olfactory system is sensitive not only to the sex pheromone compound but also to (3E,13Z)‐octadecadien‐1‐ol as well. (3E,13Z)‐octadecadien‐1‐ol is attributed to putative pheromone antagonists. During the analysis of genetic diversity, six mitotypes of N. variatella CO1‐5′ were detected and preliminary named Mit1–Mit6. The obtained high haplotype diversity (Hd = 0.796) and low nucleotide diversity (Pi = 0.007) suggest that this species may have undergone a recent population expansion.

Frog call transmission in forestry monocultures: Are there drawbacks?

AbstractAnuran males emit advertisement calls for the purpose of attracting females and repelling conspecific males. Call transmission is influenced by the acoustics of the propagation environment, including vegetation. Thus, forestry monocultures of non‐native trees represent artificial environments that could modify call transmission. These monocultures have substituted large areas of Atlantic Forest in southern Brazil, representing a conservation challenge. Considering this context, we hypothesized that anurans have calls that are less attenuated in their native environment than in forest plantations. To test it, we performed sound transmission experiments using calls of three anuran species native to southern Brazil: Boana bischoffi, B. leptolineata, and Hylodes meridionalis. We compared sound attenuation between the native forest and forestry monocultures (Eucalyptus sp. and Pinus sp. forests), and included distance from the sound source, air temperature, humidity, and vegetation density as cofactors in linear mixed models. Our results show that the advertisement calls of the two tree frogs (Boana) attenuate less in tree plantations than in native forests, while the third species shows either no differences or less attenuation in native forests. Our results can be understood according to differences in natural history and microhabitat use among the studied species. The two tree frog species vocalize perched from vegetation so their calls become better transmitted in forestry monocultures, which have fewer trees than the native forest. On the other hand, H. meridionalis calls from rocks on streams and the propagation is finely tuned to this peculiar microhabitat. We discuss the conservation implications of our findings for anurans and for the Atlantic Forest. Abstract in Portuguese and Spanish are available with online material.

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.

Two new Thereus species from Peru, with notes on ecological niche partitioning (Lepidoptera: Lycaenidae: Theclinae).

Male hairstreaks (Theclinae) are often territorial, known for occupying their observation posts at a specific time during the day and at a certain height in the vegetation. The time and height for their perch vary from species to species but remain surprisingly stable year after year in any particular location. From these strategic observation posts males can check the passage of virgin females and chase away competing males. Consequently, these posts act as resources for the males and are not only vigorously defended from conspecific males but also form males of other species. In Peru, males of two closely related species belonging to the genus Thereus Hübner were observed in situ, defending their territories. One of them, Thereus tierralinda Faynel & Fåhraeus, sp. nov., is described here based on five male specimens from Cosñipata valley, Cusco, southern Peru. Further, it is compared to Thereus columbicola (Strand) and other close species in the Thereus eryssus species group. Observations over several years of T. columbicola and this new species show that they have different spatial and temporal characteristics for their perches, presumably to avoid competition with one another. Additionally, as a result of the partial revision of the group, a second new species, Thereus vicens Faynel & Fåhraeus, sp. nov., is also described based on one male and two females from Tingo María, Huánuco, central Peru. We also note that French Guiana is a new country record for Thereus eryssus (Herbst).

Unraveling female communication through scent marks in the Norway rat

Chemical communication by females remains poorly understood, with most attention focused on female advertisement of sexual receptivity to males or mother-offspring communication. However, in social species, scents are likely to be important for mediating competition and cooperation between females determining individual reproductive success. Here, we explore chemical signaling by female laboratory rats (Rattus norvegicus) to test i) whether females target their deployment of scent information differentially according to their sexual receptivity and the genetic identity of both female and male conspecifics signaling in the local environment and ii) whether females are attracted to gain the same or different information from female scents compared to males. Consistent with targeting of scent information to colony members of similar genetic background, female rats increased scent marking in response to scents from females of the same strain. Females also suppressed scent marking in response to male scent from a genetically foreign strain while sexually receptive. Proteomic analysis of female scent deposits revealed a complex protein profile, contributed from several sources but dominated by clitoral gland secretion. In particular, female scent marks contained a series of clitoral-derived hydrolases and proteolytically truncated major urinary proteins (MUPs). Manipulated blends of clitoral secretion and urine from estrus females were strongly attractive to both sexes, while voided urine alone stimulated no interest. Our study reveals that information about female receptive status is shared between females as well as with males, while clitoral secretions containing a complex set of truncated MUPs and other proteins play a key role in female communication.

Environmental stressors differentially modulate anxiety-like behaviour in male and female zebrafish

How differently male and female responds in a stressful situation is a matter of curiosity. Apart from curiosity, this opens a new arena to the synthesis of personalized/individualized medications. Here, we used zebrafish, a suitable experimental animal model to study stress and anxiety. We evaluated the differential responses in adult male and female zebrafish on the acute exposure of three different stressors: Caffeine (100 mg/L), Conspecific alarm substance (3.5 ml/L), and sight of sympatric predators (Leaf fish and Snakehead) with the help of two different behavioural paradigms (Novel tank test & Predator exposure). Behavioural responses were captured over 6 minutes and quantified using Smart 3.0. Male zebrafish were found to be more responsive to caffeine treatment. Conspecific alarm substance-challenged males and females showed robust alarm reactions whereas females were found to be more prone to it. Female zebrafish showed statistically significant aversion to the visual representation of sympatric predators. Taken together, each stressor induced differential responses in male and female zebrafish.

Selective selfishness in alarm calling behaviour by some members of wintering mixed-species groups of crested tits and willow tits.

Animals adjust their use of alarm calls depending on social environments. We tested whether dominant (adult) and subordinate (juvenile non-kin) male crested tits (Lophophanes cristatus) warn each other and heterospecific willow tits (Poecile montanus) across the wintering season. Birds rarely alarm called when feeding alone. Both adult and juvenile crested tits warned each other in early winter, and adults did so in the middle of wintering season. However, juvenile males rarely warned conspecific adult males in the middle of the winter. Both adult and juvenile males stopped giving alarm calls when feeding together at the end of wintering season. The results suggest that the mid-winter reduction of juvenile alarms could increase the likelihood of successful predator attacks on adults, increasing the chances for juveniles to replace adults and acquire their territories. By contrast, both adult and juvenile males produced alarm calls throughout the season when foraging together with willow tits. Whether juvenile male crested tits could be selectively altering alarm call propensity to endanger adult males, thereby selfishly enhancing their own succession to territory ownership, is discussed. The results add to the understanding of the origin of mixed-species groups and explain the dynamics of social communication. This article is part of the theme issue 'Mixed-species groups and aggregations: shaping ecological and behavioural patterns and processes'.

Male Guppies Recognize Familiar Conspecific Males by Their Face

Individual recognition is a necessary cognitive ability for the maintenance of stable social relationships. Recent studies have shown that like primates, some fish species can distinguish familiar fish from unfamiliar strangers via face-recognition. However, the taxa of the studied fish species are restricted (within Perciformes) and the visual signal used for the recognition of fish remains unclear. Here, we investigated the visual signal for individual-recognition in males of a sexually dichromatic guppy (Poecilia reticulata, Cyprinodontiformes). Using guppy males, we examined the hypothesis that fish distinguish between familiar individuals and unknown strangers by their faces rather than by body coloration. We randomly presented focal fish with four types of composite photo-models: familiar (familiar-face and familiar-body = F/F), stranger (stranger-face and stranger-body = S/S), familiar face combined with stranger body (F/S) and stranger face combined with familiar body (S/F). Focal males infrequently attacked familiar-face models but frequently attacked stranger-face models, regardless of body types. These behavioral reactions indicate that guppy males discriminate between familiar and stranger males by their face, not body coloration with wide variation. Importantly, male faces contain clear individual-variation in white/metallic colored patches on the operculum visible for humans. Considering the photo-model, our results suggest that these patches might be an important visual stimulus for face-recognition in guppy males, like some cichlids. Comparative examination among males of different guppy variants, including wild type phenotype, suggests that the face color-patch is stable regardless of variation in body color, with a different genetic mechanism potentially underlying face and body colors.

Zootechnical Brown Trout (Salmo trutta L. 1758) Ovarian Fluid Fails to Upregulate the Swimming Performances of Native Mediterranean Brown Trout (Salmo cettii Rafinesque, 1810) Sperm in the Biferno River

In external fertilizer fish, ovarian fluid (OF) seems to play a key role in fertilization success, improving spermatozoa swimming performance. These OF/sperm interaction mechanisms are frequently species-specific and/or population-specific and could decrease the risk of genetic introgression of wild populations from introduced or escaped zootechnical individuals. The Mediterranean brown trout (Salmo cettii) is threatened by genetic introgression with strains of domestic brown trout (Salmo trutta) that were introduced for recreational purposes. The aim of our study was to test if native S. cettii females, rather than zootechnical S. trutta, produce OF with a greater ability to upregulate the sperm motility of conspecific males. Thus, we compared the sperm swimming performances of males inhabiting the Biferno River (Molise region—Southern Italy) activated in native S. cettii vs. zootechnical S. trutta female’s OFs. In our study, native females’ OFs (20% diluted), compared to spring water, has the ability to significantly boost the sperm performance of the autochthonous males, while zootechnical S. trutta fails. These preliminary results suggest that OF-sperm interactions could potentially influence or direct the hybridization mechanisms involving the native Mediterranean trout inhabiting the Biferno River and the domestic lineage of brown trout introduced in the past.

Half a century of evolutionary games: a synthesis of theory, application and future directions.

Half a century of evolutionary games: a synthesis of theory, application and future directions.

Display Responses of Galápagos Lava Lizards ( Microlophus bivittatus) to Manipulation of Male Shoulder Epaulets on Conspecific-Mimicking Robots

Many animal taxa perform stereotyped displays during ritualized contests over territory and mates. Such displays facilitate assessment of an opponent's prowess and reduce the probability of physical injury. Color patterns likewise can serve as indicators of an individual's body condition. Male San Cristóbal Lava Lizards (Microlophus bivittatus) of the Galápagos Islands possess a black epaulet on each shoulder that contrasts with their surrounding body coloration. We created robots with the appearance of conspecific males to explore the potential function(s) of the epaulets in a series of four experiments. Our experiments focused on epaulet presence/absence, body location, and location relative to the axis of body motion (pectoral versus pelvic girdle) during pushups. Paired trials were conducted with adult males and females using a robot that performed M. bivittatus signature displays and that differed on its two sides in epaulet attributes. We quantified two kinds of bobbing displays in subjects: signature displays and two-bob displays. We also computed a composite response (CR) score, in which point values were assigned to types of nonbobbing displays and display combinations according to the apparent level of subject aggression. Results of Experiment 1 showed that males (but not females) performed more signature displays to the side of the robot with the epaulet present than to the side with the epaulet absent, suggesting that epaulets serve a signaling function. Composite response scores did not differ between treatments for either sex. In Experiment 2 neither males nor females performed more bobbing displays to an enlarged epaulet than to an epaulet reduced in size. However, males exhibited greater CR scores to the enlarged epaulet, suggesting that epaulet size or conspicuousness is important to its function. In Experiment 3 neither sex produced more bobbing displays to an epaulet on the robot's shoulder than to one on its hip. Yet, males accumulated significantly larger CR scores in response to the shoulder epaulet than to the hip epaulet, indicating that males were aware of normal epaulet location. Finally, in Experiment 4, where our robot performed hindlimb rather than forelimb pushups, subjects failed to exhibit more bobbing displays in response to a shoulder epaulet than to a hip epaulet. CR scores also did not differ between treatments for either sex, perhaps due to the striking abnormality of hindlimb-driven bobbing displays. Although the function of M. bivittatus epaulets remains elusive, these markings clearly are salient to male conspecifics.