Copulation Attempts Research Articles

No evidence that male sexual experience increases mating success in a coercive mating system

Several studies have shown that sexual experience can alter a male's mating behaviour to increase his future mating success. One explanation is that experienced males are better at courting females and inducing them to mate. Experienced males might also be better at identifying higher quality mates, although fewer studies have tested for this benefit. In both cases, however, these potential benefits of sexual experience might be partially offset by the energetic costs of courting and mating, which tend to reduce a male's subsequent ability to invest in sexually selected traits, and thereby reduce his future attractiveness and mating success (i.e. hasten the onset of reproductive senescence). Here we used the eastern mosquitofish, Gambusia holbrooki, to test whether sexual experience elevates male mating success. We housed recently matured males either with full mating access to females (experienced males) or in the visual and olfactory presence of females with which they could not mate (naive males). We then measured the strength of male mate choice for larger, more fecund, females, male mating behaviour (time spent chasing females and the number of copulation attempts) and insemination success. Experienced and naive males did not differ significantly in their mating behaviour and there was no effect of sexual experience on the likelihood of mating or on the number of sperm inseminated (although experienced males had a tendency to be less successful when performing gonopodial thrusts). Experienced males in two-choice trials were, however, significantly more likely to ‘inspect’ both females and had a significantly stronger preference for larger females. Finally, we measured male immune response and growth to test for any costs of the increased mating effort that is concomitant with greater experience. Experienced males had significantly slower postmaturation growth and a significantly weaker immune response than naive males.

Effects of cadmium on the behaviour of Cnesterodon decemmaculatus

Fil: Ferro, Juan Pablo. Universidad Nacional de Lujan. Departamento de Ciencias Basicas. Programa de Ecofisiologia Aplicada; Argentina. Universidad Nacional de Lujan. Instituto de Ecologia y Desarrollo Sustentable. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Parque Centenario. Instituto de Ecologia y Desarrollo Sustentable; Argentina

How do familiarity and relatedness influence mate choice in Armadillidium vulgare?

Mate choice is an important process in sexual selection and usually prevents inbreeding depression in populations. In the terrestrial isopod Armadillidium vulgare, the close physical proximity between individuals may increase the risk of reproducing with siblings. Moreover, individuals of this species can be infected with the feminizing bacteria of Wolbachia, which influence male mate choice. However, little is known about the kinship or familiarity assessment of the selected partner that occurs when a male can choose between females with or without Wolbachia. To investigate the potential mechanisms leading to mate choice and the potential impact of the parasite, we performed behavioral choice tests on males where they could choose between sibling vs. nonsibling females, familiar vs. unfamiliar females, and sibling familiar vs. unfamiliar nonsibling females. To investigate the costs of inbreeding, we compared the reproductive success of both sibling and nonsibling mates. Our results revealed that male copulation attempts were higher for familiar females and for nonsibling females when both females were Wolbachia-infected, but the duration was longer when both females were Wolbachia-free. When males mated with a sibling female, their fecundity was severely decreased, consistent with inbreeding depression. Overall, we observed copulations with all types of females and demonstrated discrimination capacities and potential preferences. We highlight the complexity of the tradeoff between kinship, familiarity and parasite transmission assessment for mate choice.

Impact of the widespread pharmaceutical pollutant fluoxetine on behaviour and sperm traits in a freshwater fish

Pharmaceutical pollutants are detected in aquatic habitats and wildlife tissues globally. One widespread contaminant of major concern is the antidepressant fluoxetine, which can affect behavioural and physiological processes in non-target species. Despite this, effects of fluoxetine on wildlife behaviour have seldom been investigated across multiple fitness-related contexts, especially at environmentally realistic concentrations. Accordingly, we examined impacts of 35-day fluoxetine exposure at two environmentally relevant concentrations (31 and 374 ng/L) across a suite of fitness-related contexts in wild-caught male mosquitofish (Gambusia holbrooki). First, we investigated anxiety-related behaviours (boldness, exploration and activity) in a novel environment (maze arena) and found no significant impacts of exposure. Second, we tested effects of fluoxetine in a reproductive context, including mating behaviour and sperm quality. We found that, relative to controls, fluoxetine exposure resulted in males spending a greater amount of time pursuing females. Further, low-exposed males were more likely to attempt copulation than unexposed males. Lastly, we investigated across-context behavioural correlations, and how fluoxetine exposure might affect such relationships. A significant positive correlation was detected in control fish between activity levels in the maze and time spent pursuing females in the reproductive assay. This relationship was disrupted by fluoxetine at both exposure levels. This is the first evidence that field-detected concentrations of a pharmaceutical pollutant can disturb across-context behavioural correlations in wildlife. Our findings provide clear evidence that fluoxetine can produce context-specific behavioural effects in fish and underscore how pharmaceutical exposure at field-detected concentrations can induce important shifts in wildlife behaviour.

Looking for the right mate—What do we really know on the courtship and mating of Lucilia sericata (Meigen)?

Lucilia sericata is well known for causing myiasis in humans, livestock, pets and wildlife. It also vectors microbial pathogens, including paratuberculosis agents. This species can be exploited in maggot therapy to treat necrotic wounds, particularly those infected by multidrug-resistant pathogens. Despite its high medical and veterinary importance, our knowledge about its courtship and mating behavior is still limited. In this study, we quantified the courtship and mating behavior of L. sericata, shedding light on the potential impact of lateralization of selected behavioral traits during sexual interactions. When a male identified a female, he approached her with head pushing followed by foreleg tapping acts. Courtship lasted 7.65 ± 0.4 s. During copulation attempts, the male continued foreleg tapping acts on the female body, and tried to achieve genital contact. Copula lasted 14.88 ± 0.41 min, while male mating success was 85%. Several courtship and mating traits were found lateralized at population-level. Most of males approached the female with head pushing acts on her left side of the body. Both during courtship and copulation attempt phases, males mainly used the right foreleg to perform leg tapping acts on females. However, the impact of lateralized head pushing and foreleg tapping on the main behavioral parameters characterizing L. sericata courtship and mating was not significant, except for a higher number of foreleg tapping acts during copulation attempts displayed by right-biased males over left-biased ones. Overall, these results contribute to improve our basic knowledge on the reproductive behavior of L. sericata. Besides, selected behavioral parameters characterized here can be exploited as benchmarks to monitor mate quality during mass-rearing, as well as to select males with boosted mating competitiveness, helping to improve the success of SIT programs and behavior-based control tools.

Brawls Bring Buzz: Male Size Influences Competition and Courtship in Diadasia rinconis (Hymenoptera: Apidae).

Sexual selection on male body size in species with a female-biased sexual size dimorphism is common yet often poorly understood. In particular, in the majority of bee species, the relative contribution of intrasexual competition and female choice to patterns of male body size is unknown. In this field study, we examined two possible components of male mating success with respect to body size in the solitary bee Diadasia rinconis Cockerell (Hymenoptera: Apidae): 1) ability to procure a mate and 2) the duration of copulation. We found that larger males were better able to procure mates and copulated for shorter periods of time. Although consistent with sperm competition theory, differences in copulation duration were slight; possibly, the shorter copulations of larger males instead reflect in copulo female choice. Consistent with this notion, males engaged in complex courtship while mounted, characterized for the first time in any bee in such detail via audio recordings and high-speed, high-definition video. The number of pulses in male courtship behavior was also positively associated with copulation duration and may have stimulated females to continue copulating, thereby potentially allowing smaller males to transfer a full ejaculate. Females were shown to be potentially polyandrous and although we did not observe precopulatory rejection in the field, captive females frequently rejected copulation attempts by captive males. Our work indicates that intrasexual competition selects for increased body size in a solitary bee.

Female receptivity affects subsequent mating effort and mate choice in male guppies

High mating effort leads to choosiness because each mating event reduces future reproductive potential. Many studies have shown that males adjust their sexual behaviour relative to female fecundity and encounter rate. However, little is known about the effects of a male's past mating experiences. We used guppies, Poecilia reticulata, to investigate how males change their sexual behaviour after experiencing high or low mating success. Each male was tested with two differently sized unreceptive females before and after encountering either four indiscriminate receptive virgin females or four nonreceptive pregnant females. Males that experienced high mating success with receptive females decreased their courtship displays but increased the frequency of sneaky behaviour, whereas low mating success males previously repetitively rejected by nonreceptive females showed an increase in courtship and a decrease in sneaky copulation attempts. Mating history also influenced male choosiness, with successful males showing stronger preferences for larger females than unsuccessful males. This overall adjustment in behaviour may be attributed to a reduction of resources, such as energy and gametes, as well as prior social interaction with receptive and nonreceptive females. Males that adjust their effort and choosiness based on their recent mating history and their own condition could optimize reproductive trade-offs.

Does the winner-loser effect determine male mating success?

Winning or losing a fight can have lasting effects on competitors. Controlling for inherent fighting ability and other factors, a history of winning often makes individuals more likely to win future contests, while the opposite is true for losers (the 'winner-loser effect'). But does the winner-loser effect also influence a male's mating success? We experimentally staged contests between male mosquitofish (Gambusia holbrooki) such that focal males either won or lost three successive encounters with stimulus males. We then placed a size-matched (to control for inherent fighting ability) winner and loser with a female and monitored their behaviour (n = 63 trios). Winners spent significantly more time associating with the female. Winners did not make more copulation attempts, nor have a greater number of successful attempts. There was, however, a significant effect of male size on the number of successful copulation attempts: success decreased with male size for losers, but size had no effect on the success rate of winners.

Chemical signalling and context dependent polyandry in the false garden mantid Pseudomantis albofimbriata (Mantodea: Mantidae)

AbstractMultiple mating by females is a common phenomenon in nature. Polyandrous females may benefit from genetically diverse progeny that may survive better in a changing environment. Males in polyandrous systems, however, may not achieve their maximum paternity. Therefore, males are predicted to carry traits that prevent or reduce female polyandry. Praying mantids are predatory insects in which females can mate multiple times, predicting the evolution of male counter‐strategies. However, the rate of polyandry and male strategies against polyandry are rarely studied in these insects. In the current study, we used false garden mantids Pseudomantis albofimbriata to quantify the rate of multiple mating when several males are present within close visual range of an unmated female. We further determined how long mated females stay unattractive after mating. We found that in a scramble scenario, the subsequent males stay with a copulating pair and attempt mating once the first male has completed copulation. These second copulation attempts are often successful. If only one male is attracted as a result of the initial pheromone plume, then polyandry is unlikely because the female will remain chemically unattractive for 8 days on average (thus preventing subsequent male attractions), which is longer than the usual latency to lay the first egg sac. From previous studies, we know that single male attraction is the most common scenario in this system in both natural and semi‐natural contexts. Therefore, polyandry depends on the number of males attracted to the initial pheromone plume of the female and is likely to be a relatively uncommon phenomenon in this system.

Scientific note on interrupted sexual behavior to virgin queens and expression of male courtship-related gene fruitless in a gynandromorph of bumblebee, Bombus ignitus

Gynandromorphy, which is characterized by the coexistence of male and female tissues in a single individual, is known in insects. Gynandromorphs exhibit diverse levels of defects in sexual behavior. The distribution pattern of both sexes within the nervous systems could be responsible for these differences in behavioral traits; however, most studies have mainly focused on the unique external morphological traits of gynandromorphs, and little attention has been paid to the evaluation of nervous systems, at least in Hymenoptera. In this study, we evaluated a gynandromorphic individual of bumblebee (Bombus ignitus) with a bilaterally dimorphic head and thorax (left side, male; right side, female) and a uniformly masculine gaster. The gynandromorph exhibited abnormal sexual behavior; it normally approached and touched virgin queens but rarely made subsequent copulation attempt. Dissection of the gaster indicated that it possessed a set of male reproductive organs. We analyzed the encephalic and antennal expression patterns of the fruitless (fru) gene, which shows sex-specific alternative splicing conserved in various insect orders and has been thought to act as the master regulator of sexual behavior in male nervous systems. The gynandromorph showed left-side-biased expression of male-type fru transcripts. We discuss a possible mechanism for the observed unusual interruption of sexual behavior of the gynandromorph by reference to the fru expression pattern and our recent findings on the sexually dimorphic response to female-derived pheromones.

Male guppies compensate for lost time when mating in turbid water

Turbidity (a measure of the cloudiness of water) decreases the visual range of organisms, altering interactions within and between species. For species that visually assess mates, turbidity may affect mating interactions and mate choice. A central question, then, is to what degree organisms plastically adjust mating behaviors to cope with visually altered environments. Here, we investigate the effect of turbidity on the mating behavior of guppies (Poecilia reticulata) in Trinidad, where some streams are increasingly turbid due to upstream rock quarrying. We collected fish from two highly turbid streams (with upstream rock quarries) and two pristine streams (no upstream quarries) in the same drainages. We then observed male mating behaviors with females from the same populations in both turbid and clear assays, recording displays and sneak copulation attempts. Males from turbid streams showed greater individual consistency in mating behaviors. But regardless of provenance, male guppies spent less time associating with females in turbid water overall. When males and females did interact, however, males greatly increased the rate of all mating behaviors in turbid as compared to clear water. Thus, even when lacking a long-term evolutionary history with high turbidity, guppies compensate for lost time when mating in a visually altered environment. Given the global nature of increases in turbidity, understanding behavioral responses to this aquatic anthropogenic change is critical. Here, we investigate the degree to which Trinidadian guppy males in affected areas are able to plastically compensate in turbid water by adjusting mating behaviors. We also test the degree to which previous chronic exposure to turbidity alters this plasticity. In contrast to predictions, we did not find population differences in plasticity; across all populations, however, we found significant plasticity in the guppy mating system in response to anthropogenically increased turbidity. While males spent less time associating with females in turbid water overall, males increased mating effort during periods of association, compensating for lost time in turbid water. We discuss this plasticity and the implications for guppy sexual selection and secondary sexual characteristics such as coloration.

A specialized fungal parasite (Massospora cicadina) hijacks the sexual signals of periodical cicadas (Hemiptera: Cicadidae: Magicicada)

Male periodical cicadas (Magicicada spp.) infected with conidiospore-producing (“Stage I”) infections of the entomopathogenic fungus Massospora cicadina exhibit precisely timed wing-flick signaling behavior normally seen only in sexually receptive female cicadas. Male wing-flicks attract copulation attempts from conspecific males in the chorus; close contact apparently spreads the infective conidiospores. In contrast, males with “Stage II” infections that produce resting spores that wait for the next cicada generation do not produce female-specific signals. We propose that these complex fungus-induced behavioral changes, which resemble apparently independently derived changes in other cicada-Massospora systems, represent a fungus “extended phenotype” that hijacks cicadas, turning them into vehicles for fungus transmission at the expense of the cicadas’ own interests.

Effect of the age of ganders on reproductive behavior and fertility in a competitive mating structure

Abstract This paper presents the study on determination of gander-goose mating activity in relation to age and the position of gander in social hierarchic rank. Moreover, levels of fertility (F) and testosterone (T) in different gander age groups were analyzed. The study was carried out on one-year-old geese (N = 64) and one- or two-year-old ganders (N = 16; 1♂: 4♀). Observations of mating activity (MA) were conducted during the 3-h period of the daylight (10 h), 2-3 times per week (102 h/ group/34 days). The recording was done with a digital recorder connected to cameras. Agonistic behavioral interactions between ganders were noted to determine a social hierarchic rank. The MA was described by frequency of courtship displays, copulation attempts, successful copulation (SCop), and interaction disrupted (DMI) with agonistic behavior. Fertility was evaluated after the artificial incubation (1×/week) in a commercial hatchery. The frequency of SCop, DMI, and T concentration were higher (P<0.05) for two- than one-year-old ganders. Moreover, F was higher by 11.2% for group of geese kept with two-year-old ganders. There was an effect of the gander’s rank in social hierarchy on the frequency of MA. For dominant ganders, frequency of courtship displays (1.0/times/3 h) and copulation attempts (0.8/times/3 h) were lower (P<0.05), but SCop (1.5/times/3 h) was higher (P<0.05) than for subordinate ones. Thus, in domestic goose flocks reproductive success was associated with age of ganders used in competitive mating system. This is probably a multifactor effect of interaction between sexual/social experience, T-dependent mating motivation, and efficiency of MA. It is recommended to keep one-year-old geese with older ganders (after sexual reactivation) for optimal fertility results with sex ratio adjusted to gander-gander antagonistic interaction dynamic.

Floral scent and species divergence in a pair of sexually deceptive orchids.

Speciation is typically accompanied by the formation of isolation barriers between lineages. Commonly, reproductive barriers are separated into pre‐ and post‐zygotic mechanisms that can evolve with different speed. In this study, we measured the strength of different reproductive barriers in two closely related, sympatric orchids of the Ophrys insectifera group, namely Ophrys insectifera and Ophrys aymoninii to infer possible mechanisms of speciation. We quantified pre‐ and post‐pollination barriers through observation of pollen flow, by performing artificial inter‐ and intraspecific crosses and analyzing scent bouquets. Additionally, we investigated differences in mycorrhizal fungi as a potential extrinsic factor of post‐zygotic isolation. Our results show that floral isolation mediated by the attraction of different pollinators acts apparently as the sole reproductive barrier between the two orchid species, with later‐acting intrinsic barriers seemingly absent. Also, the two orchids share most of their fungal mycorrhizal partners in sympatry, suggesting little or no importance of mycorrhizal symbiosis in reproductive isolation. Key traits underlying floral isolation were two alkenes and wax ester, present predominantly in the floral scent of O. aymoninii. These compounds, when applied to flowers of O. insectifera, triggered attraction and a copulation attempt of the bee pollinator of O. aymoninii and thus led to the (partial) breakdown of floral isolation. Based on our results, we suggest that adaptation to different pollinators, mediated by floral scent, underlies species isolation in this plant group. Pollinator switches may be promoted by low pollination success of individuals in dense patches of plants, an assumption that we also confirmed in our study.

Subharmonics increase the auditory impact of female koala rejection calls

AbstractAlthough non‐linear phenomena are common in human and non‐human animal vocalisations, their functional relevance remains poorly understood. One theory posits that non‐linear phenomena generate unpredictability in vocalisations, which increases the auditory impact of vocal signals, and makes animals less likely to habituate to call repetition. Female koalas (Phascolarctos cinereus) produce vocal signals when they reject male copulation attempts that contain relatively high levels of non‐linear phenomena, and thus may function as attention grabbing vocal signals during the breeding season. To test this hypothesis, we used playback experiments: firstly, to determine whether female rejection calls induce heightened behavioural responses in free‐ranging male koalas during the breeding season, and secondly, to examine how the relative amount of non‐linear phenomena in rejection calls influences male behavioural response. The results show that male koalas look for longer towards speakers broadcasting playback sequences of male bellows followed by a series of female rejection calls than those broadcasting only male bellows. In addition, female rejection call sequences with more subharmonics, higher harmonics‐to‐noise ratios, and less biphonation produced the greatest male looking responses. Our findings support the hypothesis that female koala rejection calls function to grab male attention during the breeding season, and indicate that subharmonics are the main acoustic feature that increases the auditory impact of these vocal signals.

<em>Drosophila</em> Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male's copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to naïve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Drosophila Courtship Conditioning As a Measure of Learning and Memory.

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male's copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to naïve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Post-mating shift towards longer-chain cuticular hydrocarbons drastically reduces female attractiveness to males in a digger wasp

Females of most aculeate Hymenoptera mate only once and males are therefore under a strong competitive pressure which is expected to favour the evolution of rapid detection of virgin females. In several bee species, the cuticular hydrocarbon (CHC) profile exhibited by virgin females elicits male copulation attempts. However, it is still unknown how widespread this type of sexual communication is within Aculeata. Here, we investigated the use of CHCs as mating cues in the digger wasp Stizus continuus, which belongs to the family (Crabronidae) from within bees arose. In field experiments, unmanipulated, recently emerged virgin female dummies promptly elicit male copulation attempts, whereas 1–4days old mated females dummies were still attractive but to a much lesser extent. In contrast, old (10–15days) mated female dummies did not attract males at all. After hexane-washing, attractiveness almost disappeared but could be achieved by adding CHC extracts from virgin females even on hexane-washed old mated females. Thus, the chemical base of recognition of females as appropriate mating partner by males is coded in their CHC profile. Accordingly, differences in CHC profiles can be detected between sexes, with males having larger amounts of alkenes and exclusive long-chain alkanes, and within females specially according to their mating status. Shortly after mating, almost all of the major hydrocarbons found on the cuticle of females undergo significant changes in their abundance, with a clear shift from short-chain to long-chain linear and methyl-branched alkanes. The timely detection of virgin females by males in S. continuus could be advantageous within the narrow period of female emergence, when male-male competition is strongest.

High occurrence of extra-pair partnerships and homosexuality in a captive Cape Vulture Gyps coprotheres colony

The Cape Vulture Gyps coprotheres is an Endangered colonial cliff-nesting species that is typically cited as monogamous. Observations of wild Cape Vulture colonies note extra-pair breeding activities but homosexual activity has never been confirmed. Observations of breeding behaviours within a captive colony were conducted at VulPro’s breeding facilities in South Africa The captive breeding programme included 10 breeding pairs and a total of 41 non-releasable, individually recognisable, and genetically sexed subadults or adults. Observations were conducted over the 2014 breeding season as part of a study investigating the social and rearing conditions of chicks intended for release in a population supplementation programme. A high percentage of total colony copulation attempts (22%) were extra-pair copulations but only 23 interactions resulted in cloacal contact or possible sperm transfer. Four paired males formed extra-pair relationships, two of which were homosexual.

Highly variable male courtship behavioral sequences in a crambid moth

Research on male courtship behavior of moths has focused on documenting stereotyped sequences for successful copulation. We characterized successful male courtship behavior among 126 virgin mating pairs of Ostrinia nubilalis. Using Markov analysis, stereotypy indices, and a novel application of ecological network analysis, we found high variability in these sequences. Fifteen courtship behaviors were described and 96 behavioral transitions were observed, 39 of which occurred only once. The number of courtship bouts ranged from one to ten, the number of behavioral transitions ranged from four to 41, and the number of copulation attempts ranged from one to 29. Only 23% of males used a common, simple behavioral sequence. Females exhibited acceptance or rejection behaviors in 40% of the sequences, but these did not explain the high variability in male courtship sequences. About half of the transitions occurred non-randomly, and stereotypy was low. Network analysis revealed that the courtship sequences started and ended with stereotyped behaviors and the high variability occurred in the middle of the sequences. Whole system analysis showed that the courtship sequences were more variable than for optimal transfer of information. Overall, these results suggest that the sequence of behaviors may be less important than the occurrence of certain behavioral elements for successful mating.