The sexual behaviour of the red-spotted newt, Notophthalmus viridescens (Amphibia: Urodela: Salamandridae)

The sexual behaviour of the red-spotted newt, Notophthalmus viridescens is described. The male can adopt one of two types of behaviour depending on the initial responsiveness of the female. If she is responsive, the male performs a brief lateral (or hula) display, at the end of which one or more spermatophores may be deposited. If she is unresponsive, the male captures the female and a lengthy period of amplexus ensues, followed by spermatophore transfer behaviour. Spermatophore transfer is most successful after amplexus, and a preliminary cost-benefit analysis of the two types of courtship is given. The behaviour is discussed in the context of other urodele amphibians, and in the wider context of male reproductive strategies.     

The sexual strategy of the central long-toed salamander, Ambystoma macrodactylum columbianum, in south-eastern Washington

The sexual strategies of salamanders are largely'unobservable'owing to the low visibility typical of their preferred breeding habitats. We conducted both field and laboratory studies to investigate the sexual strategy of the North American central long-toed salamander, Ambystoma macrodactylum columbianum. Capture-recapture and cohort-marking of salamanders at an aquatic breeding site indicated that males arrive before females and remain in the water for longer periods of time (measured in weeks, rather than days as for females). We estimate that the overall duration of the breeding season is short, three weeks or less. Courtship between single males and females is characterized by repeated bouts of axillary amplexus, separated by multiple episodes of spermatophore deposition. Amplectant males provide females with considerable tactile stimulation in the form of head-rubbing. Unpaired males attempt to displace amplectant males from their partners by wrestling with them. In addition, unpaired males appear to interfere in ongoing courtship encounters during the spermatophore deposition stage, perhaps attempting to 'steal'inseminations. We suggest that explosive scramble-competition polygyny best describes the mating system of A. m. columbianum. Operational sex ratios are probably male-biased throughout the short breeding season, leading to intense competition among males for mates. Mate choice (by either sex) probably is of little importance as a determinant of mating success.     

Competitive PCR reveals the complexity of postcopulatory sexual selection in Teleogryllus commodus

The outcome of mate choice depends on complex interactions between males and females both before and after copulation. Although the competition between males for access to mates and premating choice by females are relatively well understood, the nature of interactions between cryptic female choice and male sperm competition within the female reproductive tract is less clear. Understanding the complexity of postcopulatory sexual selection requires an understanding of how anatomy, physiology and behaviour mediate sperm transfer and storage within multiply mated females. Here we use a newly developed molecular technique to directly quantify mixed sperm stores in multiple mating females of the black field cricket, Teleogryllus commodus. In this species, female postcopulatory choice is easily observed and manipulated as females delay the removal of the spermatophore in favour of preferred males. Using twice‐mated females, we find that the proportion of sperm in the spermatheca attributed to the second male to mate with a female (S₂) increases linearly with the time of spermatophore attachment. Moreover, we show that the insemination success of a male increases with its attractiveness and decreases with the size of the female. The effect of male attractiveness in this context suggests a previously unknown episode of mate choice in this species that reinforces the sexual selection imposed by premating choice and conflicts with the outcome of postmating male harassment. Our results provide some of the clearest evidence yet for how sperm transfer and displacement in multiply mated females can lead directly to cryptic female choice, and that three distinct periods of sexual selection operate in black field crickets.     

Sexual conflict and cryptic female choice in the black field cricket, Teleogryllus commodus

The prevalence and evolutionary consequences of cryptic female choice (CFC) remain highly controversial, not least because the processes underlying its expression are often concealed within the female reproductive tract. However, even when female discrimination is relatively easy to observe, as in numerous insect species with externally attached spermatophores, it is often difficult to demonstrate directional CFC for certain male phenotypes over others. Using a biological assay to separate male crickets into attractive or unattractive categories, we demonstrate that females strongly discriminate against unattractive males by removing their spermatophores before insemination can be completed. This results in significantly more sperm being transferred by attractive males than unattractive males. Males respond to CFC by mate guarding females after copulation, which increases the spermatophore retention of both attractive and unattractive males. Interestingly, unattractive males who suffered earlier interruption of sperm transfer benefited more from mate guarding, and they guarded females more vigilantly than attractive males. Our results suggest that postcopulatory mate guarding has evolved via sexual conflict over insemination times rather than through genetic benefits of biasing paternity toward vigorous males, as has been previously suggested.     

The effect of the presence of quiescent female nymphs, males and their spermatophores on spermatophore placement in two species of eriophyoid mites

Under sex dissociated sperm transfer, females seek spermatophores and pick up sperm without male assistance. In several species males adjust spermatophore deposition rate to the presence of conspecifics. It is not known, however, which factors could favor such elasticity in non-pairing males. In this paper, we compare male response towards conspecifics between the sex dissociated eriophyoid mites Aculus fockeui (Nalepa and Trouessart) and Aculops allotrichus (Nalepa). The species differ significantly in male reproductive strategies and, consequently, the intensity of male–male-competition. Aculus fockeui males deposit spematophores all over the leaves and occasionally leave single spermatophores beside quiescent female nymphs (QFNs). In contrast, A. allotrichus males guard QFNs and encircle them with spermatophores. In this study, males of both species deposited spermatophores close to and apart from the rival spermatophores. Aculops allotrichus males had similar spermatophore output whether they were kept alone or in a group of seven males. They did not change spermatophore output in the presence of five rival spermatophores, a QFN or a QFN and varying number of rivals, either. In contrast, A. fockeui males increased spermatophore output in the presence of rival spermatophores or when on the arena with a QFN the male number increased to eight males. They did not respond, however, to the presence of a QFN and one rival or a QFN alone. The possible effect of the species-specific intensity of male–male competition, population density, the availability of receptive females and the rate of spermatophore output on the flexibility of eriophyoid spermatophore deposition is discussed.     

Sexual dimorphism and age of Mediterranean salamanders

We analysed sexual size dimorphism (SSD) for two Mediterranean species of the “true” salamander clade possessing distinct life histories (Salamandra algira and Mertensiella caucasica) and equilibrated the morphometric approach to individual age by using skeletochronology. For species that have a short breeding season and live at high altitudes, such as Mediterranean amphibians, the fecundity advantage hypothesis predicts female-biased SSD to maximise reproductive success. Our results showed no SSD in either species; however, morphometric data indicated a male-biased dimorphism in limb (arm and leg) dimensions in both species when compared to body size. Limb dimorphisms are likely related to the particular mating system, which involves an amplexus during spermatophore transfer. Arm length appeared sexually dimorphic during ontogeny both in viviparous S. algira and oviparous M. caucasica. A review on SSD indicated monomorphy of body size as a common lineage-specific pattern among the “true” salamander clade, but also the common presence of other traits such as sexually dimorphic limb proportions.     

Molecular dissection of nuptial gifts in divergent strains of Ostrinia moths

Seminal fluid proteins (SFPs) produced in the male accessory glands and ejaculatory duct are subject to strong sexual selection, often evolve rapidly and therefore may play a key role in reproductive isolation and species formation. However, little is known about reproductive proteins for species in which males transfer ejaculate to females using a spermatophore package. By combining RNA sequencing and proteomics, we characterize putative SFPs, identify proteins transferred in the male spermatophore and identify candidate genes contributing to a one‐way gametic incompatibility between Z and E strains of the European corn borer moth Ostrinia nubilalis. We find that the accessory glands and ejaculatory duct secrete over 200 highly expressed gene products, including peptidases, peptidase regulators and odourant‐binding proteins. A comparison between Ostrinia strains reveals that accessory gland and ejaculatory duct sequences with hormone degradation and peptidase activity are among the most extremely differentially expressed. However, most spermatophore peptides lack reproductive tissue bias or canonical secretory signal motifs and aproximately one‐quarter may be produced elsewhere before being sequestered by the male accessory glands during spermatophore production. In addition, most potential gene candidates for postmating reproductive isolation do not meet standard criteria for predicted SFPs and almost three‐quarters are novel, suggesting that both postmating sexual interactions and gametic isolation likely involve molecular products beyond traditionally recognized SFPs.     

Female Choice and the Consistency of Courtship Feeding in Black-horned Tree Crickets Oecanthus nigricornis Walker (Orthoptera: Gryllidae: Oecanthinae)

Courtship feeding in insects is often strongly correlated with insemination duration and therefore provides a potential postcopulatory episode of sexual selection. We tested whether courtship feeding and other courtship traits in the black-horned tree cricket Oecanthus nigricornis showed sufficient consistency potentially to respond to sexual selection by testing whether they differed significantly among males. Duration of courtship feeding differed among males when measured repeatedly and this caused significant differences in the duration of spermatophore attachment, a trait that determines the maximum duration of insemination and thus has important fitness consequences in crickets. We also partitioned variance in courtship behaviour between the sexes to test whether differences in courtship behaviour were attributable primarily to males, females or both sexes. Duration of spermatophore attachment was controlled by females and therefore represents a mechanism of female mate choice. Significant variation in duration of spermatophore attachment was associated with differences between individuals of both sexes. Differences among males indicate that females agree in their preference of certain males whereas differences among females indicate that females differ in their receptivity to postcopulatory courtship and insemination. The fact that differences among males in duration of spermatophore attachment were due to significant differences solely in the period of courtship feeding indicates that postcopulatory female choice was mediated through courtship feeding. Whether males manipulate female choices by allocating more or fewer resources requires further testing, but we found that males court some females more vigorously than others after females dismount. The number of previous mates had opposite effects on the duration of courtship feeding for the sexes, decreasing it for males but increasing it for females, and we discuss the possible causes of these results.     

Extreme Costs of Mating for Male Two-Spot Ladybird Beetles

Male costs of mating are now thought to be widespread. The two-spot ladybird beetle (Adalia bipunctata) has been the focus of many studies of mating and sexual selection, yet the costs of mating for males are unknown. The mating system of A. bipunctata involves a spermatophore nuptial gift ingested by females after copulation. In this study, we investigate the cost to males of mating and of transferring spermatophores in terms of lifespan, ejaculate production and depletion of nutritional reserves. We found that males faced a strong trade-off between mating and survival, with males that were randomly assigned to mate a single time experiencing a 53% reduction in post-mating lifespan compared to non-mating males. This is among the most severe survival costs of a single mating yet reported. However, spermatophore transfer did not impact male survival. Instead, the costs associated with spermatophores appeared as a reduced ability to transfer spermatophores in successive matings. Furthermore, males ingested more food following spermatophore transfer than after matings without spermatophores, suggesting that spermatophore transfer depletes male nutritional reserves. This is to our knowledge the first report of an effect of variation in copulatory behaviour on male foraging behaviour. Overall, our study highlights the advantages of assessing mating costs using multiple currencies, and suggests that male A. bipunctata should exhibit mate choice.     

Dimorphic ejaculates and sperm release strategies associated with alternative mating behaviors in the squid

Sperm competition is a powerful postcopulatory selective force influencing male adaptations associated with increasing fertilization success, and it is usually related to the evolution of different strategies of ejaculate expenditure between individuals. Ejaculates may also be influenced by additional selective pressures associated with sperm competition, such as timing between insemination and fertilization, female reproductive tract morphology, and fertilization environment. Also, males that adopt alternative mating tactics may face distinct sperm competition pressures, which may lead to the evolution of intraspecific diversity in ejaculates. In loliginid squids, males with alternative reproductive tactics (sneakers and consorts) differ not only in mating behavior, but also transfer spermatophores into two distinct sites within the female. Here, we compared structure and functioning of spermatophores between sneakers and consorts in the squid Doryteuthis plei applying microscopy techniques and in vitro experiments. Sneakers and consorts exhibit differences in spermatophore structure that lead to distinct spermatophoric reactions and spermatangium morphologies. Moreover, in sneakers, sperm release lasts longer and their sperm show an aggregative behavior not detected in consorts. Slow sperm release may be a strategy to guarantee longer sperm provision, given the wide interval between sneaker mating and egg release. For consorts, in turn, intense and quick sperm discharge may be advantageous, as timing between mating and egg‐laying is relatively short. Within the complex squid mating system, factors such as (i) different fertilization sites and (ii) interval between mating and egg release may also influence sperm competition, and ultimately shape the evolution of divergent ejaculates between dimorphic males.     

Sexual dimorphism in calanoid copepods: morphology and function

Mate location and recognition are essentially asymmetrical processes in the reproductive biology of calanoid copepods with the active partner (the male) locating and catching the largely passive partner (the female). This behavioural asymmetry has led to the evolution of sexual dimorphism in copepods, playing many pivotal roles during the various successive phases of copulatory and post-copulatory behaviour. Sexually dimorphic appendages and structures are engaged in (1) mate recognition by the male; (2) capture of the female by the male; (3) transfer and attachment of a spermatophore to the female by the male; (4) removal of discharged spermatophore(s) by the female; and (5) fertilization and release of the eggs by the female. In many male calanoids, the antennulary chemosensory system is enhanced at the final moult and this enhancement appears to be strongly linked to their mate-locating role, i.e. detection of sex pheromones released by the female. It can be extreme in calanoids inhabiting oceanic waters, taking the form of a doubling in the number of aesthetascs on almost every segment, and is less expressed in forms residing in turbulent, neritic waters. Mate recognition is a process where chemoreception and mechanoreception presumably work in conjunction. The less elaborate male chemosensory system in the Centropagoidea is counterbalanced by females playing a more active role in generating hydromechanical cues. This is reflected in females in the shape of the posterior prosomal margin, the complexity of urosomal morphology and the size of the caudal setae. Visual mate recognition may be important in the Pontellidae, which typically show sexual dimorphism in eye design. The most distinctive sexual dimorphism is the atrophy of the mouthparts of non-feeding males, illustrating how copepod detection systems can be shifted to a new modality at the final moult. In the next phase, the male captures the female using the geniculate antennule and/or other appendages. Three types of antennulary geniculations are recognized, and their detailed morphology suggests that they have originated independently. Grasping efficiency can be enhanced by the development of supplemental hinges. The scanty data on capture mechanisms in males lacking geniculate antennules are reviewed. It is suggested that the loss of the antennulary geniculation in many non-centropagoidean calanoids has evolved in response to increasing predator pressure imposed on pairs in amplexus. Spermatophore transfer and placement are generally accomplished by the modified leg 5 of the male. In some males, leg 5 consists of both a chelate grasping leg and a spermatophore-transferring leg, whereas in others, only the latter is developed. Tufts of fine setules/spinules and/or sclerotized elements on the terminal portion of the leg are involved in the transfer and attachment of the spermatophore. The configuration of gonopores, copulatory pores and their connecting ducts in the female genital double-somite is diversified in the early calanoid offshoots such as Arietellidae and Metridinidae, whereas in more derived groups, it is constant and invariable, with paired gonopores and copulatory pores located beneath a single genital operculum. The absence of seminal receptacles in most Centropagoidea limits the female's ability to store sufficient sperm for multiple egg batches, suggesting that repeated mating is necessary for sustained egg production. Discharged spermatophores are usually removed by the female leg 5 and/or specialized elements on other legs. In Tortanus (Atortus) Ohtsuka, which has rudimentary fifth legs in the female and complex coupling devices in the male, a spermatophore supposedly remains on the female urosome, since eggs appear to be released from a ventral opening of the spermatophore. The type of sexual dimorphism is closely related to habitat and biology. Some hyperbenthic families never show multiplication of aesthetascs on the male antennule, whereas families of the open pelagic realm such as the Aetideidae always have non-feeding males exhibiting secondary multiplication of antennulary aesthetascs. The various aspects and diversity of calanoid sexual dimorphism are herein considered in an evolutionary context.     

Age‐Related Male Reproductive Investment in Courtship Display and Nuptial Gifts in a Moth,Ostrinia scapulalis

Due to a trade‐off between current and future reproduction, costly reproductive investments should be increased towards the end of a lifespan when the probability of reproduction becomes low (terminal investment hypothesis). We investigated age‐related changes in male reproductive investment towards courtship display and the spermatophore in three age classes (young, middle‐aged and old) of a monandrous moth, Ostrinia scapulalis. As predicted, old males had higher mating success than young and middle‐aged males in no‐choice tests. Moreover, two‐choice tests revealed that middle‐aged males had a higher success rate than young males because of their higher courtship frequency rather than any female preference for them. It was found that old males produced a larger spermatophore than young and middle‐aged males, suggesting greater reproductive effort. The protein content of spermatophores also tended to increase with male age. Despite the age‐related variation in spermatophore size and protein content, age did not affect female fecundity or longevity. A decrease in the number of sperm in the older males might counteract the nutritional benefit of larger spermatophores. Alternatively, fitness components other than longevity and fecundity may be influenced by male age.     

d-Isoascorbic acid fed to Spodoptera littoralis moths,induces sterility due to spermatophore malformation

d-Isoascorbic acid (d-araboascorbic acid; d-erythorbic acid) fed to Spodoptera littoralis (Boisduval) moths causes males to produce malformed spermatophores with no reduction in their mating capacity. Males fed daily on the analogue, induced sterility in females paired with them. Female fertility and fecundity were markedly reduced compared with the reproductive potential of females mated to males fed either l-ascorbic acid or sucrose only. Spermatophore pathology originated in failure to inflate the spermatophore corpus at the time of mating. This failure was produced by a single male in two females independently, when males only had access to the analogue. Thus, the effect on the spermatophore seems linked to male ingestion of the analogue. When d-isoascorbic acid-feeding of males was discontinued after 48 hr, egg fertility was restored. When l-ascorbic acid followed the analogue, the effect of spermatophore malformation was not completely erased. The mode of action of this ascorbic acid analogue is discussed.     

父方投资与性角色逆转现象: 螽斯类昆虫的婚礼食物及对性选择方向的影响

大多数动物在繁殖过程中,雌性在繁殖过程中要比雄性付出更大投资,如相对于精子较大的卵子细胞,较长的育幼时间等,因而在交配过程中,雌性具有选择权,而雄性之间相互竞争以取得与雌性的交配权。然而自然世界中并不总是竞争的雄性（competitive male）-选择的雌性（selective female）这种婚配形式。在螽斯类昆虫中,雄性同样具有较大的父方投资。在繁殖期间,雄性螽斯争相鸣叫,求偶,且在交配后要给予雌性特殊的营养物质-精包,供雌性取食。因此在特定情况下,雌性之间将进行竞争以获取雄性配偶,雄性变得更具有选择性。影响这种性角色逆转的主要因素是可获得资源的紧缺。父方投资理论和性选择理论预测雄性显著地对后代投资时,雌性将表现出典型的雄性特征,她们竞争追求性活跃的雄性,而雄性将表现出典型的雌性特征,对配偶具有选择性。螽斯类昆虫中这种特殊的性角色逆转现象符合性选择理论和父方投资理论的预测。     

How the length of genital parts affects copulation performance in a carabid beetle: implications for correlated genital evolution between the sexes

To identify factors leading to the correlated evolution of exaggerated male and female genitalia, we studied the effects of the variable dimensions of corresponding functional genital parts (male copulatory piece and female vaginal appendix) on copulatory performance in the polygamous carabid beetle Carabus (Ohomopterus) maiyasanus. We used mating pairs of individuals from two populations to increase the variances in genital dimensions and determined the copulation performance (insemination and spermatophore replacement, and copulation time) in single‐ and double‐mating situations. In single mating, insemination success was not affected by genital dimensions, although the copulation time was significantly shorter when the male aedeagus was longer. In the double‐mating experiment, insemination and replacement of spermatophores by the second male succeeded more frequently when the copulatory piece was shorter and the vaginal appendix was longer, and when the difference between the length of the copulatory piece and the vaginal appendix was smaller. Thus, a matching of the corresponding genital parts between the sexes increases the male's reproductive success in sperm competition, but elongation of the copulatory piece cannot be explained simply by the improvement in male reproductive success. We discuss possible factors for the elongation of genital parts in terms of sexual conflict and reproductive interference through interspecific copulation.     

Pathology and ultrastructure of the insect virus,Hz-2V,infecting agonadal male corn earworms,Helicoverpa zea

The pathology of the reproductive tract of Hz-2V-infected agonadal male corn earworm moths, Helicoverpa zea, was studied. The examination of the reproductive tissues of adult agonadal males infected with Hz-2V during different lifestages allowed us to positively correlate the grossly malformed tissues of typical agonadal male moths to the corresponding normal tissues in uninfected males. The reproductive tissues responsible for producing sperm, a pheromonostatic peptide (PSP), and the spermatophore in normal male moths were absent or grossly malformed in the agonadal male moths. Hz-2V was observed replicating in one area of these malformed reproductive tissues in pharate adult males as early as 7 days post-pupation. Interestingly, reproductive tissues essential for initiation of copulation and transfer of reproductive fluids into a female moth during mating appear to be intact and may be functional. These data suggest that agonadal adult males are able to mate with healthy female moths and transfer Hz-2V particles, without fertilizing female moths or altering their sexual receptivity to further mating with other male moths.     

Mate Choice by Non-Virgin Females Contributes to Reproductive Isolation between Populations of the Harlequin Beetle-Riding Pseudoscorpion

Determining whether reproductive isolation evolves through mate choice and/or gametic factors that prevent fertilization or through the post‐zygotic mechanisms of hybrid sterility or inviability is fundamental to understanding speciation. Investigation of the pre‐ and post‐zygotic components of reproductive isolation is facilitated in the pseudoscorpion, Cordylochernes scorpioides, by its indirect method of sperm transfer and viviparous embryonic development. Previous research on this species, in which mate discrimination was assessed in virgin females, suggested that female choice played only a minor role in reproductive isolation between populations from French Guiana and Panamá. Here, in a study of three allopatric populations of C. scorpioides from Panamá, we assessed mating‐stage isolation in both virgin and once‐mated females, and found that female discrimination depends critically on mating status. Virgin females were almost invariably receptive, showing no tendency to discriminate against males from allopatric populations. By contrast, non‐virgin females were significantly more likely to reject foreign males than males from their own population. Male sexual motivation could not account for differences in either female sexual receptivity or male success in sperm transfer. Allopatric and sympatric males did not differ in number of spermatophores deposited as either a female’s first or second mate. Nonetheless, allopatric males achieved significantly lower sperm transfer success not only with choosy, non‐virgin females but also with virgin females. Given the lack of behavioral discrimination by virgin females, female receptivity was not the only factor influencing differences in sperm transfer success. Multivariate analysis of spermatophore morphology suggests that the higher failure rate of matings between allopatric males and virgin females resulted from population differences in sperm packet architecture. Overall, our findings indicate that assessment of discrimination against allopatric males that is limited to virgin females may seriously underestimate the contribution of female mate choice to reproductive isolation between populations.     

Satyrization without evidence of successful insemination from interspecific mating between invasive mosquitoes

Previous research has documented low frequencies of interspecific mating in nature between the invasive vectors Aedes aegypti and Aedes albopictus. It is also known that heterospecific male accessory gland substances transferred during mating sterilize A. aegypti but not A. albopictus females, leading to satyrization, a form of reproductive interference. This paper demonstrates that satyrization of A. aegypti by A. albopictus may occur without evidence of successful insemination. Our results show that A. aegypti females, previously exposed to A. albopictus males, are rendered refractory to subsequent conspecific mating even though their spermathecae contain no heterospecific sperm. Additional experiments demonstrating transfer of labelled semen from A. albopictus males to A. aegypti females and low production of viable eggs of females housed with conspecific males, following exposure to A. albopictus males, confirm higher incidences of satyrization than expected, based on heterospecific insemination rates. We conclude that frequencies of satyrization based on detection of interspecific sperm in spermathecae may underestimate the impact of this form of reproductive interference.     

Reproductive interference between Rana dalmatina and Rana temporaria affects reproductive success in natural populations

Experimental evidence suggests that reproductive interference between heterospecifics can seriously affect individual fitness; support from field studies for such an effect has, however, remained scarce. We studied reproductive interference in 25 natural breeding ponds in an area where two ranid frogs, Rana dalmatina and Rana temporaria, co-occur. The breeding seasons of the two species usually overlap and males of both species are often found in amplexus with heterospecific females, even though matings between heterospecifics produce no viable offspring. We estimated species abundance ratios based on the number of clutches laid and evaluated fertilization success. In ponds with low spatial complexity and a species abundance ratio biased towards R. temporaria, the average fertilization success of R. dalmatina eggs decreased, while this relationship was not detectable in spatially more complex ponds. Fertilization success of R. temporaria did not decrease with increasing relative numbers of heterospecifics. This asymmetry in fitness effects of reproductive interference may be attributed to R. temporaria males being more competitive in scramble competition for females than R. dalmatina males. Our study is among the first to demonstrate that in natural breeding populations of vertebrates interference among heterospecifics has the potential to substantially lower reproductive success at the population level, which may in turn affect population dynamics.     

Male Courtship Pheromones Induce Cloacal Gaping in Female Newts (Salamandridae)

Pheromones are an important component of sexual communication in courting salamanders, but the number of species in which their use has been demonstrated with behavioral evidence remains limited. Here we developed a behavioral assay for demonstrating courtship pheromone use in the aquatically courting Iberian ribbed newt Pleurodeles waltl. By performing an in-depth study of the courtship behavior, we show that females invariably open their cloaca (cloacal gaping) before engaging in pinwheel behavior, the circling movement that is the prelude to spermatophore uptake. In contrast, cloacal gaping was not observed in failed courtships, where females escaped or displayed thanatosis. Since gaping mainly occurred during male amplexus and cloacal imposition, which is the obvious period of pheromone transfer, we next investigated whether male courtship water (i.e., water holding courtship pheromones) alone was able to induce this reaction in females. These tests showed that courtship water induced cloacal gaping significantly more than water, even in the absence of a male. Cloacal gaping thus provides a simple and robust test for demonstrating courtship pheromone use in the Iberian ribbed newt. Since opening the cloaca is an essential prerequisite for spermatophore pick-up in all internally fertilizing salamanders, we hypothesize that variations on this assay will also be useful in several other species.