Love Is Blind: Indiscriminate Female Mating Responses to Male Courtship Pheromones in Newts (Salamandridae)

Internal fertilization without copulation or prolonged physical contact is a rare reproductive mode among vertebrates. In many newts (Salamandridae), the male deposits a spermatophore on the substrate in the water, which the female subsequently takes up with her cloaca. Because such an insemination requires intense coordination of both sexes, male newts have evolved a courtship display, essentially consisting of sending pheromones under water by tail-fanning towards their potential partner. Behavioral experiments until now mostly focused on an attractant function, i.e. showing that olfactory cues are able to bring both sexes together. However, since males start their display only after an initial contact phase, courtship pheromones are expected to have an alternative function. Here we developed a series of intraspecific and interspecific two-female experiments with alpine newt (Ichthyosaura alpestris) and palmate newt (Lissotriton helveticus) females, comparing behavior in male courtship water and control water. We show that male olfactory cues emitted during tail-fanning are pheromones that can induce all typical features of natural female mating behavior. Interestingly, females exposed to male pheromones of their own species show indiscriminate mating responses to conspecific and heterospecific females, indicating that visual cues are subordinate to olfactory cues during courtship.     

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.     

Conspecific and heterospecific pheromone effects on female receptivity

Courtship pheromones play an important role in salamander reproductive behaviour. In salamanders of the family Plethodontidae, males deliver specialized pheromones to females during courtship interactions. These courtship pheromones increase female receptivity and may be involved in mate discrimination. In order to test hypotheses related to mate discrimination, we staged courtship encounters between male-female Plethodon shermani pairs in which the female received pheromones obtained from either conspecific (P. shermani) or heterospecific (P. yonahlossee orP. montanus ) males. Both conspecific and heterospecific pheromones increased female receptivity. Moreover, pheromones from both heterospecific species were as effective as the conspecific pheromone in increasing female receptivity inP. shermani females. Our results suggest that the courtship pheromone signal and function may be conserved across related species, with mate discrimination occurring before pheromone delivery. Copyright 2003 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.     

Rapid Evolution of Plethodontid Modulating Factor, a Hypervariable Salamander Courtship Pheromone, is Driven by Positive Selection

Sexual communication in plethodontid salamanders is mediated by a proteinaceous pheromone that a male delivers to a female during courtship, boosting her receptivity. The pheromone consists of three proteins from three unrelated protein families. These proteins are among a small group of pheromones known to affect female receptivity in vertebrates. Previously, we showed that the genes of two of these proteins (PRF and SPF) are prone to incessant evolution driven by positive selection, presumably as a consequence of coevolution with female receptors. In this report, we focus on the evolution of the third pheromone protein gene family, plethodontid modulating factor (PMF), to determine whether it shows the same pattern of diversification. We used RT-PCR in mental gland cDNA to survey PMF sequences from three genera of plethodontid salamanders (27 spp.) to measure rates of evolution, level of gene diversification, modes of selection, and types of amino acid substitution. Like PRF and SPF, PMF is produced by a multigene family characterized by gene duplication and high levels of polymorphism. PMF evolution is rapid, incessant, and driven by positive selection. PMF is more extreme in these dimensions than both PRF and SPF. Nestled within this extraordinary variation, however, is a signature of purifying selection, acting to preserve important structural and biochemical features of the PMF protein (i.e., secretion signal, cysteine residues, and pI). Although a pattern of persistent diversification exists at the molecular level, the morphological and behavioral aspects of the pheromone delivery system show evolutionary stasis over millions of years.     

The courtship behaviour of the spotted dusky salamander, Desmognathus fuscus conanti (Amphibia: Caudata: Plethodontidae)

The courtship behaviour of the North American southern dusky salamander, Desmognathus fuscus conanti (Plethodontidae), is described for the first time. The male is the most active participant, as is typical for caudate amphibians, and much of his behaviour can be understood in terms of orientation to and stimulation (or persuasion) of the female. The latter is largely accomplished by the transfer of courtship pheromones from the male's mental (chin) gland. The orientation and persuasion phases of courtship appear necessary if sperm transfer by means of a spermatophore deposited upon the substratum is to be successful. The courtship of D. fuscus conanti is qualitatively identical to that of D. santeetlah , with which it is sexually compatible in the laboratory and with which it may hybridize in nature. However, a broader comparison of species in the genus Desmognathus reveals that the greatest interspecific differences occur during the orientation and persuasion phases of courtship. A complete understanding of the evolution of courtship behaviour in desmognathine salamanders awaits availability of ethograms for all other taxa and a robust, independent phytogeny for the entire subfamily.     

Male Courtship Pheromones Affect Female Behaviour in the Swordtail Characin (Corynopoma riisei)

Pheromones constitute an important cue used by both males and females during courtship. Here, we investigate the effect of male pheromones on female behaviour in the swordtail characin (Corynopoma riisei), a species of fish where males have a caudal pheromone gland which has been suggested to affect female behaviour during courtship. We subjected female C. riisei to male courtship pheromones and investigated the effect on both female behaviour and brain serotonergic activity levels compared to a control group. While no difference in serotonergic activity was found, the pheromone‐treated females showed lower stress levels compared to the control group. Furthermore, pheromone‐treated females increased locomotor activity over time, while a decrease in locomotor activity was observed in the control group. These results suggest that the male courtship pheromones may serve to reduce female stress and increase female activity, possibly to aid males in gaining access to females and facilitating sperm transfer.     

The role of silk in courtship and chemical communication of the false widow spider, <Emphasis Type="Italic">Steatoda grossa</Emphasis> (Araneae: Theridiidae)

In spiders, sex pheromones are often associated with silk produced by females, and function in mate attraction, recognition, and evaluation. Silk-bound pheromones typically elicit courtship behaviour in web-building spiders. Here we (1) describe courtship interactions of Steatoda grossa males with virgin or mated females, and (2) show that silk and methanol extracts of silk produced by virgin females trigger courtship behaviour (silk production) by males, whereas silk of mated females does not. Our results indicate that (1) virgin females produce a silk-bound sex pheromone, (2) males discriminate between virgin and mated females based on silk cues, and (3) male silk likely functions in sexual communication.     

Drosophila Pheromone-Sensing Neurons Expressing the ppk25 Ion Channel Subunit Stimulate Male Courtship and Female Receptivity

As in many species, gustatory pheromones regulate the mating behavior of Drosophila. Recently, several ppk genes, encoding ion channel subunits of the DEG/ENaC family, have been implicated in this process, leading to the identification of gustatory neurons that detect specific pheromones. In a subset of taste hairs on the legs of Drosophila, there are two ppk23-expressing, pheromone-sensing neurons with complementary response profiles; one neuron detects female pheromones that stimulate male courtship, the other detects male pheromones that inhibit male-male courtship. In contrast to ppk23, ppk25, is only expressed in a single gustatory neuron per taste hair, and males with impaired ppk25 function court females at reduced rates but do not display abnormal courtship of other males. These findings raised the possibility that ppk25 expression defines a subset of pheromone-sensing neurons. Here we show that ppk25 is expressed and functions in neurons that detect female-specific pheromones and mediates their stimulatory effect on male courtship. Furthermore, the role of ppk25 and ppk25-expressing neurons is not restricted to responses to female-specific pheromones. ppk25 is also required in the same subset of neurons for stimulation of male courtship by young males, males of the Tai2 strain, and by synthetic 7-pentacosene (7-P), a hydrocarbon normally found at low levels in both males and females. Finally, we unexpectedly find that, in females, ppk25 and ppk25-expressing cells regulate receptivity to mating. In the absence of the third antennal segment, which has both olfactory and auditory functions, mutations in ppk25 or silencing of ppk25-expressing neurons block female receptivity to males. Together these results indicate that ppk25 identifies a functionally specialized subset of pheromone-sensing neurons. While ppk25 neurons are required for the responses to multiple pheromones, in both males and females these neurons are specifically involved in stimulating courtship and mating.     

Courtship and mating in the whip spider Phrynichus orientalis Weygoldt, 1998 (Chelicerata: Amblypygi)

Mating behaviour and spermatophore morphology of the south-east Asian whip spider Phrynichus orientalis is described. During courtship there is less bodily contact between mates than in other studied species of the same genus. However, a peculiarity for the species is that the pair often pauses for minutes, sitting front to front without any movement. For spermatophore formation, the male shifts his body under that of the female like in many other species. However, after spermatophore formation, the male lures the female to the spermatophore instead of pulling her as in most other species studied. The spermatophore differs from those of most other species of the genus by the fact that the cushions in front of the sperm masses are replaced by a hard, transparent mass which has the same function as the cushions. Mating behaviour and spermatophore morphology of P. orientalis thus show peculiarities that so far have not been described for other species of the genus. It is not clear whether these characters should be considered plesiomorphic or apomorphic. But it does clearly show that there is more diversity within this genus than expected.     

Release of male courtship display in Periplaneta americana: evidence for female contact sex pheromone

Male American cockroaches (Periplaneta americana) are attracted to virgin females by volatile sex pheromones. After antennal contact with the female they turn through 180° and spread their wings in courtship display. A chemical contact stimulus releasing male courtship is demonstrated in the female cuticle. Experiments with standardized olfactory stimulation by volatile sex pheromones revealed that the contact stimulus is sex-specific and species-specific. It can be washed off the cuticle with non-polar solvents and was successfully transferred to glass dummies. However, it is not effective in the absence of volatile sex pheromones. Thus volatile sex pheromones are responsible for male attraction and sexual motivation, while mate recognition is accomplished through the contact pheromone.     

A Drosophila protein specific to pheromone-sensing gustatory hairs delays males' copulation attempts

In insects, increasing evidence suggests that small secreted pheromone binding proteins (PBPs) and odorant binding proteins (OBPs) are important for normal olfactory detection of airborne pheromones and odorants far from their source. In contrast, it is unknown whether extracellular ligand binding proteins participate in perception of less volatile chemicals, including many pheromones, that are detected by direct contact with chemosensory organs. CheB42a, a small Drosophila melanogaster protein unrelated to known PBPs or OBPs, is expressed and likely secreted in only a small subset of gustatory sensilla on males' front legs, the site of gustatory perception of contact pheromones. Here we show that CheB42a is expressed specifically in the sheath cells surrounding the taste neurons expressing Gr68a, a putative gustatory pheromone receptor for female cuticular hydrocarbons that stimulate male courtship. Surprisingly, however, CheB42a mutant males attempt to copulate with females earlier and more frequently than control males. Furthermore, CheB42a mutant males also attempt to copulate more frequently with other males that secrete female-specific cuticular hydrocarbon pheromones, but not with females lacking cuticular hydrocarbons. Together, these data indicate that CheB42a is required for a normal gustatory response to female cuticular hydrocarbon pheromones that modulate male courtship.     

fruitless splicing specifies male courtship behavior in Drosophila

All animals exhibit innate behaviors that are specified during their development. Drosophila melanogaster males (but not females) perform an elaborate and innate courtship ritual directed toward females (but not males). Male courtship requires products of the fruitless (fru) gene, which is spliced differently in males and females. We have generated alleles of fru that are constitutively spliced in either the male or the female mode. We show that male splicing is essential for male courtship behavior and sexual orientation. More importantly, male splicing is also sufficient to generate male behavior in otherwise normal females. These females direct their courtship toward other females (or males engineered to produce female pheromones). The splicing of a single neuronal gene thus specifies essentially all aspects of a complex innate behavior.     

Mating behaviour and reproductive isolation of three species of reptile tick

The mating behaviour of three species of reptile tick, Aponomma hydrosauri, Amblyomma albolimbatum and Amb. limbatum is similar and involves a female sex pheromone which activates males to detach from their hosts and search. After contacting females, a stereotyped six-phase courtship sequence occurs. There are qualitative differences between the species in courtship behaviour at phase 3 (reversal of position by dorsally mounted males) and phase 6 (the copulation position). On-host observations of non-conspecific mating show that females of the three species have species-specific activation pheromones, which is contrary to reports in other species of tick. Such specificity should result in reproductive isolation of the three species; however, under certain circumstances it may not prevent non-conspecific contacts between the sexes. Off-host observations of courtship behaviour show that once males of the three species contact non-conspecific females, they attempt courtship and are persistent with their courtship. Rarely did non-conspecific courtships proceed beyond phase 4 of the courtship sequence, as non-conspecific females did not lift their bodies to allow males venter contact. Differences between the species in leg orientation in the copulation position, together with body size differences, are responsible for a complete barrier to successful non-conspecific copulation. The observations illustrate the role that behavioural mechanisms play in reproductively isolating these three species of tick.     

Breeding in the crayfish, Austropotamobius pallipes: mating patterns, mate choice and intermale competition

1. The breeding behaviour of the white-clawed crayfish, Austropotamobius pallipes, which is in decline throughout Europe, was analysed. 2. Observations and experiments focused on: (i) several aspects of mating behaviour, showing the primary role of the female during courtship, the patterns of mature male receptivity and the possible existence of a mating stimulus produced by either a mating pair or a receptive female; (ii) the potential for mate choice either by males (males were not choosy, mating with the first receptive female they met) or by females (that rejected the smallest males and males with only one cheliped) and (iii) intermale fighting before copulation (larger males copulated more often, with the exception of some ’sneakers‘). 3. Sperm competition occurred when a new male was presented with a female after a previous male‘s spermatophore had been deposited; the new male cleaned the rival male‘s spermatophore by feeding on it before copulation. This behaviour has also been observed in some pseudoscorpions, millipedes and collembolans, but always in cases where the spermatophore is deposited on the substratum. Introduction     

Flash signals, nuptial gifts and female preference in photinus fireflies

The evolution of male courtship signals such as the bioluminescentflashes of fireflies may be shaped, at least in part, by femalepreference for particular characteristics of the male signal.These female preferences for male courtship signals may ariseas a result of the benefits of choosing males with particulartraits. One possible benefit of mate choice occurs if femalescan use male courtship signals as an honest indicator of malenutritional contributions at mating, nuptial gifts. This paperreviews female preference for male flash characteristics inPhotinus fireflies (Coleoptera: Lampyridae), and the potentialfor females to use male flash characteristics to predict nuptialgift quality. In Photinus firefly species with single pulseflashes females preferentially respond to flashes of greaterintensity and duration. Male Photinus provide a nuptial giftto females at mating in the form of a spermatophore and flashduration serves as a good predictor of spermatophore mass formales collected early in the season. However, Photinus firefliesdo not feed as adults, so spermatophore mass decreases withsubsequent matings. In response, nutrient-limited females maystop preferentially responding to longer duration flashes, increasingtheir overall responsiveness later in the mating season as theyforage for spermatophores. Therefore, the evolution of malecourtship signals in Photinus fireflies is the product not onlyof female preference for male flash characteristics, but alsothe costs and benefits of female choice that shape these preferences.     

Changes in courtship behaviour following rejection: The influence of female phenotype in Drosophila melanogaster

Courtship can be costly and so selection should favour individual males that reduce courtship towards female types that have a low probability of resulting in copulation. One way males can do this is by associating previous courtship failure with the traits of particular rejecting females. We characterised changes in male Drosophila melanogaster courtship behaviour following a failed mating attempt with one of the four female phenotypes that varied in size, age or mating status. To do this, we assessed individual courtship behaviour for each male presented again with a female of the same phenotype that previously rejected him. Males reduced subsequent courtship most strongly for recently mated (sexually non‐receptive) females. More interestingly, males also significantly reduced courtship activity following a failed mating experience from old females but did not do so for control (large, young, virgin) or small females. As such, males significantly reduced courtship towards both female types possessing chemical cues associated with their phenotype (age and mating status), but not towards a female phenotype based on physical characteristics (body size). Our results suggest that males are able to modify their courtship behaviour following experience, but that they are better prepared to associate chemical traits that may be more reliable indicators of the likelihood of courtship failure.     

Ejaculate expenditure by malebush crickets decreases with sperm competition intensity

Male bushcrickets transfer a spermatophore at mating that consists of a sperm-containing ampulla and a sperm-free mass, the spermatophylax, that is consumed by the female during insemination. The costs of spermatophore production for males and benefits of consumption for females result in reversals in courtship roles in nutrient limited populations that increase both the risk and intensity of sperm competition. Here we show that under conditions characteristic of courtship role reversal, male expenditure on the spermatophore is dependent on female size. When mating with small females, males increase the amount of spermatophylax material and sperm, as expected from the increased sperm competition risk associated with courtship role reversal. However, males reduce the amount of spermatophylax material and sperm transferred to larger females. Since larger females have a higher mating success when competing for nurturant males, the intensity of sperm competition covaries with female size. Reduced ejaculate expenditure under increased sperm competition intensity is in accord with theoretical expectation.     

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.