A recombinant courtship pheromone affects sexual receptivity in a plethodontid salamander

Pheromones are important chemical signals for many vertebrates, particularly during reproductive interactions. In the terrestrial salamander Plethodon shermani, a male delivers proteinaceous pheromones to the female as part of their ritualistic courtship behavior. These pheromones increase the female's receptivity to mating, as shown by a reduction in courtship duration. One pheromone component in particular is plethodontid receptivity factor (PRF), a 22-kDa protein with multiple isoforms. This protein alone can act as a courtship pheromone that causes the female to be more receptive. We used a bacterial expression system to synthesize a single recombinant isoform of PRF. The recombinant protein was identical to the native PRF, based on mass spectrometry, circular dichroism spectra, and a behavioral bioassay that tested the effects of recombinant PRF (rPRF) on female receptivity (21% reduction in courtship duration). The rPRF appears to mimic the activity of a mixture of PRF isoforms, as well as a mixture of multiple different proteins that comprise the male courtship gland extract. Pheromones that are peptides have been characterized for some vertebrates; to date, however, rPRF is one of only 2 synthesized vertebrate proteins to retain full biological activity.     

Peptide and protein pheromones in amphibians

Purification, characterization and biological activity of urodele and anuran sex-pheromones were reviewed. Female-attracting pheromones obtained from the abdominal gland of Cynops pyrrhogaster and C. ensicauda males are peptides consisting of 10 amino acid residues being designated sodefrin and silefrin, respectively. Each pheromone attracted only conspecific females. Molecular cloning of cDNAs encoding sodefrin and silefrin revealed that both are generated from precursor proteins. Synthesis of these pheromones is regulated by prolactin (PRL) and androgen. Responsiveness of the female vomeronasal epithelium to sodefrin is enhanced by PRL and estrogen. The submandibular gland of the male terrestrial salamander, Plethodon jardani secretes a 22-kD proteinaceous pheromone that enhances female receptivity. It was revealed that every salamander synthesizes multiple isoforms of this pheromone, Plethodontid receptivity factor. The magnificent tree frog, Litoria splendida breed in an aquatic environment. The skin glands of the male secrete a female-attracting peptide pheromone, splendipherin, comprising 25 amino acid residues. The significance of the structure of the amphibian sex-pheromone as peptide and protein is discussed in terms of their species specificity.     

Evolutionary replacement of components in a salamander pheromone signaling complex: more evidence for phenotypic-molecular decoupling

In this article we explore the evolutionary history of a functional complex at the molecular level in plethodontid salamanders. The complex consists of a proteinaceous courtship pheromone, a pheromone-producing gland on the male's chin, and a set of behaviors for delivering the pheromone to the female. Long-term evolutionary stasis is the defining feature of this complex at both the morphological and behavioral levels. However, our previous assessment of the pheromone gene, plethodontid receptivity factor (PRF), revealed rapid evolution at the molecular level despite stasis at higher levels of organization. Analysis of a second pheromone gene, sodefrin precursor-like factor (SPF), now indicates that evolutionary decoupling in this complex is pervasive. The evolutionary profiles of SPF and PRF are remarkably similar in that: (a) both genes exhibit high levels of sequence diversity both within and across taxa, (b) genetic diversity has been driven by strong positive selection, and (c) the genes have evolved heterogeneously in different salamander lineages. The composition of the pheromone signal as a whole, however, has experienced an extraordinary evolutionary transition. Whereas SPF has been retained throughout the 100 MY radiation of salamanders, PRF has only recently been recruited to a pheromone function (27 million years ago). When SPF and PRF coexist in the same clade, they show contrasting patterns of evolution. When one shows rapid evolution driven by positive selection, the other shows neutral divergence restrained by purifying selection. In one clade, the origin and subsequent rapid evolution of PRF appear to have interfered with the evolution and persistence of SPF, leading to a pattern of evolutionary replacement. Overall, these two pheromone genes provide a revealing window on the dynamics that drive the evolution of multiple traits in a signaling complex.     

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.     

Endocrine regulation of reproductive behavior in the newt Cynops pyrrhogaster

Hormonal control of the expression of courtship behavior and of secretion of the female-attracting pheromone sodefrin by the male red-bellied newt, Cynops pyrrhogaster, together with the hormonal influence on the responsiveness to the pheromone in the female, is reviewed.Expression of the initial stage of the courtship behavior, i.e., tail vibration by the male in front of the female, is dependent on prolactin (PRL) and androgen. During the courtship, sodefrin seems to be released from the cloaca through the ducts of the abdominal gland. Both content of immunoreactive sodefrin and preprosodefrin mRNA levels in the abdominal gland are elevated by a combination of PRL and androgen, indicating that the pheromone synthesis is stimulated by these two hormones. On the other hand, the discharge of sodefrin is accelerated by AVT, its action being mediated by V1 receptor. In female newts, responsiveness of the vomeronasal epithelium to the pheromone is elevated by a combination of PRL and estrogen. Thus, it can be concluded that PRL, AVT, and sex steroids are key hormones for the reproductive performance in the red-bellied newt. In this article, the significance of the structure of the pheromone molecule as a peptide is also discussed in terms of its species-specificity and its effectiveness in an aquatic environment.     

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.     

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.     

Involvement of arginine vasotocin in reproductive events in the male newt Cynops pyrrhogaster

Effects of arginine vasotocin (AVT) on reproductive events such as courtship behavior, pheromone release, and spermatophore discharge were investigated in the male newt Cynops pyrrhogaster. AVT enhanced the incidence and frequency of androgen-induced courtship behavior. In this case, AVT was likely to act centrally because the behavior was evoked with a much smaller amount of AVT when the hormone was administered intracerebroventricularly than when given intraperitoneally. Involvement of endogenous AVT in spontaneously occurring courtship behavior was also evidenced by the fact that administration of a V1 (vasopressor) receptor antagonist, [d(CH2)5(1), Tyr(Me)2, Arg8-vasopressin] suppressed the expression of the courtship behavior. The water in which AVT-treated males had been kept showed considerable female-attracting activity as compared with the water in which saline-injected males had been kept. Moreover, the content of sodefrin, a female-attracting pheromone in the abdominal gland, was decreased by the intraperitoneal injection of AVT, suggesting that the neurohypophyseal hormone stimulated the release of sodefrin from the abdominal gland into the water. AVT induced contraction of the excised abdominal gland concentration-dependently, and, again, the V1 receptor antagonist suppressed the AVT-induced contraction. Thus, we concluded that AVT induces the pheromone discharge, acting peripherally on a contractile structure of the abdominal gland. AVT was also found to induce spermatophore deposition in the male kept in the absence of the female. Administration of the V1 receptor blocker to the sexually developed males suppressed the spermatophore deposition. All these results indicate the involvement of AVT in reproductive events acting centrally and peripherally.     

Peptide pheromones in newts

This article reviews the current state of understanding of reproductive pheromones in amphibians, focusing mainly on the purification and characterization of peptide pheromones in newts of the genus Cynops, molecular cloning of cDNAs encoding the pheromone molecules, and hormonal control of secretion of these pheromones. Pheromones that attract sexually developed female Cynops pyrrhogaster and C. ensicauda newts were isolated from the male abdominal glands. The C. pyrrhogaster and C. ensicauda pheromones are peptides, designated sodefrin and silefrin, with the amino acid sequences SIPSKDALLK and SILSKDAQLK, respectively. Each pheromone attracts only conspecific females. Molecular cloning of cDNAs encoding sodefrin and silefrin revealed the presence of precursor proteins that are considered to generate these pheromone peptides. Pheromone precursor mRNA levels and radioimmunoassayable pheromone concentrations in the abdominal glands were elevated by prolactin and androgen. Sexual dimorphism and hormone dependency of the responsiveness of vomeronasal epithelium to sodefrin were noted. Significance of pheromones in the form of peptide for those performing reproductive behavior in an aquatic environment was also discussed.     

Pheromonal inhibition of male courtship behaviour in the brown tree snake, Boiga irregularis: a mechanism for the rejection of potential mates

Pheromones play a central role in coordinating the events leading up to copulation in snakes. We report here a novel pheromone system in the brown tree snake in which females release a pheromone that inhibits male courtship behaviour. In a previous study, we made observations of female brown tree snakes releasing cloacal secretions (CS) during courtship that appeared to cause courting males to cease courtship. All snakes have glands that release CS through ducts located along the cloacal orifice. Although CS have been studied for many years, their function in the mediation of snake behaviour has not been experimentally well determined. We examined the role of CS in the reproductive behaviour of male and female brown tree snakes. We conducted four experiments to test the effect of both male and female CS on brown tree snake behaviour under two behavioural contexts, courtship and male-male ritualized combat. Within each experiment, we compared the effects of CS to a control. Female CS caused a decrease in the time that males spent courting females and a decrease in the intensity of courtship compared with the control treatment. Male CS did not, however, affect the time that males spent displaying courtship or the intensity of that courtship. Neither male nor female CS had significant effects on male ritualized combat behaviour, including time that males spent in combat or the intensity of combat behaviours displayed. Furthermore, neither female nor male CS had an effect on female courtship versus controls. The inhibition of brown tree snake reproductive behaviours is specific to female CS inhibiting male courtship behaviour. This pheromone acts in concert with the female sex pheromone to regulate the events leading to copulation.Copyright 2003 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour.      

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.     

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.     

The receptor channel formed by ppk25, ppk29 and ppk23 can sense the Drosophila female pheromone 7,11‐heptacosadiene

In Drosophila, pheromones play a crucial role in regulating courtship behaviors. In males, female aphrodisiac pheromones promote male‐female courtship, and male antiaphrodisiac pheromones inhibit male‐male courtship. Previous studies have reported that receptor proteins belonging to the pickpocket (ppk) family, ionotropic receptor family and gustatory receptor family are required for pheromone detection and normal courtship. However, none of them has been shown to be sufficient for sensing pheromones after ectopic expression in originally unresponsive cells. “M” cells are activated by male antiaphrodisiac pheromones but not female aphrodisiac pheromones, and the activated cells inhibit male‐male courtship. In our study, male flies with ectopic expression of ppk25, ppk29 and ppk23 in “M” cells showed decreased male‐female courtship. Using an in vivo calcium imaging approach, we found that the “M” cells expressing these three ppks were significantly activated by the female aphrodisiac pheromone 7,11‐heptacosadiene (7,11‐HD). Our results indicate that a sodium channel consisting, at minimum, of ppk25, ppk29 and ppk23, can sense 7,11‐HD, most likely as a receptor. Our findings may help us gain insights into the molecular mechanisms of pheromonal functions.     

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.     

Courtship in the Zig-Zag Salamander (Plethodon dorsalis): Insights into a Transition in Pheromone-Delivery Behavior

Courtship in plethodontid salamanders includes the delivery of male courtship pheromones by two distinct modes. Within the eastern Plethodon clade of the tribe Plethodontini, members of the Plethodon cinereus species group use an ancestral ‘vaccination’ mode of delivery, while members of the P. glutinosus group use an olfactory delivery mode. In order to shed light on this transition in delivery mode, I observed courtship behavior in P. dorsalis, a species that is phylogenetically intermediate to the P. cinereus and P. glutinosus groups. My observations indicate that P. dorsalis also is intermediate to the P. cinereus and P. glutinosus species groups in terms of courtship behavior. The context of delivery of male courtship pheromones in P. dorsalis is similar to that of the P. cinereus species group; however, the mode of pheromone delivery in P. dorsalis is olfactory. Thus, a transition in the context of pheromone delivery underlies the more obvious change in pheromone delivery mode. I discuss these findings in terms of the evolution of courtship pheromone delivery across the eastern Plethodon clade. I also report the first observations of ‘premature’ spermatophore deposition by male plethodontids.     

A putative Drosophila pheromone receptor expressed in male-specific taste neurons is required for efficient courtship

Propagation in higher animals requires the efficient and accurate display of innate mating behaviors. In Drosophila melanogaster, male courtship consists of a stereotypic sequence of behaviors involving multiple sensory modalities, such as vision, audition, and chemosensation. For example, taste bristles located in the male forelegs and the labial palps are thought to recognize nonvolatile pheromones secreted by the female. Here, we report the identification of the putative pheromone receptor GR68a, which is expressed in chemosensory neurons of about 20 male-specific gustatory bristles in the forelegs. Gr68a expression is dependent on the sex determination gene doublesex, which controls many aspects of sexual differentiation and is necessary for normal courtship behavior. Tetanus toxin-mediated inactivation of Gr68a-expressing neurons or transgene-mediated RNA interference of Gr68a RNA leads to a significant reduction in male courtship performance, suggesting that GR68a protein is an essential component of pheromone-driven courtship behavior in Drosophila.     

Plethodontid modulating factor, a hypervariable salamander courtship pheromone in the three-finger protein superfamily

The soluble members of the three-finger protein superfamily all share a relatively simple 'three-finger' structure, yet perform radically different functions. Plethodontid modulating factor (PMF), a pheromone protein produced by the lungless salamander, Plethodon shermani, is a new and unusual member of this group. It affects female receptivity when delivered to the female's nares during courtship. As with other plethodontid pheromone genes, PMF is hyperexpressed in a specialized male mental (chin) gland. Unlike other plethodontid pheromone genes, however, PMF is also expressed at low levels in the skin, liver, intestine and kidneys of both sexes. The PMF sequences obtained from all tissue types were highly variable, with 103 unique haplotypes identified which averaged 35% sequence dissimilarity (range 1-60%) at the protein level. Despite this variation, however, all PMF sequences contained a conserved approximately 20-amino-acid secretion signal sequence and a pattern of eight cysteines that is also found in cytotoxins and short neurotoxins from snake venoms, as well as xenoxins from Xenopus. Although they share a common cysteine pattern, PMF isoforms differ from other three-finger proteins in: (a) amino-acid composition outside of the conserved motif; (b) length of the three distinguishing 'fingers'; (c) net charge at neutral pH. Whereas most three-finger proteins have a net positive charge at pH 7.0, PMF has a high net negative charge at neutral pH (pI range of most PMFs 3.5-4.0). Sequence comparisons suggest that PMF belongs to a distinct multigene subfamily within the three-finger protein superfamily.     

Species specificity of methyl ketone profiles in the skin lipids of female garter snakes,genus Thamnophis

One of the relatively few vertebrate pheromones to be chemically identified, the female sex pheromone of the red-sided garter snake (Thamnophis sirtalis parietalis) is a series of saturated and monounsaturated methyl ketones contained within female skin lipids. During the breeding season, this pheromone is responsible for eliciting male courtship behaviors and males are able to utilize pheromonal variation to discriminate among females. While the pheromone system of the red-sided garter snake has been the subject of many studies, relatively little is known about the pheromone systems of other garter snakes. Through chemical analyses, we demonstrate that female skin lipids of the red-spotted garter snake (Thamnophis sirtalis concinnus), northwestern garter snake (Thamnophis ordinoides), and plains garter snake (Thamnophis radix) contain similar methyl ketones. The methyl ketone profiles of these snakes differ qualitatively from one another and from the methyl ketone profiles of red-sided garter snakes with differences particularly pronounced between sympatric species. Our results provide evidence that the use of methyl ketones in sexual signaling may be ubiquitous for Thamnophis species and suggest that these compounds could play a role in reproductive isolation between species in this genus.