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.     

Effect of the mating plug on female chemical attractiveness and mating acceptance in a scorpion

After mating, females may experience a decline in sexual receptivity and attractiveness that may be associated with changes in the production and emission of sex pheromones. In some cases, these changes are produced by chemical substances or structures (e.g., mating plugs) produced by males as a strategy to avoid or reduce sperm competition. In scorpions, sex pheromones may be involved in finding potential mates and starting courtship. Here, we tested the hypothesis that the males of Urophonius brachycentrus, a species that produces a mating plug, use chemical communication (sex pheromones) to detect, localize, and discriminate females according to their mating status (virgin or inseminated), aided by chemical signaling. We also explored the effect of extracting of the mating plug on chemical communication and mating acceptance. We used Y‐maze olfactometers with different stimuli to analyze male choice and exploration time. To evaluate mating acceptance, we measured the attractiveness and receptivity of females of different mating status. We found that chemical communication occurs through volatile pheromones, but not contact pheromones. Males equally preferred sites with virgin or inseminated females with removed mating plug. In turn, females with these mating statuses were more attractive and receptive for males than inseminated females. This study suggests that the mating plug significantly affects female chemical attractiveness with an effect on volatile pheromones and decreasing sexual mating acceptance of females. The decline in the female's sexual receptivity is a complex process that may respond to several non‐exclusive mechanisms imposed by males and strategically modulated by females.     

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.     

An airborne sex pheromone in snakes

Most reptile sex pheromones so far described are lipid molecules too large to diffuse through the air; instead, they are detected via direct contact (tongue-flicking) with another animal's body or substrate-deposited trails, using the vomeronasal system. The only non-lipid pheromone reported in snakes involves courtship termination in red-sided gartersnakes (Thamnophis sirtalis parietalis): males that encounter copulatory fluids cease courtship, presumably reflecting the futility of courting an already-mating female. Our field experiments at a communal den in Manitoba show that this pheromone can work via olfaction: courtship is terminated by exposure to airborne scents from mating conspecifics, and does not require direct contact (tongue-flicking). Hence, the sexual behaviour of reptiles can be affected by airborne as well as substrate-bound pheromones.     

Pheromonal communication involved in courtship behavior in Diptera

Sex pheromones are known for many dipteran species and play an important role in courtship behavior, together with visual, tactile, acoustic and other factors. Pheromones for a number of dipterans have been recently identified. This survey covers a number of species in all the families that have been studied. The review discusses diverse courtship behaviors in Diptera, with a special focus on the sex pheromones involved. In the Nematocera suborder, pheromones are volatile components, which act at a distance. They are derived from short-chain alkanes with acetoxy groups (Cecidomyidae) or terpenes (Psychodidae). In the Cyclorrhapha, pheromones may be volatile, derived from alkanes (Tephritidae) or terpenes (Agromyzidae), or non-volatile, unsaturated or methyl-branched hydrocarbons, which act by contact (the other subgenera). The behavioral roles and regulation of these pheromones are described, and their importance in species recognition is discussed.     

Spider sex pheromones: emission, reception, structures, and functions

Spiders and their mating systems are useful study subjects with which to investigate questions of widespread interest about sexual selection, pre- and post-copulatory mate choice, sperm competition, mating strategies, and sexual conflict. Conclusions drawn from such studies are broadly applicable to a range of taxa, but rely on accurate understanding of spider sexual interactions. Extensive behavioural experimentation demonstrates the presence of sex pheromones in many spider species, and recent major advances in the identification of spider sex pheromones merit review. Synthesised here are the emission, transmission, structures, and functions of spider sex pheromones, with emphasis on the crucial and dynamic role of sex pheromones in female and male mating strategies generally. Techniques for behavioural, chemical and electrophysiological study are summarised, and I aim to provide guidelines for incorporating sex pheromones into future studies of spider mating. In the spiders, pheromones are generally emitted by females and received by males, but this pattern is not universal. Female spiders emit cuticular and/or silk-based sex pheromones, which can be airborne or received via contact with chemoreceptors on male pedipalps. Airborne pheromones primarily attract males or elicit male searching behaviour. Contact pheromones stimulate male courtship behaviour and provide specific information about the emitter's identity. Male spiders are generally choosy and are often most attracted to adult virgin females and juvenile females prior to their final moult. This suggests the first male to mate with a female has significant advantages, perhaps due to sperm priority patterns, or mated female disinterest. Both sexes may attempt to control female pheromone emission, and thus dictate the frequency and timing of female mating, reflecting the potentially different costs of female signalling and/or polyandry to both sexes. Spider sex pheromones are likely to be lipids or lipid soluble, may be closely related to primary metabolites, and are not necessarily species specific, although they can still assist with species recognition. Newer electrophysiological techniques coupled with chemical analyses assist with the identification of sex pheromone compounds. This provides opportunities for more targeted behavioural experimentation, perhaps with synthetic pheromones, and for theorising about the biosynthesis and evolution of chemical signals generally. Given the intriguing biology of spiders, and the critical role of chemical signals for spiders and many other animal taxa, a deeper understanding of spider sex pheromones should prove productive.     

Peripheral, central and behavioral responses to the cuticular pheromone bouquet in Drosophila melanogaster males

Pheromonal communication is crucial with regard to mate choice in many animals including insects. Drosophila melanogaster flies produce a pheromonal bouquet with many cuticular hydrocarbons some of which diverge between the sexes and differently affect male courtship behavior. Cuticular pheromones have a relatively high weight and are thought to be -- mostly but not only -- detected by gustatory contact. However, the response of the peripheral and central gustatory systems to these substances remains poorly explored. We measured the effect induced by pheromonal cuticular mixtures on (i) the electrophysiological response of peripheral gustatory receptor neurons, (ii) the calcium variation in brain centers receiving these gustatory inputs and (iii) the behavioral reaction induced in control males and in mutant desat1 males, which show abnormal pheromone production and perception. While male and female pheromones induced inhibitory-like effects on taste receptor neurons, the contact of male pheromones on male fore-tarsi elicits a long-lasting response of higher intensity in the dedicated gustatory brain center. We found that the behavior of control males was more strongly inhibited by male pheromones than by female pheromones, but this difference disappeared in anosmic males. Mutant desat1 males showed an increased sensitivity of their peripheral gustatory neurons to contact pheromones and a behavioral incapacity to discriminate sex pheromones. Together our data indicate that cuticular hydrocarbons induce long-lasting inhibitory effects on the relevant taste pathway which may interact with the olfactory pathway to modulate pheromonal perception.     

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.     

Pheromonal and behavioral cues trigger male-to-female aggression in Drosophila

Appropriate displays of aggression rely on the ability to recognize potential competitors. As in most species, Drosophila males fight with other males and do not attack females. In insects, sex recognition is strongly dependent on chemosensory communication, mediated by cuticular hydrocarbons acting as pheromones. While the roles of chemical and other sensory cues in stimulating male to female courtship have been well characterized in Drosophila, the signals that elicit aggression remain unclear. Here we show that when female pheromones or behavior are masculinized, males recognize females as competitors and switch from courtship to aggression. To masculinize female pheromones, a transgene carrying dsRNA for the sex determination factor transformer (traIR) was targeted to the pheromone producing cells, the oenocytes. Shortly after copulation males attacked these females, indicating that pheromonal cues can override other sensory cues. Surprisingly, masculinization of female behavior by targeting traIR to the nervous system in an otherwise normal female also was sufficient to trigger male aggression. Simultaneous masculinization of both pheromones and behavior induced a complete switch in the normal male response to a female. Control males now fought rather than copulated with these females. In a reciprocal experiment, feminization of the oenocytes and nervous system in males by expression of transformer (traF) elicited high levels of courtship and little or no aggression from control males. Finally, when confronted with flies devoid of pheromones, control males attacked male but not female opponents, suggesting that aggression is not a default behavior in the absence of pheromonal cues. Thus, our results show that masculinization of either pheromones or behavior in females is sufficient to trigger male-to-female aggression. Moreover, by manipulating both the pheromonal profile and the fighting patterns displayed by the opponent, male behavioral responses towards males and females can be completely reversed. Therefore, both pheromonal and behavioral cues are used by Drosophila males in recognizing a conspecific as a competitor.     

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.     

The male sex pheromone of the butterfly Bicyclus anynana: towards an evolutionary analysis

Background

Female sex pheromones attracting mating partners over long distances are a major determinant of reproductive isolation and speciation in Lepidoptera. Males can also produce sex pheromones but their study, particularly in butterflies, has received little attention. A detailed comparison of sex pheromones in male butterflies with those of female moths would reveal patterns of conservation versus novelty in the associated behaviours, biosynthetic pathways, compounds, scent-releasing structures and receiving systems. Here we assess whether the African butterfly Bicyclus anynana, for which genetic, genomic, phylogenetic, ecological and ethological tools are available, represents a relevant model to contribute to such comparative studies.

Methodology/Principal Findings

Using a multidisciplinary approach, we determined the chemical composition of the male sex pheromone (MSP) in the African butterfly B. anynana, and demonstrated its behavioural activity. First, we identified three compounds forming the presumptive MSP, namely (Z)-9-tetradecenol (Z9-14:OH), hexadecanal (16:Ald ) and 6,10,14-trimethylpentadecan-2-ol (6,10,14-trime-15-2-ol), and produced by the male secondary sexual structures, the androconia. Second, we described the male courtship sequence and found that males with artificially reduced amounts of MSP have a reduced mating success in semi-field conditions. Finally, we could restore the mating success of these males by perfuming them with the synthetic MSP.

Conclusions/Significance

This study provides one of the first integrative analyses of a MSP in butterflies. The toolkit it has developed will enable the investigation of the type of information about male quality that is conveyed by the MSP in intraspecific communication. Interestingly, the chemical structure of B. anynana MSP is similar to some sex pheromones of female moths making a direct comparison of pheromone biosynthesis between male butterflies and female moths relevant to future research. Such a comparison will in turn contribute to understanding the evolution of sex pheromone production and reception in butterflies.     

The different effects on courtship of volatile compounds from mated and virgin Drosophila females

Virgin Drosophila melanogaster females, which are courted vigorously, emit pheromones which stimulate males to court each other (Tompkinset al., 1980). Females which have recently copulated are courted less vigrously, and volatile compounds produced by mated females stimulate less courtship between males. Analysis of these compounds from fertilized females by gas chromatography and behavioral assays indicates that mated females emit less of the sex attractant made by virgins and may also produce material which inhibits courtship. These changes in pheromone production are initiated after the first few minutes of copulation.     

The effect of an androgen inhibitor on behavior and testicular morphology in the stickleback Gasterosteus aculeatus

It is generally held that the decline in courtship which is seen at the beginning of incubation during the reproductive cycle of the male ring dove (Streptopelia risoria) is the result of changes in endocrine secretion rather than changes in response to the external situation. To test this hypothesis, male ring doves proceeding through a cycle with their mates were tested at selected intervals to determine whether there was a decline in courtship behavior in the course of a reproductive cycle. Four conditions were examined. Inexperienced and experienced males were tested with a stimulus female in two situations; once after the mate and nest were removed from the home cage, and once after the mate only was removed. In the former situation the male courted a stimulus female irrespective of the stage of the cycle he had reached with his mate. In the latter situation, as incubation with the mate progressed, males continued to incubate when the stimulus female was introduced. Thus, the male's display of courtship during the reproductive cycle is influenced by the external stimulus situation presented by the female and the nest.     

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.     

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.     

Acoustic response to a pheromonal cue in the arctiid moth Pyrrharctia Isabella

ABSTRACT. The acoustic properties of the clicks emitted in response to male courtship pheromone by female Pyrrharctia Isabella (J. E. Smith) (Lepidoptera: Arctiidae) have been investigated. Extracts of male scent organs or synthetic male pheromones can be applied to a glass rod and used to stimulate females to produce these sounds. Power spectra, sound pressure readings, and oscillographic analyses show that the acoustic signals elicited by male extracts or synthetic male pheromones are not distinguishable from those produced in response to disturbance (handling). This is the first reported example of a sound produced by a female moth in a sexual context, and is the first reported example in moths of an acoustic response to a pheromonal stimulus.     

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.