Exaptation as a Mechanism for Functional Reinforcement of an Animal Pheromone System.

Animal sex pheromone systems often exist as multicomponent signals [1-11] to which chemical cues have been added over evolutionary time. Little is known on why and how additional molecules become recruited and conserved in an already functional pheromone system. Here, we investigated the evolutionary trajectory of a series of 15 kDa proteins-termed persuasins-that were co-opted more recently alongside the ancient sodefrin precursor-like factor (SPF) courtship pheromone system in salamanders [9, 12]. Expression, genomic, and molecular phylogenetic analyses show that persuasins originated from a gene that is expressed as a multi-domain protein in internal organs where it has no pheromone function but underwent gene duplication and neofunctionalization. The subsequent evolution combined domain loss and the introduction of a proteolytic cleavage site in the duplicated gene to give rise to two-domain cysteine rich proteins with structural properties similar to SPF pheromones [12]. An expression shift to the pheromone-producing glands, where expression of persuasins was immediately spatiotemporally synchronized with the already available pheromone system, completed the birth of a new pheromone. Electrostatic forces between members of both protein families likely enhance co-localization and simultaneous activation of different female olfactory neurons, explaining why persuasins immediately had a selective advantage. In line with this, behavioral assays show that persuasins increase female receptivity on their own but also exert a cumulative or synergistic effect in combination with SPF, clearly reinforcing the pheromone system as a whole. Our study reveals molecular remodeling of an existing protein architecture as an evolutionary mechanism for functional reinforcement of animal pheromone systems.

Characterization of Spodoptera litura (Lepidoptera: Noctuidae) Takeout Genes and Their Differential Responses to Insecticides and Sex Pheromone.

Spodoptera litura (S. litura) is one of the most serious agricultural insect pests worldwide. Takeout (TO) is involved in a variety of physiological and biochemical pathways and performs various biological functions. We characterized 18 S. litura TO genes and investigated their differential responses to insecticides and sex pheromones. All predicted TO proteins have two Cysteines that are unique to the N-terminal of the TO family proteins and contain four highly conserved Prolines, two Glycines, and one Tyrosine. The expression levels of seven TO genes in the male antennae were higher than those in the female antennae, although the expression levels of 10 TO genes in the female were higher than those in the male. We investigated the effects of the sex pheromone and three insecticides, that is, chlorpyrifos (Ch), emamectin benzoate (EB), and fipronil (Fi), on the expression levels of the TO genes in the antennae. The results showed that the insecticides and sex pheromone affect the expression levels of the TO genes. One day after the treatment, the expression levels of SlTO15 and SlTO4 were significantly induced by the Ch/EB treatment. Two days after the S. litura moths were treated with Fi, the expression of SlTO4 was significantly induced (28.35-fold). The expression of SlTO10 changed significantly after the Ch and EB treatment, although the expression of SlTO12 and SlTO15 was inhibited by the three insecticides after two days of treatment. Our results lay a foundation for studying the role of TO genes in the interaction between insecticides and sex pheromone.

[The role of the vomeronasal system in the formation of the human sexual behaviour]. / Rol' vomeronazal'noi sistemy v formirovanii seksual'nogo povedeniia cheloveka.

This review deals with the structure and function of the vomeronasal system and evaluation of its influence on the sexual sphere of humans and animals. Special attention is given to the role of pheromones in the regulation of the sexual behaviour. The data concerning the function of the vomeronasal organ following surgical interventions in the nasal cavity are discussed.

Evidence for a role of oestrogen receptor-related receptor in the regulation of male sexual behaviour in the moth Agrotis ipsilon.

The oestrogen receptor-related receptors (ERRs) are orphan nuclear receptors that were originally identified on the basis of their close homology to the oestrogen receptors. The three mammalian ERR genes participate in the regulation of vital physiological processes including reproduction, development and metabolic homeostasis. Although unique ERRs have been found in insects, data on the function and regulation of these receptors remain sparse. In the present study, a 2095-bp full-length cDNA encoding an ERR, termed AiERR, was isolated from males of the moth Agrotis ipsilon and deposited in the GenBank database under the accession number KT944662. The predicted AiERR protein shared an overall identity of 47-82% with other known insect and mammalian ERR homologues. AiERR exhibited a broad tissue expression pattern with the detection of one transcript of approximately 2 kb in the primary olfactory centres, the antennal lobes (AL). In adult males, the amount of AiERR mRNA in the AL increased concomitantly with age and responses to the female-emitted sex pheromone. Moreover, AiERR knockdown induced an inhibition in the sex pheromone-orientated flight of male. Using A. ipsilon as a model, our study demonstrates that the insect ERR is critical for the performance of male sexual behaviour, probably by acting on central pheromone processing.

A cytochrome P450 gene plays a role in the recognition of sex pheromones in the tobacco cutworm, Spodoptera litura.

Cytochrome P450 (P450 or CYP) genes are involved in fundamental physiological functions, and might be also associated with the olfactory recognition of sex pheromones in beetles and moths. A P450 gene, Spodoptera litura CYP4L4 (SlituCYP4L4), was cloned for the first time from the antennae of S. litura. SlituCYP4L4 was almost exclusively expressed in the adult stage and predominantly expressed in the adult antennae. In situ hybridization showed that SlituCYP4L4 localized mainly at the base of the long sensilla trichoidea, which responds to sex pheromone components. Pretreatment with an S. litura sex pheromone significantly reduced the expression levels of SlituCYP4L4, consistent with other genes involved in sex pheromone recognition. The expression level of SlituCYP4L4 was different in moths collected with different ratios of sex pheromone lures and collected in different geographical locations. After gene knockdown of SlituCYP4L4 in the antennae, the electroantennogram (EAG) responses of male and female moths to (9Z,11E)-tetradecadienyl acetate or (9Z,12E)-tetradecadienyl acetate were significantly decreased. In contrast, EAG responses to plant volatiles and sex pheromones of other moth species were not significantly influenced in these moths. SlituCYP4L4 was also expressed in the gustatory tissues and sensilla, which suggests that SlituCYP4L4 may have other functions in the chemosensory system. Our results have shown for the first time the function of a CYP gene with appendage-specific expression in insect sex pheromone recognition, especially in adult moths.

Ready for a fight? The physiological effects of detecting an opponent's pheromone cues prior to a contest.

Reception of pheromone cues can elicit significant physiological (e.g. steroid hormone levels) changes in the recipient. These pheromone-induced physiological changes have been well documented for male-female interactions, but scarcely in same-sex interactions (male-male and female-female). We sought to address this dearth in the current literature and examine whether mangrove rivulus fish (Kryptolebias marmoratus) could detect and, ultimately, mount a physiological response to the pheromone signature of a potential, same-sex competitor. We examined steroid hormone levels in mangrove rivulus exposed to one of three treatments: 1) isolation, 2) exposure to pheromones of a size-matched partner, and 3) pheromone exposure to a size-matched opponent followed by a physical encounter with the opponent. We found that exposure to a competitor's pheromone cues elicited a significant increase in testosterone levels. Increases in testosterone were similar across genetically distinct lineages derived from geographically distinct populations. Further, testosterone levels were similar between individuals only exposed to pheromone cues and individuals exposed to both pheromone cues and a subsequent physical encounter. Our findings led us to generate a number of testable predictions regarding how mangrove rivulus utilize pheromone signals in social interactions, the molecular mechanisms linking social stimuli and hormonal responses, and the possible adaptive benefits of hormonal responsiveness to receiving a potential competitor's pheromone cues.

Absence of Female-Typical Pheromone-Induced Hypothalamic Neural Responses and Kisspeptin Neuronal Activity in α-Fetoprotein Knockout Female Mice.

Pheromones induce sexually dimorphic neuroendocrine responses, such as LH secretion. However, the neuronal network by which pheromones are converted into signals that will initiate and modulate endocrine changes remains unclear. We asked whether 2 sexually dimorphic populations in the anteroventral periventricular and periventricular nuclei that express kisspeptin and tyrosine hydroxylase (TH) are potential candidates that will transduce the olfactory signal to the neuroendocrine system. Furthermore, we assessed whether this transduction is sensitive to perinatal actions of estradiol by using female mice deficient in α-fetoprotein (AfpKO), which lack the protective actions of Afp against maternal estradiol. Wild-type (WT) and AfpKO male and female mice were exposed to same- versus opposite-sex odors and the expression of Fos (the protein product of the immediate early gene c-Fos) was analyzed along the olfactory projection pathways as well as whether kisspeptin, TH, and GnRH neurons are responsive to opposite-sex odors. Male odors induced a female-typical Fos expression in target forebrain sites of olfactory inputs involved in reproduction in WT, but not in AfpKO females, whereas female odors induced a male-typical Fos expression in males of both genotypes. In WT females, opposite-sex odors induced Fos in kisspeptin and TH neurons, whereas in AfpKO females and WT males, only a lower, but still significant, Fos expression was observed in TH but not in kisspeptin neurons. Finally, opposite-sex odors did not induce any significant Fos expression in GnRH neurons of both sexes or genotypes. Our results strongly suggest a role for fetal estrogen in the sexual differentiation of neural responses to sex-related olfactory cues.

'Do you remember the first time?' Host plant preference in a moth is modulated by experiences during larval feeding and adult mating.

In insects, like in other animals, experience-based modulation of preference, a form of phenotypic plasticity, is common in heterogeneous environments. However, the role of multiple fitness-relevant experiences on insect preference remains largely unexplored. For the multivoltine polyphagous moth Spodoptera littoralis we investigated effects of larval and adult experiences on subsequent reproductive behaviours. We demonstrate, for the first time in male and female insects, that mating experience on a plant modulates plant preference in subsequent reproductive behaviours, whereas exposure to the plant alone or plant together with sex pheromone does not affect this preference. When including larval feeding experiences, we found that both larval rearing and adult mating experiences modulate host plant preference. These findings represent the first evidence that host plant preferences in polyphagous insects are determined by a combination of innate preferences modulated by sensory feedback triggered by multiple rewarding experiences throughout their lifetime.

Activation of the cerebellum by olfactory stimulation in sexually naive male rats.

INTRODUCTION: The cerebellum has been linked to multiple functions, such as motor control, cognition, memory, and emotional processing. As for its involvement in the sensory systems, the role of the cerebellum in the sense of smell remains unclear. We suggest that sexually naive male rats will present increased neuronal activity in the cerebellar vermis after being stimulated with almond odour or oestrous odour from receptive females. METHODS: We compared activity in the cerebellar vermis using Fos immunoreactivity after olfactory stimulation. Stimulation took place during 60 min in a cube-shaped acrylic chamber with a double bottom. Stimuli were clean woodchip bedding, bedding with almond extract, and bedding taken from a cage of receptive females. Male rats were subsequently anaesthetised with intraperitoneal sodium pentobarbital. Cerebellar tissue was fixed with paraformaldehyde for later immunohistochemical analysis. RESULTS: The number of Fos immunoreactive cells in all lobes of the cerebellar vermis was similar between groups stimulated with almond extract and with oestrous odour, and higher than in the clean woodchip group. CONCLUSIONS: Stimulation of the main olfactory system (almond) and the accessory system (oestrous odour) increases Fos protein production in the granular layer of the cortex of the cerebellar vermis in naive male rats.

Integrated action of pheromone signals in promoting courtship behavior in male mice.

The mammalian vomeronasal organ encodes pheromone information about gender, reproductive status, genetic background and individual differences. It remains unknown how pheromone information interacts to trigger innate behaviors. In this study, we identify vomeronasal receptors responsible for detecting female pheromones. A sub-group of V1re clade members recognizes gender-identifying cues in female urine. Multiple members of the V1rj clade are cognate receptors for urinary estrus signals, as well as for sulfated estrogen (SE) compounds. In both cases, the same cue activates multiple homologous receptors, suggesting redundancy in encoding female pheromone cues. Neither gender-specific cues nor SEs alone are sufficient to promote courtship behavior in male mice, whereas robust courtship behavior can be induced when the two cues are applied together. Thus, integrated action of different female cues is required in pheromone-triggered mating behavior. These results suggest a gating mechanism in the vomeronasal circuit in promoting specific innate behavior.DOI: http://dx.doi.org/10.7554/eLife.03025.001.

Identification of an olfactory signal molecule that activates the central regulator of reproduction in goats.

Pheromone signals regulate conspecific behavior and physiology [1]. Releaser pheromones induce specific behavior by exerting acute effects on the neuronal response, whereas primer pheromones induce physiological changes with long-lasting effects by changing the neuroendocrine status of the recipients. In mammals, although several types of releaser pheromones have been identified [2-5], the identities of primer pheromones, as well as their mechanisms of action, remain largely unknown [6]. In sheep and goats, the seasonally anestrous endocrine state of females is changed to the estrous state upon exposure to male scents [7, 8]. This so-called "male effect" is one of the most conspicuous primer pheromone effects in mammals [9, 10]. In this study, we have identified an olfactory signal molecule that activates the central regulator of reproduction, the gonadotropin-releasing hormone (GnRH) pulse generator, in goats. Using gas chromatography-mass spectrometry to analyze male goat headspace volatiles, we identified several ethyl-branched aldehydes and ketones. We electrophysiologically demonstrated that one of these compounds, 4-ethyloctanal, activates the GnRH pulse generator in female goats. This is the first report of an olfactory molecule that has been shown to activate the central reproductive axis, and this discovery will provide a new direction for primer pheromone research.

Pheromones: the scent of a male.

There has been an enduring fascination with discovering biological odours which can evoke behavioral and physiological responses in mammals. New findings in goats have now identified a key molecule involved in the effect male odours have on female reproductive cycles.

Adolescent gain in positive valence of a socially relevant stimulus: engagement of the mesocorticolimbic reward circuitry.

A successful transition from childhood to adulthood requires adolescent maturation of social information processing. The neurobiological underpinnings of this maturational process remain elusive. This research employed the male Syrian hamster as a tractable animal model for investigating the neural circuitry involved in this critical transition. In this species, adult and juvenile males display different behavioral and neural responses to vaginal secretions, which contain pheromones essential for expression of sexual behavior in adulthood. These studies tested the hypothesis that vaginal secretions acquire positive valence over adolescent development via remodeling of neural circuits underlying sexual reward. Sexually naïve adult, but not juvenile, hamsters showed a conditioned place preference for vaginal secretions. Differences in behavioral response to vaginal secretions between juveniles and adults correlated with a difference in the vaginal secretion-induced neural activation pattern in mesocorticolimbic reward circuitry. Fos immunoreactivity increased in response to vaginal secretions in the medial amygdala and ventral tegmental dopaminergic cells of both juvenile and adult males. However, only in adults was there a Fos response to vaginal secretions in non-dopaminergic cells in interfascicular ventral tegmental area, nucleus accumbens core and infralimbic medial prefrontal cortex. These results demonstrate that a socially relevant chemosensory stimulus acquires the status of an unconditioned reward during adolescence, and that this adolescent gain in social reward is correlated with experience-independent engagement of specific cell groups in reward circuitry.

A complex set of sex pheromones identified in the cuttlefish Sepia officinalis.

BACKGROUND: The cephalopod mollusk Sepia officinalis can be considered as a relevant model for studying reproduction strategies associated to seasonal migrations. Using transcriptomic and peptidomic approaches, we aim to identify peptide sex pheromones that are thought to induce the aggregation of mature cuttlefish in their egg-laying areas. RESULTS: To facilitate the identification of sex pheromones, 576 5'-expressed sequence tags (ESTs) were sequenced from a single cDNA library generated from accessory sex glands of female cuttlefish. Our analysis yielded 223 unique sequences composed of 186 singletons and 37 contigs. Three major redundant ESTs called SPα, SPα' and SPß were identified as good candidates for putative sex pheromone transcripts and are part of the 87 unique sequences classified as unknown. The alignment of translated SPα and SPα' revealed a high level of conservation, with 98.4% identity. Translation led to a 248-amino acid precursor containing six peptides with multiple putative disulfide bonds. The alignment of SPα-α' with SPß revealed a partial structural conservation, with 37.3% identity. Translation of SPß led to a 252-amino acid precursor containing five peptides. The occurrence of a signal peptide on SPα, SPα' and SPß showed that the peptides were secreted. RT-PCR and mass spectrometry analyses revealed a co-localization of transcripts and expression products in the oviduct gland. Preliminary in vitro experiments performed on gills and penises revealed target organs involved in mating and ventilation. CONCLUSIONS: The analysis of the accessory sex gland transcriptome of Sepia officinalis led to the identification of peptidic sex pheromones. Although preliminary functional tests suggested the involvement of the α3 and ß2 peptides in ventilation and mating stimulation, further functional investigations will make it possible to identify the complete set of biological activities expected from waterborne pheromones.

Algunos aspectos fisiológicos de la selección sexual / Some physiological issues of the sexual selection

La atracción y el cortejo sexual integran el contexto de la selección sexual. En los humanos, complejos mecanismos psicológicos y sociales son probablemente los mayores determinantes de la atracción sexual. Si bien se postula la participación de factores de índole genética ­-que podrían explicar la importancia de los rasgos fisicos en la elección del compañero sexual (teoría de los buenos genes)-, está por otra parte comprobado que los cambios fisiológicos (espontáneos en la mujer e inducidos en los hombres) juegan un preponderante papel a los fines de la reproducción.

The attraction and courtship are inclusive parts of the sexual selection. Very important psychological and social mechanisms are possibly the main determinants of the sexual attraction in humans. The genetic participation is assumed as the explication of the preeminent importance of the physical characteristics (the good gene theory). The physiologic changes associated with the sexual selection (spontaneous in women and induced in males) play a very important role in the essence of the sexual selection: the reproductive possibility.

Dung as a potential medium for inter-sexual chemical signaling in Asian elephants (Elephas maximus).

Chemical signaling is a prominent mode of male-female communication among elephants, especially during their sexually active periods. Studies on the Asian elephant in zoos have shown the significance of a urinary pheromone (Z7-12:Ac) in conveying the reproductive status of a female toward the opposite sex. We investigated the additional possibility of an inter-sexual chemical signal being conveyed through dung. Sixteen semi-captive adult male elephants were presented with dung samples of three female elephants in different reproductive phases. Each male was tested in 3 separate trials, within an interval of 1-3 days. The trials followed a double-blind pattern as the male and female elephants used in the trials were strangers, and the observer was not aware of the reproductive status of females during the period of bioassays. Males responded preferentially (P<0.005), in terms of higher frequency of sniff, check and place behavior toward the dung of females close to pre-ovulatory period (follicular-phase) as compared to those in post-ovulatory period (luteal-phase). The response toward the follicular phase samples declined over repeated trials though was still significantly higher than the corresponding response toward the non-ovulatory phase in each of the trials performed. This is the first study to show that male Asian elephants were able to distinguish the reproductive phase of the female by possibly detecting a pre-ovulatory pheromone released in dung.

Male primer endocrine responses to preovulatory female cyprinids under natural conditions in Sweden.

This study investigated two related aspects of male-female reproductive interactions in the family Cyprinidae: (1) whether ovulating female rudd Scardinius erythrophthalmus (subfamily Leuciscinae) induce endocrine and gonadal priming responses in conspecific males, a phenomenon which has been described only in species from the subfamily Cyprininae such as goldfish, Carassius auratus, crucian carp Carassius carassius and common carp, Cyprinus carpio and (2) whether the stimuli mediating these responses are species-specific. Field studies of three sympatric European cyprinids, two leuciscins (S. erythrophthalmus and white bream Blicca bjoerkna) and one cyprinin (C. carassius), were conducted on fishes captured in Sweden in the spawning season and held in net pens under natural conditions. As previously reported in C. carassius, male S. erythrophthalmus increased milt (sperm and seminal fluid) volume and plasma concentrations of the sperm maturation hormone 4-pregnen-17,20ß-diol-3-one (17,20ß-P) when they were held with female S. erythrophthalmus induced to ovulate by injection of Ovaprim (GnRH analogue plus dopamine antagonist). Male S. erythrophthalmus had larger milt volumes than male C. carassius prior to and following exposure to ovulatory conspecifics, but exhibited a smaller proportional milt increase in response to stimulation, suggesting species differences in sperm allocation at spawning. The presence of female S. erythrophthalmus and B. bjoerkna did not affect milt volumes of C. carassius under two experimental conditions: (1) ovulating S. erythrophthalmus and B. bjoerkna did not increase the milt volumes of C. carassius and (2) S. erythrophthalmus and B. bjoerkna did not interfere with the milt volume increase induced in male C. carassius by ovulating conspecifics. These results suggest that, as in C. auratus, C. carassius and C. carpio (subfamily Cyprininae), female S. erythrophthalmus (subfamily Leuciscinae) release a preovulatory pheromone that exerts priming effects on male hormones and sperm allocation. The findings also indicate that C. carassius discriminate between the reproductive odours of conspecifics and heterospecifics.

Interaction of cellular and network mechanisms for efficient pheromone coding in moths.

Sensory systems, both in the living and in machines, have to be optimized with respect to their environmental conditions. The pheromone subsystem of the olfactory system of moths is a particularly well-defined example in which rapid variations of odor content in turbulent plumes require fast, concentration-invariant neural representations. It is not clear how cellular and network mechanisms in the moth antennal lobe contribute to coding efficiency. Using computational modeling, we show that intrinsic potassium currents (I(A) and I(SK)) in projection neurons may combine with extrinsic inhibition from local interneurons to implement a dual latency code for both pheromone identity and intensity. The mean latency reflects stimulus intensity, whereas latency differences carry concentration-invariant information about stimulus identity. In accordance with physiological results, the projection neurons exhibit a multiphasic response of inhibition-excitation-inhibition. Together with synaptic inhibition, intrinsic currents I(A) and I(SK) account for the first and second inhibitory phases and contribute to a rapid encoding of pheromone information. The first inhibition plays the role of a reset to limit variability in the time to first spike. The second inhibition prevents responses of excessive duration to allow tracking of intermittent stimuli.

Effects of brief exposure of male pheromone on multiple-unit activity at close proximity to kisspeptin neurons in the goat arcuate nucleus.

Exposure of females to the male pheromone induces pulsatile release of gonadotropin-releasing hormone (GnRH) in goats. Recently, kisspeptin neurons in the arcuate nucleus (ARC) have been suggested to represent the proximate source of the GnRH pulse generator. In this study, we examined the effects of the pheromone on multiple-unit activity (MUA) in female goats fitted with recording electrodes aimed at the ARC kisspeptin neurons. In all eight goats, periodic bursts in MUA (MUA volleys), which were considered to be electrophysiological manifestations of the GnRH pulse generator, were observed. The mean intervolley interval (T) during the control period was calculated in each goat that was then exposed to the male pheromone for 1 sec at timings of 1/4 T, 1/2 T or 3/4 T after one regularly occurring MUA volley. An instantaneous rise in MUA was observed immediately after the exposure regardless of timing. Exposure at a timing of 3/4 T resulted in an MUA volley within 60 sec following the instantaneous rise in all goats. In contrast, an MUA volley was induced in only 2 goats by exposure at 1/2 T, while exposure at 1/4 T failed to induce an MUA volley in any goats. These results suggest that transmission of the pheromone signal to the ARC, represented by an instantaneous rise, activates the GnRH pulse generator. Moreover, the timing-dependent pheromone action in inducing an MUA volley indicates that the GnRH pulse generator has a refractory period for the pheromone signal after the burst.

Reproduction: a new venue for studying function of adult neurogenesis?

Adult neurogenesis has been a focus within the past few years because it is a newly recognized form of neuroplasticity that may play significant roles in behaviors and recovery process after disease. Mammalian adult neurogenesis could be found in two brain regions: hippocampus and subventricular zone (SVZ). While it is well established that hippocampal neurogenesis participates in memory formation and anxiety, the physiological function of SVZ neurogenesis is still under intense investigation. Recent studies disclose that SVZ neurogenesis is under regulation of reproductive cues like pheromones. Reciprocally, the newborn neurons may exert their effect on reproductive and maternal behaviors. This review discusses recent understanding of the interrelationship between neurogenesis and reproduction. The studies highlighted in this review illustrate the potential importance of neurogenesis in reproductive function and will provide new insights for the significance of adult neurogenesis.