Generating parallel representations of position and identity in the olfactory system.

In Drosophila, a dedicated olfactory channel senses a male pheromone, cis-vaccenyl acetate (cVA), promoting female courtship while repelling males. Here, we show that separate cVA-processing streams extract qualitative and positional information. cVA sensory neurons respond to concentration differences in a 5-mm range around a male. Second-order projection neurons encode the angular position of a male by detecting inter-antennal differences in cVA concentration, which are amplified through contralateral inhibition. At the third circuit layer, we identify 47 cell types with diverse input-output connectivity. One population responds tonically to male flies, a second is tuned to olfactory looming, while a third integrates cVA and taste to coincidentally promote female mating. The separation of olfactory features resembles the mammalian what and where visual streams; together with multisensory integration, this enables behavioral responses appropriate to specific ethological contexts.

Multisensory Logic of Infant-Directed Aggression by Males.

Newborn mice emit signals that promote parenting from mothers and fathers but trigger aggressive responses from virgin males. Although pup-directed attacks by males require vomeronasal function, the specific infant cues that elicit this behavior are unknown. We developed a behavioral paradigm based on reconstituted pup cues and showed that discrete infant morphological features combined with salivary chemosignals elicit robust male aggression. Seven vomeronasal receptors were identified based on infant-mediated activity, and the involvement of two receptors, Vmn2r65 and Vmn2r88, in infant-directed aggression was demonstrated by genetic deletion. Using the activation of these receptors as readouts for biochemical fractionation, we isolated two pheromonal compounds, the submandibular gland protein C and hemoglobins. Unexpectedly, none of the identified vomeronasal receptors and associated cues were specific to pups. Thus, infant-mediated aggression by virgin males relies on the recognition of pup's physical traits in addition to parental and infant chemical cues.

orco Mutagenesis Causes Loss of Antennal Lobe Glomeruli and Impaired Social Behavior in Ants.

Life inside ant colonies is orchestrated with diverse pheromones, but it is not clear how ants perceive these social signals. It has been proposed that pheromone perception in ants evolved via expansions in the numbers of odorant receptors (ORs) and antennal lobe glomeruli. Here, we generate the first mutant lines in the clonal raider ant, Ooceraea biroi, by disrupting orco, a gene required for the function of all ORs. We find that orco mutants exhibit severe deficiencies in social behavior and fitness, suggesting they are unable to perceive pheromones. Surprisingly, unlike in Drosophila melanogaster, orco mutant ants also lack most of the ∼500 antennal lobe glomeruli found in wild-type ants. These results illustrate that ORs are essential for ant social organization and raise the possibility that, similar to mammals, receptor function is required for the development and/or maintenance of the highly complex olfactory processing areas in the ant brain. VIDEO ABSTRACT.

Accelerated hermaphrodite maturation on male pheromones suggests a general principle of coordination between larval behavior and development.

Environment in general and social signals in particular could alter development. In Caenorhabditis elegans, male pheromones hasten development of hermaphrodite larvae. We show that this involves acceleration of growth and both somatic and germline development during the last larval stage (L4). Larvae exposed to male pheromones spend more time in L3 and less in the quiescent period between L3 and L4. This behavioral alteration improves provision in early L4, likely allowing for faster development. Larvae must be exposed to male pheromones in late L3 for behavioral and developmental effects to occur. Latter portions of other larval stages also contain periods of heightened sensitivity to environmental signals. Behavior during the early part of the larval stages is biased toward exploration, whereas later the emphasis shifts to food consumption. We argue that this organization allows assessment of the environment to identify the most suitable patch of resources, followed by acquisition of sufficient nutrition and salient information for the developmental events in the next larval stage. Evidence from other species indicates that such coordination of behavior and development may be a general feature of larval development.

Chemical species recognition in an adaptive radiation of Hawaiian Tetragnatha spiders (Araneae: Tetragnathidae).

Studies of adaptive radiations have played a central role in our understanding of reproductive isolation. Yet the focus has been on human-biased visual and auditory signals, leaving gaps in our knowledge of other modalities. To date, studies on chemical signals in adaptive radiations have focused on systems with multimodal signalling, making it difficult to isolate the role chemicals play in reproductive isolation. In this study we examine the use of chemical signals in the species recognition and adaptive radiation of Hawaiian Tetragnatha spiders by focusing on entire communities of co-occurring species, and conducting behavioural assays in conjunction with chemical analysis of their silks using gas chromatography-mass spectrometry. Male spiders significantly preferred the silk extracts of conspecific mates over those of sympatric heterospecifics. The compounds found in the silk extracts, long chain alkyl methyl ethers, were remarkably species-specific in the combination and quantity. The differences in the profile were greatest between co-occurring species and between closely related sibling species. Lastly, there were significant differences in the chemical profile between two populations of a particular species. These findings provide key insights into the role chemical signals play in the attainment and maintenance of reproductive barriers between closely related co-occurring species.

The vomeronasal system of the wolf (Canis lupus signatus): The singularities of a wild canid.

Wolves, akin to their fellow canids, extensively employ chemical signals for various aspects of communication, including territory maintenance, reproductive synchronisation and social hierarchy signalling. Pheromone-mediated chemical communication operates unconsciously among individuals, serving as an innate sensory modality that regulates both their physiology and behaviour. Despite its crucial role in the life of the wolf, there is a lacuna in comprehensive research on the neuroanatomical and physiological underpinnings of chemical communication within this species. This study investigates the vomeronasal system (VNS) of the Iberian wolf, simultaneously probing potential alterations brought about by dog domestication. Our findings demonstrate the presence of a fully functional VNS, vital for pheromone-mediated communication, in the Iberian wolf. While macroscopic similarities between the VNS of the wolf and the domestic dog are discernible, notable microscopic differences emerge. These distinctions include the presence of neuronal clusters associated with the sensory epithelium of the vomeronasal organ (VNO) and a heightened degree of differentiation of the accessory olfactory bulb (AOB). Immunohistochemical analyses reveal the expression of the two primary families of vomeronasal receptors (V1R and V2R) within the VNO. However, only the V1R family is expressed in the AOB. These findings not only yield profound insights into the VNS of the wolf but also hint at how domestication might have altered neural configurations that underpin species-specific behaviours. This understanding holds implications for the development of innovative strategies, such as the application of semiochemicals for wolf population management, aligning with contemporary conservation goals.

'Speed-ageing' of human skin in serum-free organ culture ex vivo: An instructive novel assay for preclinical human skin ageing research demonstrates senolytic effects of caffeine and 2,5-dimethylpyrazine.

Preclinical human skin ageing research has been limited by the paucity of instructive and clinically relevant models. In this pilot study, we report that healthy human skin of different age groups undergoes extremely accelerated ageing within only 3 days, if organ-cultured in a defined serum-free medium. Quantitative (immuno-)histomorphometry documented this unexpected ex vivo phenotype on the basis of ageing-associated biomarkers: the epidermis showed significantly reduced rete ridges and keratinocyte proliferation, sirtuin-1, MTCO1 and collagen 17a1 protein levels; this contrasted with significantly increased expression of the DNA-damage marker, γH2A.X. In the dermis, collagen 1 and 3 and hyaluronic acid content were significantly reduced compared to Day 0 skin. qRT-PCR of whole skin RNA extracts also showed up-regulated mRNA levels of several (inflamm-) ageing biomarkers (MMP-1, -2, -3, -9; IL6, IL8, CXCL10 and CDKN1). Caffeine, a methylxanthine with recognized anti-ageing properties, counteracted the dermal collagen 1 and 3 reduction, the epidermal accumulation of γH2A.X, and the up-regulation of CXCL10, IL6, IL8, MMP2 and CDKN1. Finally, we present novel anti-ageing effects of topical 2,5-dimethylpyrazine, a natural pheromone TRPM5 ion channel activator. Thus, this instructive, clinically relevant "speed-ageing" assay provides a simple, but powerful new research tool for dissecting skin ageing and rejuvenation, and is well-suited to identify novel anti-ageing actives directly in the human target organ.

The transcriptomic signature of adaptations associated with perfume collection in orchid bees.

Secondary sexual traits can convey information on mate quality with the signal honesty maintained by the costly nature of trait expression. Mating signals are also often underpinned by physiological, morphological, and behavioural adaptations, which may require the evolution of novelty, but the genetic basis in many cases is unknown. In orchid bees, males acquire chemical compounds from the environment that act as pheromone-like bouquets (perfumes) during courtship displays. This process could be costly, potentially due to the cognitive demands of learning and the physiological demands of collecting a mix of extrinsic chemical compounds that may require detoxification. Furthermore, a novel trait, a specialized perfume pouch in the hind leg, is required for compound storage. We studied gene expression in the brain, hind leg, and Malpighian tubules-a tissue involved in detoxification-to investigate changes in gene expression following perfume collection. We detected upregulation of genes enriched in functions related to transcription, odorant binding, and receptor activity in the Malpighian tubules. On the other hand, we did not find any evidence for learning processes following perfume collection, or gene expression changes in the hind leg, perhaps due to constitutive expression, or the age of the sampled bees. We did identify high expression of chemosensory proteins in the hind legs, which we suggest could play a role in perfume collection or storage, with further functional studies necessary to determine their binding properties and potential physiological importance. Los rasgos sexuales secundarios pueden servir como indicadores de calidad de la pareja, y en algunos casos la honestidad de la señal se mantiene por el costo de expresar el rasgo. A menudo las señales sexuales están respaldadas por adaptaciones fisiológicas, morfológicas y de comportamiento por lo tanto pueden requerir la evolución de nuevos rasgos, pero en muchos casos se desconoce la base genética. En las abejas de las orquídeas, los machos recolectan compuestos químicos del medio ambiente, los cuales actúan como feromonas (perfumes) durante el despliegue de cortejo. Este proceso podría ser costoso, posiblemente debido a las demandas cognitivas del aprendizaje y las demandas fisiológicas de recolectar una mezcla de compuestos químicos extrínsecos que pueden requerir desintoxicación. Además, se requiere la evolución de un contenedor para almacenar perfumes en la pata trasera. Para investigar los cambios en la expresión génica después de la recolección de perfume, estudiamos la expresión génica en el cerebro, la pata trasera y los túbulos de Malpighi (tejido involucrado en la desintoxicación). Encontramos varios genes regulados positivamente en los túbulos de Malpighi después de la recolección que están enriquecidos en factores de transcripción, proteínas de fijación de olores, y proteínas con actividad de receptor. Por otro lado, no encontramos ninguna evidencia de procesos de aprendizaje posteriores a la recolección de perfumes, o cambios en la expresión génica en la pata trasera, esto quizás debido a la expresión constitutiva o la edad de las abejas muestreadas. Además, identificamos una alta expresión de proteínas quimio-sensoriales en las patas traseras, que podría desempeñar un papel en la recolección o almacenamiento de perfumes. Más estudios funcionales son necesarios para determinar las propiedades de fijación de las proteínas y su potencial importancia fisiológica.

Complexity of Chemical Emissions Increases Concurrently with Sexual Maturity in Heliconius Butterflies.

Pheromone communication is widespread among animals. Since it is often involved in mate choice, pheromone production is often tightly controlled. Although male sex pheromones (MSPs) and anti-aphrodisiacs have been studied in some Heliconius butterfly species, little is known about the factors affecting their production and release in these long-lived butterflies. Here, we investigate the effect of post-eclosion age on chemical blends from pheromone-emitting tissues in Heliconius atthis and Heliconius charithonia, exhibiting respectively free-mating and pupal-mating strategies that are hypothesised to differently affect the timing of their pheromone emissions. We focus on two different tissues: the wing androconia, responsible for MSPs used in courtship, and the genital tip, the production site for anti-aphrodisiac pheromones that affect post-mating behaviour. Gas chromatography-mass spectrometric analysis of tissue extracts from virgin males and females of both species from day 0 to 8 post-eclosion demonstrates the following. Some ubiquitous fatty acid precursors are already detectable at day 0. The complexity of the chemical blends increases with age regardless of tissue or sex. No obvious difference in the time course of blend production was evident between the two species, but female tissues in H. charithonia were more affected by age than in H. atthis. We suggest that compounds unique to male androconia and genitals and whose amount increases with age are potential candidates for future investigation into their roles as pheromones. While this analysis revealed some of the complexity in Heliconius chemical ecology, the effects of other factors, such as the time of day, remain unknown.

Individual Variation in Male Pheromone Production in Xylocopa sonorina Correlates with size and Gland Color.

Sex pheromones are species-specific chemical signals that facilitate the location, identification, and selection of mating partners. These pheromones can vary between individuals, and act as signals of mate quality. Here, we investigate the variation of male pheromones in the mesosomal glands of the large carpenter bee Xylocopa sonorina, within a Northern California population. We tested the hypothesis that morphological traits are correlated with the observed variation in chemical blend composition of these bees. We also conducted behavioral assays to test whether these male pheromones act as long-range attractants to conspecifics. We found that larger males with darker mesosomal glands have a higher pheromone amount in their glands. Our analysis also suggests that this pheromone blend functions as a long-range attractant to both males and females. We show that both male body size and sexual maturation are important factors influencing pheromone abundance, and that this pheromone blend acts as a long-range attractant. We hypothesize that this recorded variation in male pheromone could be important for female choice.

Evolution of Acquired Perfumes and Endogenous Lipid Secretions in Orchid Bees.

Male orchid bees are unique in the animal kingdom for making perfumes that function as sex pheromone. Males collect volatile chemicals from the environment in the neotropical forests, including floral and non-floral sources, creating complex but species-specific blends. Male orchid bees exhibit several adaptations to facilitate perfume collection and storage. When collecting volatile compounds, males apply lipid substances that they secrete from cephalic labial glands onto the fragrant substrate. These lipids help dissolve and retain the volatiles, similar to the process of 'enfleurage' in the traditional perfume industry. We investigated how the chemical composition of acquired perfume and labial gland secretions varied across the phylogeny of orchid bees, including 65 species in five genera from Central and South America. Perfumes showed rapid evolution as revealed by low overall phylogenetic signal, in agreement with the idea that perfume compounds diverge rapidly and substantially among closely related species due to their role in species recognition. A possible exception were perfumes in the genus Eulaema, clustering closely in chemospace, partly mediated by high proportions of carvone and trans-carvone oxide. Labial gland secretions, in contrast, showed a strong phylogenetic signal at the genus level, with secretions of Eufriesea and Exaerete dominated by fatty acids and Eulaema dominated by saturated acetates of chain lengths 12 to 16 C-atoms. Secretions of the majority of Euglossa were heavily dominated by one unsaturated long chain diacetate, (9Z)-Eicosen-1,20-diyldiacetate. However, we also identified few highly divergent species of Euglossa in four subclades (11 species) that appear to have secondarily replaced the diacetate with other compounds. In comparison with environment-derived perfumes, the evolution of labial gland secretion is much slower, likely constrained by the underlying biochemical pathways, but perhaps influenced by perfume-solvent chemical interactions.

Extreme olfactory sensitivity of silver and bighead carp to overlapping suites of 21-carbon steroids suggests that these species, and likely all other Cyprinoidei, employ them as pheromones.

Although well established that several fishes including goldfish in the suborder Cypinoidei within the family Cypriniformes use the maturation-inducing steroid 17,20ß-dihydroxy-pregn-4-ene-3-one (17,20ßP) and its metabolites as a priming pheromone which they detect with sensitivity and specificity, it is unclear whether and how other Cypriniformes might have evolved to do so. This study examined this question in the family Xenocyprididae. Using electro-olfactogram recording we tested the olfactory sensitivity of silver (Hypophthalmichthys molitrix) and bighead carp (H. nobilis) to a range of 213 steroids in 21 mixtures at 10-9M. While silver carp detected 6 of 21 mixtures, bighead carp detected 5 (p< 0.05). Silver carp were sensitive to 13 21-carbon steroids in these mixtures including 17,20ßP while bighead carp detected 9, including 8 detected by silver carp. This assortment of steroids overlapped that detected by goldfish (family Cyprinidae) but no non-Cyprinoid, suggesting common evolutionary origin and function with differences characteristic of species-specificity.

Enhancing entomopathogenic nematode efficacy with Pheromones: A field study targeting the pecan weevil.

Consistent efficacy is required for entomopathogenic nematodes to gain wider adoption as biocontrol agents. Recently, we demonstrated that when exposed to nematode pheromone blends, entomopathogenic nematodes showed increased dispersal, infectivity, and efficacy under laboratory and greenhouse conditions. Prior to this study, the impact of entomopathogenic nematode-pheromone combinations on field efficacy had yet to be studied. Steinernema feltiae is a commercially available entomopathogenic nematode that has been shown to increase mortality in insect pests such as the pecan weevil Curculio caryae. In this study, the pecan weevil was used as a model system to evaluate changes in S. feltiae efficacy when treated with a partially purified ascaroside pheromone blend. Following exposure to the pheromone blend, the efficacy of S. feltiae significantly increased as measured with decreased C. caryae survival despite unfavorable environmental conditions. The results of this study highlight a potential new avenue for using entomopathogenic nematodes in field conditions. With increased efficacy, using entomopathogenic nematodes will reduce reliance on conventional management methods in pecan production, translating into more environmentally acceptable practices.

Cuticular lipid profiles of selected species of cyclocephaline beetles (Melolonthidae, Cyclocephalini).

Neotropical cyclocephaline beetles, a diverse group of flower-loving insects, significantly impact natural and agricultural ecosystems. In particular, the genus Cyclocephala, with over 350 species, displays polymorphism and cryptic complexes. Lacking a comprehensive DNA barcoding framework, accessible tools for species differentiation are needed for research in taxonomy, ecology, and crop management. Moreover, cuticular hydrocarbons are believed to be involved in sexual recognition mechanisms in these beetles. In the present study we examined the cuticular chemical profiles of six species from the genus Cyclocephala and two populations of Erioscelis emarginata and assessed their efficiency in population, species, and sex differentiation. Overall we identified 74 compounds in cuticular extracts of the selected taxa. Linear alkanes and unsaturated hydrocarbons were prominent, with ten compounds between them explaining 85.6% of species dissimilarity. Although the cuticular chemical profiles efficiently differentiated all investigated taxa, only C. ohausiana showed significant cuticular profile differences between sexes. Our analysis also revealed two E. emarginata clades within a larger group of 'Cyclocephala' species, but they were not aligned with the two studied populations. Our research underscores the significance of cuticular lipid profiles in distinguishing selected cyclocephaline beetle species and contemplates their potential impact as contact pheromones on sexual segregation and speciation.

Fatty Acid Origin of Insect Pheromones.

Pheromones are utilized to a great extent in insects. Many of these pheromones are biosynthesized through a pathway involving fatty acids. This chapter will provide examples where the biosynthetic pathways of fatty acid-derived pheromones have been studied in detail. These include pheromones from Lepidoptera, Coleoptera, and Hymenoptera. Many species of Lepidoptera utilize fatty acids as precursors to pheromones with a functional group that include aldehydes, alcohols, and acetate esters. In addition, the biosynthesis of hydrocarbons will be briefly examined because many insects utilize hydrocarbons or modified hydrocarbons as pheromones.

Binding properties of chemosensory protein 4 in Riptortus pedestris to aggregation pheromones.

In insects, chemosensory proteins (CSPs) play an important role in the perception of the external environment and have been widely used for protein-binding characterization. Riptortus pedestris has received increased attention as a potential cause of soybean staygreen syndrome in recent years. In this study, we found that RpedCSP4 expression in the antennae of adult R. pedestris increased with age, with no significant difference in expression level observed between males and females, as determined through quantitative real-time polymerase chain reaction (qRT-PCR). Subsequently, we investigated the ability of RpedCSP4 to bind various ligands (five aggregated pheromone components and 13 soybean volatiles) using a prokaryotic expression system and fluorescence competitive binding assays. We found that RpedCSP4 binds to three aggregated pheromone components of R. pedestris, namely, ((E)-2-hexenyl (Z)-3-hexenoate (E2Z3), (E)-2-hexenyl (E)-2-hexenoate (E2E2), and (E)-2-hexenyl hexenoate (E2HH)), and that its binding capacities are most stable under acidic condition. Finally, the structure and protein-ligand interactions of RpedCSP4 were further analyzed via homology modeling, molecular docking, and targeted mutagenesis experiments. The L29A mutant exhibited a loss of binding ability to these three aggregated pheromone components. Our results show that the olfactory function of RpedCSP4 provides new insights into the binding mechanism of RpedCSPs to aggregation pheromones and contributes to discover new target candidates that will provide a theoretical basis for future population control of R. pedestris.

Organotransition Metal Chemistry of Terpenes: Syntheses, Structures, Reactivity and Molecular Rearrangements.

The impact of organometallic chemistry on the terpene field only really blossomed in the 1960s and 1970s with the realisation that carbon-carbon bond formation under mild conditions could be achieved by using nickel or iron carbonyls as synthetic reagents. Concomitantly, the development of palladium derivatives capable of the controlled coupling of isoprene units attracted the attention of numerous highly talented researchers, including future Nobel laureates. We discuss briefly how early work on the syntheses of simple monoterpenes soon progressed to sesquiterpenes and diterpenes of increasing complexity, such as humulene, flexibilene, vitamin A, or pheromones of commercial value, in particular those used in perfumery (muscone, lavandulol), or grandisol and red scale pheromone as replacements for harmful pesticides. As the field progressed, there has been more emphasis on developing organometallic routes to enantiopure rather than racemic products, as well as gaining precise mechanistic data on the transformations, notably the course of metal-promoted molecular rearrangements that have long been a feature of terpene chemistry. We note the impact of the enormously enhanced analytical techniques, high-field NMR spectroscopy and X-ray crystallography, and their use to re-examine the originally proposed structures of terpenes and their organometallic derivatives. Finally, we highlight the very recent ground-breaking use of the crystalline sponge method to acquire structural data on low-melting or volatile terpenes. The literature cited herein covers the period 1959 to 2023.

Structure and number of mating pheromone genes is closely linked to sexual reproductive strategy in Huntiella.

BACKGROUND: Huntiella resides in the Ceratocystidaceae, a family of fungi that accommodates important plant pathogens and insect-associated saprotrophs. Species in the genus have either heterothallic or unisexual (a form of homothallism) mating systems, providing an opportunity to investigate the genetic mechanisms that enable transitions between reproductive strategies in related species. Two newly sequenced Huntiella genomes are introduced in this study and comparative genomics and transcriptomics tools are used to investigate the differences between heterothallism and unisexuality across the genus. RESULTS: Heterothallic species harbored up to seven copies of the a-factor pheromone, each of which possessed numerous mature peptide repeats. In comparison, unisexual Huntiella species had only two or three copies of this gene, each with fewer repeats. Similarly, while the heterothallic species expressed up to 12 copies of the mature α-factor pheromone, unisexual species had up to six copies. These significant differences imply that unisexual Huntiella species do not rely on a mating partner recognition system in the same way that heterothallic fungi do. CONCLUSION: While it is suspected that mating type-independent pheromone expression is the mechanism allowing for unisexual reproduction in Huntiella species, our results suggest that the transition to unisexuality may also have been associated with changes in the genes governing the pheromone pathway. While these results are specifically related to Huntiella, they provide clues leading to a better understanding of sexual reproduction and the fluidity of mating strategies in fungi more broadly.

Reverse Chemical Ecology Suggests Putative Primate Pheromones.

Pheromonal communication is widespread among living organisms, but in apes and particularly in humans there is currently no strong evidence for such phenomenon. Among primates, lemurs use pheromones to communicate within members of the same species, whereas in some monkeys such capabilities seem to be lost. Chemical communication in humans appears to be impaired by the lack or malfunctioning of biochemical tools and anatomical structures mediating detection of pheromones. Here, we report on a pheromone-carrier protein (SAL) adopting a "reverse chemical ecology" approach to get insights on the structures of potential pheromones in a representative species of lemurs (Microcebus murinus) known to use pheromones, Old-World monkeys (Cercocebus atys) for which chemical communication has been observed, and humans (Homo sapiens), where pheromones and chemical communication are still questioned. We have expressed the SAL orthologous proteins of these primate species, after reconstructing the gene encoding the human SAL, which is disrupted due to a single base mutation preventing its translation into RNA. Ligand-binding experiments with the recombinant SALs revealed macrocyclic ketones and lactones as the best ligands for all three proteins, suggesting cyclopentadecanone, pentadecanolide, and closely related compounds as the best candidates for potential pheromones. Such hypothesis agrees with the presence of a chemical very similar to hexadecanolide in the gland secretions of Mandrillus sphinx, a species closely related to C. atys. Our results indicate that the function of this carrier protein has not changed much during evolution from lemurs to humans, although its physiological role has been certainly impaired in humans.

Variation of Clasper Scent Gland Composition of Heliconius Butterflies from a Biodiversity Hotspot.

Male Heliconius butterflies possess two pheromone emitting structures, wing androconia and abdominal clasper scent glands. The composition of the clasper scent gland of males of 17 Heliconius and Eueides species from an overlapping area in Ecuador, comprising three mimicry groups, was investigated by GC/MS. The chemical signal serves as an anti-aphrodisiac signal that is transferred from males to females during mating, indicating the mating status of the female to prevent them from harassment by other males. In addition, the odour may also serve in predator defence. There is potential for convergence driven by mimicry, although, such convergence might be detrimental for species recognition of the butterflies within the mimicry ring, making mating more difficult. More than 500 compounds were detected, consisting of volatile, semi-volatile or non-volatile compounds, including terpenes, fatty acid esters or aromatic compounds. Several novel esters were identified by GC/MS and GC/IR data, microderivatisation and synthesis, including butyl (Z)-3-dodecenoate and other (Z)-3-alkenoates, 3-oxohexyl citronellate and 5-methylhexa-3,5-dienyl (E)-2,3-dihydrofarnesoate. The secretions were found to be species specific, potentially allowing for species differentiation. Statistical analysis of the compounds showed differentiation by phylogenetic clade and species, but not by mimicry group.