Backbone and side chain NMR assignments and secondary structure calculation of the pheromone binding protein3 of Ostrinia nubilalis, an agricultural pest.

Ostrinia nubilalis, also known as European Corn Borer (ECB), is a serious pest in Europe and North America, as well as in Central Asia and Northern Africa. It damages a variety of agricultural crops such as corn, oats, buckwheat, millet, and soybeans. causing annually at least one billion dollars in loss. The Ostrinia nubilalis pheromone-binding protein3 (OnubPBP3), preferentially expressed in the male moth antenna, has been implicated in the detection of the female-secreted pheromone blend during the mating process. Understanding the structure of and function of OnubPBP3, including the mechanism of pheromone binding and its release at the dendritic olfactory neuron (ORN), is essential if integrated pest management through sensory inhibition is to be achieved. We report here the backbone and side-chain resonance assignments of OnubPBP3 at pH 6.5 using various triple resonance NMR experiments on a 13C, 15N-labeled protein sample. The secondary structure of OnubPBP3 consists of six α-helices and an unstructured C-terminus based on backbone chemical shifts.

Sublethal Effects of Four Insecticides Targeting Cholinergic Neurons on Partner and Host Finding in the Parasitic Wasp Nasonia vitripennis.

Lethal and sublethal effects of pesticides on nontarget organisms are one of the causes of the current decline of many insect species. However, research in the past decades has focused primarily on pollinators, although other beneficial nontarget organisms such as parasitic wasps may also be affected. We studied the sublethal effects of the four insecticides acetamiprid, dimethoate, flupyradifurone, and sulfoxaflor on pheromone-mediated sexual communication and olfactory host finding of the parasitic wasp Nasonia vitripennis. All agents target cholinergic neurons, which are involved in the processing of chemical information by insects. We applied insecticide doses topically and tested the response of treated wasps to sex pheromones and host-associated chemical cues. In addition, we investigated the mating rate of insecticide-treated wasps. The pheromone response of females surviving insecticide treatment was disrupted by acetamiprid (≥0.63 ng), dimethoate (≥0.105 ng), and flupyradifurone (≥21 ng), whereas sulfoxaflor had no significant effects at the tested doses. Olfactory host finding was affected by all insecticides (acetamiprid ≥1.05 ng, dimethoate ≥0.105 ng, flupyradifurone ≥5.25 ng, sulfoxaflor ≥0.52 ng). Remarkably, females treated with ≥0.21 ng dimethoate even avoided host odor. The mating rate of treated N. vitripennis couples was decreased by acetamiprid (6.3 ng), flupyradifurone (≥2.63 ng), and sulfoxaflor (2.63 ng), whereas dimethoate showed only minor effects. Finally, we determined the amount of artificial nectar consumed by N. vitripennis females within 48 h. Considering this amount (∼2 µL) and the maximum concentrations of the insecticides reported in nectar, tested doses can be considered field-realistic. Our results suggest that exposure of parasitic wasps to field-realistic doses of insecticides targeting the cholinergic system reduces their effectiveness as natural enemies by impairing the olfactory sense. Environ Toxicol Chem 2023;42:2400-2411. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Electrophysiological and Behavioral Responses of Spotted Stem Borer, Chilo partellus, to Sex Pheromone Components and Their Blends.

The spotted stem borer, Chilo partellus (Lepidoptera: Crambidae) is the major insect pest of maize and sorghum crops during the rainy season. The pheromone released by adult virgin females has been reported to consist of (Z)-11-hexadecenal (Z11-16:Ald) as major component and (Z)-11-hexadecenol (Z11-16OH) as minor component. The latter has been reported to reduce the efficiency of major component to trap the male moths. We studied the electrophysiological and behavioral response of male C. partellus moths to Z11-16:Ald, Z11-16OH, their E-isomers (E)-11-hexadecenal and (E)-11-hexadecen-1-ol, and blends. Electroantennogram (EAG) studies revealed that male C. partellus antennae elicited significantly greater responses to both (Z)-11-hexadecenal and (Z)-11-hexadecenol compared to their respective E-isomers. Behavioral response studies through wind tunnel and cage bioassay showed that blends of Z11-16:Ald and Z11-16OH in the proportion of 100:100 and 100:95 elicited significantly higher responses from male moths. The attractiveness of these pheromone components and blends in field also revealed that traps baited with 100:100 proportion was most effective. Our studies clearly showed that the minor alcohol component Z11-16OH is important for enhancing attractiveness of the pheromone and provides a more effective blend for monitoring of this pest.

Chemical notes of tsetse fly mating.

Volatile pheromones offer a means to control flies that spread disease.

A volatile sex attractant of tsetse flies.

Tsetse flies transmit trypanosomes-parasites that cause devastating diseases in humans and livestock-across much of sub-Saharan Africa. Chemical communication through volatile pheromones is common among insects; however, it remains unknown if and how such chemical communication occurs in tsetse flies. We identified methyl palmitoleate (MPO), methyl oleate, and methyl palmitate as compounds that are produced by the tsetse fly Glossina morsitans and elicit strong behavioral responses. MPO evoked a behavioral response in male-but not virgin female-G. morsitans. G. morsitans males mounted females of another species, Glossina fuscipes, when they were treated with MPO. We further identified a subpopulation of olfactory neurons in G. morsitans that increase their firing rate in response to MPO and showed that infecting flies with African trypanosomes alters the flies' chemical profile and mating behavior. The identification of volatile attractants in tsetse flies may be useful for reducing disease spread.

Anthropogenic noise may impair the mating behaviour of the Shore Crab Carcinus Maenas.

Anthropogenic noise is a recent addition to the list of human-made threats to the environment, with potential and established negative impacts on a wide range of animals. Despite their economic and ecological significance, few studies have considered the impact of anthropogenic noise on crustaceans, though past studies have shown that it can cause significant effects to crustacean physiology, anatomy, and behaviour. Mating behaviour in crustaceans could potentially be severely affected by anthropogenic noise, given that noise has been demonstrated to impact some crustacean's ability to detect and respond to chemical, visual, and acoustic cues, all of which are vital in courtship rituals. To explore if noise has an impact on crustacean mating, we tested the responses of male green shore crabs (Carcinus maenas) from the southwest UK coast by exposing them to ship noise recordings while simultaneously presenting them with a dummy-female soaked in the female-sex pheromone uridine diphosphate (UDP) in an experimental tank setup (recording treatment: n = 15, control treatment: n = 15). We found a significant, negative effect of noise on the occurrence of mating behaviour compared to no noise conditions, though no significant effect of noise on the time it took for a crab to respond to the pheromone. Such effects suggest reproductive impairment due to anthropogenic noise, which could potentially contribute to decreased crustacean populations and subsequent ecological and economic repercussions. Given the findings of our preliminary study, more research should be undertaken that includes larger sample sizes, double blind setups, and controlled laboratory trials in order to more fully extrapolate the potential impact of noise on mating in the natural environment.

A male pheromone that improves the quality of the oogenic germline.

Pheromones exchanged by conspecifics are a major class of chemical signals that can alter behavior, physiology, and development. In particular, males and females communicate with potential mating partners via sex pheromones to promote reproductive success. Physiological and developmental mechanisms by which pheromones facilitate progeny production remain largely enigmatic. Here, we describe how a Caenorhabditis elegans male pheromone, ascr#10, improves the oogenic germline. Before most signs of aging become evident, C. elegans hermaphrodites start producing lower-quality gametes characterized by abnormal morphology, increased rates of chromosomal nondisjunction, and higher penetrance of deleterious alleles. We show that exposure to the male pheromone substantially ameliorates these defects and reduces embryonic lethality. ascr#10 stimulates proliferation of germline precursor cells in adult hermaphrodites. Coupled to the greater precursor supply is increased physiological germline cell death, which is required to improve oocyte quality in older mothers. The hermaphrodite germline is sensitive to the pheromone only during a time window, comparable in duration to a larval stage, in early adulthood. During this period, prereproductive adults assess the suitability of the environment for reproduction. Our results identify developmental events that occur in the oogenic germline in response to a male pheromone. They also suggest that the opposite effects of the pheromone on gamete quality and maternal longevity arise from competition over resource allocation between soma and the germline.

A closer look at sex pheromone autodetection in the Oriental fruit moth.

Female moths emit sex pheromone to attracts males, and although they are not attracted to their own sex pheromone, they appear to detect it as it affects their behavior. In order to elucidate the mechanism of pheromone "autodetection" we compared responses of olfactory receptor neurons (ORNs) of male and female Grapholita molesta, a species with reported pheromone autodetection. Two concentrations of the major (Z8-12:Ac) and minor (E8-12:Ac) sex pheromone components, a plant-volatile blend containing methyl salicylate, terpinyl acetate and (E)-ß-farnesene, and the male-produced hair-pencil (i.e., courtship) pheromone (ethyl trans-cinnamate) were tested in 45 male and 305 female ORNs. Hierarchical cluster analysis showed radically different peripheral olfactory systems between sexes that could be linked to their specific roles. In males 63% of the ORNs were tuned specifically to the major or minor female sex pheromone components, and 4% to the plant volatile blend, while the remaining 33% showed unspecific responses to the stimulus panel. In females 3% of the ORNs were specifically tuned to the male hair-pencil pheromone, 6% to the plant volatile blend, 91% were unspecific, and no ORN was tuned their own sex pheromone components. The lack of sex pheromone-specific ORNs in females suggests that they are not able to discriminate pheromone blends, and thus pheromone autodetection is unlikely in this species. We discuss our results in the context of the methodological limitations inherent to odor stimulation studies.

Pheromone Receptor Knock-Out Affects Pheromone Detection and Brain Structure in a Moth.

Sex pheromone receptors are crucial in insects for mate finding and contribute to species premating isolation. Many pheromone receptors have been functionally characterized, especially in moths, but loss of function studies are rare. Notably, the potential role of pheromone receptors in the development of the macroglomeruli in the antennal lobe (the brain structures processing pheromone signals) is not known. Here, we used CRISPR-Cas9 to knock-out the receptor for the major component of the sex pheromone of the noctuid moth Spodoptera littoralis, and investigated the resulting effects on electrophysiological responses of peripheral pheromone-sensitive neurons and on the structure of the macroglomeruli. We show that the inactivation of the receptor specifically affected the responses of the corresponding antennal neurons did not impact the number of macroglomeruli in the antennal lobe but reduced the size of the macroglomerulus processing input from neurons tuned to the main pheromone component. We suggest that this mutant neuroanatomical phenotype results from a lack of neuronal activity due to the absence of the pheromone receptor and potentially reduced neural connectivity between peripheral and antennal lobe neurons. This is the first evidence of the role of a moth pheromone receptor in macroglomerulus development and extends our knowledge of the different functions odorant receptors can have in insect neurodevelopment.

Male and female developmental temperature modulate post-copulatory interactions in a beetle.

Sexual selection theory has proven to be fundamental to our understanding of the male-female (sperm-egg) interactions that characterise fertilisation. However, sexual selection does not operate in a void and abiotic environmental factors have been shown to modulate the outcome of pre-copularory sexual interactions. Environmental modulation of post-copulatory interactions are particularly likely because the form and function of primary reproductive traits appears to be acutely sensitive to temperature stress. Here we report the effects of developmental temperature on female reproductive architecture and the interaction between male and female developmental temperature on the outcome of sperm competition in the bruchid beetle Callosobruchus maculatus. When females were reared at developmental temperatures above and below typical temperatures the bursa copulatrix (site of spermatophore deposition) were smaller and, were either shorter and broader (high temperatures) or longer and thinner (low temperatures) than those reared at intermediate temperatures. Males and females reared at low developmental temperatures were less likely to mate than those reared at higher temperatures. Where copulation occurred, females reared at the highest temperature copulated for longest, whilst males reared at the lowest temperature spent longer in copula. Male developmental temperature had a significant impact on the outcome of sperm competition: males reared at 17 °C were largely unsuccessful in sperm competition against control (27 °C) males, although some of the variation in the outcome of sperm competition was a product of the interaction between male and female developmental temperature. Our results demonstrate that male-female interactions that characterise pre- and post-copulatory outcomes are sensitive to developmental temperature and that plasticity in cryptic female preferences could lead to heterogeneous selection on the male reproductive phenotype.

Large-scale characterization of sex pheromone communication systems in Drosophila.

Insects use sex pheromones as a reproductive isolating mechanism to attract conspecifics and repel heterospecifics. Despite the profound knowledge of sex pheromones, little is known about the coevolutionary mechanisms and constraints on their production and detection. Using whole-genome sequences to infer the kinship among 99 drosophilids, we investigate how phylogenetic and chemical traits have interacted at a wide evolutionary timescale. Through a series of chemical syntheses and electrophysiological recordings, we identify 52 sex-specific compounds, many of which are detected via olfaction. Behavioral analyses reveal that many of the 43 male-specific compounds are transferred to the female during copulation and mediate female receptivity and/or male courtship inhibition. Measurement of phylogenetic signals demonstrates that sex pheromones and their cognate olfactory channels evolve rapidly and independently over evolutionary time to guarantee efficient intra- and inter-specific communication systems. Our results show how sexual isolation barriers between species can be reinforced by species-specific olfactory signals.

Hormonal Prostaglandin F2α Mediates Behavioral Responsiveness to a Species-Specific Multi-component Male Hormonal Sex Pheromone in a Female Fish.

Although hormonally-derived female sex pheromones have been well described in approximately a dozen species of teleost fish, only a few male sex pheromones have been characterized and the neuroendocrine underpinnings of behavioral responsiveness to them is not understood. Herein, we describe a study that addresses this question using the goldfish, Carassius auratus, an important model species of how hormones drive behavior in egg-laying teleost fishes. Our study had four components. First, we examined behavioral responsiveness of female goldfish and found that when injected with prostaglandin F2α (PGF2α), a treatment that drives female sexual receptivity, and found that they became strongly and uniquely attracted to the odor of conspecific mature males, while non-PGF2α-treated goldfish did not discern males from females. Next, we characterized the complexity and specificity of the male pheromone by examining the responsiveness of PGF2α-treated females to the odor of either mature male conspecifics or male common carp odor, as well as their nonpolar and polar fractions. We found that the odor of male goldfish was more attractive than that of male common carp, and that its activity was attributable to both its nonpolar and polar fractions with the later conveying information on species-identity. Third, we hypothesized that androstenedione (AD), a 19-carbon sex steroid produced by all male fish might be the nonpolar fraction and tested whether PGF2α-treated goldfish were attracted to either AD alone or as part of a mixture in conspecific water. We found that while AD was inactive on its own, it became highly attractive when added to previously unattractive female conspecific water. Lastly, in a test of whether nonhormonal conspecific odor might determine species-specificity, we added AD to water of three species of fish and found that while AD made goldfish water strongly attractive, its effects on other species holding water were small. We conclude that circulating PGF2α produced at the time of ovulation induces behavioral sensitivity to a male sex pheromone in female goldfish and that this male pheromone is comprised of AD and a mixture of body metabolites. Because PGF2α commonly mediates ovulation and female sexual behavior in egg-laying fishes, and AD is universally produced by male fishes as a precursor to testosterone, we suggest that these two hormones may have similar roles mediating male-female behavior and communication in many species of fish.

bric à brac controls sex pheromone choice by male European corn borer moths.

The sex pheromone system of ~160,000 moth species acts as a powerful form of assortative mating whereby females attract conspecific males with a species-specific blend of volatile compounds. Understanding how female pheromone production and male preference coevolve to produce this diversity requires knowledge of the genes underlying change in both traits. In the European corn borer moth, pheromone blend variation is controlled by two alleles of an autosomal fatty-acyl reductase gene expressed in the female pheromone gland (pgFAR). Here we show that asymmetric male preference is controlled by cis-acting variation in a sex-linked transcription factor expressed in the developing male antenna, bric à brac (bab). A genome-wide association study of preference using pheromone-trapped males implicates variation in the 293 kb bab intron 1, rather than the coding sequence. Linkage disequilibrium between bab intron 1 and pgFAR further validates bab as the preference locus, and demonstrates that the two genes interact to contribute to assortative mating. Thus, lack of physical linkage is not a constraint for coevolutionary divergence of female pheromone production and male behavioral response genes, in contrast to what is often predicted by evolutionary theory.

Sex peptide receptor mediates the post-mating switch in Helicoverpa armigera (Lepidoptera: Noctuidae) female reproductive behavior.

BACKGROUND: In Drosophila, the sex peptide receptor (SPR) in females to detect male sex peptide (SP) and trigger changes in female behavior including mating rejection and oviposition. In moths, the SPR had been identified and investigated the limited function by using RNA interference (RNAi). However, the detailed function of SPR after a normal mating in moths, especially the role in female sexual receptivity, remains to be elucidated. RESULTS: In this study, we use Helicoverpa armigera, an economically important pest worldwide, as a case study to verify the function of SPR in vivo by CRISPR/Cas9 gene editing system. Analyzing the post-mating behaviors in mutant females, we investigated that HarmSPR could mediate the long-term sexual receptivity (remating, sex pheromone production and calling behavior) switch in female, but not affect the short-term sexual receptivity and the duration time of remating. The oviposition of mated HarmSPR-/- females was significantly decreased by ~70% comparing to wild-type (WT) mated females and there were more eggs staying in the ovary of mutant females. SPR also showed effect on female longevity, which is that the mated HarmSPR-/- females showed longer longevity than mated WT females. CONCLUSION: The results detailedly demonstrated the functional role of SPR in female post-mating behaviors and we could propose a pest control strategy in which developing an antagonist of SPR to reduce the oviposition of female. © 2021 Society of Chemical Industry.

Limoniic Acid - Major Component of the Sex Pheromones of the Click Beetles Limonius canus and L. californicus.

Wireworms, the larvae of click beetles (Coleoptera: Elateridae), are soil-dwelling insect pests inflicting major economic damage on many types of agricultural crops worldwide. The objective of this work was to identify the female-produced sex pheromones of the Pacific Coast wireworm, Limonius canus LeConte, and the sugarbeet wireworm, L. californicus (Mannerheim) (Coleoptera: Elateridae). Headspace volatiles from separate groups of female L. canus and L. californicus were collected on Porapak Q and analyzed by gas chromatography with electroantennographic detection (GC-EAD) and GC-mass spectrometry. GC-EAD recordings revealed strong responses from male L. canus and male L. californicus antennae to the same compound, which appeared below GC detection threshold. The structure of this candidate pheromone component was deduced from the results of micro-analytical treatments of extracts, retention index calculations on four GC columns, and by syntheses of more than 25 model compounds which were assessed for their GC retention characteristics and electrophysiological activity. The EAD-active compound was identified as (E)-4-ethyloct-4-enoic acid, which we name limoniic acid. In field experiments in British Columbia and Alberta, Canada, traps baited with synthetic limoniic acid captured large numbers of male Limonius click beetles, whereas unbaited control traps captured few. Compared to traps baited with the analogue, (E)-5-ethyloct-4-enoic acid, traps baited with limoniic acid captured 9-times more male L. californicus, and 6.5-times more male western field wireworms, L. infuscatus Motschulsky, but 2.3-times fewer male L. canus. Limoniic acid can now be developed for detection, monitoring and possibly control of L. californicus, L. infuscatus and L. canus populations.

Bioactive Male-Produced Volatiles from Anastrepha obliqua and their Role in Attraction of Conspecific Females.

The study of insect semiochemicals, especially pheromones, is of fundamental importance for the development of strategies for controlling agricultural pests. In this study, volatile compounds involved in the communication between males and females of the fruit fly, Anastrepha obliqua (Diptera: Tephritidae), for mating purposes were characterized to develop attractant formulations for females of this species. Extracts containing volatile compounds released by males of A. obliqua were obtained by the dynamic headspace technique and analyzed by gas chromatography coupled with an electroantennographic detector (GC-EAD) and gas chromatography coupled with mass spectrometry (GC-MS). Twenty-one volatile compounds were identified in the aeration extracts of males. Five of them caused EAD responses from the antennae of females: 1-heptanol, linalool, (Z)-3-nonen-1-ol, (E,Z)-3,6-nonadien-1-ol, and (Z,E)-α-farnesene. Six synthetic mixtures of these compounds, including the five-component blend and all possible four-component blends, were formulated in a biopolymer and used in behavioral bioassays conducted in the laboratory arena with conspecific virgin females. One blend of 1-heptanol, linalool, (Z)-3-nonen-1-ol, and (Z,E)-α-farnesene attracted more females than the collection of volatiles from virgin males used as control. The other mixtures were as attractive to A. obliqua females as the control treatment. This study indicates potential for use of these compounds in monitoring and control strategies for this pest.

Deployment of Aggregation-Sex Pheromones of Longhorned Beetles (Coleoptera: Cerambycidae) Facilitates the Discovery and Identification of their Parasitoids.

Longhorned beetles (Coleoptera: Cerambycidae) include many species that are among the most damaging pests of managed and natural forest ecosystems worldwide. Many species of cerambycids use volatile chemical signals (i.e., pheromones) to locate mates. Pheromones are often used by natural enemies, including parasitoids, to locate hosts and therefore can be useful tools for identifying host-parasitoid relationships. In two field experiments, we baited linear transects of sticky traps with pheromones of cerambycid beetles in the subfamily Cerambycinae. Enantiomeric mixtures of four linear alkanes or four linear alkanes and a ketol were tested separately to evaluate their attractiveness to hymenopteran parasitoids. We hypothesized that parasitoids would be attracted to these pheromones. Significant treatment effects were found for 10 species of parasitoids. Notably, Wroughtonia ligator (Say) (Hymenoptera: Braconidae) was attracted to syn-hexanediols, the pheromone constituents of its host, Neoclytus acuminatus acuminatus (F.) (Coleoptera: Cerambycidae). Location and time of sampling also significantly affected responses for multiple species of parasitoids. These findings contribute to the basic understanding of cues that parasitoids use to locate hosts and suggest that pheromones can be used to hypothesize host relationships between some species of cerambycids and their parasitoids. Future work should evaluate response by known species of parasitoids to the complete blends of pheromones used by the cerambycids they attack, as well as other odors that are associated with host trees of cerambycids.

Female-Based Patterns and Social Function in Avian Chemical Communication.

Much of the growing interest in avian chemical signals has focused on the role of kin recognition or mate attraction, often with an emphasis on males, with uropygial gland secretions perhaps providing information about an individual's identity and quality. Yet, data collected to date suggest sexual dimorphism in uropygial glands and secretions are often emphasized in female, rather than in male birds. That is, when a sexual difference occurs (often during the breeding season only), it is the female that typically exhibits one of three patterns: (1) a larger uropygial gland, (2) a greater abundance of volatile or semi-volatile preen oil compounds and/or (3) greater diversity of preen oil compounds or associated microbes. These patterns fit a majority of birds studied to date (23 of 30 chemically dimorphic species exhibit a female emphasis). Multiple species that do not fit are confounded by a lack of data for seasonal effects or proper quantitative measures of chemical compounds. We propose several social functions for these secretions in female-based patterns, similar to those reported in mammals, but which are largely unstudied in birds. These include: (1) intersexual advertisement of female receptivity or quality, including priming effects on male physiology, (2) intrasexual competition, including scent marking and reproductive suppression or (3) parental behaviors, such as parent-offspring recognition and chemical protection of eggs and nestlings. Revisiting the gaps of chemical studies to quantify the existence of female social chemosignals and any fitness benefit(s) during breeding are potentially fruitful but overlooked areas of future research.

Small molecule signals mediate social behaviors in C. elegans.

The last few decades have seen the structural and functional elucidation of small-molecule chemical signals called ascarosides in C. elegans. Ascarosides mediate several biological processes in worms, ranging from development, to behavior. These signals are modular in their design architecture, with their building blocks derived from metabolic pathways. Behavioral responses are not only concentration dependent, but also are influenced by the current physiological state of the animal. Cellular and circuit-level analyses suggest that these signals constitute a complex communication system, employing both synergistic molecular elements and sex-specific neuronal circuits governing the response. In this review, we discuss research from multiple laboratories, including our own, that detail how these chemical signals govern several different social behaviors in C. elegans. We propose that the ascaroside repertoire represents a link between diverse metabolic and neurobiological life-history traits and governs the survival of C. elegans in its natural environment.

How to maintain underground social relationships? Chemosensory sex, partner and self recognition in a fossorial amphisbaenian.

Maintaining social relationships depends on the ability to recognize partners or group members against other individuals. This is especially important in animals with relatively stable social groups. The amphisbaenian Trogonophis wiegmanni is a semi blind fossorial reptile that spends its entire life underground where it interacts with mates and social partners. In this environment, visual cues are limited. Chemosensory cues may rather allow conspecific social and partner recognition. We recorded the number of tongue-flick (TF) rates of T. wiegmanni amphisbaenians to scents of both sexes with different pairing social bonds (familiar vs. unfamiliar) presented on cotton swabs to test discrimination of social groups. As seen from a rise in the number of TFs, males discriminated unfamiliar females from unfamiliar males. This suggests that chemical cues may be used by males to locate new mates. In contrast, females detected scent of unfamiliar conspecifics, but did not show sex discrimination. Both males and females discriminated the scent of an individual with which they had formed a pair bond from an unfamiliar individual of the same sex as the partner. Also, males, but not females, were capable of self-recognition, suggesting that scent marks of males in home ranges may provide individual information in intrasexual relationships. We conclude that conspecific discrimination based on chemical cues may allow the maintenance of social relationships and relatively stable pairs in fossorial reptiles inhabiting visually restricted environments.