Beyond their impressive repertoire of camouflage as the predominant primary defensive strategy, stick and leaf insects (Phasmatodea) can actively defend themselves chemically with deterrent substances emitted from a pair of prothoracic repellent glands. The Vietnamese Prickly Stick Insect Neohirasea catbaensis emits a conspicuous secretion when disturbed, often described as smoky, which constitutes a unique characteristic not known from any other stick and leaf insect. Here we investigate the anatomy and the chemical components of the repellent glands in this species. Via micro-computed tomography (µ-CT), we observed comparatively small sac-like glands with no ejaculatory ducts present. The anatomy of the repellent glands of both male and female is described in detail. By applying gas chromatography (GC) in combination with high-resolution accurate mass spectrometry (HRAM-MS), we found 4-vinylphenol as the major component of the secretion, accompanied by 2-methoxy-4-vinylphenol and eugenol as minor constituents. This is the first time that these molecules have been identified as repellent substances from an animal. We discuss these findings in relation to the chemical diversity and ecological function of phasmatodean repellent secretions.
Bark beetles are among the most important invasive pests affecting pine forestry worldwide, yet the chemical ecology of many introduced species remains largely unknown. Cyrtogenius luteus is an invasive scolytine established in pine plantations in South America, for which no pheromone had previously been identified. Here, we report the identification and biological evaluation of a male-associated aggregation pheromone in C. luteus. Volatiles from male-infested, female-infested, and unattacked Pinus taeda wood were collected and analyzed by thermal desorption GC–MS. A compound consistently detected only in male-infested pine wood was identified as p-mentha-1,8-dien-4-ol through comparison with a synthetic standard. In Y-olfactometer assays, both males and females were attracted to volatiles from unattacked pine relative to clean air and showed a significant preference for male-attacked pine over unattacked pine, as indicated by both first choice and residence time. Field trials with synthetic p-mentha-1,8-dien-4-ol confirmed its biological activity: cross-vane traps baited with a combination of synthetic p-mentha-1,8-dien-4-ol and host attractants increased adult captures more than 20-fold, in comparison with the conventional host-attractant baited traps. Males and females were captured in similar proportions. Together, the male-specific occurrence of p-mentha-1,8-dien-4-ol, the attraction of both sexes to male-attacked host material, and the response of both sexes to the synthetic compound in the field, identify the racemate of this oxygenated monoterpene as a male-aggregation pheromone of C. luteus. This is the first pheromone structure reported for the species and provides new insight into the chemical ecology of an invasive bark beetle established in South American pine plantations.
Three different diastereomers of 6-ethyl-3,5-dimethyltetrahydro-2H-pyran-2-one (henceforth lactone) were identified from male-specific volatiles produced by the neotropical cerambycid beetles (subfamily Lamiinae) Atrypanius lignarius (Bates), Oedopeza umbrosa (Germar), and Oreodera glauca glauca (L.). Atrypanius lignarius produced one of the enantiomers of the (3R*,5S*,6S*)-diastereomer, whereas O. umbrosa produced the (3S,5S,6R)-lactone, and O. glauca glauca produced the (3S,5R,6S)-lactone. In field bioassays, the O. umbrosa (3S,5S,6R)-lactone attracted only conspecific adults of both sexes. The racemic (3R*,5S*,6S*)-lactone was strongly attractive to A. lignarius, while also eliciting significant cross-attraction in O. umbrosa and also the lamiine cerambycid Aegomorphus jaspideus (Germar). Although O. glauca glauca was not captured during the trials, possibly because we were unable to carry out bioassays where it had originally been captured, the racemic (3S*,5R*,6S*)-lactone attracted the congener Oreodera quinquetuberculata (Drapiez), as well as A. lignarius, A. jaspideus, and O. umbrosa. These results suggest that the 6-ethyl-3,5-dimethyltetrahydro-2H-pyran-2-one structural motif, which attracted species of three tribes, may be widely shared within the Lamiinae, with species exploiting different stereoisomers to provide some degree of species specificity to their signals.
For the control of pests by mating disruption (MD), passive pheromone dispensers are now widely used and have proved their efficacy. Although the release rate of dispensers and its change over time are usually well assessed, the airborne pheromone concentrations generated in a treated area are generally unknown. This hinders the development and optimisation of the treatment by MD. The present work reports the results of a field campaign of measurements carried out with the aim of quantifying airborne concentrations of dodecyl acetate released by passive dispensers distributed over a covered crop area. Dodecyl acetate is used as a model pheromone due to its low reactivity in the air allowing the release and dispersion processes to be characterized. The relationship between emissions and ambient air concentrations was investigated using both an empirical approach and an atmospheric dispersion model. Ambient air concentrations in the crop area were predicted by combining a previously developed emission model with temperature and wind speed data. The measured airborne concentrations ranged from 16 to 113 ng m−3 depending on the location of sampling and the release rate of dispensers at the time of sampling. The treatment-level concentrations persisted all along the sampling period for at least 44 days, while temporal changes in airborne concentration were mainly driven by the release rate of dispensers and its dependence on temperature.
Insects communicate identity, health, and reproductive state through diverse chemicals secreted from multiple glands and production sites. Although these exocrine sites often contain a similar profile of compounds, they are usually studied in isolation. Here, we tested the hypothesis that changes in the identity and abundance of chemical signals from different exocrine sites within the same individual are coordinated, suggesting they may be governed by shared regulatory processes. To do so, we quantified the cuticular lipids and the glandular secretion of three exocrine glands in bumble bee queens across three key life stages: unmated newly-emerged queen (gynes), young founders shortly after nest initiation and old founders towards the end of the life cycle. We found that as queens aged and transitioned into reproduction, they produced hydrocarbons and esters with shorter chain length and shifted toward a greater investment in alkenes over alkanes across several exocrine sites. Terpenoid-, acetate- and wax-esters were produced across multiple exocrine sites with partially overlapping patterns. These remarkably coordinated shifts across glands that differ in both function and anatomical origin suggest that the overall chemical profile of an insect is coordinated across glandular sites and may convey more biologically relevant information than any single glandular secretion alone. Insect chemical communication is likely shaped by a shared regulatory architecture or coordinated responses to environmental conditions, highlighting a unifying mechanism underlying complex social signals.
Visually conspicuous sexually dimorphic traits such as body size, coloration, weapons, and ornaments are less effective in nocturnal mammals, favoring the evolution of signals in alternative sensory modalities. Acoustic and chemical signals, in contrast, function well in low-light environments, and chemical cues in particular may act as honest indicators of individual quality, as physiological condition and immune responses can alter odor profiles. Reproductively active male fringe-lipped bats, Trachops cirrhosus, exhibit a sexually dimorphic forearm crust characterized by a strong odor and seasonal enlargement coincident with female fertility. Crust size is positively correlated with testosterone levels, and males with larger crusts are more frequently found roosting with multiple females, suggesting a role in sexual signaling. Previous work identified six major chemical components of the crust. Here, we evaluated inter-individual variation in both chemical composition and compound concentrations of the crust. Gas chromatography – mass spectrometry (GC–MS) analyses revealed twelve volatile organic compounds detected in crust samples, with individual males containing an average of 7 ± 3 chemical compounds. Chemical composition and chemical concentrations varied substantially among individuals, indicating that male crusts are not composed by a determined set or standardized ratios of compounds. Instead, chemical profiles were highly individualized. Crust size was positively associated with both chemical diversity and compound abundance. Larger crusts (> 10 mg) contained more compounds and higher overall concentrations than smaller crusts. Cholesterol and Cholest-7-en-3-ol were the most consistently detected compounds across males. Five of six previously reported compounds were confirmed. Concentrations of individual compounds differed by several orders of magnitude among males, and some compounds were absent in certain individuals. These results indicate pronounced individual chemical variability in male forearm crusts, supporting the hypothesis that this trait functions as a complex and individualized chemical signal potentially relevant to mate choice and sexual selection.
Spotted-wing drosophila, Drosophila suzukii (Diptera: Drosophilidae), is a destructive pest of ripening berries and stone fruits. D. suzukii is attracted to ripening fruit volatiles, allowing it to occupy a distinct ecological niche from other Drosophila species. Ripening fruit volatiles act as relatively specific cues, which can be leveraged for pest management. Here, we tested whether volatiles from damaged host fruit serve as attractive adult host-finding cues. Using multivariate analysis, we characterized the headspace of mechanically damaged or undamaged blueberries and strawberries, finding distinct volatile organic chemical profiles. In no-choice assays, D. suzukii adults responded similarly between damaged and undamaged treatments at 24 and 48 h; however, when presented with a choice, adults were more strongly attracted to damaged than undamaged fruit volatiles. Adults exhibited a preference for volatiles from an individual damaged fruit over those from many undamaged fruit, despite higher total volatile release by the undamaged treatment as quantified by chromatography. In oviposition assays, females held in constant darkness laid significantly more eggs on damaged than undamaged fruit, whereas those exposed to a light cycle showed no preference. Infestation by early-instar D. suzukii induced a volatile profile distinct from mechanical damage, which attracted females but not males. Together, these results suggest that adult D. suzukii exhibit a preference for the specific ratio of volatiles from mechanically damaged fruit, while additional sensory inputs likely modulate subsequent behavioral decisions. Practically, damaged fruit volatile profiles identified here provide a chemical basis for developing more attractive lures to improve D. suzukii control and monitoring.
Pheromone-mediated behaviors are crucial for the survival and reproduction of Drosophila melanogaster, governing activities such as courtship, mating, and aggression. While many studies have focused on short-range interactions involving direct contact between flies, the role of olfactory cues alone in this attraction remains poorly understood. In this study, we investigated the behavioral responses of virgin male and female D. melanogaster to conspecific headspace using a Y-maze. We tested fly responses under various environmental conditions (e.g., in the presence or absence of food odors and with or without social isolation of the tested animals before the experiments). Finally, we repeated some of the experiments in a still-air trap assay. Our results suggest that fly odors may play only a limited role in mate-seeking behavior in D. melanogaster.
Haematobia irritans (L.) (Diptera: Muscidae), commonly known as the horn fly, is one of the most economically significant ectoparasites of cattle worldwide, causing substantial losses in meat, milk, and hide production. Its management has historically relied on broad-spectrum insecticides, whose overuse has driven the emergence of resistant populations and raised environmental concerns, emphasizing the urgent need to alternative, ecofriendly control strategies. A key yet underexplored aspect of horn fly biology is behind one of its characteristic behaviors, oviposition behavior: gravid females deposit eggs almost exclusively in very fresh cattle dung, a preference that diminishes rapidly with time post-defecation. Although volatile organic compounds (VOCs) from dung are known to mediate this behavior, the biological origin of these chemical cues remains poorly characterized. This review proposes that microbial volatile organic compounds (mVOCs) produced by the cattle dung microbiota-particularly anaerobic bacteria derived from the bovine rumen-are major elicitors of horn fly oviposition behavior, constituting a transkingdom chemical signaling system. By systematically cross-referencing VOCs reported in fresh cattle dung with compounds known to elicit electrophysiological and behavioral responses in H. irritans, we identified four key semiochemicals of probable microbial origin: phenol, p-cresol, indole, and α-pinene, produced by bacterial families including Enterobacteriaceae, Clostridiaceae, Paenibacillaceae, and Lactobacillaceae. The temporal decline in dung attractiveness is proposed to reflect the succession of the microbial community from anaerobic to aerobic dominance, with a concomitant shift in the mVOC profile. This hypothesis is further supported by the observation that the early developmental stages of H. irritans harbor an anaerobe-dominated microbiota. This framework may provide opportunities for the development of environmentally friendly blends for horn fly management.
Herbivorous insects must identify suitable host plants for egg-laying and reliably reject unsuitable ones, but whether non-host plants are actively rejected through deterrent cues, or simply fail to trigger host-recognition signals, remains unclear. We addressed this question in the swallowtail butterfly Papilio xuthus by combining oviposition behavior trials with electrophysiological recordings from taste sensilla on the female forelegs. Females primed with a Citrus (host plant) extract laid eggs on a plain water control 51.5
Herbicide exposure can alter plant secondary metabolism with consequences for insect host selection, yet its effects on volatile organic compound (VOC) signaling remain poorly understood. Red maple (Acer rubrum L.) is a widely planted ornamental tree frequently attacked by flatheaded borers (Chrysobothris spp.), and herbicide injury has been associated with increased borer activity. We investigated how trunk application of two commercial herbicides, one contact herbicide (pelargonic acid) and one systemic herbicide (glyphosate), influence VOC emissions and associated nutritional and defensive chemistry in two red maple cultivars differing in susceptibility to Chrysobothris. Across a four-week field experiment, foliar VOCs were quantified alongside sugars, polyphenols, and tannins, and related to borer oviposition attempts and larval success. Herbicide exposure induced compound-specific and cultivar-dependent VOC responses, with the susceptible cultivar (‘Brandywine’) exhibiting greater temporal variability and elevated emissions of select monoterpenes, including β-myrcene. These VOC shifts coincided with increased foliar polyphenols and trunk sugar concentrations. In contrast, the less susceptible cultivar (‘Autumn Blaze’) showed comparatively stable chemical profiles across treatments. Oviposition attempts and larval occurrence were concentrated on herbicide-treated ‘Brandywine’ trees, particularly following contact herbicide exposure. Together, these results suggest that herbicide-induced modulation of VOC signaling, coupled with changes in nutritional and defensive chemistry, may influence adult host selection, larval performance following oviposition, and ultimately enhance host suitability and apparency to Chrysobothris.
The spotted lanternfly (Lycorma delicatula), an invasive planthopper in the United States, threatens vineyards by reducing yield and compromising plant health, yet grapevine immune responses to its feeding remain poorly understood. This study evaluated systemic grapevine responses to spotted lanternfly feeding under varying insect densities and feeding durations. Potted Vitis vinifera cultivar Cabernet Franc vines were infested with 0, 5, 10, or 15 adults per vine for 1, 3, 5, 7, 14, 21, or 29 days, using separate plants at each time point. Leaf tissue was analyzed for chlorophyll content, polyphenol oxidase (PPO) and peroxidase (POX) activity, protein content, total phenolics, and condensed tannins. Bioassays assessed spotted lanternfly survival and weight gain on previously infested plants. Chlorophyll content declined after 21–29 days in vines infested with 15 insects. PPO activity increased throughout the experiment (except on day 7) under infestations of 10–15 insects, whereas POX activity rose after 7 days but declined after 29 days of feeding. Leaf protein content increased in vines infested with 15 insects after 7 and 29 days, and phenolic concentrations increased consistently from 7 to 29 days in vines infested with 10–15 insects. Condensed tannin concentrations were unaffected by density treatments. Bioassays showed reduced survival and weight gain of lanternflies when fed on vines previously infested with 15 insects for 29 days. Collectively, these results suggest that prolonged, high-density spotted lanternfly feeding induces coordinated, time- and density-dependent systemic grapevine defenses associated with reduced host suitability. Future studies should identify the mechanisms underlying these responses and their persistence across grapevine cultivars.
We describe the identification, synthesis, and field testing of an aggregation-sex pheromone produced by male Acalolepta aesthetica (Olliff 1890) (Coleoptera: Cerambycidae: Lamiinae), a cerambycid beetle native to Australia that has recently become established in Hawai‘i. Comparing extracts of headspace volatiles from both sexes revealed that males emitted two sex-specific volatiles, (3E,5Z)-octadien-2-ol and (3E,5Z)-octadien-2-one. Both compounds were synthesized and their bioactivity was tested in field bioassays. Only (3E,5Z)-octadien-2-ol was attractive to beetles of both sexes in these trials, and the corresponding ketone appeared to antagonize attraction when combined with the alcohol. In a dose-response study, traps baited with all doses of (3E,5Z)-octadien-2-ol except 33.3 mg captured more beetles than solvent-control traps, with no significant differences among the effective doses. In a subsequent field trial, beetle capture did not differ between 100 mg of a racemic blend and the insect-produced (R)-enantiomer of this compound, indicating that the unnatural (S)-enantiomer is not antagonistic. No other cerambycid species were captured during our field trials. The structural motif of (3E,5Z)-octadien-2-ol is unique among pheromones identified from lamiines to date. Together, our findings provide a foundation for developing pheromone-based tools to detect, delimit and potentially manage A. aesthetica.
In evolutionary terms, chemical senses are the oldest sensory modality and play key roles in ecological functions such as orientation, foraging, and socio-sexual communication. Chemical perception encompasses olfaction, taste, and chemesthesis, and has been widely studied across vertebrates. However, these senses remain poorly documented in marine mammals, particularly cetaceans. The transition from land to water profoundly reshaped cetacean sensory systems, and for decades they were considered incapable of chemoreception, implying a complete regression. Recent studies challenge this view, suggesting a more complex and incomplete regression. In this review, we synthesize current knowledge from different perspectives, using an integrative framework. First, we examine the anatomical basis of chemoreception, identifying relevant structures enabling this process and their development across early life stages. Second, we explore genetic and molecular foundations, focusing on genes underlying the development and function of chemical receptors. Third, we review behavioural evidence, highlighting experimental studies on cetacean responses to chemical cues in ecological contexts such as predator–prey interactions, foraging, and social behaviour. This approach allows us to characterize and quantify chemosensory regression in cetaceans and confirms divergent evolutionary trajectories between odontocetes and mysticetes. We also identify key unresolved questions: (1) cetaceans appear to use social chemical signals, but the mechanisms remain unknown; (2) reliance on taste is still debated, with conflicting findings; (3) the ontogeny of chemical senses remains poorly understood; and (4) alternative chemical modalities such as chemesthesis have been largely overlooked despite hypothesis on their potential ecological importance.
Japanese beetle, Popillia japonica Newman, is an invasive species that attacks many field and ornamental crops. Intercropping soybean with sorghum is suggested as a strategy to reduce the abundance of P. japonica throughout soybean fields. The mechanism by which this affects P. japonica is unknown but may arise from a difference in the host plant compounds emitted by the intercropped plants because P. japonica responds to olfactory cues. In this study, we investigated a semiochemical-based mechanism for the behavioral response of P. japonica to intercropping soybean with sorghum by (1) evaluating P. japonica behavioral responses to solvent extracts from monocropped soy, intercropped soy, and a mixture of soy and sorghum extracts in no-choice video-tracking and preference in a release-recapture dual-choice assay to host stimuli from plants; and (2) applying solid-phase microextraction (SPME) in conjunction with gas chromatography coupled with mass spectrometry (GC-MS) to characterize the semiochemical profiles of each extract. We found unique semiochemical profiles among our treatments, with 1-octen-3-ol and 1-octanol characteristic of monocropped soybean and dodecane-1-iodo primarily in sorghum extracts. Nevertheless, our treatments did not significantly affect movement or orientation by P. japonica compared to controls, nor did conspecifics exhibit a significant preference for any of the treatments in a dual-choice assay at a local scale. Therefore, if intercropping soybean with sorghum significantly affects the behavior of P. japonica in the field, it may occur at a different scale, or in response to non-olfactory stimuli (e.g., visual, habitat, or landscape cues) rather than semiochemical cues.
Quercus aquifolioides is an ecologically important oak species on the Qinghai–Tibet Plateau that experiences significant damage from Soritia leptatina. To clarify the behavioral response of S. leptatina to leaf volatiles of Q. aquifolioides, we analyzed leaf-associated volatile organic compounds using solid-phase microextraction coupled with gas chromatography–mass spectrometry (SPME–GC–MS), measured electrophysiological and behavioral responses of adult S. leptatina to selected leaf volatiles using electroantennography (EAG) and Y-tube olfactometer assays, and evaluated the field-trapping potential of bioactive volatiles. The detected leaf-associated profile included C6 green-leaf-volatile derivatives, long-chain fatty acids and fatty-acid esters, terpenes, and aromatic compounds. Unmated male and female adults showed strong EAG responses to leaf acetate, palmitic acid, and myristic acid, with the highest response recorded for 10 µg/µL leaf acetate (0.97 mV). Y-tube assays indicated that leaf acetate, myristic acid, and naphthalene elicited significant behavioral attraction at specific concentrations (P < 0.05), whereas palmitic acid did not show significant attraction in this assay. Field trapping experiments further showed that palmitic acid and myristic acid increased adult captures, with 100 µg/µL myristic acid producing the highest catch (137 individuals). These results suggest that myristic acid and palmitic acid may be useful for monitoring, early warning, and environmentally friendly management of S. leptatina, although their behavioral roles should be interpreted according to assay type and concentration.
Fall armyworm (Spodoptera frugiperda) has emerged as a major pest of maize across the Americas, Africa and Asia. Current management relies heavily on synthetic insecticides, which are environmentally unsustainable. However, pheromone-based monitoring, a sustainable and highly promising alternative, often shows variable field efficacy, highlighting the need to identify host plant volatiles that can enhance pheromone-mediated attraction. In this study, we demonstrate that specific maize-derived volatiles play a key role in influencing male behavioral responses. Among the identified compounds, benzaldehyde, myrcene, (Z)-3-hexenyl acetate, nonanal, and ocimene elicited male antennal responses. In laboratory bio-assays, benzaldehyde, (Z)-3-hexenyl acetate, and nonanal induced significantly higher male preference. Field application of ternary pheromone blend consisting of (Z)-9-tetradecenyl acetate, (Z)-7-dodecenyl acetate, and (Z)-11-hexadecenyl acetate at (0.88, 0.01, and 0.11 mg, respectively), supplemented with benzaldehyde and nonanal (0.1 and 0.2 mg, respectively), or nonanal alone at 0.2 mg, significantly enhanced trap captures. In contrast, (Z)-3-hexenyl acetate did not alter trap efficacy, whereas higher doses of either volatile reduced trap captures. These findings provide evidence that host plant volatiles particularly nonanal, and benzaldehyde can synergize with sex pheromone compounds of S. frugiperda, offering a promising strategy to improve semiochemical-based monitoring and integrated pest management.
Cryptic differentiation poses a significant challenge to taxonomy, particularly in morphologically conserved lineages whose divergence is not immediately apparent. In eusocial insects like ants, where species boundaries are often maintained through chemical rather than morphological differentiation, integrative methodologies are essential for resolving taxonomic complexity. Here, we present compelling evidence for the recent divergence of African carpenter ants initially identified as Camponotus maculatus into two distinct sub-populations. This divergence is supported through an integrative approach combining chemical, behavioral, and genetic analyses. Most strikingly, we identify two sharply contrasting chemotypes characterized by categorically different cuticular hydrocarbon (CHC) profiles that unambiguously separate the investigated ant populations. Concordantly, behavioral assays reveal that worker ants consistently exhibit aggression toward individuals with opposing chemotypes, while displaying affiliative behaviors toward shared chemotypes irrespective of colony affiliation. Genetic barcoding further corroborates these findings, indicated by phylogenetic clusters largely corresponding with the two chemotypes. Our results highlight the primary role of chemical differentiation within morphologically indistinguishable populations to resolve cryptic sub-population structures. These findings also provide valuable insights into potential early-stage chemical differentiation mechanisms in eusocial insect populations.
Plants available to wild herbivores, especially browsers, often contain plant secondary metabolites (PSMs). Herbivores have evolved behavioral, physiological, and microbial mechanisms for avoiding and detoxifying PSMs. The detoxification limitation hypothesis suggests that herbivores can reduce toxicity by consuming a mixture of PSMs to avoid overloading a particular detoxification pathway. Although this hypothesis has been examined for smaller mammalian hindgut-fermenters, less is known about responses to PSM mixtures in wild ruminants. To assess the role of host and microbial responses to PSM composition, we used controlled feeding trials to measure voluntary dry matter and PSM intake, urinary excretion of glucuronic acid (GA, a byproduct of PSM detoxification through conjugation), and the diversity and relative abundance of gastrointestinal bacterial families in the feces of two species of captive-raised deer (Odocoileus hemionus, O. virginianus). Deer were fed five mixtures of four purified PSMs that included two same-chemical class mixtures, two different-class mixtures, and one 4-way mixture of all chemicals. Overall, we found that PSM composition had minimal effect on intake, that GA was a consistent physiological biomarker of PSM intake regardless of PSM composition, and that dietary phenolics may influence microbial communities more than monoterpenes. Our results partially conformed to the detoxification limitation hypothesis, where deer consumed less of one same-class mixture (monoterpenes) than different-class mixtures. Our results point to the complexity of the interplay between different behavioral, physiological, and microbial mechanisms that can mediate the consequences of PSMs.
