Sulforaphane (SFN), an isothiocyanate produced by the mustard oil bomb defense system of cruciferous plants, is a natural inhibitor of class I and II histone deacetylases (HDACs). We previously showed that dietary SFN inhibits HDAC activity, delays larval development, and alters the expression of more than 1,600 genes in the agricultural pests Spodoptera exigua and Trichoplusia ni. Here we investigated the mechanisms underlying these responses and whether their effects extend across generations. RNA-seq analyses revealed that SFN rapidly disrupted the normal developmental progression of gene expression in both somatic and germline tissues. ATAC-seq analyses indicated that these transcriptional changes in S. exigua were accompanied by widespread alterations in chromatin accessibility. In this species, we detected the suppression of genes involved in energy metabolism, consistent with the observed developmental delays. We observed that many SFN-induced changes in chromatin accessibility and gene expression persisted in the offspring of exposed insects despite the offspring never consuming SFN themselves. These findings demonstrate that SFN acts through chromatin-associated mechanisms to alter transcriptional programs during development, with intergenerational effects. We propose that SFN, and perhaps other HDAC inhibitors produced by plants in nature, may act as an "epigenetic weapon" with the potential to influence herbivore gene expression across generations.

Araecerus fasciculatus (Coleoptera: Anthribidae) is a cosmopolitan insect that infests various stored grains, including cocoa beans, producing significant economic losses. Cocoa beans are marketed under two processing methods: washed or fermented. However, during storage they may become infested by insects. In this study, the olfactory response of A. fasciculatus to volatile compounds emitted by unfermented washed, fermented, and infested cocoa beans was evaluated. Volatile compounds from cocoa beans were collected using dynamic aeration and analyzed by gas chromatography-mass spectrometry (GC-MS). Behavioral responses to beans, extracts, synthetic blends, 3-methylbutanoic acid, heptanal, and a binary blend (3-methylbutanoic acid plus heptanal) were evaluated in a Y-tube olfactometer. Antennal activity was determined by electroantennography (EAG). Chemical analysis revealed the presence of ketones, alcohols, esters, terpenes, acids, hydrocarbons, aldehydes, and pyrazines, with volatile profiles varying according to postharvest handling. In behavioral bioassays, unfermented washed beans did not attract either sex, fermented beans attracted both males and females, and infested beans attracted only males. The EAG analysis demonstrated that the volatile extract from fermented cocoa beans, as well as 3-methylbutanoic acid and heptanal, elicited the antennal responses. The binary blend did not elicit a significant attraction compared with the control, whereas each compound tested individually produced stronger responses. Males and females were attracted to 3-methylbutanoic acid at 0.001 µg, while heptanal attracted females at 0.1 and 0.01 µg and males at 1 and 10 µg. These findings identify key compounds in relation to the doses tested, providing insights for future management of A. fasciculatus in grain storage facilities.

Various plant-related factors have been shown to influence volatile organic compound (VOC)-mediated plant-to-plant interactions, but effects of plant ontogeny have been rarely considered. Here, we tested if these interactions differ between two ontogenetic stages in wild cotton, Gossypium hirsutum. We used 2.5- and 4.5-month-old plants (seedlings and juveniles, respectively), arranged in triplets where one plant acted as an emitter and two as receivers (all plants within a cage were of the same developmental stage). Half of the emitters were subjected to herbivory by Alabama argillacea caterpillars, while the emitters of the other triplets were left unharmed. After 48 h of VOC exposure, we collected the VOCs from a subset of the emitter plants (for 2 h), and one receiver from each cage was removed to measure extrafloral nectar (EFN) production and to collect leaves for total phenolic analyses. The remaining receivers were subjected to herbivory to assess signaling effects on defense induction. Emitter seedlings released larger quantities of VOCs in response to caterpillar damage than juveniles, and ontogenetic stages also differed in the composition of the emitted VOC blend. Accordingly, we found trends for ontogenetic variation in signaling effects: seedling receivers exposed to damaged emitters tended to produce more EFN both prior to and following damage compared to those exposed to undamaged emitters, whereas juvenile receivers showed no EFN response to emitter damage. Similarly, seedling receivers exposed to damaged emitters exhibited higher phenolic concentrations, whereas no such signaling effect was observed in juveniles. These findings indicate that VOC-mediated signaling of defenses is strong in young cotton plants but weakens during development in pre-reproductive juvenile plants, and that this is reflected in both direct and indirect defense responses.

The endophytic fungus Alternaria oxytropis is vertically transmitted through silky crazyweed (Oxytropis sericea Nutt.) and produces the neurotoxin swainsonine, yet its influence on host chemical signaling remains unclear. Here we characterize the floral and foliar volatile organic compound (VOC) profiles of endophyte-infected (E+) and endophyte-free (E-) O. sericea using dynamic headspace GC-MS on greenhouse- and field-grown plants - the first characterization of volatile profiles in this species. Endophyte infection had a negligible effect on VOC composition, explaining less than 1% of compositional variance in both greenhouse and field settings, with no compound differing consistently by infection status. Seven volatiles were reliably detected across both environments. The dominant axes of variation (PCA) instead reflected differences among individuals and between size-matched E+/E- plant across years. These accounted for most compositional variance (72.6% greenhouse, 64.9% field), and VOC profiles differed markedly between collection years, likely reflecting fine-scale environmental and physiological variation. Ancillary nectar assays and pollinator observations revealed no consistent differences between E + and E- plants, and seed set was unaffected regardless of endophyte status. Swainsonine in floral organs was roughly an order of magnitude lower than in leaves and near-trace in nectar, indicating that any localized chemical defense is likely decoupled from scent chemistry. Together, these findings position the Oxytropis-Alternaria association toward the commensal end of the symbiotic continuum: the fungus maintains vertical transmission while the host shows no detectable effect on volatile signaling, pollinator attraction, or reproductive output, despite the resources required to sustain fungal alkaloid biosynthesis. This system offers a tractable model for understanding how endophyte associations persist in the absence of measurable host benefit.

Insect development involves profound physiological reorganization that can alter the assimilation and allocation of metabolites across life stages. In herbivorous insects, these processes are tightly linked to diet-derived chemical exposure, yet how metabolic organization shifts throughout ontogeny remains poorly understood. Here, we investigated ontogenetic metabolomic variation in Battus polydamas feeding on Aristolochia gigantea, a system characterized by a specialized ecological interaction between insect and host plant. We examined how chemical profiles vary across larval, pupal, and adult stages, as well as in the host plant and larval frass, to determine whether metamorphosis is associated with stage-specific reorganization of the insect metabolome. Multivariate analyses revealed clear separation among sample types, and PERMANOVA confirmed significant differences among groups. Pupae and adult bodies showed the greatest similarity, whereas plant and frass formed distinct clusters. Wings exhibited a differentiated metabolomic profile. Molecular networking revealed a diverse set of primary and secondary metabolites, including carbohydrates, amino acids, phenolics, fatty acids, and sterol-related compounds. Nine metabolites differed significantly among plant, insect stages, and frass. Lipid- and sterol-related compounds showed stage-specific variation, particularly in pupae and wings, while phenolic derivatives and fatty acids distinguished insect tissues from plant and frass samples. Frass displayed marked chemical divergence, consistent with differential assimilation and metabolic processing. In summary, the metabolomic profile of B. polydamas is reorganized across ontogeny, reflecting stage-specific patterns of metabolite retention and transformation throughout development.

Plant chemical defenses are known to influence herbivore performance, yet their effects on herbivore immunity are poorly understood. In this study, we manipulated both the mean and variance of the concentration of a plant toxin in an artificial diet to assess the effects on herbivore immune function and survival. Our results revealed a biphasic immune response across increasing mean toxin concentrations, with exposure to low toxin concentrations enhancing immune function, creating a 'window of enhanced immunity', and exposure to higher concentrations suppressing it relative to toxin-free diets. Encountering variable toxin levels may alter this relationship by mixing toxin levels from within and outside this window. Importantly, we found that when the mean toxin concentration is just outside of the window of enhanced immunity, exposure to low variability in toxin concentrations enhanced herbivore immunocompetence compared to when they were exposed to high or no variation. Elevated immunity correlated with increased survival under pathogen challenge.

Plant hybridization often alters herbivore-relevant traits, yet the effects of hybrid feeding on specialist herbivores, especially those that co-opt host-plant defenses, are largely unknown. We examined how naturally derived hybrids between Asclepias syriaca and A. speciosa (A. syriaca x speciosa) affect monarch butterfly (Danaus plexippus) performance and cardenolide sequestration. Across three independent greenhouse experiments, monarch caterpillars reared on genetically heterogeneous A. syriaca x speciosa hybrid milkweeds grew more slowly than caterpillars reared on either parental species. Monarchs reared on hybrids also, on average, sequestered lower total cardenolide concentrations into adult tissues. However, plant measures of cardenolide chemistry did not differ significantly among parental and hybrid host groups, and measured physical defenses (trichome and latex) showed only partial differences among groups, and none in the expected direction. Element analyses of plant leaf tissue, although preliminary, revealed a negative relationship between caterpillar growth and manganese level, with hybrids containing between two- to three-fold higher levels of manganese as either parent species. These results indicate that host-plant hybridization can alter both specialist herbivore performance and acquired chemical defense, even when broad measures of host defenses do not explain those effects. Hybridization may therefore influence plant-herbivore interactions not only by changing host quality, but also by altering how specialist herbivores acquire plant-derived defenses.

Chemical signalling and endocrine state are known to covary with social environment, but the speed of their response to social transitions under natural conditions remains unclear. We examined how major urinary proteins (MUPs) and faecal steroid hormone metabolites respond to shifts between solitary housing and a complex social setting in two house mouse subspecies, Mus musculus musculus and M. m. domesticus. Using repeated sampling in semi-natural enclosures, we measured MUP, testosterone, and corticosterone metabolite concentrations across three contexts: before social exposure, during the social phase, and after return to isolation. Reproductive output was quantified within a 44-day window around each sampling event. Entry into the enclosures triggered a strong, coordinated response: urinary MUP concentrations increased in both sexes and subspecies, paralleled by rises in testosterone and corticosterone metabolites during early social exposure. In males, this pattern reversed quite quickly after removal, with MUPs and both hormone metabolites declining within 24 h of renewed isolation. Under stable social conditions, urinary MUP concentrations increased with current reproductive output, whereas testosterone and corticosterone metabolites showed no such association. MUP concentrations were also unrelated to either hormone, suggesting that socially responsive MUP investment is not directly coupled to steroid levels. Despite baseline differences between subspecies and the expected male bias in MUP production, the main context-dependent patterns were consistent across taxa. Our results demonstrate pronounced social plasticity in chemical signalling and endocrine state, and indicate that MUP concentrations more closely reflect reproductive outcomes than steroid metabolite concentrations.

Interspecific chemical communication in insects is quintessential in mediating ecological interactions across trophic levels. Kairomones are semiochemicals emitted by one species (the emitter) and are inadvertently beneficial to another species (the receiver). These chemical markers are particularly critical in prey-predator or host-parasitoid relationships, such as host/prey location and selection. Host/prey bodies and their by-products emit pheromones, volatile and non-volatile cuticular compounds, and induce plant volatiles by oviposition, zoophytophagy, and herbivory, which are copious sources of kairomone signaling. Advances in analytical chemistry and behavioral ecology have enabled the identification and synthesis of such kairomones for integration into integrated pest management (IPM) systems. As semiochemicals are ubiquitous in insects, the literature is robust, and prior reviews often focus on a subset of applications. Here, we aim to consolidate the breadth of current and emerging knowledge on plant- and insect-derived kairomones and highlight their diverse origins, significance in host/prey selection and behavioral orientation, and high specificity in multitrophic interactions. We discuss the compatibility of kairomone-based tools with novel agricultural technologies for pest control, suggesting their potential to enhance crop protection while reducing reliance on broad-spectrum pesticides. We also delineate the limitations of such tools for future research efforts and the feasibility of sampling realistic field headspace volatiles, rather than in laboratories, to identify kairomones, and the formulation of kairomone blends that attract multiple natural enemies simultaneously, rather than kairomones for single natural enemy species, which currently dominate the literature. Most experimental field studies tested kairomones as a standalone pest control tool. We emphasize that combining kairomone use with other IPM components, such as natural enemy-rewarding plants (attract and reward), push-pull companion plants, and beneficial microbial inoculants, would be beneficial. Although complexities abound in formulating and synthesizing kairomones, applying kairomones remains a frontier in sustainable pest management, and opportunities for optimization and integration into IPM programs are abundant.

Olfaction plays a prominent role in the evolution of animal behaviour, facilitating both intraspecies and interspecies communication, and predator-prey interactions. Volatile organic compounds (VOCs) underpin these odours, and their analysis is therefore central to characterising and replicating cues such as pheromones, allelochemicals, and other released signals used to attract, deter, or condition animal responses. Thus, this review aims to introduce the reader to the applications of animal odours in animal management and behavioural control, and to provide a critical, practice-oriented guide to selecting and combining VOC sampling and analytical techniques for animal odour studies. Animal odour profiling typically involves three steps: (1) sampling, (2) transfer to the instrument, and (3) detection/identification of VOCs. Gas chromatography-mass spectrometry (GC-MS) is the gold standard for VOC analysis, and is often paired with headspace techniques, including static headspace, dynamic headspace, and solid-phase microextraction (SPME) to facilitate sampling and transfer of VOCs. In addition, liquid and distillation-based extraction methods are discussed, which remain important for capturing less volatile or matrix-bound components in animal odour and semiochemical research. On the analytical side we discuss GC-MS alongside emerging and complementary techniques, including proton transfer reaction-mass spectrometry (PTR-MS), selected ion flow tube-mass spectrometry (SIFT-MS), gas chromatography-olfactometry (GC-O), and electronic noses, emphasising their respective strengths and limitations for qualitative and quantitative VOC analysis. Collectively, the literature shows that no single sampling or analytical approach is universally optimal. Effective animal odour profiling therefore requires matching extraction and detection strategies to the volatility, stability, and behavioural relevance of target VOCs. Careful consideration should also be given to how these methodological choices influence the odour profiles used in ecological, behavioural, and applied management contexts.

A mixture of hydrocarbons of different molecular weights covers the Coleoptera cuticle, protecting the insects from desiccation and, in some cases, providing information about their physiological state. Courtship and mating in Cyclocephala barrerai Martínez begin when the male's antennae touch the female's thorax and elytra. The male antennae of C. barrerai have sensilla that detect hydrocarbons; however, the role of these compounds in the mating behavior of this beetle species remains unknown. Here, we analyzed the behavioral responses of both sexes of C. barrerai to conspecific cuticular hydrocarbons (CHCs) and identified them to determine how their composition varies by sex and age. CHCs were extracted from laboratory-reared C. barrerai individuals by immersion in hexane. We measured the percentage of adults that responded to CHCs extracts applied to a minimal visual component, a filter paper, an inert decoy, and a conspecific carcass stripped of CHCs. We identified and quantified the CHCs using gas chromatography coupled with mass spectrometry. Males exhibited arrestment, courtship, and copulation behaviors in response to CHCs extracted from females. The extracts contained nine compounds, seven of which were identified and six quantified. Insects emerged from the pupa with a similar amount of CHCs, and they differentiated as the gonads matured. Our results suggest that CHCs from C. barrerai females function as a contact sex pheromone.

The spotted wing drosophila, Drosophila suzukii, is an economically significant invasive pest affecting berry crops worldwide, with effective management reliant on comprehending its reproductive behaviour. This study evaluated the effects of age, sex, reproductive status, and irradiation on the cuticular hydrocarbon (CHC) profile of D. suzukii. Samples from irradiated and non-irradiated adults were analyzed using gas chromatography coupled with mass spectrometry. Heneicosene, (Z)-9-tricosene, and hexacosene were identified in the samples. The abundance of these compounds varied mainly as a function of age and mating condition, especially in females. Heneicosene increased with age and mating, whereas (Z)-9-tricosene showed a contrary trend. Irradiation did not affect most CHCs, although an increase in hexacosene was detected in older irradiated females. In males, the profiles were more stable. The results indicate that CHCs in D. suzukii are highly plastic and that irradiation has specific and limited effects, suggesting that the Sterile Insect Technique may be implemented without significantly disrupting the chemical communication essential for mating.

Stable flies, Stomoxys calcitrans, are severe blood-feeding pests of cattle. As previously reported, fecal deposits of stable flies attract conspecific flies. Here we report the identification and behavioral testing of fly fecal semiochemicals. In laboratory two-choice bioassays, CO2-activated flies landed more often on sites baited with stable fly feces, or water extract thereof, than on unbaited control sites. Water extracts of feces attracted flies, whereas non-polar solvent extracts did not. Fecal deposits only of blood-fed, but not sugar-fed, flies elicited preferential responses, indicating that the feces-derived semiochemicals are metabolites of blood protein digestion. Analyses of freeze-dried, purified water extract - following derivatization of polar compounds - by gas chromatography-mass spectrometry revealed 13 polar candidate semiochemicals. A synthetic blend of formamide, ethylamine, ammonium carbonate, urea, valine, 3-methylbutyric acid, lactic acid, ketoisocaproic acid, and oxalic acid attracted flies as effectively as water extract of feces, but not all constituents of this synthetic lure may be needed for fly attraction. Further research is needed to determine whether the synthetic blend attracts flies in field settings, and whether a less complex - but still highly effective - lure can be developed to augment the efficacy of current vision-based stable fly traps.

In this study, we chronically exposed virgin male and female moths of two closely related noctuid species, Helicoverpa armigera (the cotton bollworm, CBW) and H. assulta (the oriental tobacco budworm, OTB), to conspecific sex pheromones, interspecific sex pheromones, or a control environment, and measured several reproductive and longevity parameters in females, as well as male longevity. The results show that pheromone exposure treatment shaped the temporal pattern of female oviposition (i.e., delayed oviposition), but had no significant effect on life-history span factor (the principal component onto which total oviposition duration and female longevity loaded most heavily, with loadings of 0.9846 and 0.9228, respectively). Exposure to conspecific or interspecific sex pheromone atmospheres shortened the longevity of OTB males relative to the control, whereas CBW male longevity was not affected. These findings suggest that, in addition to their well-known role in mate attraction, sex pheromones may asymmetrically shape life-history traits in the two sexes. However, because this study was conducted under confined laboratory conditions without measuring mating success or population growth, the potential implication that pheromone exposure could regulate population dynamics by affecting reproductive synchrony between the sexes remains hypothetical and awaits validation under more ecologically realistic conditions.

Predatory oyster mushrooms (Pleurotus spp.) immobilize nematode prey using toxin droplets. The chemical composition and regulation of toxin droplet production have remained incompletely characterized since their initial observation over 40 years ago, though 3-octanone has been identified as a possible contributor. We quantified toxin droplet abundance in twenty Pleurotus strains spanning six species and compared their chemical profiles using GC-MS headspace and liquid sampling. Droplet abundance varied among strains and, in most, increased following the addition of washed nematodes. Strains that produced many toxin droplets had distinct volatile and liquid metabolite profiles compared with low-droplet-producing strains and control groups. Our targeted analysis identified 3-octanone in seven Pleurotus strains, including its first reported detection in P. eryngii and P. populinus. Our untargeted analysis putatively identified several additional candidate nematicidal molecules that were significantly enriched in high-droplet producing strains. Here, we provide evidence consistent with the hypothesis that Pleurotus may use a cocktail of secondary metabolites, rather than 3-octanone alone, to paralyze nematode prey, supporting an alternative to the one-molecule mechanism of paralysis.