Queen pheromones are chemical signals that mediate reproductive division of labor in eusocial animals. Remarkably, queen pheromones are composed of identical or chemically similar compounds in some ants, wasps and bees, even though these taxa diverged >150MYA and evolved queens and workers independently. Here, we measure the transcriptomic consequences of experimental exposure to queen pheromones in workers from two ant and two bee species (genera: Lasius, Apis, Bombus), and test whether they are similar across species. Queen pheromone exposure affected transcription and splicing at many loci. Many genes responded consistently in multiple species, and the set of pheromone-sensitive genes was enriched for functions relating to lipid biosynthesis and transport, olfaction, production of cuticle, oogenesis, and histone (de)acetylation. Pheromone-sensitive genes tend to be evolutionarily ancient, positively selected, peripheral in the gene coexpression network, hypomethylated, and caste-specific in their expression. Our results reveal how queen pheromones achieve their effects, and suggest that ants and bees use similar genetic modules to achieve reproductive division of labor.

Microorganisms and nematodes in the rhizosphere profoundly impact plant health, and small-molecule signaling is presumed to play a central role in plant rhizosphere interactions. However, the nature of the signals and underlying mechanisms are poorly understood. Here we show that the ascaroside ascr#18, a pheromone secreted by plant-parasitic nematodes, is metabolized by plants to generate chemical signals that repel nematodes and reduce infection. Comparative metabolomics of plant tissues and excretions revealed that ascr#18 is converted into shorter side-chained ascarosides that confer repellency. An Arabidopsis mutant defective in two peroxisomal acyl-CoA oxidases does not metabolize ascr#18 and does not repel nematodes, indicating that plants, like nematodes, employ conserved peroxisomal β-oxidation to edit ascarosides and change their message. Our results suggest that plant-editing of nematode pheromones serves as a defense mechanism that acts in parallel to conventional pattern-triggered immunity, demonstrating that plants may actively manipulate chemical signaling of soil organisms.

Female fruit flies (Drosophila melanogaster) oviposit at communal sites where the larvae may cooperate or compete for resources depending on group size. This offers a model system to determine how females assess quantitative social information. We show that the concentration of pheromones found on a substrate increases linearly with the number of adult flies that have visited that site. Females prefer oviposition sites with pheromone concentrations corresponding to an intermediate number of previous visitors, whereas sites with low or high concentrations are unattractive. This dose-dependent decision is based on a blend of 11-cis-Vaccenyl Acetate (cVA) indicating the number of previous visitors and heptanal (a novel pheromone deriving from the oxidation of 7-Tricosene), which acts as a dose-independent co-factor. This response is mediated by detection of cVA by odorant receptor neurons Or67d and Or65a, and at least five different odorant receptor neurons for heptanal. Our results identify a mechanism allowing individuals to transform a linear increase of pheromones into a non-linear behavioral response.

Eusocial insects use cuticular hydrocarbons as components of pheromones that mediate social behaviours, such as caste and nestmate recognition, and regulation of reproduction. In ants such as Harpegnathos saltator, the queen produces a pheromone which suppresses the development of workers' ovaries and if she is removed, workers can transition to a reproductive state known as gamergate. Here we functionally characterize a subfamily of odorant receptors (Ors) with a nine-exon gene structure that have undergone a massive expansion in ants and other eusocial insects. We deorphanize 22 representative members and find they can detect cuticular hydrocarbons from different ant castes, with one (HsOr263) that responds strongly to gamergate extract and a candidate queen pheromone component. After systematic testing with a diverse panel of hydrocarbons, we find that most Harpegnathos saltator Ors are narrowly tuned, suggesting that several receptors must contribute to detection and discrimination of different cuticular hydrocarbons important in mediating eusocial behaviour.Cuticular hydrocarbons (CHC) mediate the interactions between individuals in eusocial insects, but the sensory receptors for CHCs are unclear. Here the authors show that in ants such as H. saltator, the 9-exon subfamily of odorant receptors (HsOrs) responds to CHCs, and ectopic expression of HsOrs in Drosophila neurons imparts responsiveness to CHCs.

Males from many species ensure paternity by preventing their mates from copulating with other males. One mate-guarding strategy involves marking females with anti-aphrodisiac pheromones (AAPs), which reduces the females' attractiveness and dissuades other males from courting. Since females benefit from polyandry, sexual conflict theory predicts that females should develop mechanisms to counteract AAPs to achieve additional copulations, but no such mechanisms have been documented. Here we show that during copulation Drosophila melanogaster males transfer two AAPs: cis-Vaccenyl Acetate (cVA) to the females' reproductive tract, and 7-Tricosene (7-T) to the females' cuticle. A few hours after copulation, females actively eject cVA from their reproductive tract, which results in increased attractiveness and re-mating. Although 7-T remains on those females, we show that it is the combination of the two chemicals that reduces attractiveness. To our knowledge, female AAP ejection provides the first example of a female mechanism that counter-acts chemical mate-guarding.

Insect sexual communication often relies upon sex pheromones. Most insect pheromones, however, contain carbon-carbon double bonds and potentially degrade by oxidation. Here, we show that frequently reported increased levels of Anthropocenic ozone can oxidize all described male-specific pheromones of Drosophila melanogaster, resulting in reduced amounts of pheromones such as cis-Vaccenyl Acetate and (Z)-7-Tricosene. At the same time female acceptance of ozone-exposed males is significantly delayed. Interestingly, groups of ozone-exposed males also exhibit significantly increased levels of male-male courtship behaviour. When repeating similar experiments with nine other drosophilid species, we observe pheromone degradation and/or disrupted sex recognition in eight of them. Our data suggest that Anthropocenic levels of ozone can extensively oxidize double bonds in a variety of insect pheromones, thereby leading to deviations in sexual recognition.

CD36 transmembrane proteins have diverse roles in lipid uptake, cell adhesion and pathogen sensing. Despite numerous in vitro studies, how they act in native cellular contexts is poorly understood. A Drosophila CD36 homologue, sensory neuron membrane protein 1 (SNMP1), was previously shown to facilitate detection of lipid-derived pheromones by their cognate receptors in olfactory cilia. Here we investigate how SNMP1 functions in vivo. Structure-activity dissection demonstrates that SNMP1's ectodomain is essential, but intracellular and transmembrane domains dispensable, for cilia localization and pheromone-evoked responses. SNMP1 can be substituted by mammalian CD36, whose ectodomain can interact with insect pheromones. Homology modelling, using the mammalian LIMP-2 structure as template, reveals a putative tunnel in the SNMP1 ectodomain that is sufficiently large to accommodate pheromone molecules. Amino-acid substitutions predicted to block this tunnel diminish pheromone sensitivity. We propose a model in which SNMP1 funnels hydrophobic pheromones from the extracellular fluid to integral membrane receptors.

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.

Moth sex pheromones are a classical model for studying sexual selection. Females typically produce a species-specific pheromone blend that attracts males. Revealing the enzymes involved in the interspecific variation in blend composition is key for understanding the evolution of these sexual communication systems. The nature of the enzymes involved in the variation of acetate esters, which are prominent compounds in moth pheromone blends, remains unclear. We identify enzymes involved in acetate degradation using two closely related moth species: Heliothis (Chloridea) subflexa and H. (C.) virescens, which have different quantities of acetate esters in their sex pheromone. Through comparative transcriptomic analyses and CRISPR/Cas9 knockouts, we show that two lipases and two esterases from H. virescens reduce the levels of pheromone acetate esters when expressed in H. subflexa females. Together, our results show that lipases and carboxylesterases are involved in tuning Lepidoptera pheromones composition.

Pathogens and parasites can manipulate their hosts to optimize their own fitness. For instance, bacterial pathogens have been shown to affect their host plants' volatile and non-volatile metabolites, which results in increased attraction of insect vectors to the plant, and, hence, to increased pathogen dispersal. Behavioral manipulation by parasites has also been shown for mice, snails and zebrafish as well as for insects. Here we show that infection by pathogenic bacteria alters the social communication system of Drosophila melanogaster. More specifically, infected flies and their frass emit dramatically increased amounts of fly odors, including the aggregation pheromones methyl laurate, methyl myristate, and methyl palmitate, attracting healthy flies, which in turn become infected and further enhance pathogen dispersal. Thus, olfactory cues for attraction and aggregation are vulnerable to pathogenic manipulation, and we show that the alteration of social pheromones can be beneficial to the microbe while detrimental to the insect host.Behavioral manipulation of host by pathogens has been observed in vertebrates, invertebrates, and plants. Here the authors show that in Drosophila, infection with pathogenic bacteria leads to increased pheromone release, which attracts healthy flies. This process benefits the pathogen since it enhances bacterial dispersal, but is detrimental to the host.

The accessory olfactory system (AOS) guides behaviours that are important for survival and reproduction, but understanding of AOS function is limited by a lack of identified natural ligands. Here we report that mouse faeces are a robust source of AOS chemosignals and identify bile acids as a class of natural AOS ligands. Single-unit electrophysiological recordings from accessory olfactory bulb neurons in ex vivo preparations show that AOS neurons are strongly and selectively activated by peripheral stimulation with mouse faecal extracts. Faecal extracts contain several unconjugated bile acids that cause concentration-dependent neuronal activity in the AOS. Many AOS neurons respond selectively to bile acids that are variably excreted in male and female mouse faeces, and others respond to bile acids absent in mouse faeces. These results identify faeces as a natural source of AOS information, and suggest that bile acids may be mammalian pheromones and kairomones.

Insect vectors are required for the transmission of many species of parasitic nematodes, but the mechanisms by which the vectors and nematodes coordinate their life cycles are poorly understood. Here, we report that ascarosides, an evolutionarily conserved family of nematode pheromones, are produced not only by a plant-parasitic nematode, but also by its vector beetle. The pinewood nematode and its vector beetle cause pine wilt disease, which threatens forest ecosystems world-wide. Ascarosides secreted by the dispersal third-stage nematode LIII larvae promote beetle pupation by inducing ecdysone production in the beetle and up-regulating ecdysone-dependent gene expression. Once the beetle develops into the adult stage, it secretes ascarosides that attract the dispersal fourth-stage nematode LIV larvae, potentially facilitating their movement into the beetle trachea for transport to the next pine tree. These results demonstrate that ascarosides play a key role in the survival and spread of pine wilt disease.

The codling moth Cydia pomonella, a major invasive pest of pome fruit, has spread around the globe in the last half century. We generated a chromosome-level scaffold assembly including the Z chromosome and a portion of the W chromosome. This assembly reveals the duplication of an olfactory receptor gene (OR3), which we demonstrate enhances the ability of C. pomonella to exploit kairomones and pheromones in locating both host plants and mates. Genome-wide association studies contrasting insecticide-resistant and susceptible strains identify hundreds of single nucleotide polymorphisms (SNPs) potentially associated with insecticide resistance, including three SNPs found in the promoter of CYP6B2. RNAi knockdown of CYP6B2 increases C. pomonella sensitivity to two insecticides, deltamethrin and azinphos methyl. The high-quality genome assembly of C. pomonella informs the genetic basis of its invasiveness, suggesting the codling moth has distinctive capabilities and adaptive potential that may explain its worldwide expansion.

Mating drive is balanced by a need to safeguard resources for offspring, yet the neural basis for negative regulation of mating remains poorly understood. In rodents, pheromones critically regulate sexual behavior. Here, we observe suppression of adult female sexual behavior in mice by exocrine gland-secreting peptide 22 (ESP22), a lacrimal protein from juvenile mice. ESP22 activates a dedicated vomeronasal receptor, V2Rp4, and V2Rp4 knockout eliminates ESP22 effects on sexual behavior. Genetic tracing of ESP22-responsive neural circuits reveals a critical limbic system connection that inhibits reproductive behavior. Furthermore, V2Rp4 counteracts a highly related vomeronasal receptor, V2Rp5, that detects the male sex pheromone ESP1. Interestingly, V2Rp4 and V2Rp5 are encoded by adjacent genes, yet couple to distinct circuits and mediate opposing effects on female sexual behavior. Collectively, our study reveals molecular and neural mechanisms underlying pheromone-mediated sexual rejection, and more generally, how inputs are routed through olfactory circuits to evoke specific behaviors.

Although phylogenetically nested within the moths, butterflies have diverged extensively in a number of life history traits. Whereas moths rely greatly on chemical signals, visual advertisement is the hallmark of mate finding in butterflies. In the context of courtship, however, male chemical signals are widespread in both groups although they likely have multiple evolutionary origins. Here, we report that in males of the butterfly Bicyclus anynana, courtship scents are produced de novo via biosynthetic pathways shared with females of many moth species. We show that two of the pheromone components that play a major role in mate choice, namely the (Z)-9-tetradecenol and hexadecanal, are produced through the activity of a fatty acyl Δ11-desaturase and two specialized alcohol-forming fatty acyl reductases. Our study provides the first evidence of conservation and sharing of ancestral genetic modules for the production of FA-derived pheromones over a long evolutionary timeframe thereby reconciling mate communication in moths and butterflies.

Early-life nourishment exerts long-term influences upon adult physiology and disease risk. These lasting effects of diet are well established but the underlying mechanisms are only partially understood. Here we show that restricting dietary yeast during Drosophila development can, depending upon the subsequent adult environment, more than double median lifespan. Developmental diet acts via a long-term influence upon the adult production of toxic molecules, which we term autotoxins, that are shed into the environment and shorten the lifespan of both sexes. Autotoxins are synthesised by oenocytes and some of them correspond to alkene hydrocarbons that also act as pheromones. This study identifies a mechanism by which the developmental dietary history of an animal regulates its own longevity and that of its conspecific neighbours. It also has important implications for the design of lifespan experiments as autotoxins can influence the regulation of longevity by other factors including diet, sex, insulin signalling and population density.

Plant-defense responses are triggered by perception of conserved microbe-associated molecular patterns (MAMPs), for example, flagellin or peptidoglycan. However, it remained unknown whether plants can detect conserved molecular patterns derived from plant-parasitic animals, including nematodes. Here we show that several genera of plant-parasitic nematodes produce small molecules called ascarosides, an evolutionarily conserved family of nematode pheromones. Picomolar to micromolar concentrations of ascr#18, the major ascaroside in plant-parasitic nematodes, induce hallmark defense responses including the expression of genes associated with MAMP-triggered immunity, activation of mitogen-activated protein kinases, as well as salicylic acid- and jasmonic acid-mediated defense signalling pathways. Ascr#18 perception increases resistance in Arabidopsis, tomato, potato and barley to viral, bacterial, oomycete, fungal and nematode infections. These results indicate that plants recognize ascarosides as a conserved molecular signature of nematodes. Using small-molecule signals such as ascarosides to activate plant immune responses has potential utility to improve economic and environmental sustainability of agriculture.

Sexual behavior is essential for the survival of many species. In female rodents, mate preference and copulatory behavior depend on pheromones and are synchronized with ovulation to ensure reproductive success. The neural circuits driving this orchestration in the brain have, however, remained elusive. Here, we demonstrate that neurons controlling ovulation in the mammalian brain are at the core of a branching neural circuit governing both mate preference and copulatory behavior. We show that male odors detected in the vomeronasal organ activate kisspeptin neurons in female mice. Classical kisspeptin/Kiss1R signaling subsequently triggers olfactory-driven mate preference. In contrast, copulatory behavior is elicited by kisspeptin neurons in a parallel circuit independent of Kiss1R involving nitric oxide signaling. Consistent with this, we find that kisspeptin neurons impinge onto nitric oxide-synthesizing neurons in the ventromedial hypothalamus. Our data establish kisspeptin neurons as a central regulatory hub orchestrating sexual behavior in the female mouse brain.

Many heritable mutualisms, in which beneficial symbionts are transmitted vertically between host generations, originate as antagonisms with parasite dispersal constrained by the host. Only after the parasite gains control over its transmission is the symbiosis expected to transition from antagonism to mutualism. Here, we explore this prediction in the mutualism between the fungus Rhizopus microsporus (Rm, Mucoromycotina) and a beta-proteobacterium Burkholderia, which controls host asexual reproduction. We show that reproductive addiction of Rm to endobacteria extends to mating, and is mediated by the symbiont gaining transcriptional control of the fungal ras2 gene, which encodes a GTPase central to fungal reproductive development. We also discover candidate G-protein-coupled receptors for the perception of trisporic acids, mating pheromones unique to Mucoromycotina. Our results demonstrate that regulating host asexual proliferation and modifying its sexual reproduction are sufficient for the symbiont's control of its own transmission, needed for antagonism-to-mutualism transition in heritable symbioses. These properties establish the Rm-Burkholderia symbiosis as a powerful system for identifying reproductive genes in Mucoromycotina.

The most frequently encountered symbiont on tree roots is the ascomycete Cenococcum geophilum, the only mycorrhizal species within the largest fungal class Dothideomycetes, a class known for devastating plant pathogens. Here we show that the symbiotic genomic idiosyncrasies of ectomycorrhizal basidiomycetes are also present in C. geophilum with symbiosis-induced, taxon-specific genes of unknown function and reduced numbers of plant cell wall-degrading enzymes. C. geophilum still holds a significant set of genes in categories known to be involved in pathogenesis and shows an increased genome size due to transposable elements proliferation. Transcript profiling revealed a striking upregulation of membrane transporters, including aquaporin water channels and sugar transporters, and mycorrhiza-induced small secreted proteins (MiSSPs) in ectomycorrhiza compared with free-living mycelium. The frequency with which this symbiont is found on tree roots and its possible role in water and nutrient transport in symbiosis calls for further studies on mechanisms of host and environmental adaptation.