A diterpene synthase from the sandfly Lutzomyia longipalpis produces the pheromone sobralene.

The phlebotomine sandfly, Lutzomyia longipalpis, a major vector of the Leishmania parasite, uses terpene pheromones to attract conspecifics for mating. Examination of the L. longipalpis genome revealed a putative terpene synthase (TPS), which-upon heterologous expression in, and purification from, Escherichia coli-yielded a functional enzyme. The TPS, termed LlTPS, converted geranyl diphosphate (GPP) into a mixture of monoterpenes with low efficiency, of which ß-ocimene was the major product. (E,E)-farnesyl diphosphate (FPP) principally produced small amounts of (E)-ß-farnesene, while (Z,E)- and (Z,Z)-FPP yielded a mixture of bisabolene isomers. None of these mono- and sesquiterpenes are known volatiles of L. longipalpis. Notably, however, when provided with (E,E,E)-geranylgeranyl diphosphate (GGPP), LlTPS gave sobralene as its major product. This diterpene pheromone is released by certain chemotypes of L. longipalpis, in particular those found in the Ceará state of Brazil. Minor diterpene components were also seen as products of the enzyme that matched those seen in a sandfly pheromone extract.

Sequential Isolation of Essential Oils Repellent to the Red Palm Weevil Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae).

The push-pull approach using semiochemicals in pest control requires both an attractant and a repellent. Many previous studies have arbitrarily tested one or more known insect repellents or plant essential oils (EOs) hoping to find repellents of an insect pest. We used a comprehensive approach that synergistically tests in the field numerous natural volatiles from commercial EOs to identify repellents of the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae), a worldwide pest of palms and date palms. Volatiles from 79 EOs in slow-release devices were divided into five groups and tested in traps with attractive pheromone compared to traps with pheromone alone. EO-treatment groups exhibiting repellency due to significant trap shutdown, were further subdivided into subgroups of four EOs each and tested further. Two groups of four EOs (cypress, desert wormwood, elemi, and Eucalyptus citriodora) and (niaouli, nutmeg, oregano, and orange sweet), or their corresponding mixtures of major volatiles, caused pheromone trap reductions of up to 92%. Further tests showed that seven of the eight EOs are similarly repellent as the corresponding subgroup. This systematic approach of successively testing sub-fractions of EOs in the field for trap shutdown should be useful to identify repellents of other insect pests of crops.

Identification of Aggregation Pheromone as an Attractant for Odontothrips loti, A Serious Thrips Pest on Alfalfa.

Odontothrips loti (Haliday) (Thysanoptera: Thripidae) is one of the most serious pests on alfalfa, causing direct damage by feeding and indirect damage by transmitting plant viruses. This damage causes significant loss in alfalfa production. Semiochemicals offer opportunities to develop new approaches to thrips management. In this study, behavioral responses of female and male adults of O. loti to headspace volatiles from live female and male conspecifics were tested in a Y-tube olfactometer. The results showed that both male and female adults of O. loti were attracted to the odors released by conspecific males but not those released by females. Headspace volatiles released by female and male adults were collected using headspace solid-phase microextraction (HS-SPME). The active compound in the volatiles was identified by gas chromatography-mass spectrometry (GC-MS). The analysis showed that there was one major compound, (R)-lavandulyl (R)-2-methylbutanoate. The attractive activity of the synthetic aggregation pheromone compound was tested under laboratory and field conditions. In an olfactometer, both male and female adults showed significant preference for synthetic (R)-lavandulyl (R)-2-methylbutanoate at certain doses. Lures with synthetic (R)-lavandulyl (R)-2-methylbutanoate significantly increased the trap catches of sticky white traps at doses of 40-80 µg in the field. This study confirmed the production of aggregation pheromone by O. loti male adults and identified its active compound as (R)-lavandulyl (R)-2-methylbutanoate, providing a basis for population monitoring and mass trapping of this pest.

RpedOBP1 plays key roles in aggregation pheromones reception of the Riptortus pedestris.

Riptortus pedestris (Hemiptera: Alydidae) is a notable soybean pest, with diapause and non-diapause individuals showing different sensitivities to aggregation pheromones. This study aimed to investigate how R. pedestris detects aggregation pheromones through electroantennogram (EAG) and behavioral experiments, transcriptome sequencing and qRT-PCR, as well as competitive fluorescence-binding assay. Results indicated that diapausing females and males of R. pedestris exhibited a heightened EAG response and were more attracted to the aggregation pheromone components compared to their non-diapause counterparts. Transcriptome sequencing and qRT-PCR analyses revealed significantly higher expression of RpedOBP1 in the antennae of diapause females and males compared to non-diapausing R. pedestris. The competitive fluorescence-binding assay demonstrated that RpedOBP1 displayed the strongest binding affinity to E2HE2H, suggesting its crucial role in recognizing the aggregation pheromone. These findings have the potential to inform the development of integrated pest management strategies utilizing behavioral approaches for bean bug control.

Binding properties of chemosensory protein 12 in Riptortus pedestris to aggregation pheromone (E)-2-hexenyl (Z)-3-hexenoate.

Riptortus pedestris (bean bug), a common soybean pest, has a highly developed olfactory system to find hosts for feeding and oviposition. Chemosensory proteins (CSPs) have been identified in many insect species; however, their functions in R. pedestris remain unknown. In this study, quantitative real time-polymerase chain reaction (qRT-PCR) revealed that the expression of RpedCSP12 in the adult antennae of R. pedestris increased with age. Moreover, a significant difference in the expression levels of RpedCSP12 was observed between male and female antennae at one and three days of age. We also investigated the binding ability of RpedCSP12 to different ligands using a prokaryotic expression system and fluorescence competitive binding assays. We found that RpedCSP12 only bound to one aggregation pheromone, (E)-2-hexenyl (Z)-3-hexenoate, and its binding decreased with increasing pH. Furthermore, homology modelling, molecular docking, and site-directed mutagenesis revealed that the Y27A, L74A, and L85A mutants lost their binding ability to (E)-2-hexenyl (Z)-3-hexenoate. Our findings highlight the olfactory roles of RpedCSP12, providing insights into the mechanism by which RpedCSPs bind to aggregation pheromones. Therefore, our study can be used as a theoretical basis for the population control of R. pedestris in the future.

Effect of Soybean Volatiles on the Behavior of the Bean Bug, Riptortus pedestris.

The bean bug, Riptortus pedestris, is a polyphagous insect that feeds primarily on leguminous plants, especially soybean (Glycine max). Although the bean bug is an economically important pest of soybean, little is known about how the insect locates soybean fields. In this study, we examined the electroantennogram responses of R. pedestris to soybean volatiles and examined the behavioral responses of the adult bean bugs. R. pedestris adults were attracted more to their host-plant soybean, even when physical contact was absent, than to air or a non-host plant. Accordingly, we hypothesized that R. pedestris can recognize soybean through a plant's volatile organic compounds (VOCs). Five VOCs were identified from intact soybean plants at the vegetative stage: (Z)-3-hexen-1-ol, (Z)-3-hexenyl acetate, 4-ethylbenzaldehyde, α-farnesene, and methyl salicylate. Response spectra of the antennae to these volatiles clearly showed that both male and female R. pedestris can detect soybean volatiles. The adult bean bugs did not show behavioral orientation to any individual compounds but showed significant orientation to a particular blend of synthetic soybean volatiles when tested under laboratory conditions. In the field, this soybean volatile blend did not significantly attract the bean bugs, but it did interact synergistically with the aggregation pheromone to attract the bean bugs. These results highlight the role of host plant volatiles in the sensory ecology of R. pedestris and help explain colonization pattern of the bean bugs in soybean fields.

Comparison and Functional Analysis of Odorant-Binding Proteins and Chemosensory Proteins in Two Closely Related Thrips Species, Frankliniella occidentalis and Frankliniella intonsa (Thysanoptera: Thripidae) Based on Antennal Transcriptome Analysis.

Two closely related thrips species, Frankliniella occidentalis and Frankliniella intonsa, are important pests on agricultural and horticultural crops. They have several similarities, including occurrence patterns, host range, and aggregation pheromone compounds. However, there are very few reports about the chemosensory genes and olfactory mechanisms in these two species. To expand our knowledge of the thrips chemosensory system, we conducted antennal transcriptome analysis of two thrips species, and identified seven odorant-binding proteins (OBPs) and eight chemosensory proteins (CSPs) in F. occidentalis, as well as six OBPs and six CSPs in F. intonsa. OBPs and CSPs showed high sequence identity between the two thrips species. The RT-qPCR results showed that the orthologous genes FoccOBP1/3/4/5/6, FintOBP1/3/4/6, FoccCSP1/2/3, and FintCSP1/2 were highly expressed in male adults. Molecular docking results suggested that orthologous pairs FoccOBP4/FintOBP4, FoccOBP6/FintOBP6, and FoccCSP2/FintCSP2 might be involved in transporting the major aggregation pheromone compound neryl (S)-2-methylbutanoate, while orthologous pairs FoccOBP6/FintOBP6, FoccCSP2/FintCSP2, and FoccCSP3/FintCSP3 might be involved in transporting the minor aggregation pheromone compound (R)-lavandulyl acetate. These results will provide a fundamental basis for understanding the molecular mechanisms of pheromone reception in the two thrips species.

Key Residues Affecting Binding Affinity of Sirex noctilio Fabricius Odorant-Binding Protein (SnocOBP9) to Aggregation Pheromone.

Sirex noctilio Fabricius (Hymenoptera Siricidae) is a major quarantine pest responsible for substantial economic losses in the pine industry. To achieve better pest control, (Z)-3-decen-ol was identified as the male pheromone and used as a field chemical trapping agent. However, the interactions between odorant-binding proteins (OBPs) and pheromones are poorly described. In this study, SnocOBP9 had a higher binding affinity with Z3D (Ki = 1.53 ± 0.09 µM) than other chemical ligands. Molecular dynamics simulation and binding mode analysis revealed that several nonpolar residues were the main drivers for hydrophobic interactions between SnocOBP9 and Z3D. Additionally, computational alanine scanning results indicated that five amino acids (MET54, PHE57, PHE71, PHE74, LEU116) in SnocOBP9 could potentially alter the binding affinity to Z3D. Finally, we used single-site-directed mutagenesis to substitute these five residues with alanine. These results imply that the five residues play crucial roles in the SnocOBP9-Z3D complex. Our research confirmed the function of SnocOBP9, uncovered the key residues involved in SnocOBP9-Z3D interactions, and provides an inspiration to improve the effects of pheromone agent traps.

Pheromone receptor of the globally invasive quarantine pest of the palm tree, the red palm weevil (Rhynchophorus ferrugineus).

Palm trees are of immense economic, sociocultural, touristic, and patrimonial significance all over the world, and date palm-related knowledge, traditions, and practices are now included in UNESCOs list of the Intangible Cultural Heritage of Humanity. Of all the pests that infest these trees, the red palm weevil (RPW), Rhynchophorus ferrugineus (Olivier), is its primary enemy. The RPW is a category-1 quarantine insect pest that causes enormous economic losses in palm tree cultivation worldwide. The RPW synchronizes mass gathering on the palm tree for feeding and mating, regulated by a male-produced pheromone composed of two methyl-branched compounds, (4RS, 5RS)-4-methylnonan-5-ol (ferrugineol) and 4(RS)-methylnonan-5-one (ferrugineone). Despite the importance of odorant detection in long-range orientation towards palm trees, palm colonization, and mating, the pheromone receptor has not been identified in this species. In this study, we report the identification and characterization of the first RPW pheromone receptor, RferOR1. Using gene silencing and functional expression in Drosophila olfactory receptor neurons, we demonstrate that RferOR1 is tuned to ferrugineol and ferrugineone and binds five other structurally related molecules. We reveal the lifetime expression of RferOR1, which correlates with adult mating success irrespective of age, a factor that could explain the wide distribution and spread of this pest. As palm weevils are challenging to control based on conventional methods, elucidation of the mechanisms of pheromone detection opens new routes for mating disruption and the early detection of this pest via the development of pheromone receptor-based biosensors.

A larval aggregation pheromone as foraging cue for insectivorous birds.

Although birds have traditionally been considered anosmic, increasing evidence indicates that olfaction plays an important role in the foraging behaviours of insectivorous birds. Recent studies have shown that birds can exploit herbivore-induced plant volatiles and sexual pheromones of adult insects to locate their prey. Many insectivorous birds prey on immature insects, providing relevant ecosystem services as pest regulators in natural and agricultural ecosystems. We asked whether birds could rely on chemical cues emitted by the immature stages of insects to prey on them. To address this question, we performed field experiments to evaluate if insectivorous birds can detect the aggregation pheromone produced by the larvae of the carpenter worm, Chilecomadia valdiviana. Groups of five artificial larvae were placed in branches of 72 adult trees in a remnant fragment of a sclerophyllous forest in central Chile. Each grouping of larvae contained a rubber septum loaded with either larval pheromone as treatment or solvent alone as control. We found that the number of larvae damaged by bird pecks was significantly higher in groups with dispensers containing the larval extract than in control groups. Our results show that birds can rely on immature insect-derived chemical cues used for larvae aggregation to prey on them.

De novo formation of an aggregation pheromone precursor by an isoprenyl diphosphate synthase-related terpene synthase in the harlequin bug.

Insects use a diverse array of specialized terpene metabolites as pheromones in intraspecific interactions. In contrast to plants and microbes, which employ enzymes called terpene synthases (TPSs) to synthesize terpene metabolites, limited information from few species is available about the enzymatic mechanisms underlying terpene pheromone biosynthesis in insects. Several stink bugs (Hemiptera: Pentatomidae), among them severe agricultural pests, release 15-carbon sesquiterpenes with a bisabolene skeleton as sex or aggregation pheromones. The harlequin bug, Murgantia histrionica, a specialist pest of crucifers, uses two stereoisomers of 10,11-epoxy-1-bisabolen-3-ol as a male-released aggregation pheromone called murgantiol. We show that MhTPS (MhIDS-1), an enzyme unrelated to plant and microbial TPSs but with similarity to trans-isoprenyl diphosphate synthases (IDS) of the core terpene biosynthetic pathway, catalyzes the formation of (1S,6S,7R)-1,10-bisaboladien-1-ol (sesquipiperitol) as a terpene intermediate in murgantiol biosynthesis. Sesquipiperitol, a so-far-unknown compound in animals, also occurs in plants, indicating convergent evolution in the biosynthesis of this sesquiterpene. RNAi-mediated knockdown of MhTPS mRNA confirmed the role of MhTPS in murgantiol biosynthesis. MhTPS expression is highly specific to tissues lining the cuticle of the abdominal sternites of mature males. Phylogenetic analysis suggests that MhTPS is derived from a trans-IDS progenitor and diverged from bona fide trans-IDS proteins including MhIDS-2, which functions as an (E,E)-farnesyl diphosphate (FPP) synthase. Structure-guided mutagenesis revealed several residues critical to MhTPS and MhFPPS activity. The emergence of an IDS-like protein with TPS activity in M. histrionica demonstrates that de novo terpene biosynthesis evolved in the Hemiptera in an adaptation for intraspecific communication.

Communicating the nutritional value of sugar in Drosophila.

Sweet-insensitive Drosophila mutants are unable to readily identify sugar. In presence of wild-type (WT) flies, however, these mutant flies demonstrated a marked increase in their preference for nutritive sugar. Real-time recordings of starved WT flies revealed that these flies discharge a drop from their gut end after consuming nutritive sugars, but not nonnutritive sugars. We proposed that the drop may contain a molecule(s) named calorie-induced secreted factor (CIF), which serves as a signal to inform other flies about its nutritional value. Consistent with this, we observed a robust preference of flies for nutritive sugar containing CIF over nutritive sugar without CIF. Feeding appears to be a prerequisite for the release of CIF, given that fed flies did not produce it. Additionally, correlation analyses and pharmacological approaches suggest that the nutritional value, rather than the taste, of the consumed sugar correlates strongly with the amount (or intensity) of the released CIF. We observed that the release of this attractant signal requires the consumption of macronutrients, specifically nutritive sugars and l-enantiomer essential amino acids (l-eAAs), but it is negligibly released when flies are fed nonnutritive sugars, unnatural d-enantiomer essential amino acids (d-eAAs), fatty acids, alcohol, or salts. Finally, CIF (i) is not detected by the olfactory system, (ii) is not influenced by the sex of the fly, and (iii) is not limited to one species of Drosophila.

Behavioral and Antennal Responses of Tribolium confusum to Varronia globosa Essential Oil and Its Main Constituents: Perspective for Their Use as Repellent.

Essential oils of aromatic plants represent an alternative to classical pest control with synthetic chemicals. They are especially promising for the alternative control of stored product pest insects. Here, we tested behavioral and electrophysiological responses of the stored product pest Tribolium confusum, to the essential oil of a Brazilian indigenous plant, Varronia globosa, collected in the Caatinga ecosystem. We analyzed the essential oil by GC-MS, tested the effects of the entire oil and its major components on the behavior of individual beetles in a four-way olfactometer, and investigated responses to these stimuli in electroantennogram recordings (EAG). We could identify 25 constituents in the essential oil of V. globosa, with anethole, caryophyllene and spathulenole as main components. The oil and its main component anethole had repellent effects already at low doses, whereas caryophyllene had only a repellent effect at a high dose. In addition, the essential oil abolished the attractive effect of the T. confusum aggregation pheromone. EAG recordings revealed dose-dependent responses to the individual components and increasing responses to the blend and even more to the entire oil. Our study reveals the potential of anethole and the essential oil of V. globosa in the management of stored product pests.

Chemical signal interactions of the bark beetle with fungal symbionts, and host/non-host trees.

The symbiosis between the bark beetle (Ips subelongatus) and its fungal symbiont (Endoconidiophora fujiensis) poses a serious threat to larch forests. However, the signaling pathways between these symbiotic partners and their host/non-host trees are not fully understood. Inoculation of the host larch (Larix principis-rupprechtii) with two strains of E. fujiensis induced a rapid and long-term release of monoterpenes. Although the fungi had a level of tolerance to these compounds, many monoterpenes inhibited fungal growth in culture. Moreover, monoterpenes with stronger inhibitory effects on fungal growth exhibited weaker synergistic effects on the attraction of I. subelongatus to aggregation pheromone. Surprisingly, individual isomers of aggregation pheromone components promoted fungal symbiont growth in a culture medium. Non-host volatiles (NHVs) were tested and shown to completely inhibit the growth of fungal symbionts in culture but had no effects on beetle responses to aggregation pheromone, with the exception of (Z)-3-hexen-1-ol. These results reveal convergence and mutualism patterns in the evolution of I. subelongatus and E. fujiensis with respect to host tree volatiles but not in response to NHVs. Ultimately, we put forward a hypothesis that host plants are ecological and evolutionary determinants of bark beetle-fungus symbioses in terms of their complex signaling interactions.

Effects of Pheromone Dose and Conspecific Density on the Use of Aggregation-Sex Pheromones by the Longhorn Beetle Phymatodes grandis and Sympatric Species (Coleoptera: Cerambycidae).

Many species of longhorn beetles (Coleoptera: Cerambycidae) utilize male-produced aggregation-sex pheromones that attract both sexes. However, the reasons why and the details of how this type of pheromone is used by cerambycids and other coleopteran species that utilize analogous male-produced pheromones remain unclear. Thus, our goals were to test the hypotheses that 1) cerambycids respond to pheromones in a dose-dependent (= release rate-dependent) manner and 2) pheromone emission is density-dependent. If true, these characteristics of pheromone use could suggest that cerambycids utilize an optimal density strategy to limit competition for scarce and ephemeral hosts, i.e., the stressed or dying trees that typically constitute their larval hosts. Attraction of beetles to a range of release rates of two common pheromone components - 2-methylbutanol and 3-hydroxyhexan-2-one - was tested in field trials. Responses, as measured by the number of beetles caught in pheromone-baited traps, increased with release rates for five endemic species, even at the highest rates tested (~1450 µg/h for 2-methylbutanol and ~720 µg/h for 3-hydroxyhexan-2-one). The effect of density of conspecific males on per capita pheromone production was tested by collecting the volatiles produced by individuals, pairs, or groups of three or four male Phymatodes grandis Casey. Frequency of pheromone production was significantly different among the treatment densities, and emission rates of the pheromone (R)-2-methylbutanol decreased with increasing density. These results are discussed in the context of a possible optimal density strategy used by cerambycids, and more broadly, in relation to the use of male-produced aggregation-sex pheromones by other coleopterans. In addition, we report the identification of the pheromones of four of our five test species, specifically, Phymatodes obliquus Casey, Brothylus conspersus LeConte, Brothylus gemmulatus LeConte, and Xylotrechus albonotatus Casey.

Isopentyl Butanoate: Aggregation Pheromone of the Brown Spiny Bug, Clavigralla tomentosicollis (Hemiptera: Coreidae), and Kairomone for the Egg Parasitoid Gryon sp. (Hymenoptera: Scelionidae).

The brown spiny bug, Clavigralla tomentosicollis Stål (Hemiptera: Coreidae) is a key pest of leguminous crops in many countries in Africa, causing significant yield losses especially in cowpea, pigeon pea and common beans. Although C. tomentosicollis uses olfaction to aggregate, little is known about the identity of the aggregation pheromone. This study aimed to identify the aggregation pheromone of C. tomentosicollis and to test its potential role in the behavior of its egg parasitoid, Gryon sp. In Y-tube olfactometer bioassays, only male volatiles strongly attracted both sexes of C. tomentosicollis. Coupled gas chromatography/electroantennographic detection (GC/EAD) and GC/mass spectrometry were used to identify antennally-active compounds from male volatiles. Antennae of both sexes detected identical components including a male-specific component, identified as isopentyl butanoate, which was also detected by antenna of the egg parasitoid. In olfactometer bioassays, both sexes of C. tomentosicollis and the egg parasitoid responded to isopentyl butanoate. These results suggest that isopentyl butanoate serves as an aggregation pheromone for both sexes of C. tomentosicollis and a useful kairomone to attract the parasitoid in the management of C. tomentosicollis.

Male-Produced Aggregation Pheromone of the Sloe Bug, Dolycoris baccarum L. (Hemiptera: Heteroptera: Pentatomidae).

The sloe bug, Dolycoris baccarum L. (Hemiptera: Heteroptera: Pentatomidae), is a Palearctic species and an important polyphagous pest. Previous studies have reported that male D. baccarum produce an aggregation pheromone, to which conspecific adults of both sexes respond. We used solid-phase microextraction (SPME) to collect volatiles released by sexually mature virgin males and females, and identified potential components of the male-produced aggregation pheromone. Analysis of the SPME samples by gas chromatography-mass spectrometry (GC-MS) revealed three male-specific components, α-bisabolol, trans-α-bergamotene, and ß-bisabolene, at a ratio of 100:10:3. A field trapping experiment showed that (±)-α-bisabolol alone attracted D. baccarum adults, and it was synergized by trans-α-bergamotene, but not (S)-ß-bisabolene. However, neither trans-α-bergamotene nor (S)-ß-bisabolene was attractive individually. A binary blend of (±)-α-bisabolol and trans-α-bergamotene in a 10:1 ratio was less attractive than caged live males, suggesting that further study is necessary to determine the absolute configuration or steroisomeric composition of the natural pheromone components. This is the first report of α-bisabolol and trans-α-bergamotene as semiochemicals among Pentatomidae, and the first to identify α-bisabolol from insects.

Species-specific aggregation pheromones contribute to coexistence in two closely related thrips species.

Pheromones play an important role in mediating interspecific interactions in insects. In an insect community, pheromones can reveal information about the senders, which could be used by other members of the food web (competitor, natural enemies, etc.) to their own advantage. The aggregation pheromones of two closely related thrips species, Frankliniella occidentalis and Frankliniella intonsa, have been identified with the same major compounds, (R)-lavandulyl acetate and neryl (S)-2-methylbutanoate, but in different ratios. However, the roles of the aggregation pheromones in the interspecific interactions between these two closely related species are unknown. Here, we investigated the roles of major aggregation pheromone compounds in interspecific interactions between F. occidentalis and F. intonsa for both long and short ranges. The results showed that, at tested doses, neither aggregation pheromone-induced long range cross-attraction nor short range cross-mating was detected between F. occidentalis and F. intonsa. Field-trapping trials showed that the species-specificity in aggregation pheromones was regulated by the ratio of two major compounds. However, species-specific blends of the two major compounds had no effect on short-range interactions between these two species. Our data from the thrips species provide support for the 'aggregation model of coexistence', explaining the species-specific pheromone-mediated coexistence of closely related species. Thus, species-specific pheromones could be one of the factors affecting population dynamics and community structure in closely related insects with similar niches.

Novel family of terpene synthases evolved from trans-isoprenyl diphosphate synthases in a flea beetle.

Sesquiterpenes play important roles in insect communication, for example as pheromones. However, no sesquiterpene synthases, the enzymes involved in construction of the basic carbon skeleton, have been identified in insects to date. We investigated the biosynthesis of the sesquiterpene (6R,7S)-himachala-9,11-diene in the crucifer flea beetle Phyllotreta striolata, a compound previously identified as a male-produced aggregation pheromone in several Phyllotreta species. A (6R,7S)-himachala-9,11-diene-producing sesquiterpene synthase activity was detected in crude beetle protein extracts, but only when (Z,E)-farnesyl diphosphate [(Z,E)-FPP] was offered as a substrate. No sequences resembling sesquiterpene synthases from plants, fungi, or bacteria were found in the P. striolata transcriptome, but we identified nine divergent putative trans-isoprenyl diphosphate synthase (trans-IDS) transcripts. Four of these putative trans-IDSs exhibited terpene synthase (TPS) activity when heterologously expressed. Recombinant PsTPS1 converted (Z,E)-FPP to (6R,7S)-himachala-9,11-diene and other sesquiterpenes observed in beetle extracts. RNAi-mediated knockdown of PsTPS1 mRNA in P. striolata males led to reduced emission of aggregation pheromone, confirming a significant role of PsTPS1 in pheromone biosynthesis. Two expressed enzymes showed genuine IDS activity, with PsIDS1 synthesizing (E,E)-FPP, whereas PsIDS3 produced neryl diphosphate, (Z,Z)-FPP, and (Z,E)-FPP. In a phylogenetic analysis, the PsTPS enzymes and PsIDS3 were clearly separated from a clade of known coleopteran trans-IDS enzymes including PsIDS1 and PsIDS2. However, the exon-intron structures of IDS and TPS genes in P. striolata are conserved, suggesting that this TPS gene family evolved from trans-IDS ancestors.

Pheromones modulate responsiveness to a noxious stimulus in honey bees.

Pheromones are chemical substances released into the environment by an individual, which trigger stereotyped behaviors and/or physiological processes in individuals of the same species. Yet, a novel hypothesis has suggested that pheromones not only elicit innate responses but also contribute to behavioral plasticity by affecting the subjective evaluation of appetitive or aversive stimuli. To test this hypothesis, we exposed bees to three pheromonal components whose valence was either negative (i.e. associated with aversive events: isopentyl acetate and 2-heptanone) or positive (i.e. associated with appetitive events: geraniol). We then determined the effect of this exposure on the subjective evaluation of aversive stimuli by quantifying responsiveness to a series of increasing electric shock voltages before and after exposure. Two experiments were conducted varying the time lapse between shock series (15 min in experiment 1, and 24 h in experiment 2). In experiment 1, we observed a general decrease of shock responsiveness caused by fatigue, due to the short lapse of time between the two series of shocks. This decrease could only be counteracted by isopentyl acetate. The enhancing effect of isopentyl acetate on shock responsiveness was also found in experiment 2. Conversely, geraniol decreased aversive responsiveness in this experiment; 2-heptanone did not affect aversive responsiveness in any experiment. Overall, our results demonstrate that certain pheromones modulate the salience of aversive stimuli according to their valence. In this way, they would affect the motivation to engage in aversive responses, thus acting as modulators of behavioral plasticity.