Sex pheromone communication in an insect parasitoid, Campoletis chlorideae Uchida.

Sex pheromones are pivotal for insect reproduction. However, the mechanism of sex pheromone communication remains enigmatic in hymenopteran parasitoids. Here we have identified the sex pheromone and elucidated the olfactory basis of sex pheromone communication in Campoletis chlorideae (Ichneumonidae), a solitary larval endoparasitoid of over 30 lepidopteran pests. Using coupled gas chromatography-electroantennogram detection, we identified two female-derived pheromone components, tetradecanal (14:Ald) and 2-heptadecanone (2-Hep) (1:4.6), eliciting strong antennal responses from males but weak responses from females. We observed that males but not females were attracted to both single components and the blend. The hexane-washed female cadavers failed to arouse males, and replenishing 14:Ald and 2-Hep could partially restore the sexual attraction of males. We further expressed six C. chlorideae male-biased odorant receptors in Drosophila T1 neurons and found that CchlOR18 and CchlOR47 were selectively tuned to 14:Ald and 2-Hep, respectively. To verify the biological significance of this data, we knocked down CchlOR18 and CchlOR47 individually or together in vivo and show that the attraction of C. chlorideae to their respective ligands was abolished. Moreover, the parasitoids defective in either of the receptors were less likely to court and copulate. Finally, we show that the sex pheromone and (Z)-jasmone, a potent female attractant, can synergistically affect behaviors of virgin males and virgin females and ultimately increase the parasitic efficiency of C. chlorideae. Our study provides new insights into the molecular mechanism of sex pheromone communication in C. chlorideae that may permit manipulation of parasitoid behavior for pest control.

Mutagenesis of the odorant receptor co-receptor (Orco) reveals severe olfactory defects in the crop pest moth Helicoverpa armigera.

BACKGROUND: Odorant receptors (ORs) as odorant-gated ion channels play a crucial role in insect olfaction. They are formed by a heteromultimeric complex of the odorant receptor co-receptor (Orco) and a ligand-selective Or. Other types of olfactory receptor proteins, such as ionotropic receptors (IRs) and some gustatory receptors (GRs), are also involved in the olfactory system of insects. Orco as an obligatory subunit of ORs is highly conserved, providing an opportunity to systematically evaluate OR-dependent olfactory responses. RESULTS: Herein, we successfully established a homozygous mutant (Orco-/-) of Helicoverpa armigera, a notorious crop pest, using the CRISPR/Cas9 gene editing technique. We then compared the olfactory response characteristics of wild type (WT) and Orco-/- adults and larvae. Orco-/- males were infertile, while Orco-/- females were fertile. The lifespan of Orco-/- females was longer than that of WT females. The expressions of most Ors, Irs, and other olfaction-related genes in adult antennae of Orco-/- moths were not obviously affected, but some of them were up- or down-regulated. In addition, there was no change in the neuroanatomical phenotype of Orco-/- moths at the level of the antennal lobe (including the macroglomerular complex region of the male). Using EAG and SSR techniques, we discovered that electrophysiological responses of Orco-/- moths to sex pheromone components and many host plant odorants were absent. The upwind flight behaviors toward sex pheromones of Orco-/- males were severely reduced in a wind tunnel experiment. The oviposition selectivity of Orco-/- females to the host plant (green pepper) has completely disappeared, and the chemotaxis toward green pepper was also lost in Orco-/- larvae. CONCLUSIONS: Our study indicates that OR-mediated olfaction is essential for pheromone communication, oviposition selection, and larval chemotaxis of H. armigera, suggesting a strategy in which mate searching and host-seeking behaviors of moth pests could be disrupted by inhibiting or silencing Orco expression.

Identification and functional characterization of chemosensory genes in olfactory and taste organs of Spodoptera litura (Lepidoptera: Noctuidae).

The tobacco cutworm Spodoptera litura is one of the most destructive polyphagous crop pests. Olfaction and taste play a crucial role in its host plant selection and sexual communication, but the expression profile of chemosensory genes remains unclear. In this study, we identified 185 chemosensory genes from 7 organs in S. litura by transcriptome sequencing, of which 72 genes were published for the first time, including 27 odorant receptors (ORs), 26 gustatory receptors (GRs), 1 ionotropic receptor (IR), 16 odorant-binding proteins (OBPs), and 2 chemosensory proteins (CSPs). Phylogenetic analyses revealed that ORs, IRs, OBPs, and sensory neuron membrane proteins (SNMPs) were mainly expressed in antennae and sequence-conserved among Noctuidae species. The most differentially expressed genes (DEGs) between sexes were ORs and OBPs, and no DEGs were found in GRs. GR transcripts were enriched in proboscis, and the expression of sugar receptors was the highest. Carbon dioxide receptors, sugar receptor-SliuGR6, and bitter GRs-SlituGR43 and SlituGR66 had higher sequence identities between Noctuidae species. CSPs were broadly expressed in various organs, and SlituCSP13 was a DEG in adult antennae. The functional analysis in the Drosophila OR67d expression system found that SlituOR50, a receptor highly expressed in female antennae, is selectively tuned to farnesyl acetate. The results provide a solid foundation for understanding the molecular mechanisms by which chemosensory genes operate to elicit behavioral responses in polyphagous insects.

Functional types of long trichoid sensilla responding to sex pheromone components in Plutella xylostella.

Sex pheromones, which consist of multiple components in specific ratios promote intraspecific sexual communications of insects. Plutella xylostella (L.) is a worldwide pest of cruciferous vegetables, the mating behavior of which is highly dependent on its olfactory system. Long trichoid sensilla on male antennae are the main olfactory sensilla that can sense sex pheromones. However, the underlying mechanisms remain unclear. In this study, 3 sex pheromone components from sex pheromone gland secretions of P. xylostella female adults were identified as Z11-16:Ald, Z11-16:Ac, and Z11-16:OH in a ratio of 9.4 : 100 : 17 using gas chromatography - mass spectrometry and gas chromatography with electroantennographic detection. Electrophysiological responses of 581 and 385 long trichoid sensilla of male adults and female adults, respectively, to the 3 components were measured by single sensillum recording. Hierarchical clustering analysis showed that the long trichoid sensilla were of 6 different types. In the male antennae, 52.32%, 5.51%, and 1.89% of the sensilla responded to Z11-16:Ald, Z11-16:Ac, and Z11-16:OH, which are named as A type, B type, and C type sensilla, respectively; 2.93% named as D type sensilla responded to both Z11-16:Ald and Z11-16:Ac, and 0.34% named as E type sensilla were sensitive to both Z11-16:Ald and Z11-16:OH. In the female antennae, only 7.53% of long trichoid sensilla responded to the sex pheromone components, A type sensilla were 3.64%, B type and C type sensilla were both 0.52%, D type sensilla were 1.30%, and 1.56% of the sensilla responded to all 3 components, which were named as F type sensilla. The responding long trichoid sensilla were located from the base to the terminal of the male antennae and from the base to the middle of the female antennae. The pheromone mixture (Z11-16:Ald : Z11-16:Ac : Z11-16:OH = 9.4 : 100 : 17) had a weakly repellent effect on female adults of P. xylostella. Our results lay the foundation for further studies on sex pheromone communications in P. xylostella.

Discovery of Insect Attractants Based on the Functional Analyses of Female-Biased Odorant Receptors and Their Orthologs in Two Closely Related Species.

Olfaction plays an instrumental role in host plant selection by phytophagous insects. Helicoverpa assulta and Helicoverpa armigera are two closely related moth species with different host plant ranges. In this study, we first comparatively analyzed the function of 11 female-biased odorant receptors (ORs) and their orthologs in the two species by the Drosophila T1 neuron expression system and then examined the electroantennography responses of the two species to the most effective OR ligands. Behavioral assays using a Y-tube olfactometer indicate that guaiene, the primary ligand of HassOR21-2 and HarmOR21-2, only attracts the females, while benzyl acetone, the main ligand of HassOR35 and HarmOR35, attracts both sexes of the two species. Oviposition preference experiments further confirm that guaiene and benzyl acetone are potent oviposition attractants for the mated females of both species. These findings deepen our understanding of the olfactory coding mechanisms of host plant selection in herbivorous insects and provide valuable attractants for managing pest populations.

Revisiting the sex pheromone of the fall armyworm Spodoptera frugiperda, a new invasive pest in South China.

The fall armyworm Spodoptera frugiperda is a worldwide serious agricultural pest, and recently invaded South China. Sex pheromone can be employed to monitor its population dynamics accurately in the field. However, the pheromone components previously reported by testing different geographic populations and strains are not consistent. On the basis of confirming that the S. frugiperda population from Yunnan Province belonged to the corn strain, we analyzed the potential sex pheromone components in the pheromone gland extracts of females using gas chromatography coupled with electroantennographic detection (GC-EAD), gas chromatography coupled with mass spectrometry (GC-MS) and electroantennography (EAG). The results show that (Z)-9-tetradecenal acetate (Z9-14:Ac), (Z)-11-hexadecenyl acetate (Z11-16:Ac), (Z)-7-dodecenyl acetate (Z7-12:Ac) or (E)-7-dodecenyl acetate (E7-12:Ac) with a ratio of 100 : 15.8 : 3.9 induced EAD responses to varying degrees: Z9-14:Ac elicited a strong EAD response, Z7-12:Ac or E7-12:Ac elicited a small but clear EAD response, while Z11-16:Ac elicited a weak EAD response. Further single sensillum recording (SSR) showed that Z9-14:Ac and Z7-12:Ac induced dose-dependent activities in two types (A and B) of sensilla in male antennae, respectively, while the sensilla in response to E7-12:Ac and Z11-16:Ac was not recorded. Finally, wind tunnel tests reveal that Z9-14:Ac and Z7-12:Ac are two principal sex pheromone components of the tested population.

Functional analysis of pheromone receptor repertoire in the fall armyworm, Spodoptera frugiperda.

BACKGROUND: The fall armyworm, Spodoptera frugiperda (J. E. Smith), is a polyphagous moth species that is spreading all around the globe. It uses (Z)-9-tetradecenyl acetate (Z9-14:Ac) and (Z)-7-dodecenyl acetate (Z7-12:Ac) (100:3.9) as essential sex pheromone components. However, our understanding of the molecular basis of pheromone detection of S. frugiperda is still incomplete. RESULTS: Herein, we identified six PRs, i.e. SfruOR6, 11, 13, 16, 56, and 62, by transcriptome sequencing. Subsequently, we heterologously expressed them in Drosophila OR67d neurons and determined their response spectra with a large panel of sex pheromones and analogs. Among them, SfruOR13-expressing neurons strongly respond to the major sex pheromone component Z9-14:Ac, but also comparably to (Z,E)-9,12-tetradecadienyl acetate (Z9,E12-14:Ac) and weakly to (Z)-9-dodecenyl acetate (Z9-12:Ac). Both SfruOR56 and SfruOR62 are specifically tuned to the minor sex pheromone component Z7-12:Ac with varying intensities and sensitivities. In addition, SfruOR6 is activated only by Z9,E12-14:Ac, and SfruOR16 by both (Z)-9-tetradecenol (Z9-14:OH) and (Z)-9-tetradecenal (Z9-14:Ald). However, the OR67d neurons expressing SfruOR11 remain silent to all compounds tested, a phenomenon commonly found in the OR11 clade of Noctuidae species. Next, using single sensillum recording, we characterized four sensilla types on the antennae of males, namely A, B, C and D types that are tuned to the ligands of PRs, thereby confirming that S. frugiperda uses both SfruOR56 and SfruOR62 to detect Z7-12:Ac. Finally, using wind tunnel assay, we demonstrate that both Z9,E12-14:Ac and Z9-14:OH act as antagonists to the sex pheromone. CONCLUSION: We have deorphanized five PRs and characterized four types of sensilla responsible for the detection of pheromone compounds, providing insights into the peripheral encoding of sex pheromones in S. frugiperda.