Glucosinolates are token stimuli in host selection of many crucifer specialist insects, but the underlying molecular basis for host selection in these insects remains enigmatic. Using a combination of behavioral, electrophysiological, and molecular methods, we investigate glucosinolate receptors in the cabbage butterflyPieris rapae. Sinigrin, as a potent feeding stimulant, elicited activity in larval maxillary lateral sensilla styloconica, as well as in adult medial tarsal sensilla. TwoP.rapaegustatory receptor genesPrapGr28andPrapGr15were identified with high expression in female tarsi, and the subsequent functional analyses showed thatXenopusoocytes only expressingPrapGr28had specific responses to sinigrin; when ectopically expressed inDrosophilasugar sensing neurons, PrapGr28 conferred sinigrin sensitivity to these neurons. RNA interference experiments further showed that knockdown ofPrapGr28reduced the sensitivity of adult medial tarsal sensilla to sinigrin. Taken together, we conclude that PrapGr28 is a gustatory receptor tuned to sinigrin inP.rapae, which paves the way for revealing the molecular basis of the relationships between crucifer plants and their specialist insects.

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.

The closely related species Helicoverpa armigera (H. armigera) and Helicoverpa assulta (H. assulta) have different host plant ranges and share two principal components of sex pheromones but with reversed ratios. The antennae are the main olfactory organ of insects and play a crucial role in host plant selection and mate seeking. However, the genetic basis for gene expression divergence in the antennae of the two species is unclear. We performed an allele-specific expression (ASE) analysis in the antennal transcriptomes of the two species and their F₁ hybrids, examining the connection between gene expression divergence and phenotypic differences. The results show that the proportion of genes classified as all cis was higher than that of all trans in males and reversed in females. The contribution of regulatory patterns to gene expression divergence in males was less than that in females, which explained the functional differentiation of male and female antennae. Among the five groups of F₁ hybrids, the fertile males from the cross of H. armigera female and H. assulta male had the lowest proportion of misexpressed genes, and the inferred regulatory patterns were more accurate. By using this group of F₁ hybrids, we discovered that cis-related regulations play a crucial role in gene expression divergence of sex pheromone perception-related proteins. These results are helpful for understanding how specific changes in the gene expression of olfactory-related genes can contribute to rapid evolutionary changes in important olfactory traits in closely related moths.

Myo-inositol, a sugar alcohol produced by most plants, serves as a nutrient and feeding stimulant for many phytophagous insects. Inositol-sensitive taste sensilla have been characterized in many Lepidoptera larvae, but their molecular bases remain unclear. In this study, we determined the gustatory receptors (GRs) for myo-inositol in larva of Helicoverpa armigera, a worldwide crop pest. First, electrophysiological analyses revealed that medial sensilla styloconica strongly responded to myo-inositol and ribose, with weaker responses to xylose, and one GRN inside sensillum may mediate the response to these three chemicals. Based on phylogenetic analysis of sugar GRs of Lepidoptera insects and previous results on Bombyx mori, we then selected two candidate GRs, HarmGR13 and HarmGR11. Using CRISPR-Cas9, we generated knockout mutants for the two GR genes. Knocking out HarmGR13 abolished the responses of the sensilla to myo-inositol, ribose, and xylose, while knocking out HarmGR11 showed no changes. Behavioral assays confirmed that larvae of HarmGR13 homozygous mutant lost the feeding preference to myo-inositol which the wild-type larvae had. Further functional analysis with Xenopus oocytes expressing system and two-electrode voltage-clamping demonstrated that myo-inositol and ribose specifically induced concentration-dependent currents in HarmGR13-expressing oocytes. Structural predictions and molecular docking of HarmGR13 revealed three amino acid residues might be involved in ligand binding. Mutation of these residues resulted in loss of oocyte responses to myo-inositol and ribose. We reveal that HarmGR13 is a receptor that mediates the activity of the cells sensitive to inositol and ribose in larvae, providing new molecular targets for the strategy of regulating the feeding behavior of pests by modifying taste.

Feeding and oviposition deterrents help phytophagous insects to identify host plants. The taste organs of phytophagous insects contain bitter gustatory receptors (GRs). To explore their function, we focused on PxylGr34, a bitter GR in Plutella xylostella (L.). We detected abundant PxylGr34 transcripts in the larval head and specific expression of PxylGr34 in the antennae of females. Analyses using the Xenopus oocyte expression system and two-electrode voltage-clamp recording showed that PxylGr34 is specifically tuned to the plant hormone brassinolide (BL) and its analog 24-epibrassinolide. Electrophysiological analyses revealed that the medial sensilla styloconica on the maxillary galea of the 4 ^th instar larvae are responsive to BL. Dual-choice bioassays demonstrated that BL inhibits larval feeding and female ovipositing. Knock-down of PxylGr34 by RNAi abolished BL-induced feeding inhibition. These results shed light on gustatory coding mechanisms and deterrence of insect feeding and ovipositing, and may be useful for designing plant hormone-based pest management strategies.