Functional analysis of a bitter gustatory receptor highly expressed in the larval maxillary galea of Helicoverpa armigera.

Many plant secondary substances are feeding deterrents for insects and play a key role in the selection of host plants. The taste sensilla of phytophagous insects contain gustatory sensory neurons sensitive to deterrents but the molecular basis of deterrent chemoreception remains unknown. We investigated the function of Gr180, the most highly expressed bitter gustatory receptor in the maxillary galea of Helicoverpa armigera larvae. Functional analyses using the Xenopus oocyte expression system and two-electrode voltage clamp revealed that the oocytes expressing Gr180 responded to coumarin. Tip recording results showed that the medial sensilla styloconica of the maxilla of fifth instar larvae exhibited electrophysiological responses to coumarin. Two-choice feeding bioassays confirmed that coumarin inhibited larval feeding. A homozygous mutant strain of H. armigera with truncated Gr180 proteins (Gr180-/-) was established using the CRISPR-Cas9 system. The responses of the medial sensilla styloconica in Gr180-/- to coumarin were almost abolished, and the responses to sinigrin and strychnine were also significantly decreased. Knockout of Gr180 alleviated the feeding deterrent effects of coumarin, sinigrin, and strychnine. Thus, we conclude that Gr180 is a receptor responding to coumarin,and also participates in sensing sinigrin and strychnine. These results enhance our understanding of the gustatory sensing mechanisms of phytophagous insects to deterrents.

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.

Identification of a gustatory receptor tuned to sinigrin in the cabbage butterfly Pieris rapae.

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 butterfly Pieris rapae. Sinigrin, as a potent feeding stimulant, elicited activity in larval maxillary lateral sensilla styloconica, as well as in adult medial tarsal sensilla. Two P. rapae gustatory receptor genes PrapGr28 and PrapGr15 were identified with high expression in female tarsi, and the subsequent functional analyses showed that Xenopus oocytes only expressing PrapGr28 had specific responses to sinigrin; when ectopically expressed in Drosophila sugar sensing neurons, PrapGr28 conferred sinigrin sensitivity to these neurons. RNA interference experiments further showed that knockdown of PrapGr28 reduced the sensitivity of adult medial tarsal sensilla to sinigrin. Taken together, we conclude that PrapGr28 is a gustatory receptor tuned to sinigrin in P. rapae, which paves the way for revealing the molecular basis of the relationships between crucifer plants and their specialist insects.

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.

The Genetic Basis of Gene Expression Divergence in Antennae of Two Closely Related Moth Species, Helicoverpa armigera and Helicoverpa assulta.

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.

Differential immunosuppression by Campoletis chlorideae eggs and ichnovirus in larvae of Helicoverpa armigera and Spodoptera exigua.

The ichneumonid wasp, Campoletis chlorideae Uchida, successfully develops in the cotton bollworm Helicoverpa armigera (Hübner), but rarely survives in the beet armyworm Spodoptera exigua (Hübner) due to the encapsulation by host immunity. In this study, we investigated the role of C. chlorideae ichnovirus (CcIV) and eggs in the evasion of the host immune system. Washed eggs of different types, immature, mature, newly laid, or pretreated with protease K, were injected alone or with the calyx fluid containing CcIV into the larvae of H. armigera and S. exigua. In H. armigera, when injected with washed eggs alone, only 9.5% of the mature eggs were encapsulated at 24h post-injection. This is much lower than that of the immature eggs (100%), mature eggs pretreated with protease K (100%) and newly laid eggs (54.4%). No encapsulation was observed when the washed eggs were co-injected with calyx fluid at 24h post-injection. Conversely, the eggs in all treatments were encapsulated in S. exigua. Electron microscopic observations of parasitoid eggs showed structural differences between the surfaces of the mature and other kinds of eggs. The injected CcIV decreased the numbers of host hemocytes and suppressed the spreading ability of plasmatocytes and granulocytes in H. armigera, but had little effect on the hemocytes from S. exigua. In conclusion, the C. chlorideae egg provides a passive protection against encapsulation by itself, and CcIV supplies an active protection through disrupting host immune responses. These coordinated protections are host-specific, implying their role in host range determination.

Unique function of a chemosensory protein in the proboscis of two Helicoverpa species.

Chemosensory proteins (CSPs) are soluble proteins found only in arthropods. Some of them fill the lumen of chemosensilla and are believed to play a role similar to that of odorant-binding proteins in the detection of semiochemicals. Other members of the CSP family have been reported to perform different functions, from delivery of pheromones to development. This report is focused on a member (CSP4) of the family that is highly and almost exclusively present in the proboscis of two sibling noctuid species, Helicoverpa armigera and H. assulta. We expressed the protein in bacteria and measured binding to terpenoids and related compounds. Using specific antibodies, we found that when the moths suck on a sugar solution, CSP4 is partly extruded from the proboscis. A solution of protein can also fill a hydrophobic tube of same length and diameter as the proboscis by capillary action. On this basis, we suggest that CSP4 acts as a wetting agent to reduce the surface tension of aqueous solutions and consequently the pressure involved in sucking.

Inheritance of electrophysiological responses to leaf saps of host- and nonhost plants in two Helicoverpa species and their hybrids.

The polyphagous cotton bollworm Helicoverpa armigera (Hübner) and the oligophagous oriental tobacco budworm Helicoverpa assulta (Guenée) (Lepidoptera: Noctuidae) display contrasting heritable feeding preferences for cotton and pepper leaves. In this study, electrophysiological response patterns to cotton and pepper leaf saps in gustatory sensilla styloconica on the maxillae of these two species, their reciprocal F1 hybrids, and backcrossed lines were investigated using the tip recording technique. The identity of the neurons responding to the two leaf saps has been established using action potential waveform analysis. The two plant leaf saps elicited neural activity in at least six of the eight taste neurons innervating the lateral and medial sensilla styloconica of the parental species and crosses. Discriminant analysis of this multineural input predicted that correct classification occurred in 87 - 92% of the cases. Differences in taste neuron responses between insect lines to the two plant saps were consistent with differences in feeding preference behaviors. Comparisons of taste neuron response patterns of parental species, F1 hybrids and backcrosses indicate that autosomal loci contributed to the difference in gustatory response patterns between the two Helicoverpa species with the H. armigera derived alleles being partly dominant to those carried by H. assulta. These findings contribute to the understanding of gustatory codes for preference and provide insight into taste evolution of lepidopteran insects.

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.

The larva and adult of Helicoverpa armigera use differential gustatory receptors to sense sucrose.

Almost all herbivorous insects feed on plants and use sucrose as a feeding stimulant, but the molecular basis of their sucrose reception remains unclear. Helicoverpa armigera as a notorious crop pest worldwide mainly feeds on reproductive organs of many plant species in the larval stage, and its adult draws nectar. In this study, we determined that the sucrose sensory neurons located in the contact chemosensilla on larval maxillary galea were 100-1000 times more sensitive to sucrose than those on adult antennae, tarsi, and proboscis. Using the Xenopus expression system, we discovered that Gr10 highly expressed in the larval sensilla was specifically tuned to sucrose, while Gr6 highly expressed in the adult sensilla responded to fucose, sucrose and fructose. Moreover, using CRISPR/Cas9, we revealed that Gr10 was mainly used by larvae to detect lower sucrose, while Gr6 was primarily used by adults to detect higher sucrose and other saccharides, which results in differences in selectivity and sensitivity between larval and adult sugar sensory neurons. Our results demonstrate the sugar receptors in this moth are evolved to adapt toward the larval and adult foods with different types and amounts of sugar, and fill in a gap in sweet taste of animals.

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.

Identification and field evaluation of pear fruit volatiles attractive to the oriental fruit moth, Cydia molesta.

Plant volatiles play a key role in host plant location of phytophagous insects. Cydia molesta is an important pest of pear fruit late in the growing season. We identified and quantified volatiles from immature and mature fruits of six pear varieties by using gas chromatography-mass spectrometry (GC-MS). Attractiveness of synthetic blends to adults based on gas chromatography-electroantennogram detection (GC-EAD) activity was investigated in both field and laboratory. Consistent electroantennographic activity was obtained for 12 compounds from headspace collections of the mature fruits of the six pear varieties. Qualitative and quantitative differences were found among six odor profiles. Among the six mixtures, the mixture of 1-hexanol, nonanal, ethyl butanoate, butyl acetate, ethyl hexanoate, hexyl acetate, hexyl butanoate, and farnesene (different isomers) with a 1:1:100:70:7:5:1:4 ratio from the variety Jimi and the mixture of nonanal, ethyl butanoate, 3-methylbutyl acetate, ethyl hexanoate, hexyl acetate, and farnesene with a 1:100:1:32:1:2 ratio from the variety Huangjin were highly attractive to both sexes in the field. However, male captures were much higher than those of females. Further wind tunnel tests proved that both sexes exhibited upwind flight to the lures, but only males landed on the source. Our finding indicates that mixtures mimicking Jimi and Huangjin volatiles attract both females and males of C. molesta, and these host volatiles may be involved in mate finding behavior.

Comparison of functions of pheromone receptor repertoires in Helicoverpa armigera and Helicoverpa assulta using a Drosophila expression system.

Helicoverpa armigera and H. assulta are sympatric closely related species sharing two sex pheromone components, (Z)-11-hexadecenal (Z11-16:Ald) and (Z)-9-hexadecenal (Z9-16:Ald) but in opposite ratios, 97:3 and 3:97 respectively. This feature makes them a feasible model for studying the evolution of pheromone coding mechanisms of lepidopteran insects. Despite a decade-long study to deorphanize the pheromone receptor (PR) repertoires of the two species, the comparison of the function of all PR orthologs between the two species is incomplete. Moreover, the ligands of OR14 and OR15 have so far not been found, likely due to the missing of the active ligand(s) in the compound panel and/or incompatibility of heterologous expression systems used. In the present study, we expressed the PR repertoires of both Helicoverpa species in Drosophila T1 neurons to comparatively study the function of PRs. Among those PRs, OR13, OR6, and OR14 of both species are functionally conserved and narrowly tuned, and the T1 neurons expressing each of them respond to Z11-16:Ald, (Z)-9-hexadecenol (Z9-16:OH), and (Z)-11-hexadecenyl acetate (Z11-16:Ac), respectively. While HarmOR16-expressing neurons respond strongly to (Z)-9-tetradecenal (Z9-14:Ald) and (Z)-11-hexadecenol (Z11-16:OH), the neurons expressing HassOR16 mainly respond to Z9-14:Ald and also weakly respond to (Z)-9-tetradecenol (Z9-14:OH). Moreover, HarmOR14b-expressing neurons are activated by Z9-14:Ald, whereas HassOR14b-expressing neurons are sensitive to Z9-16:Ald, Z9-14:Ald, and (Z)-9-hexadecenol (Z9-16:OH). In addition, HarmOR15-expressing neurons are selectively responsive to Z9-14:Ald. However, the Drosophila T1 neurons expressing either HarmOR11 or HassOR11 are silent to all of the compounds tested. In summary, except for OR11, we have deorphanized all the PRs of these two Helicoverpa species using a Drosophila expression system and a large panel of pheromone compounds, thereby providing a valuable reference for parsing the code of peripheral coding of pheromones.

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.

Contribution of odorant binding proteins to olfactory detection of (Z)-11-hexadecenal in Helicoverpa armigera.

Helicoverpa armigera utilizes (Z)-11-hexadecenal (Z11-16:Ald) as its major sex pheromone component. Three pheromone binding proteins (PBPs) and two general odorant binding proteins (GOBPs) are abundantly expressed in the male antennae of H. armigera. However, their precise roles in the olfactory detection of Z11-16:Ald remain enigmatic. To answer this question, we first synthesized the antibody against HarmOR13, an olfactory receptor (OR) primarily responding to Z11-16:Ald and mapped the local associations between PBPs/GOBPs and HarmOR13. Immunostaining showed that HarmPBPs and HarmGOBPs were localized in the supporting cells of trichoid sensilla and basiconic sensilla respectively. In particular, HarmPBP1 and HarmPBP2 were colocalized in the cells surrounding the olfactory receptor neurons (ORNs) expressing HarmOR13. Next, using two noninterfering binary expression tools, we heterologously expressed HarmPBP1, HarmPBP2 and HarmOR13 in Drosophila T1 sensilla to validate the functional interplay between PBPs and HarmOR13. We found that the addition of HarmPBP1 or HarmPBP2, not HarmPBP3, significantly increased HarmOR13's response to Z11-16:Ald. However, the presence of either HarmPBP1 or HarmPBP2 was ineffective to change the tuning breadth of HarmOR13 and modulate the response kinetics of this receptor. Taken together, this work demonstrates both HarmPBP1 and HarmPBP2 are involved in Z11-16:Ald detection. Our results support the idea that PBPs can contribute to the peripheral olfactory sensitivity but do little in modulating the selectivity and the response kinetics of corresponding ORs.

A moth odorant receptor highly expressed in the ovipositor is involved in detecting host-plant volatiles.

Antennae are often considered to be the nostrils of insects. Here, we sequenced the transcriptome of the pheromone gland-ovipositor complex of Helicoverpa assulta and discovered that an odorant receptor (OR) gene, HassOR31, had much higher expression in the ovipositor than in antennae or other tissues. To determine whether the ovipositor was involved in odorant detection, we co-expressed HassOR31 and its co-receptor, HassORco, in a Xenopus oocyte model system, and demonstrated that the OR was responsive to 12 plant odorants, especially Z-3-hexenyl butyrate. These odorants elicited electrophysiological responses of some sensilla in the ovipositor, and HassOR31 and HassORco were co-expressed within ovipositor sensilla. Two oviposition preference experiments showed that female moths lacking antennae still preferentially selected oviposition sites containing plant volatiles. We suggest that the expression of HassOR31 in the ovipositor of H. assulta helps females to determine precise egg-laying sites in host plants.

When most insects reproduce they lay eggs that hatch into juveniles known as larvae. To provide good sources of food for the larvae, the adult insects have to carefully select where to lay the eggs. Host plants produce specific sets of chemicals known as odorants that the adult insects are able to smell using proteins called odorant receptors. It is generally thought that odorant receptors in the antennae on the head are responsible for guiding adult insects to good egg-laying sites. However, recent studies have reported that odorant receptors are also present in the egg-laying organs of several different species of moth. It remains unclear what role these odorant receptors may play in egg-laying. The oriental tobacco budworm (Helicoverpa assulta) is considered a serious pest in agriculture. The adult moths lay their eggs on a narrow range of plants in the nightshade family including tobacco and hot pepper. Li et al. have now investigated the odorant receptors of H. assulta and found that one gene for an odorant receptor called HassOR31 was expressed much more in the egg-laying organs of the moths than in the antennae. Further experiments showed that this receptor was tuned to respond to 12 odorants that also stimulated responses in the egg-laying organ of H. assulta. Together these findings suggest that this odorant receptor in the egg-laying organ helps the moths find suitable host plants to lay their eggs on. The work of Li et al. may help us understand how H. assulta evolved to lay its eggs on specific members of the nightshade family and lead to new methods of controlling this pest. An insect's sense of smell guides many other behaviors including finding food, mates and avoiding enemies. Therefore, these findings may inspire researchers to investigate whether odorant receptors in the antennae or other organs guide these behaviors.

Olfactory coding of intra- and interspecific pheromonal messages by the male Mythimna separata in North China.

Moths often use multi-component pheromones with fixed ratios to keep intraspecific communication and interspecific isolation. Unusually, the Oriental armyworm Mythimna separata in North China use only Z11-16:Ald as the essential component of its sex pheromone to find mates. To understand how this species keeps behavioral isolation from other species sharing Z11-16:Ald as a major pheromone component, we study the olfactory coding of intra- and interspecific pheromonal messages in the males of M. separata. Firstly, we functionally characterized the long trichoid sensilla in male antennae by single sensillum recording. Two types of sensilla were classified: the A type sensilla responded to Z11-16:Ald and Z9-14:Ald, and the B type sensilla mainly to Z9-14:Ald, and also to Z11-16:Ac, Z11-16:OH, and Z9-16:Ald. Next, we examined the glomerulus responses in the antennal lobes to these compounds by using in vivo optical imaging. The results showed that among the three subunits of the macroglomerular complex (MGC), Z11-16:Ald activated the cumulus, Z9-14:Ald activated the dorso-anterior and the cumulus, Z11-16:OH and Z11-16:Ac activated the dorso-anterior and dorso-posterior, respectively. However, Z9-16:Ald activated an ordinary glomerulus. Thirdly, we tested the behavioral responses of the males to these compounds in the wind tunnel. Addition of Z9-14:Ald at the ratio of 1:10 greatly reduced the attractiveness of Z11-16:Ald, addition of Z9-16:Ald or Z11-16:OH at the ratio of 1:1 also had behavioral antagonistic effects, while addition of Z11-16:Ac had no effect on the attractiveness of Z11-16:Ald. Finally, we used antennal transcriptome data and the Xenopus expression system to identify the receptor of Z9-14:Ald in M. separata. The Xenopus oocytes co-expressing MsepOR2 and MsepORco showed a strong response to Z9-14:Ald. Two-color fluorescence in situ hybridization validated that the cells expressing MsepOR2 and MsepOR3, tuned to Z9-14:Ald and Z11-16:Ald respectively, were localized in the different sensilla of male antennae. Comparing the sex pheromone communication channel of the related species, our results suggest that the conserved olfactory pathways for behavioral antagonists play a crucial role in behavioral isolation of noctuid species.

A gustatory receptor tuned to the steroid plant hormone brassinolide in Plutella xylostella (Lepidoptera: Plutellidae).

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, the GRs in Plutella xylostella were analyzed. Through RNA sequencing and qPCR, we detected abundant PxylGr34 transcripts in the larval head and adult antennae. Functional analyses using the Xenopus oocyte expression system and 24 diverse phytochemicals showed that PxylGr34 is tuned to the canonical plant hormones brassinolide (BL) and 24-epibrassinolide (EBL). Electrophysiological analyses revealed that the medial sensilla styloconica of 4th instar larvae are responsive to BL and EBL. Dual-choice bioassays demonstrated that BL inhibits larval feeding and female oviposition. Knock-down of PxylGr34 by RNAi attenuates the taste responses to BL, and abolishes BL-induced feeding inhibition. These results increase our understanding of how herbivorous insects detect compounds that deter feeding and oviposition, and may be useful for designing plant hormone-based pest management strategies.

Plant-eating insects use their sense of taste to decide where to feed and where to lay their eggs. They do this using taste sensors called gustatory receptors which reside in the antennae and legs of adults, and in the mouthparts of larvae. Some of these sensors detect sugars which signal to the insect that the plant is a nutritious source of food. While others detect bitter compounds, such as poisons released by plants in self-defense. One of the most widespread plant-eating insects is the diamondback moth, which feeds and lays its eggs on cruciferous vegetable crops, like cabbage, oilseed rape and broccoli. Before laying its eggs, female diamondback moths pat the vegetable's leaves with their antennae, tasting for the presence of chemicals. But little was known about the identity of these chemicals. Cabbages produce large amounts of a hormone called brassinolide, which is known to play a role in plant growth. To find out whether diamondback moths can taste this hormone, Yang et al. examined all their known gustatory receptors. This revealed that the adult antennae and larval mouthparts of these moths make high levels of a receptor called PxylGr34. To investigate the role of PxylGr34, Yang et al. genetically modified frog eggs to produce this receptor. Various tests on these receptors, as well as receptors in the mouthparts of diamondback larvae, showed that PxylGr34 is able to sense the hormone brassinolide. To find out how this affects the behavior of the moths, Yang et al. investigated how adults and larvae responded to different levels of the hormone. This revealed that the presence of brassinolide significantly decreased both larval feeding and the amount of eggs laid by adult moths. Farmers already use brassinolide to enhance plant growth and protect crops from stress. These results suggest that the hormone might also help to shield plants from insect damage. However, more research is needed to understand how this hormone acts as a deterrent. Further studies could improve understanding of insect behavior and potentially identify more chemicals that can be used for pest control.

The olfactory reception of acetic acid and ionotropic receptors in the Oriental armyworm, Mythimna separata Walker.

Various insect species including moths have shown significant foraging preference to acetic acid. However, the olfactory reception and behavioral outputs of acetic acid in moths remain unsolved. The female adults of Oriental armyworm, Mythimna separata, exhibit high preference to acetic acid enriched sweet vinegar solutions, making them good targets for exploration of acid reception and performance. We first proved that acetic acid is an essential component which elicited electrophysiological responses from volatiles of the sweet vinegar solution. Successive single sensillum recording tests showed that at least 4 types (as1, as2, as3, and as4) of sensilla were involved in acetic acid reception in the antennae. The low dosages of acetic acid elicited upwind flight and close search, and pre-contact proboscis extension responses of the fasted females, indicating it serves as a food related olfactory cue. In vivo optical imaging data showed that low dosages of acetic acid activated one ordinary glomerulus (DC3), and high dosages evoked additional two glomeruli (DC1 and AC1) in the antennal lobe. A systematic survey on olfaction related receptors in three related transcriptomes has yielded 67 olfactory receptors (ORs) and 19 ionotropic receptors (IRs). Among, MsepIR8a, MsepIR64a, MsepIR75q1, and MsepIR75q2 were chosen as putative acid receptors by blasting against known acid IRs in Drosophila and comparing essential amino acid residues which related to acid sensing. Later in situ hybridization revealed that MsepIr8a was co-expressed with each of the other 3 Irs, suggesting its putative co-receptor role. This study reveals olfactory reception of acetic acid as an attractant in M. separata, and it provides the solid basis for later deorphanization of relevant receptors.

An odorant receptor and glomerulus responding to farnesene in Helicoverpa assulta (Lepidoptera: Noctuidae).

Terpenoids emitted from herbivore-damaged plants were found to play an important role in regulating tritrophic interactions. How herbivores and their natural enemies perceive terpenoids has not been thoroughly elucidated to date. Using in vivo calcium imaging, we found in this study that farnesene activates one glomerulus in the antennal lobe of female Helicoverpa assulta. The response induced by a mixture of farnesene isomers is stronger than that elicited by E-ß-farnesene alone. In the Xenopus oocyte expression system, HassOR23/ORco is narrowly tuned to farnesene isomers and compounds with similar structures. Finally, the behavioral studies showed that the farnesene isomers have an inhibitory effect on oviposition of female H. assulta, but have an attractive effect on host searching of Campoletis chlorideae, the key endoparasitoid of H. assulta larvae. These results demonstrate that farnesene isomers are encoded by a labeled-line mode in the olfactory system of female H. assulta, suggesting that farnesene as a chemical signal from plants has important behavioral relevance and evolutionary implications in the tritrophic context.