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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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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-β-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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The Oriental armyworm, Mythimna separata, has been described to emit geographic population-specific sex pheromones, with either Z11-16:Ald or Z11-16:Ac as the major component. Using a comprehensive set of electrophysiological, behavioral, and genetic analyses, we study the sex pheromone communication of M. separata in North China from pheromone receptors and antennal lobe to behavior. GC-EAD results show that Z11-16:Ald is the only compound eliciting electrophysiological responses in pheromone gland extracts. Further in vivo optical imaging studies reveal that Z11-16:Ald activates the cumulus of the MGC and show dose-dependent responses. The wind tunnel tests demonstrate that Z11-16:Ald alone is sufficient to induce the entire sequence of male sexual behaviors. Transcriptome and q-PCR results show that MsepOR3 is specifically and abundantly expressed in male antennae. By using the Xenopus oocytes and two-electrode voltage-clamp recording, we finally validate that the oocytes expressing MsepOR3/ORco gave dose dependent responses to Z11-16:Ald. We suggest single Z11-16:Ald could be used for monitoring the population of M. separata in North China., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The sympatric closely related species Helicoverpa armigera and Helicoverpa assulta use 97:3 and 7:93 of (Z)-11-hexadecenal and (Z)-9-hexadecenal, respectively, as their sex pheromone to find/locate correct sex mates. Moreover, (Z)-11-hexadecenyl alcohol and (Z)-9-hexadecenyl alcohol are more abundant in the pheromone gland of H. assulta than in that of H. armigera. To clarify the molecular basis of these differences, we sequenced the pheromone gland transcriptomes of the two species and compared the expression patterns of the candidate enzyme genes involved in the pheromone biosynthetic pathways by FPKM values and quantitative RT-PCR analysis. We found that the desaturase gene LPAQ expressed about 70 times higher in H. armigera than in H. assulta, whereas another desaturase gene NPVE expressed about 60 times higher in H. assulta than in H. armigera. We also observed significantly higher expression of the fatty acyl reductase (FAR) gene FAR1 and the aldehyde reductase (AR) gene AR3 in H. assulta than in H. armigera. Examination of the pheromone glands of the backcross offspring of their hybrids to H. assulta showed a positive linear correlation between the expression level of LPAQ and the amount of Z11-16:Ald and between the expression level of NPVE and the amount of Z9-16:Ald in the pheromone glands. Taken together, these data demonstrate that the expressional divergences of LPAQ and NPVE determine the opposite sex pheromone component ratios in the two species and the divergent expression of FAR1 and AR3 may account for the greater accumulation of alcohols in the pheromone gland of H. assulta., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Lepidopteran caterpillars rely on olfaction and gustation to discriminate among food sources. Compared to the larval gustation, the larval olfaction has been poorly investigated. To uncover the molecular basis of olfaction in Helicoverpa armigera larvae, we identified 17 odorant receptor (Or) genes in larval antennae and maxillae using transcriptome sequencing, and functionally identified the response spectra of seven Ors to ecologically relevant odorants. Innate behavioural responses of larvae to active odorants were evaluated in chemotaxis assays. Several odorant blends were formulated based on the Ors tuning spectra and caterpillar chemotaxis. A four-component blend strongly attracted H. armigera larvae, and cis-jasmone and 1-pentanol were identified as essential components. Localization analyses showed that the two Ors detecting these components (Or41 and Or52) were expressed in the same sensory neurons. This is the first evidence that Ors in a polyphagous caterpillar respond to odorants in a combinatorial manner. The design of attractants to target specific olfactory pathways may promote the development of new baits for pest management., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Adult moths possess an organ in their labial palps, the labial-palp pit organ, which is specialized for sensing carbon dioxide (CO2). They use CO2 as a cue to detect healthy plants and find food or lay eggs on them. The molecular bases of the CO2 receptor in Drosophila melanogaster and Aedes aegypti have been reported, but the molecular mechanisms of the CO2 receptor in Lepidoptera remains elusive. In this study, we first re-examined three putative Helicoverpa armigera CO2 gustatory receptor genes (HarmGr1, HarmGr2, and HarmGr3), and then analyzed expression patterns of them. RT-PCR results verified they were predominantly expressed in the labial palps of H. armigera. Thus, we used in situ hybridization to localize the expression of three genes in the labial palps. We found that all three genes were co-expressed in the same cells of the labial palps. Next, we employed the Xenopus laevis oocyte expression system and the two-electrode voltage-clamp recording to study the function of the three genes. Results showed that only oocytes co-expressing HarmGr1 and HarmGr3 or co-expressing HarmGr1, HarmGr2 and HarmGr3 gave robust responses to NaHCO3. Finally, we confirmed that the sensory cells in labial palps of both females and males show dose dependent responses to CO2 stimuli by using single sensillum recording. Our work uncovers that HarmGr1 and HarmGr3 are indispensable and sufficient for CO2 sensing in labial palps of H. armigera., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Insect gustatory systems play important roles in food selection and feeding behaviors. In spite of the enormous progress in understanding gustation in Drosophila, for other insects one of the key elements in gustatory signaling, the gustatory receptor (GR), is still elusive. In this study, we report that fructose elicits behavioral and physiological responses in Helicoverpa armigera (Harm) to fructose and identify the gustatory receptor for this sugar. Using the proboscis extension reflex (PER) assays we found that females respond to fructose following stimulation of the distal part of the antenna, where we have identified contact chemosensilla tuned to fructose in tip recording experiments. We isolated three full-length cDNAs encoding candidate HarmGRs based on comparison with orthologous GR sequences in Heliothis virescens and functionally characterized the responses of HarmGR4 to 15 chemicals when this receptor was expressed in Xenopus oocytes with two-electrode voltage-clamp recording. Among the tastants tested, the oocytes dose-dependently responded only to D-fructose (EC50 = 0.045 M). By combining behavioral, electrophysiological and molecular approaches, these results provide basic knowledge for further research on the molecular mechanisms of gustatory reception., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The olfactory system of moth species in subfamily Heliothinae is an attractive model to study the evolution of the pheromone reception because they show distinct differentiation in sex pheromone components or ratios that activate pheromone receptors (PRs). However, functional assessment of PRs in closely related species remains largely untried. Here we present a special cloning strategy to isolate full-length cDNAs encoding candidate odorant receptors (ORs) from Helicoverpa armigera (Harm) and Helicoverpa assulta (Hass) on the basis of Heliothis virescens ORs, and investigate the functional properties of PRs to determine how the evolution of moth PRs contribute to intraspecific mating choice and speciation extension. We cloned 11 OR orthologs from H. armigera and 10 from H. assulta. We functionally characterized the responses of PRs of both species to seven pheromone compounds using the heterologous expression system of Xenopus ooctyes. HassOR13 was found to be highly tuned to the sex pheromone component Z11-16:Ald, and unexpectedly, both HarmOR14b and HassOR16 were specific for Z9-14:Ald. However, HarmOR6 and HassOR6 showed much higher specificity to Z9-16:OH than to Z9-16:Ald or Z9-14:Ald. HarmOR11, HarmOR14a, HassOR11 and HassOR14b failed to respond to the tested chemicals. Based on our results and previous research, we can show that some PR orthologs from H. armigera, H. assulta and H. virescens such as OR13s have similar ligand selectivity, but others have different ligand specificity. The combined PR function and sex pheromone component analysis suggests that the evolution of PRs can meet species-specific demands., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The two sibling species Helicoverpa armigera and Helicoverpa assulta utilise the same two aldehydes as their sex pheromones, but in opposite ratios. In both species three odorant-binding proteins (OBPs) can be classified as pheromone-binding proteins (PBPs). To investigate the role of these three PBPs in chemical communication between sexes and their mode of action, we have expressed the proteins in bacteria and prepared mutants lacking their C-terminal regions. Using polyclonal antibodies we found that the expression of the three PBPs is basically confined to the antennae of both sexes and both species. Binding experiments with the fluorescent probe N-phenyl-1-naphthylamine across a pH range indicated that, the affinity of wild-type proteins decreases at low pH, while that of the mutants is not or less affected, suggesting that a conformational change of the C-terminus occurs in these proteins, as reported for other lepidopteran OBPs. All three proteins bind with similar strength both pheromone components, as well as their corresponding alcohols and acetates. However, they exhibit significant selectivity to linear alcohols and aldehydes of different length, with optimal affinities to the ligand of 13-15 carbon atoms for PBP1 and 12-14 carbon atoms for PBP2. We suggest that all three PBPs might cooperate to build a unique olfactory image, that could help avoiding cross-mating between the two species and with other noctuids., (Copyright © 2012 Elsevier Ltd. All rights reserved.)