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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., (© 2024 Institute of Zoology, Chinese Academy of Sciences.)

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.

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.

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., (© 2022. The Author(s).)

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 F 1 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 F 1 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 F 1 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.

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., (© 2021 Institute of Zoology, Chinese Academy of Sciences.)

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., (© 2022 Society of Chemical Industry.)

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.)

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.)

Two sympatric species Helicoverpa armigera and Helicoverpa assulta use (Z)-11-hexadecenal and (Z)-9-hexadecenal as sex pheromone components in reverse ratio. They also share several other pheromone gland components (PGCs). We present a comparative study on the olfactory coding mechanism and behavioral effects of these additional PGCs in pheromone communication of the two species using single sensillum recording, in situ hybridization, calcium imaging, and wind tunnel. We classify antennal sensilla types A, B and C into A, B1, B2, C1, C2 and C3 based on the response profiles, and identify the glomeruli responsible for antagonist detection in both species. The abundance of these sensilla types when compared with the number of OSNs expressing each of six pheromone receptors suggests that HarmOR13 and HassOR13 are expressed in OSNs housed within A type sensilla, HarmOR14b within B and C type sensilla, while HassOR6 and HassOR16 within some of C type sensilla. We find that for H. armigera, (Z)-11-hexadecenol and (Z)-11-hexadecenyl acetate act as behavioral antagonists. For H. assulta, instead, (Z)-11-hexadecenyl acetate acts as an agonist, while (Z)-9-hexadecenol, (Z)-11-hexadecenol and (Z)-9-hexadecenyl acetate are antagonists. The results provide an overall picture of intra- and interspecific olfactory and behavioral responses to all PGCs in two sister species.

The relative proportions of components in a pheromone blend play a major role in sexual recognition in moths. Two sympatric species, Helicoverpa armigera and Helicoverpa assulta, use (Z)-11-hexadecenal (Z11-16: Ald) and (Z)-9-hexadecenal (Z9-16: Ald) as essential sex pheromone components but in very different ratios, 97∶3 and 7∶93 respectively. Using wind tunnel tests, single sensillum recording and in vivo calcium imaging, we comparatively studied behavioral responses and physiological activities at the level of antennal sensilla and antennal lobe (AL) in males of the two species to blends of the two pheromone components in different ratios (100∶0, 97∶3, 50∶50, 7∶93, 0∶100). Z11-16: Ald and Z9-16: Ald were recognized by two populations of olfactory sensory neurons (OSNs) in different trichoid sensilla on antennae of both species. The ratios of OSNs responding to Z11-16:Ald and Z9-16:Ald OSNs were 100∶28.9 and 21.9∶100 in H. armigera and H. assulta, respectively. The Z11-16:Ald OSNs in H. armigera exhibited higher sensitivity and efficacy than those in H. assulta, while the Z9-16:Ald OSNs in H. armigera had the same sensitivity but lower efficacy than those in H. assulta. At the dosage of 10 µg, Z11-16: Ald and Z9-16: Ald evoked calcium activity in 8.5% and 3.0% of the AL surface in H. armigera, while 5.4% and 8.6% of AL in H. assulta, respectively. The calcium activities in the AL reflected the peripheral input signals of the binary pheromone mixtures and correlated with the behavioral output. These results demonstrate that the binary pheromone blends were precisely coded by the firing frequency of individual OSNs tuned to Z11-16: Ald or Z9-16: Ald, as well as their population sizes. Such information was then accurately reported to ALs of H. armigera and H. assulta, eventually producing different behaviors.

The two closely related moth species, Helicoverpa armigera and H. assulta, are sympatric in China. Both species use a mixture of (Z)-11-hexadecenal (Z11-16:Ald) and (Z)-9-hexadecenal (Z9-16:Ald) as their sex pheromones but in widely different ratios. Hybridization and backcrossing experiments between H. armigera and H. assulta were conducted and sex pheromone compositions of the parent species, their F(1) hybrids and backcrosses were compared to study the genetic basis of the production of their sex pheromone blend composition. Results show that the difference in sex pheromone blend ratios of these Helicoverpa species is mainly controlled by an autosomal locus with two alleles, with the allele from H. armigera being almost completely dominant over that derived from H. assulta.

Two sibling species, Helicoverpa assulta and Helicoverpa armigera both use (Z)-9-hexadecenal and (Z)-11-hexadecenal as their sex pheromone components but in almost reversed ratios, 93:7 and 3:97, respectively. H. assulta and H. armigera males performed upwind flight in response to the H. assulta sex pheromone blend (93:7). H. armigera responded strongly to the H. armigera blend (3:97), whereas H. assulta males remained inactive upon exposure to this blend. Both species gave clear dose-dependent electrophysiological responses to (Z)-11-hexadecenal. However, (Z)-9-hexadecenal evoked strong dose-dependent electrophysiological responses in H. assulta males but not in H. armigera. The two male F1 hybrids exhibited similar behavioral responses to two sex pheromone blends and electrophysiological responses to two pheromone components as H. armigera males. This indicated that H. armigera genes appear dominant in determining the behavioral response and electrophysiological responses. Behavioral and electrophysiological responses of backcrosses of male F1 hybrids (H. armigera female x H. assulta male) with female H. assulta and H. armigera were close to that of H. assulta and H. armigera, respectively. However, backcrosses of female F1 hybrids (H. assulta female x H. armigera male) with male H. assulta and H. armigera showed reduced behavioral responses but normal electrophysiological responses compared to males of the respective parental line.

Two Helicoverpa species, H. armigera and H. assulta use (Z)-11-hexadecenal and (Z)-9-hexadecenal as their sex attractant pheromone components but in opposite ratios. Since both female and male interspecific hybrids produced by female H. assulta and male H. armigera have been obtained in our laboratory, we can make a comparative study of sex pheromone composition and biosynthesis in the two species and their hybrid. With GC and GC-MS analyses using single gland extracts, the ratio of (Z)-9-hexadecenal to (Z)-11-hexadecenal was determined as 2.1:100 in H. armigera, and 1739:100 in H. assulta. The hybrid has a ratio of 4.0: 100, which is closer to that of H. armigera, but significantly different from H. armigera. We investigated pheromone biosynthesis with labeling experiments, using various fatty acid precursors in H. armigera, H. assulta and the hybrid. In H. armigera, (Z)-11-hexadecenal is produced by delta11 desaturation of palmitic acid, followed by reduction and terminal oxidation; (Z)-9-hexadecenal results from delta11 desaturation of stearic acid, followed by one cycle of chain shortening, reduction and terminal oxidation. delta11 desaturase is the unique desaturase for the production of the two pheromone components. In our Chinese strain of H. assulta, palmitic acid is used as the substrate to form both the major pheromone component, (Z)-9-hexadecenal and the minor one, (Z)-11-hexadecenal. Our data suggest that delta9 desaturase is the major desaturase, and delta11 desaturase is responsible for the minor component in H. assulta, which is consistent with previous work. However, the weak chain shortening acting on (Z)-9 and (Z)-11-octadecenoic acid, which is present in the pheromone glands, does occur in this species to produce (Z)-7 and (Z)-9-hexadecenoic acid. In the hybrid, the major pheromone component, (Z)-11-hexadecenal is produced by delta11 desaturation of palmitic acid, followed by reduction and terminal oxidation. The direct fatty acid precursor of the minor component, (Z)-9-hexadecenoic acid is mainly produced by delta9 desaturation of palmitic acid, but also by delta11 desaturation of stearic acid and one cycle of chain shortening. The greater relative amounts of (Z)-9-hexadecenal in the hybrid are due to the fact that both palmitic and stearic acids are used as substrates, whereas only stearic acid is used as substrate in H. armigera. The evolutionary relationships between the desaturases in several Helicoverpa species are also discussed in this paper.