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    Current Researches

    In the strenuous efforts of preventing the destruction of crops by insects, chemical control serves as a main weapon. However, this approach also has led to many serious problems both in the contamination of the environment with the pesticides and the development of resistance to insecticides in insect populations. So much so that, there is an urgent need in developing new techniques for pest control. Through the understanding of how insects recognize particular chemical cues, we are able to develop novel methods to control insects by disrupting the ability of insects to findtheir host plants or mates.

    Our researches focus on insect olfaction and gustation. Taking the insect olfaction system as an example, olfactory sensory neurons (OSNs) are usually confined within multiporous basiconic or trichoid sensilla located on the antennae and maxillary palps. There are two main components in sensilla: (1) odorant-binding proteins (OBPs) including a subclass named PBPs (pheromone-binding proteins) that are necessary to bind odorants and ferry them across the hydrophobic sensillum lymph to the proximity of receptors; (2) odorant receptors (ORs) which are believed to interact with the odorants and serve as the major determinant for the specificity of a given OSNs. OSNs from insect sensilla project to the antennal lobe of the deuterocerebrum where they terminate in glomeruli. Generally, one OSN has one type of olfactory receptors, and one glomerulus is innervated with one type of OSN although there are some exceptions. Thus, the pattern of activity in the antennal lobe reflects the various dimensions, both qualitatively and quantitatively, of the chemical environment surrounding the insects. A further set of fibers, called projection neurons, relay the olfactory information up to the mushroom bodies and the lateral horns located in the protocerebum. Insects use this complex machinery to discriminate between very closely related chemical structures as well as between different mixtures of the same components through sensing a wide range of chemical signals including pheromones andplant volatiles.

    The ongoing projects in the lab address how insects navigate such a complex milieu to locate the potential mates and appropriate host plants by advancing the understanding of the role of the major chemosensory proteins in olfactory and taste recognition. One system developed for such a study involves two closely related species of moths, Helicoverpa armigera and H. assulta. The two species share some host plants but have very different host plant ranges: H. armigera is a typical generalist whereas H. assulta a specialist. They also use the same two components of the sex pheromone, (Z)-11-hexadecenal (Z11-16: Ald) and (Z)-9-hexadecenal (Z9-16: Ald) but in reversed ratios, 100:2.5 and 6:100, respectively. Another system we embark on is specialist herbivorous insects on cruciferous plants, in which glucosinolates represent classical plant secondary compounds mediating insect-plant interactions. When plants are injured, glucosinolates can be converted to the toxic isothiocyanates by the enzyme myrosinase, which is stored in specialized plant cells. The specialists such as diamondback moths and cabbage butterflies own specific sensory neurons responding to these chemicals, but the receptors tuned to glucosinolates and isothiocyanates are still unclear.

    Current researches are being developed with the following emphases:

    1. By the use of electrophysiological techniques such as single cell recording, tip recording, GC-EAD and mass spectrometry for chemical identification, we characterize the OSNs and gustatory sensory neurons (GRNs) responding to phytochemicals and pheromone components, and classify them into distinct types according to their molecular receptive ranges. We are also identifying olfactory receptor and gustatory receptor genes by using the computer algorithm to search the insect genome databases, transcriptome analysis, and degenerate RT-PCR and RACE.
    2. We express olfactory receptors and gustatory receptors in Xenopus oocytes and Drosophila mutants, and analyze their functions by using two electrode voltage clamping and single sensillum recording, respectively. We also use in situ hybridization to localize their expressions in chemosensory sensilla.
    3. Using optical imaging, neuron labeling, and intracellular recording, we map out the associations between olfactory receptors, OSNs, and antennal lobe glomeruli. We also characterize the antennal lobe morphological atlas and document olfactory response patterns across the antennal lobe glomeruli to stimuli and their combination.
    4. With next-generation sequencing and CRISPR-Cas9 genome editing, we in-depth explore the molecular basis of chemical cues-directed insect behaviours.

     

     

    Recent Progress
    (#authors contributed equally;*corresponding author)

    Review of pheromone receptors in heliothine species: expression, function, and evolution
    Ke Yang, Chen-Zhu Wang*
    Entomologia Experimentalis et Applicata 2020, doi:10.1111/eea.12982.
      Heliothine species (Lepidoptera: Noctuidae, Heliothinae) include some of the world’s most devastating pest species, such as Heliothis virescens (Fabricius) in the Americas and Helicoverpa armigera (Hubner) in the Old World. Sex pheromone communication of these agricultural pest species has been investigated from pheromone receptors (PRs) to brains and behavior in detail. In recent years, great progress has been made in the mechanistic dissection of pheromone reception, demonstrating that PRs play a key role in determining the response characteristics of pheromone-sensitive olfactory receptor neurons. In this review, we focus on the functional characterization of PRs in heliothine species, and summarize recent progress in the identification of receptors tuned to principal sex pheromone components including Z11-16:Ald, Z9-16:Ald, Z9-14:Ald, and other related alcohols and acetates. Evolution of PRs in ligand selectivity is also discussed. The efficiency and faithfulness of three frequently used heterologous expression systems – Xenopus laevis Daudin oocytes, modified HEK 293 cells, and Drosophila ‘empty neuron’ mutants – are compared, and the research strategies, skills, and perspectives in the studies are envisioned. Finally, we propose future research topics on PRs in heliothine species.

    A moth odorant receptor highly expressed in the ovipositor is involved in detecting host-plant volatiles
    Rui-Ting Li, Ling-Qiao Huang, Jun-Feng Dong, Chen-Zhu Wang*
    eLife 2020, 9:e53706
      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.

    Olfactory coding of intra- and interspecific pheromonal messages by the male Mythimna separata in North China
    Nan-Ji Jiang, Rui Tang, Hao Guo, Chao Ning, Jian-Cheng Li, Han Wu, Ling-Qiao Huang, Chen-Zhu Wang*
    Insect Biochemistry and Molecular Biology 2020, 125: 103439
      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.

    The olfactory reception of acetic acid and ionotropic receptors in the Oriental armyworm, Mythimna separata Walker
    Rui Tang, Nan-Ji Jiang, Chao Ning, Guo-Cheng Li, Ling-Qiao Huang, Chen-Zhu Wang*
    Insect Biochemistry and Molecular Biology 2020, 118: 103312
      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)
    Han Wu, Rui-Ting Li, Jun-Feng Dong, Nan-Ji Jiang, Ling-Qiao Huang, Chen-Zhu Wang*
    Insect Biochemistry and Molecular Biology 2019, 115: 103106
      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.

    Dissecting sex pheromone communication of Mythimna separata (Walker) in North China from receptor molecules and antennal lobes to behavior
    Nan-Ji Jiang, Rui Tang, Han Wu, Meng Xu, Chao Ning, Ling-Qiao Huang, Chen-Zhu Wang*
    Insect Biochemistry and Molecular Biology 2019, 111: 103176
      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.

    An odorant receptor mediates the attractiveness of cis-jasmone to Campoletis chlorideae, the endoparasitoid of Helicoverpa armigera
    Ya-Lan Sun, Jun-Feng Dong, Ping-Ping Ding, Ling-Qiao Huang, Jiu-Guang Sun, Chen-Zhu Wang*
    Insect Molecular Biology 2019, 28, 23-34
      Parasitic wasps rely on olfaction to locate their hosts in complex chemical environments. Odorant receptors (ORs) function together with well-conserved odorant coreceptors (ORcos) to determine the sensitivity and specificity of olfactory reception. Campoletis chlorideae (Hymenoptera: Ichneunmonidae) is a solitary larval endoparasitoid of the cotton bollworm, Helicoverpa armigera, and some other noctuid species. To understand the molecular basis of C. chlorideae's olfactory reception, we sequenced the transcriptome of adult male and female heads (including antennae) and identified 211 OR transcripts, with 95 being putatively full length. The tissue expression profiles, as assessed by reverse-transcription PCR, showed that seven ORs were expressed only or more highly in female antennae. Their functions were analysed using the Xenopus laevis oocyte expression system and two-electrode voltage-clamp recordings. CchlOR62 was tuned to cis-jasmone, which was attractive to female C. chlorideae adults and H. armigera larvae in the subsequent behavioural assays. Further bioassays using caged plants showed that the parasitism rate of H. armigera larvae by C. chlorideae on cis-jasmone-treated tobacco plants was higher than on the control plants. Thus, cis-jasmone appears to be an important infochemical involved in the interactions of plants, H. armigera and C. chlorideae, and CchlOR62 mediates the attractiveness of cis-jasmone to C. chlorideae.

    Two single-point mutations shift the ligand selectivity of a pheromone receptor between two closely related moth species
    Ke Yang, Ling-Qiao Huang, Chao Ning, Chen-Zhu Wang*
    eLife, 2017, 6: e29100 doi: 10.7554/eLife.29100
      Male moths possess highly sensitive and selective olfactory systems that detect sex pheromones produced by their females. Pheromone receptors (PRs) play a key role in this process. The PR HassOr14b is found to be tuned to (Z)?9-hexadecenal, the major sex-pheromone component, in Helicoverpa assulta. HassOr14b is co-localized with HassOr6 or HassOr16 in two olfactory sensory neurons within the same sensilla. As HarmOr14b, the ortholog of HassOr14b in the closely related species Helicoverpa armigera, is tuned to another chemical (Z)?9-tetradecenal, we study the amino acid residues that determine their ligand selectivity. Two amino acids located in the intracellular domains F232I and T355I together determine the functional difference between the two orthologs. We conclude that species-specific changes in the tuning specificity of the PRs in the two Helicoverpa moth species could be achieved with just a few amino acid substitutions, which provides new insights into the evolution of closely related moth species.

    Expressional divergences of two desaturase genes determine the opposite ratios of two sex pheromone components in Helicoverpa armigera and Helicoverpa assulta
    Rui-Ting Li, Chao Ning, Ling-Qiao Huang, Jun-Feng Dong, Xianchun Li, Chen-Zhu Wang*
    Insect Biochemistry and Molecular Biology 2017, 90: 90-100
      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.

    Design of larval chemical attractants based on odorant response spectra of odorant receptors in the cotton bollworm
    Chang Di, Chao Ning, Ling-Qiao Huang, Chen-Zhu Wang*
    Insect Biochemistry and Molecular Biology 2017, 84: 48-62
      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 behavioral 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.

    The inheritance of the pheromone sensory system in two Helicoverpa species: dominance of H. armigera and possible introgression from H. assulta
    Meng Xu, Jun-Feng Dong, Han Wu, Xin-Cheng Zhao, Ling-Qiao Huang, Chen-Zhu Wang*
    Frontiers in Cellular Neuroscience 2017, 10: 302
      Hybridization of sympatric closely related species may sometimes lead to introgression and speciation. The sister species Helicoverpa armigera and Helicoverpa assulta both use (Z)-11-hexadecenal and (Z)-9-hexadecenal as sex pheromone components but in reversed ratios. Female H. armigera and male H. assulta could hybridize and produce fertile male hybrids, which can then backcross with females of the two parent species to get backcross lines in the laboratory. In this study, we compared the olfactory responses to pheromone compounds in the periphery and in the antennal lobes of males of the two species, as well as of their hybrids and backcrosses. Single-sensillum recordings were carried out to explore characteristics of male-specific sensilla in antennae, and in vivo calcium imaging and digital 3D-reconstruction were used to describe what happens in the macroglomerular complex (MGC) of antennal lobes. The results show that the population ratio of the two olfactory sensory neurons responding to two sex pheromone components in male antennae are controlled by a major gene, and the allele of H. armigera is dominant. Consistently, the study of the representative areas activated by sex pheromone components in antennal lobes further support the dominance of H. armigera in pheromone detection. However, the topological structure of the MGC in the hybrid was similar but not identical to that in H. armigera. All the subtypes of male-specific sensilla in the two species were found in the male hybrids and backcrosses. Moreover, two new subtypes with broader response spectra (the expanded A subtype and the expanded C subtype) emerged in the hybrids. Based on the inheritance pattern of the pheromone sensory system, we predict that when hybridization of female H. armigera and male H. assulta occurs in the field, male hybrids would readily backcross with female H. armigera, and introgression might occur from H. assulta into H. armigera through repeated backcrossing.

    Functional validation of the carbon dioxide receptor in labial palps of Helicoverpa armigera moths
    Chao Ning, Ke Yang, Meng Xu, Ling-Qiao Huang, Chen-Zhu Wang*
    Insect Biochemistry and Molecular Biology 2016, 73: 12-19
      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.

    Olfactory perception and behavioral effects of sex pheromone gland components in Helicoverpa armigera and Helicoverpa assulta
    Meng Xu#, Hao Guo#, Chao Hou#, Han Wu, Ling-Qiao Huang, Chen-Zhu Wang*
    Scientific Reports 2016, 6: 22998
      Two sympatric species Helicoverpa armigera and Helicoverpa assulta use (Z)-11-hexadecenal and (Z)-9hexadecenal 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)-11hexadecenol 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.

    Specific olfactory neurons and glomeruli are associated to differences in behavioral responses to pheromone components between two Helicoverpa species
    Han Wu, MengXu, ChaoHou, Ling-QiaoHuang, Jun-FengDong, Chen-Zhu Wang*
    Frontiers in Behavioral Neuroscience 2015, 9
      Sex pheromone communication of moths helps to understand the mechanisms underlying reproductive isolation and speciation. Helicoverpa armigera and Helicoverpa assulta use (Z)-11-hexadecenal (Z11-16:Ald) and (Z)-9-hexadecenal (Z9-16:Ald) as pheromone components in reversed ratios, 97:3 and 5:95, respectively. H. armigera also produces trace amount of (Z)-9-tetradecenal (Z9-14:Ald) in the sex pheromone gland, but H. assulta does not. Wind tunnel studies revealed that the addition of small amounts (0.3%) of Z9-14:Ald to the main pheromone blend of H. armigera increased the males' attraction, but at higher doses (1%, 10%) the same compound acted as an inhibitor. In H. assulta, Z9-14:Ald reduced male attraction when presented as 1% to the pheromone blend, but was ineffective at lower concentrations (0.3%). Three types (A-C) of sensilla trichodea in antennae were identified by single sensillum recording, responding to Z11-16:Ald, Z9-14:Ald, and both Z9-16:Ald and Z9-14:Ald, respectively. Calcium imaging in the antennal lobes (ALs) revealed that the input information of the three chemicals was transmitted to three units of the macroglomerular complex (MGC) in ALs in both species: a large glomerulus for the major pheromone components, a small one for the minor pheromone components, and a third one for the behavioral antagonists. The type A and C neurons tuned to Z11-16:Ald and Z9-16:Ald had a reversed target in the MGC between the two species. In H. armigera, low doses (1, 10 μg) of Z9-14:Ald dominantly activated the glomerulus which processes the minor pheromone component, while a higher dose (100 μg) also evoked an equal activity in the antagonistic glomerulus. In H. assulta, instead, Z9-14:Ald always strongly activated the antagonistic glomerulus. These results suggest that Z9-14:Ald plays different roles in the sexual communication of two Helicoverpa species through activation of functionally different olfactory pathways.

    A gustatory receptor tuned to d-fructose in antennal sensilla chaetica of Helicoverpa armigera
    Xiao-Jing Jiang, Chao Ning, Hao Guo, Yan-Yan Jia, Ling-Qiao Huang, Ming-Jing Qu, Chen-Zhu Wang*
    Insect Biochemistry and Molecular Biology 2015, 60: 39-46
      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.

    Differential immunosuppression by Campoletis chlorideae eggs and ichnovirus in larvae of Helicoverpa armigera and Spodoptera exigua
    Li-Bin Han#, Li-Hong Yin#, Ling-Qiao Huang, Chen-Zhu Wang
    Journal of Invertebrate Pathology 2015, 130: 88-96
      The ichneumonid wasp, Campoletis chlorideae Uchida, successfully develops in the cotton bollworm Helicoverpa armigera (Hubner), but rarely survives in the beet armyworm Spodoptera exigua (Hubner) 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.

    Sequence similarity and functional comparisons of pheromone receptor orthologs in two closely related Helicoverpa species
    Xiao-Jing Jiang, Hao Guo, Chang Di, Shanlin Yu, Ligui Zhu, Ling-Qiao Huang, Chen-Zhu Wang*
    Insect Biochemistry and Molecular Biology 2014, 48: 63-74
      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.

    Unique function of a chemosensory protein in the proboscis of two Helicoverpa species
    Yu-Ling Liu#, Hao Guo#, Ling-Qiao Huang, Paolo Pelosi*, Chen-Zhu Wang*
    The Journal of Experimental Biology 2014, 217: 1821-1826
      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.

    Aldehyde reductase activity in the antennae of Helicoverpa armigera
    Hao Guo, Antonella Del Corso,Ling-Qiao Huang, Umberto Mura, Paolo Pelosi*, Chen-Zhu Wang*
    Insect Molecular Biology 2014, 23: 330-340
      In the present study, we identified two aldehyde reductase activities in the antennae of Helicoverpa species, NADH and NADPH-dependent activity. We expressed one of these proteins of H. armigera, aldo-keto reductase (AKR), which bears 56% identity to bovine aldose reductase, displays a NADPH-dependent activity and is mainly expressed in the antennae of adults. Whole-mount immunostaining showed that the enzyme is concentrated in the cells at the base of chemosensilla and in the nerves. The enzyme activity of H. armigera AKR is markedly different from those of mammalian enzymes. The best substrates are linear aliphatic aldehydes of 8-10 carbon atoms, but not hydroxyaldehydes. Both pheromone components of H. armigera, which are unsaturated aldehydes of 16 carbons, are very poor substrates. Unlike mammalian AKRs, the H. armigera enzyme is weakly affected by common inhibitors and exhibits a different behavior from the action of thiols. A model of the enzyme suggests that the four cysteines are in their reduced form, as are the seven cysteines of mammalian enzymes. The occurrence of orthologous proteins in other insect species, that do not use aldehydes as pheromones, excludes the possibility of classifying this enzyme among the pheromone-degrading enzymes, as has been previously described in other insect species.

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