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

  1. Rui-Ting Li
  2. Ling-Qiao Huang
  3. Jun-Feng Dong
  4. Chen-Zhu Wang  Is a corresponding author
  1. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, China
  2. CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, China
  3. Forestry College, Henan University of Science and Technology, China
Research Article
Cite this article as: eLife 2020;9:e53706 doi: 10.7554/eLife.53706
6 figures, 2 videos, 1 table, 2 data sets and 4 additional files

Figures

Figure 1 with 1 supplement
Gene expression of HassOR31 in different tissues of H. assulta.

(A) Tissue expression profiles of putative chemosensory receptor genes identified in the pheromone gland-ovipositor complex (PG-OV) of H. assulta. (B) TPM values of HassOR31 and HassORco in different tissues. Ant, antennae; Proboscis, mixed female and male proboscis; Forelegs, mixed female and male forelegs. (C) qRT-PCR results of HassOR31 and HassORco in different tissues relative to housekeeping gene 18S. Tho, thorax; Abd, abdomen.

Figure 1—figure supplement 1
Tissue expression profiles of some putative chemosensory related genes identified in the pheromone gland-ovipositor complex of H. assulta.

TPM values are listed below different tissues. OBP, odorant binding protein; CSP, chemosensory protein; GOBP, general odorant binding protein; PBP, pheromone binding protein; SNMP, sensory neuron membrane protein.

Figure 2 with 1 supplement
Localization of HassOR31 and HassORco expression in cells of H. assulta ovipositors.

Double-FISH with female ovipositors using combinations of labeled OR probes and visualization of cells bearing distinct HassOR31 transcripts by red (DIG) (A1, B1, C1) and HassORco transcripts by green (biotin) (A2, B2, C2) fluorescence, respectively. Co-labelling of cells by both OR probes appear as yellow/orange color in the overlay of the red and green fluorescence channels (A3, B3, C3). Bright-field images are presented as references (A4, B4, C4). Arrows indicate the cell location. Scale bars: 10 µm. (A1-3) The HassOR31 and HassORco probes label the same cell. (B1-3, C1-3) Only the HassOR31 probe is detected. Scale bars: 10 μm.

Figure 2—figure supplement 1
More examples of two-color in situ hybridization visualizing the combinations of HassOR31 (red) and HassORco (green) in the ovipositor of H. assulta.

Arrows indicate the cell location. Scale bars: 10 μm.

Functional analyses of chemosensory receptors in Xenopus oocytes.

(A) Inward current responses of Xenopus oocytes with expressed HassOR31/HassORco, HassOR31 alone, or HassOR31/HassiGluR7 in response to odorants (10−4 M solution). Odorants were applied for 8 s at times indicated by arrowheads; (B) Odorant-response spectra of Xenopus oocytes with expressed HassOR31/HassORco. Responses were measured as induced inward currents, expressed in nA. Error bars show standard error of the mean (n = 3–5), columns with different letters are significantly different at p<0.05 (One-way ANOVA followed by post hoc analysis with Turkey test); (C) Dose responses of Xenopus oocytes with expressed HassOR31/HassORco to the most effective ligand Z-3-hexenyl butyrate (n = 4), The EC50 value for Z-3-hexenyl butyrate was 1.606 × 10–5 M.

Scanning electron micrographs (SEM) of the ovipositor of H. assulta and its associated sensilla.

(A) An overview of the extended tip of the ovipositor of H. assulta, which contains two anal papillae surrounding the ovipore. (B) Details of the papilla close to the ovipore. There are four types of sensilla distributed on each papilla: Type I (C), putative mechanical sensilla widely distributed on the papilla, with raised pocket-like bases and a smooth surface (D); Type II (E), putative mechanical sensilla with sockets, showing flexible areas and non-porous surfaces (F); Type III (G), putative olfactory/taste sensilla shorter than Type I sensilla and morphologically similar to the trichoid on antennae, with pores on both the surface and the tip (H); Type IV (I), putative olfactory/taste sensilla located near the ovipore and morphologically similar to the basiconic sensilla, with pores on the surface and a large terminal pore (J).

Single sensillum recordings (SSR) of putative chemosensory sensilla in an H. assulta ovipositor.

(A) The firing rate of a putative chemosensory sensilla to several plant volatile and sex pheromone components. Chemicals with * induced significantly electrophysiological responses at p<0.05 relative to PO (paraffin oil) (One-way ANOVA followed by post hoc analysis with Turkey test, n = 3–4; Z-3-Hexenyl butyrate, p=0.0224). (B) The exemplary recordings of electrophysiological activities in a putative chemosensory sensilla to different load doses (1 µg, 10 µg, 100 µg and 1000 µg) of Z-3-hexenyl butyrate. (C) SSR dose responses of putative chemosensory sensilla to Z-3-hexenyl butyrate (n = 5), EC50 = 86.50 μg.

Figure 6 with 1 supplement
Oviposition preference of the antennae amputated and intact mated females of H. assulta.

(A) Number of eggs on the hot pepper treated gauze and control gauze. Two and three asterisks indicate significant difference (p<0.01 and p<0.001, paired t test). (B) Oviposition preference index between the hot pepper treated gauze and control gauze. Three asterisks indicate significant difference (p<0.001, unpaired t test). (C) Number of eggs on the Z-3-hexenyl butyrate treated fake leaves and control fake leaves. One and two asterisks indicate significant difference (p<0.05 and p<0.01, paired t test). (D) The oviposition preference index between the Z-3-hexenyl butyrate treated fake leaves and control fake leaves. NS indicates no significant difference (p>0.05, unpaired t test). All experiments were carried out with five to seven biological replications, with 10–15 mated female moths used in each replicate.

Figure 6—figure supplement 1
The set-up of oviposition choice tests and the spread of eggs laid by mated females of H. assulta.

(A) The choice test 1, the pepper treated gauzes vs control gauzes. The gauze was equally divided into four sections. The diameter of the hot pepper fruit discs is 1.5 cm. To avoid the moths directly contacting the hot pepper fruit discs, a stainless-steel cloth was put on the gauze. (B) The choice test 2, Z-3-hexenyl butyrate treated fake plants vs paraffin oil treated fake plants. Z-3-Hexenyl butyrate dissolved paraffin oil or paraffin oil were dropped into rubber heads, and one rubber head was placed on the leaf of one fake plant. (C and D) The spread of eggs laid by intact and antennae amputated mated females in the choice test 1, respectively. Eggs were marked in red.

Videos

Video 1
Z-stack video of two-color in situ hybridization visualizing the combinations of HassOR31 (red) and HassORco (green) in the ovipositor of H. assulta.

White arrows indicate the cells only expressing HassOR31, and yellow arrow the cell co-expressing HassOR31 and HassORco.

Video 2
An exemplary SSR experiment video.

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Gene (Helicoverpa assulta)18S ribosomal RNA geneNCBIGenBank: EU057177.1
Commercial assay, kitRNeasy Plus Universal Mini KitQiagenCat# 73404
Commercial assay, kitDynabeads mRNA purification kitInvitrogenCat# 61006
Commercial assay, kitQ5 High-Fidelity DNA PolymeraseNEBCat# M0491
Commercial assay, kitM-MLV reverse transcriptasePromegaCat# M1701
Commercial assay, kitSYBR Premix Ex TaqIITakaraCat# RR820
Commercial assay, kitT7/SP6 RNA transcription systemRocheCat# 10999644001
Commercial assay, kitmMESSAGE mMACHINE SP6AmbionCat# AM1340
Software, algorithmTrimmomaticTrimmomaticRRID:SCR_011848
Software, algorithmFastQCFastQCRRID:SCR_014583
Software, algorithmTrinityTrinityRRID:SCR_013048
Software, algorithmRSEMRSEMRRID:SCR_013027
Software, algorithmGraphPad PrismGraphPad PrismRRID:SCR_0027987.0
Software, algorithmZEN Digital Imaging for Light MicroscopyZEN Digital Imaging for Light MicroscopyRRID:SCR_0136722012
Software, algorithmAdobe IllustratorAdobe systemsRRID:SCR_014198CS6
Software, algorithmpCLAMP softwarepCLAMP softwareRRID:SCR_011323

Data availability

Sequencing data has been uploaded to SRA database under PRJNA592822 and SAMN13440804.

The following data sets were generated
  1. 1
    NCBI BioSample
    1. Li Rui-Ting
    2. H Ling-Qiao
    3. D Jun-Feng
    4. W Chen-Zhu
    (2020)
    ID SAMN13440804. Model organism or animal sample from Helicoverpa assulta.
  2. 2
    NCBI BioProject
    1. Li Rui-Ting
    2. H Ling-Qiao
    3. D Jun-Feng
    4. W Chen-Zhu
    (2020)
    ID PRJNA592822. Transcriptome sequencing of pheromone gland- ovipositor of Helicoverpa. assulta (a moth).

Additional files

Supplementary file 1

Expression values of putative chemosensory receptors in the pheromone gland-ovipositor complex of H. assulta (Hass) and H. armigera (Harm).

OR, odorant receptor; GR, gustatory receptor; IR, antennal ionotropic receptor; iGluR, ionotropic glutamate receptor.

https://cdn.elifesciences.org/articles/53706/elife-53706-supp1-v2.docx
Supplementary file 2

Primers used for qRT-PCR, RT-PCR, probe synthesis and full-length cDNA cloning.

https://cdn.elifesciences.org/articles/53706/elife-53706-supp2-v2.docx
Supplementary file 3

Tested compounds for functional analysis of HassOR31.

https://cdn.elifesciences.org/articles/53706/elife-53706-supp3-v2.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/53706/elife-53706-transrepform-v2.docx

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