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The Lepidoptera is the second largest insect order, which has the most extensive knowledge of sex pheromones and mechanisms of pheromone communication since the identification of the first insect pheromone in Bombyx mori. In the past 15 years, pheromone receptors have been identified and functionally characterized in many moth species. HarmOR14 is a typical pheromone receptor of Helicoverpa armigera which showed no response to the tested pheromones in Xenopus oocyte expression system, but its orthologous gene in Heliothis virescens, HvirOR14 could be activated by pheromones in the same expression system. To assess the possible functions of OR14 in vivo, in this study, we knocked out this gene using CRISPR/Cas9 system and compared the mating behaviors and EAG response to pheromones between the wild type and mutant strains. Our results showed that OR14 mutants did not affect the mating rate or the EAG response to pheromones but could prolong the mating duration and change the mating time in undefined manners, which extends our understanding to this kind of pheromone receptors.
FIGURE 1. Genetic mapping of pheromone receptors in H. armigera genome. Five PR genes including OR14b, OR14, OR15, OR16, and OR6 are located in a same scaffold (Harm_1.0 scaffold_53, NW_018395443.1) in tandem arrays. The visible map was generated using the tool GSDS2.0 (http://gsds.cbi.pku.edu.cn) according to the loci information of PR genes.
FIGURE 2. CRISPR/Cas9-based knock out of H. armigera OR14. (A) The HarmOR14 protein possesses seven transmembrane domains. The seven transmembrane domains were predicted on TOPCONS website (http://topcons.net/). The nucleotide deletion was generated in the first transmembrane domain (green circle). (B) The HarmOR14 gene contains nine exons in genome and the exon 2 was targeted for CRISPR single-guide RNA (sgRNA) design (red region). PAM site was indicated in purple and the deletion was indicated as dash lines. The mutation caused a frameshift at codon 75 and a premature stop codon, giving rise to a truncated protein of 80 amino acids. (C) The three sequencing chromatograms indicated three genotypes, WT (top), heterozygote (middle) and homozygote (bottom), respectively, and the mutation site was shown with a red frame.
FIGURE 3. The mating behavior of wild type and OR14 mutant strains. (A) Schematic representation of behavioral experiment design. (B) Scatter-dot plot comparing the mating rate of wild type (left, red) and OR14 mutant (right, green) strains. Each dot represents the mating rate of one group. Error bars indicate SEM. n = 22 and 13, respectively. Studentâs t-test, df = 33, t = â0.05, P = 0.962. (C) Scatter-dot plot comparing the mating duration of wild type (left, red) and OR14 mutant (right, green) strains. Each dot represents the mating duration of one pair of successful mating moths. n = 96 and 102, respectively. Studentâs t-test, df = 184, t = 6.23, P < 0.001. n.s., no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001. (D) Line graph showing the mating rate in each hour from 20:00 to 06:00 of wild type (red) and OR14 mutant (green) strains.
FIGURE 4. EAG responses of wild type and OR14 knockout males to pheromone blends in different concentrations. (A–C) Dose-response curves of wild type (green) and OR14 mutated male (red) antennae stimulated with Z11-16:Ald, Z9-16:Ald and Z9-14:Ald, respectively. Error bars indicate SEM. n = 18–20. *P < 0.05; **P < 0.01; ***P < 0.001.
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