Pharmacological Properties and Function of the PxOctβ3 Octopamine Receptor in Plutella xylostella (L.)

Abstract

The diamondback moth (Plutella xylostella) is one of the most destructive lepidopteran pests of cruciferous vegetables, and insights into regulation of its physiological processes contribute towards the development of new pesticides against it. Thus, we investigated the regulatory functions of its β-adrenergic-like octopamine receptor (PxOctβ3). The open reading frame (ORF) of PxOctβ3 was phylogenetically analyzed, and the levels of expression of the receptor mRNA were determined. This ORF was also cloned and expressed in HEK-293 cells. A series of octopamine receptor agonists and antagonists were tested against PxOctβ3. We showed that the receptor is a member of the Octβ3 protein family, and an analysis using quantitative PCR showed that it was expressed at all developmental stages of P. xylostella. Octopamine activated PxOctβ3, resulting in increased levels of intracellular cAMP. Furthermore, the agonists naphazoline, clonidine, 2-phenethylamine, and amitraz activated the PxOctβ3 receptor, and naphazoline was the most effective. Only metoclopramide and mianserin had significant antagonistic effects on PxOctβ3, whereas yohimbine, phentolamine, and chlorpromazine lacked obvious antagonistic effects. The injection of double-stranded RNA in an RNA interference assay indicated that PxOctβ3 regulates development in P. xylostella. This study demonstrated the pharmacological properties and functions of PxOctβ3 in P. xylostella, thus, providing a theoretical basis for the design of pesticides that target octopamine receptors.

Keywords: Plutella xylostella; RNAi; gene expression; octopamine receptor; pharmacology.

Figure 1

Amino acid sequence alignment of PxOctβ3 and orthologous receptors from Apis mellifera (XP_006557730.1), Drosophila melanogaster (NP_001034043.2), and Tribolium castaneum (NP_001280505.1). The seven predicted transmembrane regions are denoted by TM1to M7. Potential N-glycosylation and phosphorylation sites by PKC are marked with filled circles and triangles, respectively. Potential palmitoylation sites are marked with empty circles. Two conserved cysteine residues are marked with empty squares. The second phenylalanine after the FxxxWxP motif in TM6, a unique feature of aminergic GPCRs, is marked with a filled square. GPCR, G-protein-coupled receptors; PKC, protein kinase C.

Figure 2

Phylogenetic tree analysis of representative members of the different OARs. Phylogenetic reconstruction was performed using the amino acid positions that were found in the TM1 to 7 regions. Numbers in the middle of the branches denote percentage bootstrap support with 1000 replications per branch. The receptor names followed by their GenBank accession numbers are listed in the tree. Bm, Bombyx mori; Pr, Pieris rapae; Cs, Chillo suppressalis; Px, Plutella xylostella; Dm, Drosophila melanogaster; Am, Apis mellifera; Tc, Tribolium castaneum; Ni, Nilaparvata lugens; OAR, octopamine receptor.

Figure 3

Relative level of expression (mean ± SE) of Octβ3 in Plutella xylostella. (A) The levels of expression of PxOctβ3 genes in different development stages (first to fourth larvae, prepupae, pupae, adults, and eggs). The level of expression of PxOctβ3 in the second-instar larvae was used for comparison. (B) The levels of expression of PxOctβ3 in the tissues (head (Hd), epidermis (Ep), midgut (Mg), and Malpighian tubules (Mt)) of the fourth-instar larvae. PxOctβ3 in the Hd was used for comparison. The data were the mean values of three independent experiments (n = 3) and normalized to the endogenous ribosomal protein L32, which served as the internal control. Different letters above the bars indicate significant differences in different stages and tissues (p < 0.05). Statistical comparisons were performed using one-way analysis of variance (ANOVA) followed by Tukey’s honest significant difference (HSD) test to determine whether significant differences occurred.

Figure 4

Effects of various biogenic amines, agonists, and antagonists on the concentration of cAMP in HEK-293 cells that overexpressed the PxOctβ3 gene. The pcDNA3.1-PxOctβ3 plasmid was stably expressed in HEK-293 cells, which were used to detect changes in the levels of intracellular cAMP after treatment with different biogenic amines, agonists, and antagonists. The relative concentration of intracellular cAMP was calculated by defining the concentration of cAMP in the control cells as 1 (4.08 ± 0.39 pmol/mg protein). (A) Effects of various biogenic amines on the levels of cAMP in HEK-293 cells that stably expressed the PxOctβ3 gene. HEK-293 cells that stably expressed PxOctβ3 were treated with forskolin as a positive control. The relative change in [cAMP] was provided as a multiple of the value obtained for the untreated control (=1). (B) Effects of different doses of octopamine on the levels of cAMP in HEK-293 cells that stably expressed PxOctβ3. (C) The effects of different antagonists (100 μM) on the levels of cAMP in HEK-293 cells that stably expressed PxOctβ3. Data represent the mean of three experiments. (D) Effects of various agonists on the levels of cAMP in HEK-293 cells that stably expressed the PxOctβ3 gene. HEK-293 cells that stably expressed PxOctβ3 were treated with forskolin as a positive control. The statistical analysis was based on a one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, ns means no significant.

Figure 5

Effect of silencing PxOctβ3 expression on pupation in Plutella xylostella. (A) The relative levels of mRNA expression of PxOctβ3 after injection with 200 ng dsOA2B3 and dsGFP. DsRNAs were injected into one-day-old fourth-instar larvae. The relative levels of mRNA expression of PxOctβ3 were determined using RT-qPCR at 24 h, 48 h, and 72 h postinjection. (B) The pupation rate of P. xylostella larvae at 48 h, 60 h, and 72 h postinjection. Error bars represent the standard error of the calculated means based on three biological replicates. Asterisks indicate significant differences (Student’s t-test, p < 0.05). dsGFP, double-stranded green fluorescence protein; dsRNA, double-stranded RNA; RT-qPCR, quantitative reverse transcription PCR. ** p ≤ 0.01, * p ≤ 0.05, ns means no significant.