Molecular and Pharmacological Characterization of β-Adrenergic-like Octopamine Receptors in the Endoparasitoid Cotesia chilonis (Hymenoptera: Braconidae)

Abstract

Octopamine (OA) is structurally and functionally similar to adrenaline/noradrenaline in vertebrates, and OA modulates diverse physiological and behavioral processes in invertebrates. OA exerts its actions by binding to specific octopamine receptors (OARs). Functional and pharmacological characterization of OARs have been investigated in several insects. However, the literature on OARs is scarce for parasitoids. Here we cloned three β-adrenergic-like OARs (CcOctβRs) from Cotesia chilonis. CcOctβRs share high similarity with their own orthologous receptors. The transcript levels of CcOctβRs were varied in different tissues. When heterologously expressed in CHO-K1 cells, CcOctβRs induced cAMP production, and were dose-dependently activated by OA, TA and putative octopaminergic agonists. Their activities were inhibited by potential antagonists and were most efficiently blocked by epinastine. Our study offers important information about the molecular and pharmacological properties of β-adrenergic-like OARs from C. chilonis that will provide the basis to reveal the contribution of individual receptors to the physiological processes and behaviors in parasitoids.

Keywords: cAMP; expression profiles; octopamine receptor; parasitoid; pharmacology.

Figure 1

Phylogenetic relationship of CcOctβRs and various biogenic amine receptors. MEGA 7.0 software was used to construct the neighbor-joining tree. Drosophila ninaE rhodopsin 1 (CG4550) and FMRFamide receptor (CG2114) were used as outgroups. CcOctβRs are in bold. The accession numbers of amino acid sequences used in the phylogenetic analysis were indicated in Supplementary Table S1. Abbreviations: Am, Apis mellifera; Dm, Drosophila melanogaster; Tc, Tribolium castaneum; Cs, Chilo suppressalis; Nl, Nilaparvata lugens; Hs, Homo sapiens; Pc, Priapulus caudatus; Pd, Priapulus caudatus; Sk, Saccoglossus kowalevskii. Hs and Sk are deuterostomian species, and the others are protostomian species.

Figure 2

Relative transcript levels of CcOctβ1R (A), CcOctβ2R (B), and CcOctβ3R (C) in different tissues of Cotesia chilonis adults. Different lowercase letters on the bars represent statistical differences in the transcript levels (p < 0.05, Tukey’s HSD test). Data are expressed as the means ± SE of three biological replicates.

Figure 3

Effects of various biogenic amines and putative synthetic agonists on intracellular cAMP production in CcOctβ1R (A)-, CcOctβ2R (B)-, and CcOctβ3R (C)-expressing CHO-K1 cells. Biogenic amines and agonists were measured with a concentration of 1 μΜ, and forskolin (10 μM) served as a positive control. The statistical differences between the basal value and the treatments are indicated by the asterisks (** p < 0.01, *** p < 0.001, Dunnett’s multiple comparison test). Data are presented as means ± SE of four experiments.

Figure 4

Concentration-response effects of OA, TA and various agonists on intracellular cAMP production in CcOctβ1R (A)-, CcOctβ2R (B)-, and CcOctβ3R (C)-expressing CHO-K1 cells. The values are calculated to the maximal cAMP response (=100%) for each agonist. Data are presented as means ± SE of four experiments.

Figure 5

Effects of putative synthetic antagonists (10 μM) on OA-stimulated intracellular cAMP production in CcOctβ1R (A)-, CcOctβ2R (B)-, and CcOctβ3R (C)-expressing CHO-K1 cells. Dunnett’s multiple comparison test was performed for the statistical analysis (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 6

Dose-response curves of 12 antagonists on OA-stimulated intracellular cAMP production in CcOctβR-expressing CHO-K1 cells. Antagonists used were chlorpromazine (A), cyproheptadine (B), metoclopramide (C), mianserin (D), phentolamine (E), epinastine (F), methiothepin (G), clozapine (H), asenapine (I), amitriptyline (J), chlorprothixene (K), and doxepin (L). Each individual curve is normalized to its respective OA stimulation in the absence of antagonist (=100%). Data are presented as means ± SE of four experiments.