Spatial Expression Analysis of Odorant Binding Proteins in Both Sexes of the Aphid Parasitoid Aphidius gifuensis and Their Ligand Binding Properties

Abstract

In China, Aphidius gifuensis is one of the most common endoparasitoids of the green peach aphid Myzus persicae and grain aphid Sitobion miscanthi in the field. Insect odorant-binding proteins (OBPs) play vital roles in odor perception during feeding, host searching, mating and oviposition. In addition, some OBPs are involved in other physiological processes such as gustation and reproduction. In the present study, a comparative antennal transcriptomic analysis was applied between male and female A. gifuensis. The spatial expression patterns among antennae, heads, thoraxes, abdomens and legs of OBPs in both sexes were further profiled. Fifteen AgifOBPs were predicted, and 14 of them were identified by gene cloning, including 12 classic OBPs and 2 min-C OBPs. As expected, all OBPs were mainly expressed at high levels in antennae, heads or legs which are sensory organs and tissues. Finally, ligand binding properties of 2 OBPs (AgifOBP7 and AgifOBP9) were further evaluated. Female leg specifically expressed AgifOBP9 displays a broad and high binding property to aphid alarm pheromones, plant green volatiles and aphid sex pheromones (Ki < 10 μΜ). However, female leg specifically expressed AgifOBP7 displays poor affinity for all tested ligands except CAU-II-11 ((E)-3,7-dimethylocta-2,6-dien-1-yl-2-hydroxy-3-methoxybenzoate), a reported (E)-β-farnesene (EBF) analog with an exceptionally high binding affinity (Ki = 1.07 ± 0.08 μΜ). In summary, we reported the spatial expression pattern of the OBP repertoire in A. gifuensis, and further studied the binding properties of OBP7 and OBP9, which are mainly expressed in female legs, laying the foundation for the dissection of the contribution of OBPs to chemosensation in A. gifuensis.

Keywords: Aphidifus gifuensis; fluorescence binding assay; odorant binding protein; spatial expression pattern; transcriptome.

FIGURE 1

Heatmap of differentially expressed OBP genes between females and males based on FPKM values of antennae transcriptomes.

FIGURE 2

Phylogenetic relationships of target parasitoid putative OBPs and 66 putative other hymenopteran OBPs; detailed relationships of the putative AgifOBPs (in red), MmedOBPs (in blue), AmelOBPs (in green), MpulOBPs (in orange), and AconOBPs (in purple). The trees were constructed with MEGA 11 using an LG + model and bootstrap support was calculated with 1,000 rapid bootstrap replicates with a 95% cutoff. The species names are abbreviated with four letters, and their full names with all accession numbers of the OBP amino acid sequences are provided in Supplementary Material S1.

FIGURE 3

The relative expression patterns of different AgifOBP genes in different tissues of males and females as measured by quantitative real-time polymerase chain reaction. The fold changes are relative to the transcript levels of OBP13 in the male thorax. The NADH and ACTIN genes were used as references to normalize the expression of each tested gene. The data are presented as the mean ± SD. The asterisk * and ** above the bars indicate significant differences at p < 0.05; and p < 0.01, respectively, according to two-way ANOVA.

FIGURE 4

Relative expression of AgifOBP genes in the different tissues of male and female A. gifuensis as measured by quantitative real-time polymerase chain reaction. Relative fold changes were normalized to the transcript levels in the male thorax. The NADH and ACTIN genes were used as references to normalize the expression of each tested gene. The standard error is represented by the error bar (n = 3) and the different lowercase letters (a, b, c, d, e) indicate significant differences in transcript abundances (two-way ANOVA followed by Tukey’s HSD multiple comparison test, p < 0.05).

FIGURE 5

Expression and binding properties of AgifOBP7 and AgifOBP9 with candidate ligands. (A) Expression and purification of AgifOBP7, (B) Expression and purification of AgifOBP9. Line M: molecular weight PR 1910 (11–180 KDa) Marker, 11, 17, 25, 35, 48, 63, 75, 100, 135, 180 KDa; IN: Induced pET-28a (+)/AgifOBP7/9; Super: pET-28a (+)/AgifOBP7/9 Supernatant; IB: pET-28a (+)/AgifOBP7/9 Inclusion body; Pur: Purified pET-28a (+)/AgifOBP7/9 without His-tag. (C,D) Binding curves of AgifOBP7 and AgifOBP9 with N-phenyl-1-naphthylamine (1-NPN) in 50 mM Tris-HCl buffer (pH 7.4). (E,F) Competitive binding curves of AgifOBP7 and AgifOBP9 to components of aphid alarm pheromones ((E)-beta-farnesene, (-)-α-pinene, (-)-β-pinene, (+)-limonene, (E)-beta-farnesene derivative (CAU-II-11); aphid sexual pheromone (4aSR 7SR 7aRS)-nepetalactone, the volatiles of wheat green leaf (cis-3-hexen-1-ol) and aphid induced plant main volatiles (methyl salicylate, 6-methyl-5-hepten-2-one). A mixture of the recombinant protein and N-phenyl-1-naphthylamine (1-NPN) in 50 mM Tris-HCl buffer (pH 7.4) at the concentration of 2 μM was titrated with 1 mM solutions of each competing ligand to a final concentration range of 2–16 μM. Fluorescence values are presented as percent of the values in the absence of competitor. Date are the means ± SD of three independent experiments.