Molecular Characterization of Chemosensory Protein (CSP) Genes and the Involvement of AgifCSP5 in the Perception of Host Location in the Aphid Parasitoid Aphidius gifuensis

Abstract

Aphidius gifuensis is the dominant parasitic natural enemy of aphids. Elucidating the molecular mechanism of host recognition of A. gifuensis would improve its biological control effect. Chemosensory proteins (CSPs) play a crucial role in insect olfactory systems and are mainly involved in host localization. In this study, a total of nine CSPs of A. gifuensis with complete open reading frames were identified based on antennal transcriptome data. Phylogenetic analysis revealed that AgifCSPs were mainly clustered into three subgroups (AgifCSP1/2/7/8, AgifCSP3/9, and AgifCSP4/5/6). AgifCSP2/5 showed high expression in the antennae of both sexes. Moreover, AgifCSP5 was found to be specifically expressed in the antennae. In addition, fluorescent binding assays revealed that AifCSP5 had greater affinities for 7 of 32 volatile odor molecules from various sources. Molecular docking and site-directed mutagenesis results revealed that the residue at which AgifCSP5 binds to these seven plant volatiles is Tyr75. Behavior tests further confirmed that trans-2-nonenal, one of the seven active volatiles in the ligand binding test, significantly attracted female adults at a relatively low concentration of 10 mg/mL. In conclusion, AgifCSP5 may be involved in locating aphid-infested crops from long distances by detecting and binding trans-2-nonenal. These findings provide a theoretical foundation for further understanding the olfactory recognition mechanisms and indirect aphid localization behavior of A. gifuensis from long distances by first identifying the host plant of aphids.

Keywords: Aphidius gifuensis; behavioral response; binding affinity; chemosensory protein; molecular docking; tissue expression profile.

Figure 1

Analysis of nine AgifCSP sequences. (A) Sequence alignment of AgifCSPs, cysteine residues highlighted by red color and conserved cysteine residues marked with asterisks (*). (B) Localization of 9 AgifCSPs on Aphidius gifuensis chromosomes. (C) Motifs, domains and gene structure analysis of nine AgifCSPs. (D) Sequence details of eight motifs.

Figure 2

Phylogenetic tree of nine AgifCSPs of A. gifuensis CSPs and other insect CSPs. Seventeen species were included: Apis mellifera: Amel; Apis cerana: Acer; Chouioia cunea: Ccun; Microplitis mediator: Mmed; Diachasma alloeum: Dall; Cephus cinctus: Ccin; Sclerodermus sp. MQW-2015: Ssp; Meteorus pulchricornis: Mpul; Aulacocentrum confusum: Acon; Camponotus japonicas: Cjap; Encarsia formosa: Efor; Tribolium castaneum: Tc/Tcas; Bombyx mori: Bmor; Myzus persicae: Mper; Aphis gossypii: Agos; and Sitobion avenae: Save. The bootstrap support was calculated with 1000 rapid bootstrap replicates. Nine AgifCSPs marked with green dots, form three clusters showed by three colored shadows.

Figure 3

Gene expression of AgifCSPs in both sexes of parasitoids. (A), Gene expression of nine AgifCSPs in antennae; FAn and MAn are antennae of female and male adults, respectively; (B,C), Gene expression profiles of AgifCSP2 and AgifCSP5 in various tissues, including the head without antenna, thorax, abdomen, antenna, leg, and wing of A. gifuensis; ‘*’ indicates that gene expression was significantly different between males and females at p < 0.05 according to a t test, and ‘ns’ indicates no difference between the sexes.

Figure 4

Analysis of AgifCSP5 protein expression, purification, and binding profiles. (A–C) Wild-type, R24A, Y75A protein purification, M: 180 kDa protein molecular marker; Lane 1: Noninduced AgifCSP5-pET30a; Lane 2: induced AgifCSP5-pET30a; Lane 3: induced AgifCSP5 supernatant lysate; Lane 4: inclusion body; Lane 5: purified protein. (D–G) Competitive binding curves of AgifCSP5 to compounds. (D) Ketones and aldehydes. (E) Alkenes. (F) Esters and alkanes. (G), Alcohols. (H–J) Binding curves and scatter plots (insert) of the wild type, R24A, and Y75 of AgifCSP5 to 1-NPN. (K) 1/Ki of all ligands to AgifCSP5; the blue bars indicate plant volatiles, the orange bars indicate aphid pheromones, and the red dashed lines indicate strong binding affinity.

Figure 5

3D and 2D structures of AgifCSP5 interacting with ligands.

Figure 6

1/Ki of ligands to mutants (R24A and Y75A) of AgifCSP5. The error bars represent the standard error of the mean (mean ± SE), and values followed by different letters are significantly different at p < 0.05 according to the LSD test.

Figure 7

Behavioral responses of A. gifuensis to various volatiles. The chi-square test (χ2) was performed to detect significant differences in behavioral responses between compounds and paraffin oil (PO).