Optimization of reverse chemical ecology method: false positive binding of Aenasius bambawalei odorant binding protein 1 caused by uncertain binding mechanism

Abstract

Odorant binding proteins (OBPs) are considered as the core molecular targets in reverse chemical ecology, which is a convenient and efficient method by which to screen potential semiochemicals. Herein, we identified a classic OBP, AbamOBP1 from Aenasius bambawalei, which showed high mRNA expression in male antennae. Fluorescence competitive binding assay (FCBA) results demonstrated that AbamOBP1 has higher binding affinity with ligands at acid pH, suggesting the physiologically inconsistent binding affinity of this protein. Amongst the four compounds with the highest binding affinities at acid pH, 2, 4, 4-trimethyl-2-pentene and 1-octen-3-one were shown to have attractant activity for male adults, whereas (-)-limonene and an analogue of 1-octen-3-ol exhibited nonbehavioural activity. Further homology modelling and fluorescence quenching experiments demonstrated that the stoichiometry of the binding of this protein to these ligands was not 1: 1, suggesting that the results of FCBA were false. In contrast, the apparent association constants (Ka) of fluorescence quenching experiments seemed to be more reliable, because 2, 4, 4-trimethyl-2-pentene and 1-octen-3-one had observably higher Ka than (-)-limonene and 1-octen-3-ol at neutral pH. Based on the characteristics of different OBPs, various approaches should be applied to study their binding affinities with ligands, which could modify and complement the results of FCBA and contribute to the application of reverse chemical ecology.

The role of chemosensory protein 10 in the detection of behaviorally active compounds in brown planthopper, Nilaparvata lugens

“…It is persuasive that NlugCSP10 may contribute to the detection of cis ‐3‐hexenyl acetate communication. It has been reported that dsRNA treatment of Aenasius bambawalei also inhibited responses to volatiles (Li et al ., ), and similarly Dastarcus helophoroides was also non‐responsive to volatiles after dsRNA injection (Yang et al ., ). On the other hand, injection of dsNlugCSP10 did not show any effect on the behavioral responses of the BPH toward eicosane and (+)‐ β ‐pinene; it is likely that there are other CSPs and OBPs also participating in olfactory recognition in insects (Pelosi et al ., ; Zeng et al ., ).…”

Functional Analysis of the Chemosensory Protein MsepCSP8 From the Oriental Armyworm Mythimna separata

“…The failure of adults displaying a preference for volatiles after RNAi treatment implies that disruption of olfaction caused by dsRNA prevented adults from detecting odors. It has been reported that dsRNA treatment of Aenasius bambawalei also inhibited responses to volatiles ( Li et al, 2018 ), and Dastarcus helophoroides was similarly non-responsive to volatiles after dsRNA injection ( Yang et al, 2017 ).…”

Odorant Receptors and Odorant-Binding Proteins as Insect Pest Control Targets: A Comparative Analysis

“…However, recent findings for the OBP1 of Aenasius bambawalei (AbamOBP1) report the binding of the protein with lower K D at acid pHs, inconsistent with the better binding at basic pHs in previous studies. The authors report that the binding stoichiometry between AbamOBP1 and tested ligands was not 1:1, which is likely caused by the presence of dimers or even trimers of OBPs and, therefore, a 100% active protein could not be assumed, suggesting false positives from the competitive binding assays (Li et al, 2018 ). It appears that a combined methodology such as fluorescence intrinsic quenching assays (Bette and Breer, 2002 ) could be in better accordance with behavioral assays.…”