HETEROLOGOUS EXPRESSION OF TWO ACETYLCHOLINESTERASES OF COLORADO POTATO BEETLE LEPTINOTARSA DECEMLINEATA IN BACTERIA ESCHERICHIA COLI AND PRODUCTION OF FORM-SPECIFIC ANTIBODIES

V V Dolgikh,S R Fasulati,S A Timofeev,D S Kireeva,Y V Volodartseva,I V Senderskiy,V S Zhuravlyov

doi:10.31993/2308-6459-2020-103-2-13291

V V Dolgikh, S R Fasulati + Show 5 more

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https://doi.org/10.31993/2308-6459-2020-103-2-13291

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Abstract

The Colorado potato beetle Leptinotarsa decemlineata is a widespread pest of plants of the Solanaceae family. Huge economic loss caused by L. decemlineata around the world is multiplied by its ability to develop resistance to all major insecticide classes. Previously, such resistance was found to be associated with mutations in the target enzyme LdAChE2, orthologous to Drosophila melanogaster acetylcholinesterase. However, discovery of the second form of L. decemlineata acetylcholinesterase LdAChE1 has changed this view. In order to compare the role of two acetylcholinesterase forms in the Colorado potato beetle physiology and in pest resistance to insecticides, gene copies were cloned and their heterologous expression in bacteria E. coli was followed by production of polyclonal antibodies against the recombinant proteins. Immunoblotting with produced antibodies demonstrated the absence of cross-reactivity, a lower content of LdAChE1 in the tissues of L. decemlineata adults compared with the second form, and the association of LdAChE2 with membranes. Further immunoaffinity purification of natural enzymes from the beetle tissues as well as their heterologous expression in insect cell cultures should help to evaluate the role of each form in physiology of the pest and in its resistance to insecticides.

Highlights

The Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), is a widespread pest of crops of the family Solanaceae, that may cause complete defoliation of potato plants
Preparation of samples from five organs and tissues of CPB adults followed by their immunoblotting with Abs against two forms of LdAChEs demonstrated specific recognition of single protein bands of about 70–75 kDa (Fig. 3)
An interesting result of this experiment was a lower intensity of LdAChE1 staining by specific Abs compared to LdAChE2 in the same samples

Summary

Introduction

The Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), is a widespread pest of crops of the family Solanaceae, that may cause complete defoliation of potato plants. Huge economic loss caused by CPB in North America, Europe and Asia is multiplied by its ability to develop resistance to all major insecticide classes (Alyokhin et al, 2008). In many cases such resistance may be associated to mutations in their target enzyme acetylcholinesterase (AChE, EC 3.1.1.7) (Yao et al, 1997; Kim et al, 2006; Malekmohammadia et al, 2012; Pang, 2014). Finding of the single gene in Drosophila melanogaster (Hall, Spierer, 1986; Harel et al, 2000) has suggested that insects have only one AChE form (Toutant, 1989). The orthologs of D. melanogaster genes are named as ace and the orthologs of A. gambiae ace gene are named as ace (Huchard et al, 2006)

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