Multiple mutations in the nicotinic acetylcholine receptor Cc\u03b16 gene associated with resistance to spinosad in medfly

Enric Ureña,Nathalia Perera,Félix Ortego,Ana Guillem-Amat,Francisco Couso-Ferrer,Beatriz Beroiz,Pedro Hernández-Crespo,Pedro Castañera,Elena López-Errasquín

doi:10.1038/s41598-019-38681-w

Abstract

Spinosad is an insecticide widely used for the control of insect pest species, including Mediterranean fruit fly, Ceratitis capitata. Its target site is the α6 subunit of the nicotinic acetylcholine receptors, and different mutations in this subunit confer resistance to spinosad in diverse insect species. The insect α6 gene contains 12 exons, with mutually exclusive versions of exons 3 (3a, 3b) and 8 (8a, 8b, 8c). We report here the selection of a medfly strain highly resistant to spinosad, JW-100 s, and we identify three recessive Ccα6 mutant alleles in the JW-100 s population: (i) Ccα63aQ68* containing a point mutation that generates a premature stop codon on exon 3a (3aQ68*); (ii) Ccα63aAG>AT containing a point mutation in the 5′ splicing site of exon 3a (3aAG > AT); and (iii) Ccα63aQ68*-K352* that contains the mutation 3aQ68* and another point mutation on exon 10 (K352*). Though our analysis of the susceptibility to spinosad in field populations indicates that resistance has not yet evolved, a better understanding of the mechanism of action of spinosad is essential to implement sustainable management practices to avoid the development of resistance in field populations.

Highlights

Spinosad is a natural product mixture of two compounds, spinosyn A and spinosyn D, produced by the bacteria Saccharopolyspora spinosa and used as insecticide since 19971
We found that LC50 values, which estimate the concentration killing 50% of the individuals, were always far below the concentration of the spinosad-based insecticide recommended for C. capitata treatments in Spain (240 ppm, Spintor Cebo 0.024% p/v, Dow Agrosciences) (Table 1)
In a number of insect species, high levels of spinosad resistance have been associated with alterations in the α6 gene affecting all the α6 isoforms

Summary

Introduction

Spinosad is a natural product mixture of two compounds, spinosyn A and spinosyn D, produced by the bacteria Saccharopolyspora spinosa and used as insecticide since 19971. Exon 3 contains the acetylcholine binding loop D, exon 8 includes part of the TM2 domain involved in the formation of the pore, and some of the editing sites described in α6 are located in the proximity of the acetylcholine binding pocket[10,11]. Whether this α6 mRNA diversity leads to functionally distinct receptors and/or affect the interaction with spinosad remains elusive. The identification of resistance mechanisms and the development of specific diagnostic tools for the early detection of resistance is essential for the implementation of appropriate resistance management strategies[34]

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