title: Insecticide resistance mediated by an exon skipping event.
abstract: Many genes increase coding capacity by alternate exon usage. The gene encoding the insect nicotinic acetylcholine receptor (nAChR) α6 subunit, target of the bio-insecticide spinosad, is one example of this and expands protein diversity via alternative splicing of mutually exclusive exons. Here, we show that spinosad resistance in the tomato leaf miner, Tuta absoluta is associated with aberrant regulation of splicing of Taα6 resulting in a novel form of insecticide resistance mediated by exon skipping. Sequencing of the α6 subunit cDNA from spinosad selected and unselected strains of T. absoluta revealed all Taα6 transcripts of the selected strain were devoid of exon 3, with comparison of genomic DNA and mRNA revealing this is a result of exon skipping. Exon skipping cosegregated with spinosad resistance in survival bioassays, and functional characterization of this alteration using modified human nAChR α7, a model of insect α6, demonstrated that exon 3 is essential for receptor function and hence spinosad sensitivity. DNA and RNA sequencing analyses suggested that exon skipping did not result from genetic alterations in intronic or exonic cis-regulatory elements, but rather was associated with a single epigenetic modification downstream of exon 3a, and quantitative changes in the expression of trans-acting proteins that have known roles in the regulation of alternative splicing. Our results demonstrate that the intrinsic capacity of the α6 gene to generate transcript diversity via alternative splicing can be readily exploited during the evolution of resistance and identifies exon skipping as a molecular alteration conferring insecticide resistance.
authors.affiliation: Rothamsted Research, Harpenden, AL5 2JQ, UK.|School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD, UK.
authors.forename: Madeleine
authors.initials: M
authors.lastname: Berger
authors.affiliation: Rothamsted Research, Harpenden, AL5 2JQ, UK.
authors.forename: Alin Mirel
authors.initials: AM
authors.lastname: Puinean
authors.affiliation: College of Life and Environmental Sciences, Biosciences, University of Exeter, Penryn Campus, Penryn, Cornwall, TR10 9FE, UK.
authors.forename: Emma
authors.initials: E
authors.lastname: Randall
authors.affiliation: College of Life and Environmental Sciences, Biosciences, University of Exeter, Penryn Campus, Penryn, Cornwall, TR10 9FE, UK.
authors.forename: Christoph T
authors.initials: CT
authors.lastname: Zimmer
authors.affiliation: Departamento de Agronomia-(Entomologia), Universidade Federal Rural de Pernambuco, 52171-900, Recife, PE, Brazil.
authors.forename: Wellington M
authors.initials: WM
authors.lastname: Silva
authors.affiliation: Departamento de Producción Vegetal, Universidad Politécnica de Cartagena, Paseo Alfonso XIII 48, Cartagena, 30203, Spain.
authors.forename: Pablo
authors.initials: P
authors.lastname: Bielza
authors.affiliation: Rothamsted Research, Harpenden, AL5 2JQ, UK.
authors.forename: Linda M
authors.initials: LM
authors.lastname: Field
authors.affiliation: Rothamsted Research, Harpenden, AL5 2JQ, UK.
authors.forename: David
authors.initials: D
authors.lastname: Hughes
authors.affiliation: School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD, UK.
authors.forename: Ian
authors.initials: I
authors.lastname: Mellor
authors.affiliation: Rothamsted Research, Harpenden, AL5 2JQ, UK.
authors.forename: Keywan
authors.initials: K
authors.lastname: Hassani-Pak
authors.affiliation: Departamento de Agronomia-(Entomologia), Universidade Federal Rural de Pernambuco, 52171-900, Recife, PE, Brazil.
authors.forename: Herbert A A
authors.initials: HA
authors.lastname: Siqueira
authors.affiliation: Rothamsted Research, Harpenden, AL5 2JQ, UK.
authors.forename: Martin S
authors.initials: MS
authors.lastname: Williamson
authors.affiliation: College of Life and Environmental Sciences, Biosciences, University of Exeter, Penryn Campus, Penryn, Cornwall, TR10 9FE, UK.
authors.forename: Chris
authors.initials: C
authors.lastname: Bass
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D004338:Drug Combinations; D005091:Exons; D006801:Humans; D007313:Insecta / Q000235:genetics*; D007305:Insecticide Resistance / Q000235:genetics*; D018942:Macrolides; D064569:alpha7 Nicotinic Acetylcholine Receptor / Q000235:genetics*
chemical_list: D004338:Drug Combinations; D018942:Macrolides; D064569:alpha7 Nicotinic Acetylcholine Receptor; C415329:spinosad
keywords: 
  Tuta absoluta
  ; alternative splicing; nicotinic acetylcholine receptor; spinosad