Ryanodine receptor point mutations confer diamide insecticide resistance in tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae).

This is the author accepted manuscript. The final version is available from Elsevier via the DOI in this record.Insect ryanodine receptors (RyR) are the molecular target-site for the recently introduced diamide insecticides. Diamides are particularly active on Lepidoptera pests, including tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae). High levels of diamide resistance were recently described in some European populations of T. absoluta, however, the mechanisms of resistance remained unknown. In this study the molecular basis of diamide resistance was investigated in a diamide resistant strain from Italy (IT-GELA-SD4), and additional resistant field populations collected in Greece, Spain and Brazil. The genetics of resistance was investigated by reciprocally crossing strain IT-GELA-SD4 with a susceptible strain and revealed an autosomal incompletely recessive mode of inheritance. To investigate the possible role of target-site mutations as known from diamondback moth (Plutella xylostella), we sequenced respective domains of the RyR gene of T. absoluta. Genotyping of individuals of IT-GELA-SD4 and field-collected strains showing different levels of diamide resistance revealed the presence of G4903E and I4746M RyR target-site mutations. These amino acid substitutions correspond to those recently described for diamide resistant diamondback moth, i.e. G4946E and I4790M. We also detected two novel mutations, G4903V and I4746T, in some of the resistant T. absoluta strains. Radioligand binding studies with thoracic membrane preparations of the IT-GELA-SD4 strain provided functional evidence that these mutations alter the affinity of the RyR to diamides. In combination with previous work on P. xylostella our study highlights the importance of position G4903 (G4946 in P. xylostella) of the insect RyR in defining sensitivity to diamides. The discovery of diamide resistance mutations in T. absoluta populations of diverse geographic origin has serious implications for the efficacy of diamides under applied conditions. The implementation of appropriate resistance management strategies is strongly advised to delay the further spread of resistance.The work of Hellenic Agricultural Organisation - 'Demeter' was partially supported by an ARIMnet2 StomP grand to A.T and E.R. This work was also partially funded from a fellowship granted to H.A.A.S. (CNPq - PQ - Proc 308461/2013-4). The Universidad Politécnica de Cartagena group would like to thank for partial financial support the Ministerio de Economía y Competitividad of Spain and FEDER (AGL2011-25164). Lidia García-Vidal holds a grant from the MECD (FPU13/01528). The Tuta absoluta strain from Gela, Sicily was collected under the frame a resistance monitoring program established among the Hellenic Agricultural Organisation - 'Demeter' and DuPont De Nemurs (data published in 2015). Finally, the Hellenic Agricultural Organisation - 'Demeter' would like to thank Fytochem S.A., Neo Mirtos, Ierapetra for supplies of plant material