Mutation (G275E) of the nicotinic acetylcholine receptor α6 subunit is associated with high levels of resistance to spinosyns in Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae).

The tomato leafminer, Tuta absoluta, now a major pest of tomato crops worldwide, is primarily controlled using chemical insecticides. Recently, high levels of resistance to the insecticide spinosad have been described in T. absoluta populations in Brazil. Selection of a resistant field-collected strain led to very high levels of resistance to spinosad and cross-resistance to spinetoram, but not to other insecticides that target the nicotinic acetylcholine receptor (nAChR). In this study the mechanisms underlying resistance to spinosad were investigated using toxicological, biochemical and molecular approaches. Inhibition of metabolic enzymes using synergists and biochemical assessment of detoxification enzyme activity provided little evidence of metabolic resistance in the selected strain. Cloning and sequencing of the nAChR α6 subunit from T. absoluta, the spinosad target-site, from susceptible and spinosad-resistant strains were done to investigate the role of a target-site mechanism in resistance. A single nucleotide change was identified in exon 9 of the α6 subunit of the resistant strain, resulting in the replacement of the glycine (G) residue at position 275 observed in susceptible T. absoluta strains with a glutamic acid (E). A high-throughput DNA-based diagnostic assay was developed and used to assess the prevalence of the G275E mutation in 17 field populations collected from different geographical regions of Brazil. The resistant allele was found at low frequency, and in the heterozygous form, in seven of these populations but at much higher frequency and in the homozygous form in a population collected in the Iraquara municipality. The frequency of the mutation was significantly correlated with the mortality of these populations in discriminating dose bioassays. In summary our results provide evidence that the G275E mutation is an important mechanism of resistance to spinosyns in T. absoluta, and may be used as a marker for resistance monitoring in field populations.

Identification of mutations associated with pyrethroid resistance in the voltage-gated sodium channel of the tomato leaf miner (Tuta absoluta).

The tomato leaf miner, Tuta absoluta (Lepidoptera) is a significant pest of tomatoes that has undergone a rapid expansion in its range during the past six years and is now present across Europe, North Africa and parts of Asia. One of the main means of controlling this pest is through the use of chemical insecticides. In the current study insecticide bioassays were used to determine the susceptibility of five T. absoluta strains established from field collections from Europe and Brazil to pyrethroids. High levels of resistance to λ cyhalothrin and tau fluvalinate were observed in all five strains tested. To investigate whether pyrethroid resistance was mediated by mutation of the para-type sodium channel in T. absoluta the IIS4-IIS6 region of the para gene, which contains many of the mutation sites previously shown to confer knock down (kdr)-type resistance to pyrethroids across a range of different arthropod species, was cloned and sequenced. This revealed that three kdr/super-kdr-type mutations (M918T, T929I and L1014F), were present at high frequencies within all five resistant strains at known resistance 'hot-spots'. This is the first description of these mutations together in any insect population. High-throughput DNA-based diagnostic assays were developed and used to assess the prevalence of these mutations in 27 field strains from 12 countries. Overall mutant allele frequencies were high (L1014F 0.98, M918T 0.35, T929I 0.60) and remarkably no individual was observed that did not carry kdr in combination with either M918T or T929I. The presence of these mutations at high frequency in T. absoluta populations across much of its range suggests pyrethroids are likely to be ineffective for control and supports the idea that the rapid expansion of this species over the last six years may be in part mediated by the resistance of this pest to chemical insecticides.