36Insect ryanodine receptors (RyR) are the molecular target-site for the recently introduced 37 diamide insecticides. Diamides are particularly active on Lepidoptera pests, including tomato 38 leafminer, Tuta absoluta (Lepidoptera: Gelechiidae). High levels of diamide resistance were 39 recently described in some European populations of T. absoluta, however, the mechanisms 40 of resistance remained unknown. In this study the molecular basis of diamide resistance was 41

The introduction of an agricultural pest species into a new environment is a potential threat to agroecosystems of the invaded area. The phytosanitary concern is even greater if the introduced pest’s phenotype expresses traits that will impair the management of that species. The invasive tomato borer, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), is one such species and the characterization of the insecticide resistance prevailing in the area of origin is important to guide management efforts in new areas of introduction. The spinosad is one the main insecticides currently used in Brazil for control of the tomato borer; Brazil is the likely source of the introduction of the tomato borer into Europe. For this reason, spinosad resistance in Brazilian populations of this species was characterized. Spinosad resistance has been reported in Brazilian field populations of this pest species, and one resistant population that was used in this study was subjected to an additional seven generations of selection for spinosad resistance reaching levels over 180,000-fold. Inheritance studies indicated that spinosad resistance is monogenic, incompletely recessive and autosomal with high heritability (h 2 = 0.71). Spinosad resistance was unstable without selection pressure with a negative rate of change in the resistance level ( = −0.51) indicating an associated adaptive cost. Esterases and cytochrome P450-dependent monooxygenases titration decreased with spinosad selection, indicating that these detoxification enzymes are not the underlying resistance mechanism. Furthermore, the cross-resistance spectrum was restricted to the insecticide spinetoram, another spinosyn, suggesting that altered target site may be the mechanism involved. Therefore, the suspension of spinosyn use against the tomato borer would be a useful component in spinosad resistance management for this species. Spinosad use against this species in introduced areas should be carefully monitored to prevent rapid selection of high levels of resistance and the potential for its spread to new areas.

32The tomato leafminer, Tuta absoluta, now a major pest of tomato crops worldwide, is 33 primarily controlled using chemical insecticides. Recently, high levels of resistance to 34 the insecticide spinosad have been described in T. absoluta populations in Brazil. 35Selection of a resistant field-collected strain led to very high levels of resistance to 36 spinosad and cross-resistance to spinetoram, but not to other insecticides that target the 37 nicotinic acetylcholine receptor (nAChR). In this study the mechanisms underlying 38 resistance to spinosad were investigated using toxicological, biochemical and molecular 39 approaches. Inhibition of metabolic enzymes using synergists and biochemical 40 assessment of detoxification enzyme activity provided little evidence of metabolic 41 resistance in the selected strain. Cloning and sequencing of the nAChR α6 subunit from 42T. absoluta, the spinosad target-site, from susceptible and spinosad-resistant strains was 43 done to investigate the role of a target-site mechanism in resistance. A single nucleotide 44 change was identified in exon 9 of the α6 subunit of the resistant strain, resulting in the 45 replacement of the glycine (G) residue at position 275 observed in susceptible T. 46 absoluta strains with a glutamic acid (E). A high-throughput DNA-based diagnostic 47 assay was developed and used to assess the prevalence of the G275E mutation in 17 48 field populations collected from different geographical regions of Brazil. The resistant 49 allele was found at low frequency, and in the heterozygous form, in seven of these 50 populations but at much higher frequency and in the homozygous form in a population 51 collected in the Iraquara municipality. The frequency of the mutation was significantly 52 correlated with the mortality of these populations in discriminating dose bioassays. In 53 summary our results provide evidence that the G275E mutation is an important 54 mechanism of resistance to spinosyns in T. absoluta, and may be used as a marker for 55 resistance monitoring in field populations. 56 3

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