Mutation Report for: G119S_tetur-ACHE

BACKGROUND: Tetranychus urticae is a notorious crop pest with world-wide distribution that has developed resistance to a wide range of acaricides. Here, we investigated the resistance levels of a T. urticae population collected from an ornamental greenhouse in Peloponnese, Greece, and analyzed its resistance mechanisms at the molecular level. RESULTS: Toxicological assays showed resistance levels against compounds with different mode of action, with resistance ratios scaling at: 89-fold for abamectin, >1000-fold for clofentezine, >5000-fold etoxazole, 27-fold for fenpyroximate and pyridaben, 20- and 36-fold for spirodiclofen and spirotetramat, respectively and 116- and >500-fold for cyenopyrafen and cyflumetofen, respectively. Bioassays with synergists indicated the involvement of detoxification enzymes in resistance to abamectin but not to cyflumetofen and spirodiclofen. RNAseq analysis showed significant over-expression of several genes encoding detoxification enzymes such as cytochrome P450 monooxygenases and UDP-glycosyltransferases, which have been previously associated with acaricide resistance. Known target-site resistance mutations were identified in acetyl-choline esterase, chitin synthase 1 and NDUFS7/psst, but also discovered putative novel resistance mutations in targets such as the glutamate-gated chloride channel subunit 3. Interestingly, target site resistance mutations against pyrethroids or bifenazate were not identified possibly indicating a recent reduced selection pressure in Greece, as well as a possible opportunity to rotate these chemistries. CONCLUSION: We identified and characterized a striking case of multiple acaricide resistance in a field population of T. urticae. Exceptionally strong resistance phenotypes, with accumulation of multiple resistance mutations and over-expression of P450s and other detoxification genes in the same field population is reported. This article is protected by copyright. All rights reserved.

The control of Tetranychus urticae, a worldwide agricultural pest, is largely dependent on pesticides. However, their efficacy is often compromised by the development of resistance. Recent molecular studies identified a number of target site resistance mutations, such as G119S, A201S, T280A, G328A, F331W in the acetylcholinesterase gene, L1024V, A1215D, F1538I in the voltage-gated sodium channel gene, G314D and G326E in glutamate-gated chloride channel genes, G126S, I136T, S141F, D161G, P262T in the cytochrome b and the I1017F in the chitin synthase 1 gene. We examined their distribution, by sequencing the relevant gene fragments in a large number of T. urticae collections from a wide geographic range. Our study revealed that most of the resistance mutations are spread worldwide, with remarkably variable frequencies. Furthermore, we analyzed the variability of the ace locus, which has been subjected to longer periods of selection pressure historically, to investigate the evolutionary origin of ace resistant alleles and determine whether they resulted from single or multiple mutation events. By sequencing a 1540 bp ace fragment, encompassing the resistance mutations and downstream introns in 139 T. urticae individuals from 27 countries, we identified 6 susceptible and 31 resistant alleles which have arisen from at least three independent mutation events. The frequency and distribution of these ace haplotypes varied geographically, suggesting an interplay between different mutational events, gene flow and local selection.

The mutations (G228S, A391T and F439W) and duplication of the acetylcholinesterase (AChE) gene (Tuace) are involved in monocrotophos resistance in the two-spotted spider mites, Tetranychus urticae (Kwon et al., 2010a, b). The overexpression of T. urticae AChE (TuAChE) as a result of Tuace duplication was confirmed in several field-collected populations by Western blotting using an AChE-specific antibody. To investigate the effects of each mutation on the insensitivity and fitness cost of AChE, eight variants of TuAChE were expressed in vitro using the baculovirus expression system. Kinetic analysis revealed that the G228S and F439W mutations confer approximately 26-fold and 99-fold increases in the insensitivity to monocrotophos, respectively, whereas the insensitivity increased over 1165-fold in the AChE with double mutations. Nevertheless, the presence of these mutations reduced the catalytic efficiency of AChE significantly. In particular, the TuAChE having both mutations together exhibited a 17.8 approximately 27.1-fold reduced catalytic efficiency, suggesting an apparent fitness cost in the monocrotophos-resistant mites. The A391T mutation did not change the kinetic properties of either the substrate or inhibitor when present alone but mitigated the negative impacts of the F439 mutation. To simulate the catalytic activity of the overexpressed TuAChE in two T. urticae strains (approximately 6 copies for AD strain vs. 2 copies for PyriF strain), appropriate TuAChE variants were combined to make up the desired AChE copies and mutation frequencies, and their enzyme kinetics were determined. The reconstituted 6-copy and 2-copy TuAChEs exhibited catalytic efficiency levels comparable to those of a single-copy wildtype TuAChE, suggesting that, if mutations are present, multiple copies of AChE are required to restore a normal level of catalytic activity in the monocrotophos-resistant mites. In summary, the present study provides clear evidence that Tuace duplication resulted in the proportional overexpression of AChE, which was necessary to compensate for the reduced catalytic activity of AChE caused by mutations.

Molecular mechanisms of monocrotophos resistance in the two-spotted spider mite (TSSM), Tetranychus urticae Koch, were investigated. A monocrotophos-resistant strain (AD) showed ca. 3568- and 47.6-fold resistance compared to a susceptible strain (UD) and a moderately resistant strain (PyriF), respectively. No significant differences in detoxification enzyme activities, except for the cytochrome P450 monooxygenase activity, were found among the three strains. The sensitivity of acetylcholinesterase (AChE) to monocrotophos, however, was 90.6- and 41.9-fold less in AD strain compared to the UD and PyriF strains, respectively, indicating that AChE insensitivity mechanism plays a major role in monocrotophos resistance. When AChE gene (Tuace) sequences were compared, three point mutations (G228S, A391T and F439W) were identified in Tuace from the AD strain that likely contribute to the AChE insensitivity as predicted by structure analysis. Frequencies of the three mutations in field populations were predicted by quantitative sequencing (QS). Correlation analysis between the mutation frequency and actual resistance levels (LC50) of nine field populations suggested that the G228S mutation plays a more crucial role in resistance (r2 = 0.712) compared to the F439W mutation (r2 = 0.419). When correlated together, however, the correlation coefficient was substantially enhanced (r2 = 0.865), indicating that both the F439W and G228S mutations may work synergistically. The A391T mutation was homogeneously present in all field populations examined, suggesting that it may confer a basal level of resistance.

Monocrotophos-resistant two-spotted spider mites (TSSMs), Tetranychus urticae, are known to possess three mutations on the acetylcholinesterase (AChE) gene (Tuace) that are involved in target site insensitivity. Cross-strain comparison of three strains (highly resistant AD, moderately resistant PyriF and susceptible UD strains) revealed that resistant strains have relatively more Tuace copies than the UD strain and that the levels of transcript were directly proportional to copy numbers. AChEs from the AD and PyriF strains had similar V(max) values to those of AChE from the UD strain but increased K(m) and reduced k(cat) constants, suggesting that the mutated, resistant form of AChE may carry a fitness cost. Relative copy numbers of Tuace in field populations varied from 2.4 to 6.1, correlating well with their levels of resistance (r(2)= 0.895). These results are suggestive of the involvement of Tuace gene duplication in resistance. Thus, monocrotophos resistance in TSSMs appears to have evolved through a combination of mutation accumulation and extensive gene duplication.

Acetylcholinesterase (AChE) from two-spotted spider mites, Tetranychus urticae was compared between an organophosphate susceptible (TKD) and a resistant (NCN) strain. The AChE of TKD had lower affinity to acetylthiocholine and propionylthiocholine than that of NCN, and the inhibition of AChE by DDVP, ambenonium, eserine and n-methyl-eserine showed that NCN was more insensitive than TKD. AChE cDNA sequence was determined, and the 687 amino acids of primary structure were deduced. There were six replacements of amino acid residues in TKD and two in NCN. #F331(439)C was the only substitution unique to NCN, however, this mutation existed homozygously in only two out of nine mites. This residue is one of the gorge lining components, and #F331(439)C might act an important role in the sensitivity of AChE to the inhibitors.