Insecticide resistance: a silent base prediction

Response to a challenging environment proceeds through adaptation, the result of stochastic processes (chance) and of the influence of history (constraint) [1]. Adaptations, such as pesticide resistance, provide an opportunity to study historical constraints. Insecticides, widely used since the mid 1950s, have elicited numerous cases of resistance. Specific amino acid changes at unique or few critical positions of the target protein explain resistance to the major classes of insecticides, such as cyclodienes, organochlorines, pyrethroids, carbamates and organophosphates (OPs) [2–6] and sometimes lead to extremely high resistance levels (>1000 fold). In mosquitoes, a single glycine (Gly) to serine (Ser) substitution at position 119 (Torpedo nomenclature) in the acetylcholinesterase (AChE1) gene confers high levels of resistance to carbamates and OPs [3]. This G119S substitution was selected at least twice independently in Culex pipiens, once in Anopheles albimanus and once in Anopheles gambiae, suggesting that there are only few possibilities to generate high AChE1 insensitivity [7]. Although heavily controlled with carbamates and OPs, the mosquito vector of dengue and yellow fever, Aedes aegypti, never developed high levels of resistance. We first checked whether the G119S mutation in AChE1 is ineffective in this species. We cloned the complete AChE1 cDNA, encoding a protein 96.4% similar to C. pipiens AChE1, (supplemental data) and produced wild-type and G119S mutant recombinant proteins. Ae. aegypti AChE1 behaved exactly like C. pipiens AChE1: wild-type proteins were inhibited at identical doses of the carbamate insecticide propoxur (IC50 = 5 x 10–7 M), while G119S proteins remained insensitive up to 10–2 M propoxur (Figure 1). It is, therefore, unlikely that the low resistance in Ae. aegypti resulted from particular biochemical properties of its AChE1. Alternatively, Ae. aegypti AChE1 may not be able to evolve to the G119S substitution. Notably, in Ae. aegypti glycine 119 of AChE1 is encoded by a GGA codon, whereas GGC was found in all the other species analysed so far [3,7]. This silent third base change represents an extraordinarily heavy constraint. It decreases the probability of a spontaneous G119S substitution by several orders of magnitude . As Ser can be encoded by AGY or TCN, substitution to Ser requires only one mutation when Gly is encoded by GGY, whereas two are required when Gly is encoded by GGR. We thus hypothesized that high levels of resistance cannot emerge if the G119S substitution requires more than a single step mutation, a situation that could be described as a ‘codon constraint’. Accordingly, knowing the sequence of codon 119 should allow the prediction of the ability of a given mosquito species to develop high OP-resistance. We first checked all known acetylcholinesterase amino acid sequences (79 animal species), and found that a glycine is present at position 119 in all species, except in ascidians and Schistosoma, which show a serine. This suggests that presence of a glycine is critical, and that no other amino acids are allowed in this position, except serine. We next analysed the sequence of codon 119 in 26 natural populations of Ae. aegypti collected in 12 countries. In all samples, the glycine was encoded by GGA (serineimmutable), which fits with the