Two major classes of target site insensitivity mutations confer resistance to organophosphate and carbamate insecticides

Two major classes of target site insensitivity mutations confer resistance to organophosphate and carbamate insecticides

PESTICIDE Biochemistry & Physiology Pesticide Biochemistry and Physiology 79 (2004) 84–93 Two major classes of target ...

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PESTICIDE Biochemistry & Physiology

Pesticide Biochemistry and Physiology 79 (2004) 84–93

Two major classes of target site insensitivity mutations confer resistance to organophosphate and carbamate insecticides Robyn J. Russell,a,* Charles Claudianos,a,b Peter M. Campbell,a Irene Horne,a Tara D. Sutherland,a and John G. Oakeshotta a


CSIRO Entomology, GPO Box 1700, Canberra, ACT 2601, Australia Research School of Biological Sciences, The Australian National University, Canberra, ACT 0200, Australia Received 17 November 2003; accepted 30 March 2004 Available online 14 May 2004

Abstract Interspecific comparisons of bioassay and biochemical data suggest two major patterns of target site resistance to carbamates and organophosphates. Pattern I resistance, which is generally more effective for carbamates, has been shown in two sub-species of mosquitoes to be due to a particular Gly-Ser mutation in the oxyanion hole within the active site of one of their two acetylcholinesterase enzymes. Intriguingly, different substitutions at the equivalent site confer organophosphate hydrolytic ability on other esterases responsible for metabolic resistance in some other species. In the case of the aphid, Myzus persicae, Pattern I resistance is due to a Ser-Phe mutation in the vicinity of the acyl pocket of acetylcholinesterase. Pattern II resistance is at least as effective for organophosphates as it is for carbamates and may even be specific to organophosphates in some cases. Molecular studies on this pattern of resistance in three higher Diptera show that it is due to changes that constrict the acetylcholinesterase active site gorge and limit binding of the insecticide to the catalytic residues at the base of the gorge. One case of Pattern II resistance in the mosquito, Culex tritaeniorhynchus, involves the same site near the acyl pocket of acetylcholinesterase, albeit a different substitution, as that involved in Pattern I resistance in M. persicae. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Acetylcholinesterase; Insects; Carbamates; Organophosphates; Target site resistance

Several studies have now identified the molecular changes in the target site for organophosphate (OP) and carbamate insecticides that are responsible for resistance to these compounds in various insect species. The target site is the enzyme, ace* Corresponding author. Fax: +64-2-6246-4173. E-mail address: [email protected] (R.J. Russell).

tylcholinesterase (AChE), which functions to clear bound acetylcholine from its receptors in cholinergic nerves. There is just one ace gene in the higher Diptera, whereas other insects and mites have two, albeit it is apparently only the product of one of them (AChE-1 in the nomenclature of Weill et al. [1]) that functions as described above in cholinergic nerves. Remarkably some of the same

0048-3575/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2004.03.002

R.J. Russell et al. / Pesticide Biochemistry and Physiology 79 (2004) 84–93

resistance mutations have been found in the cholinergic AChE of different species, suggesting very finite options for evolving resistance. In this paper, we show how similarities and differences in the resistance mutations relate to variation in the resistance profiles of these cases as assessed on bioassay and biochemical criteria. We suggest that two major patterns of resistance to OPs/carbamates resulting from an insensitive AChE have been emerging from the bioassay and biochemical literature (Table 1). Pattern I resistance is characterised by much greater resistance ratios (and/or, at the biochemical level, much greater reduction in the sensitivity of AChE) to carbamates such as propoxur, pirimicarb or carbosulfan, than to OPs such as malaoxon, paraoxon, chlorpyriphos or temephos. We can identify 12 cases of Pattern I resistance, including three (Culex pipiens pipiens , Culex pipiens quinquefasciatus and Myzus persicae) for which the molecular basis of resistance has been described. Pattern II resistance is characterised by resistance ratios (and/ or reductions in the sensitivity of AChE) that are approximately equivalent for both carbamates and OPs, or, in the case of Bactrocera oleae, specific for OPs. We identify ten cases of Pattern II resistance, four (Drosophila melanogaster, Musca domestica, B. oleae, and Culex tritaeniorhynchus) for which the molecular basis of resistance is known. There are also a few species for which an insensitive AChE has been reported and for which molecular data have been collected, but for which the resistance profiles for both OPs and carbamates have not been reported. These include two additional mosquito species, Anopheles gambiae and Anopheles albimanus [1,2] found to contain one of the mutations in AChE-1 implicated in Pattern I resistance above. This suggests that the list of species belonging to the two major classes will grow as full data come in for more species (Table 1). The likely generality of the two patterns of resistance is also supported by the observations that they are already represented in both Holo- and Hemimetabola and in different radiations of the AChE phylogeny, as shown in Fig. 1. Two cases of Pattern II resistance involve mites (Table 1). Interestingly, both Patterns have been described in different populations of Aphis gossypii (Table 1).


The mutations underlying Pattern II resistance (Table 1) in the three higher dipterans, D. melanogaster, M. domestica, and B. oleae, have been quite thoroughly investigated biochemically and show a significant degree of overlap [3–5]. Across the three species there are a total of 11 mutations at six sites that contribute to resistance. Two of the five mutations identified in D. melanogaster were shared by M. domestica and one was shared with B. oleae. They have not been recorded in any case of Pattern I resistance, or any Pattern II resistance outside the higher Diptera. It is clear from structural modelling studies that several of the Pattern II resistance mutations in the three higher Diptera result in the constriction of the neck of the active site gorge, thereby limiting access of the pesticide to the catalytic residues at the base of the gorge [3,4]. The other case of Pattern II resistance characterised at a molecular level involves an F331W mutation (all numbering herein is according to that of Torpedo AChE; [6]) in C. tritaeniorhynchus [7]. This mutation is close to the catalytic residues at the base of the active site gorge, at a site that has been implicated in substrate guidance and binding. Like most of the higher dipteran cases above, it involves a change to a bulkier residue that may restrict access or binding of the insecticides to the base of the gorge. Intriguingly, a different substitution (S331F) at the same site is responsible for one of the cases of Pattern I resistance, involving M. persicae [8,9]. This substitution is also a change to a bulkier amino acid. Why it should result in Pattern II resistance while the C. tritaeniorhynchus mutation affected Pattern I resistance is unclear, but it may be because of fine differences in the geometry of the active site in this region. It is also notable that the M. persicae resistance is rather specific to the dimethyl carbamates like pirimicarb and the dimethyl cabamoyltriazole, triazamate, whereas most other cases of Pattern I resistance are diagnosed with the monomethyl carbamate, propoxur (see Table 1). The Pattern I resistance mutation in C. pipiens (and presumably, A. gambiae [1] and A. albimanus [2]) is a G119S change in the oxyanion hole of AChE-1, at the base of the gorge itself. It is not yet clear how such a mutation affects the sensitivity of


Table 1 Patterns of resistance to OPs/carbamates resulting from an insensitive AChE in various invertebrate species Species

Resistance profile

Pattern IIa : Carbamate resistance  Carbamate resistance Drosophila melanogaster Similar levels of resistance to OPs (paraoxon, malaoxon) and carbamates (carbaryl, propoxur)



2 ace genes cloned and sequenced; all R strains: G119S in AChE-1 is responsible for resistance


2 ace genes cloned and sequenced; no resistance mutations identified in ace-2 S331F mutation in AChE-1 is associated with resistance

[26]; Menozzi (thesis, 2000, cited in [24]); [27]

No sequence data


No sequence data


No sequence data


No sequence data No sequence data No sequence data

[32] [33] [34]

No sequence data No sequence data

[33,35] [36]

No sequence data


No sequence data


Only 1 ace gene (ace-2)c ; 5 resistance mutations identified



R.J. Russell et al. / Pesticide Biochemistry and Physiology 79 (2004) 84–93

Pattern Ia : Carbamate resistance >OP resistance Culex pipiens pipiens;Culex 4 Rb strains: details of resistance profile unpublished; 3 R strains: ki 1000-fold less for propoxur than pipiens quinquefasciatus malaoxon, paraoxon, and aldicarb 1 R strain: R factors 1600 for propoxur, 18 for chlorpyriphos, 140 for malaoxon and 5 for temephos; 2 R strains: overproduced esterases in the populations as well as insensitive AChE. Aphis gossypii of various Carbamate resistance (pirimicarb) much greater than authors OP resistance (demeton-S-methyl, methamidiphos, monocrotophos, omethoate, pirimiphos-methyl) Myzus persicae of various Marked insensitivity to pirimicarb (>100); authors no insensitivity to a range of other carbamates (e.g., methomyl, carbofuran, aldicarb and ethiofencarb) and OPs (demeton-S-methyl, dichlorvos and paraoxon) Helicoverpa armigera 35-fold resistance to thiodicarb, 75-fold to methomyl and no cross resistance to OPs Leptinotarsa decemlineata >550-fold resistance to carbofuran and ninefold of Wierenga and resistance to azinphosmethyl Hollingworth [30] Scirtothrips citri AChE insensitivity is greater for propoxur than paraoxon in a resistant strain Anopheles albimanus ki values 100-fold less for propoxur than paraoxon of various authors ki values 18-fold less for propoxur than for malaoxon; Resistance levels >1000 for propoxur, 73 for carbaryl, 37.5-1.2 for OPs Anopheles sacharovi ki values 10-fold less for propoxur than for malaoxon; Anopheles nigerrimus High level of carbamate and relatively low level of OP resistance Heliothis virescens ki values 10- to 100-fold less for propoxur than for several OPs Nephotettix cincticeps of 43-fold resistance to propoxur, 43-fold to carbaryl, Iwata and Hama [38] sixfold to malathion, 18-fold to malaoxon.

Similar ki values for OPs and carbamates

Bactrocera oleae

ki data indicate insensitivity to omethoate and paraoxon, but not to propoxur

Culex tritaeniorhynchus

AChE from a resistant strain has 870-fold decrease in fenitrooxon sensitivity (and a lesser extent to carbamates) Resistance ratios up to 10-fold higher for OPs (methamidophos and monocrotophos) than for carbamates (pirimicarb and thiodicarb)

Aphis gossypii of Li and Han [40]

Bemisia tabaci Spodoptera frugiperda Amblyseius potentillae Tetranychus kanzawai Diabrotica virgifera

Unknownd Anopheles albimanus of Weill et al. [2]

Similar ki values for OPs and carbamates Similar ki values for OPs and carbamates Similar ki values for paraoxon and propoxur OP-resistant strains showed marked cross resistance to OPs but low cross resistance to carbamates Slight decrease in AChE sensitivity to paraoxon in strain resistant to a range of OPs and carbaryl

ki data indicate high insensitivity to propoxur (OP data not reported)

Anopheles gambiae

Resistance to carbosulfan (resistance to OPs not reported)

Tetranychus urticae


Boophilus microplus

Resistance to OPs (resistance to carbamates not reported)

Musca domestica of Kozaki et al. [48]

>40-fold decrease in AChE sensitivity to fenitrooxon in resistant individuals; sensitivity to carbamates not reported

Only 1 ace gene (ace-2); 5 resistance mutations identified (2 shared with D. melanogaster AChE) Only 1 ace gene (ace-2); 2 resistance mutations identified (1 shared with D. melanogaster AChE) F331W mutation in AChE-1 is responsible for resistance


Several candidate amino acid polymorphisms among one susceptible and 3 resistant strains but association with resistance is inconclusive No sequence data No sequence data No sequence data No sequence data


[41] [42] [43] [44]

No sequence data


G119S mutation in AChE-1 is correlated with AChE insensitivity 2 ace genes in genome; G119S in AChE-1 is responsible for resistance R strain has AChE that is more (not less) sensitive to OPs/ carbamates than S strain; S strain: Ser/Gly at 119 R strain: Ser at 119 Sequenced ace-1 and ace-2 cDNAs; have not yet identified resistance mutations 2 resistance mutations identified (both found in the M. domestica of Walsh et al. [4])






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Musca domestica of Walsh et al. [4]




Residues are numbered according to the nomenclature of Toutant [6] for Torpedo AChE. a Species were assigned to Pattern I or II resistance phenotypes where bioassay and/or biochemical data were available for both OPs and carbamates. See Fournier and Mutero [51] and Oakeshott et al. [52] for comprehensive reviews. b R, resistant; S, susceptible. c The nomenclature of Weill et al. [1,24] has been followed for insect ace genes (i.e., ace-2 is the homologue of D. melanogaster ace). d The species for which an insensitive AChE has been reported and for which molecular data have been collected, but for which the resistance profiles for both OPs and carbamates have not been reported. e Weill et al. [24] re-analysed the data and suggest that the association with resistance is not significant.

[50] Resistance to azinphosmethyl (resistance to carbamates not reported) Leptinotarsa decemlineata of Zhu et al. [50]

Cloned ace-2 using PCR but could not find a resistance polymorphism; The authors conclude that S238G in AChE-2 is responsible for resistancee Only looked at AChE inhibition by propoxur Nephotettix cincticeps of Tomita et al. [49]

Comments Resistance profile Species

Table 1 (continued)


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the AChE active site to the insecticide inhibitors. Significantly however, different mutations at the same oxyanion hole site in some closely related carboxylesterases confer OP protection, albeit not by insensitivity but by hydrolysis of the insecticide [10–12]. It is remarkable enough that both target site and metabolic resistance to OPs could be due to different mutations at the equivalent sites of AChEs and carboxylesterases, respectively. But does this also teach us anything about the mechanism of action of the G119S mutation in the AChEs? OPs and carbamates act as insecticides by being essentially irreversible inhibitors (suicide substrates) for esterases that normally act on carboxylester substrates (of which choline esters are a subset). Lethality results from inhibition of esterases with essential functions like AChE. In the case of OPs, substitution of Gly119 with an Asp, Glu or His residue can confer on the mutant enzyme an ability to reverse the inhibition ([10–15]; L. Schopfer, O. Lockridge, C.A. Broomfield, pers. comm.) The most probable mechanism is that those residues act as general bases to activate a water molecule to hydrolyse the bond between the enzyme and the OP [14]. Thus, the free enzyme is regenerated, the OP is broken down, and resistance can result. In other words, the OP suicide substrate becomes an ordinary substrate. The downside is that the mutant enzymes lose their original carboxyl/cholinesterase activity so that resistance is only manifest if this original activity is dispensable. This in fact is the case for some socalled ali-esterases in insects and variants of these enzymes carrying these mutations have indeed been found to confer OP resistance in blowflies and houseflies [10–12]. These mutations have also been made in human butyrylcholinesterase (BuChE) in vitro, in successful attempts to make enzymes that would detoxify OP nerve agents, which are structurally very similar to OP insecticides ([13]; L. Schopfer, O. Lockridge, C.A. Broomfield, pers. comm.). However, we think that this mechanism cannot explain Pattern I resistance in the culicines, as it is unlikely that a G119S mutation would enhance carbamate turnover in an analogous way. One reason is based on the structural difference

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Fig. 1. Unrooted distance neighbour-joining tree showing the phylogenetic relationships of vertebrate and invertebrate AChEs and vertebrate BuChE. Parsimony analysis using the same dataset produced a congruent tree (data not shown). Bootstrap percentage values of 1000 replicates are indicated at nodes. Also asterisked are the relative positions of four putative gene duplication events and one gene deletion, with the five radiations subsequent to these events separately shaded. Scale bar indicates a distance of 0.1 amino acid substitutions per position in the sequence. Sequences are named with respect to species (abbreviated) and enzyme radiation. The translated sequences with accession details are: An g AChE-1, Anopheles gambiae (COEace5o); Cp AChE-1, Culex pipiens (CAD56155); Ag AChE-1, Aphis gossypii (AAM94376); Sg AChE-1, Schizaphis graminum (AF321574); Mp AChE-1, Myzus persicae ([8]); Am AChE-1, Apis mellifera (partial sequence, Amellifera1 2048407555BCM; Ce ace-2, Caenorhabditis elegans (Y44E3A.2); Ce ace-3, Caenorhabditis elegans (Y48B6A.8); Ce ace-4, Caenorhabditis elegans (Y48B6A.7); Bm AChE-2, Boophilus microplus (AF067771); Ha AChE, Helicoverpa armigera (AAN37403); Px AChE, Plutella xylostella (AAL33820); Ld AChE, Leptinotarsa decemlineata (Q27677); Nc AChE, Nephotettix cincticeps (AF145235); Am AChE-2, Apis mellifera (AAG43568); Md AChE, Musca domestica (AF281161); Lc AChE, Lucilia cuprina, (AAC02779); Dm AChE, Drosophila melanogaster (CG17907); Bo AChE, Bactrocera oleae (AF452052); An g AChE-2, Anopheles gambiae (COEace16o); Ag AChE-2, Aphis gossypii (AAM94375); Mp AChE-2, Myzus persicae (AF287291); Bm AChE-1, Boophilus microplus (AJ223965); Dr AChE, Danio rerio (NM_131846); Tc AChE, Torpedo californica (X03439); h AChE, Homo sapiens (M55040); Gg AChE Gallus gallus (U03472); h BuChE, Homo sapiens (AAA99296); Gg BuChE, Gallus gallus (AJ306928); and Ce ace-1, Caenorhabditis elegans (WO9B12.1).

between carbamates and carboxylesters (which are both planar around the carbonyl carbon) versus OPs (which are tetrahedral around the corre-

sponding phosphorus), which requires a different direction of attack for hydrolysis of the enzyme-inhibitor bond [16], so the general base


R.J. Russell et al. / Pesticide Biochemistry and Physiology 79 (2004) 84–93

substitutions at G119 would only be expected to benefit OP turnover. Indeed, the G119D mutant esterase from the sheep blowfly has been tested for carbamate (carbofuran, pirimicarb) hydrolysis and found to be inactive (C.A. Broomfield, R.J. Russell, and J.G. Oakeshott, unpublished). Secondly, while a G119S blowfly mutant esterase has not yet been tested for OP or carbamate hydrolytic activity, a G119S human BuChE mutant was unable to turn over OPs (L. Schopfer, O. Lockridge, C.A. Broomfield, pers. comm.). Also relevant here is another type of carboxylesterase mutation that causes OP resistance due to enhanced detoxification of OPs. This mutation does not provide a new catalytic residue, but instead is suggested to enhance the usually slow reactivation rate of the phosphorylated enzyme by allowing some steric shift in the active site. Examples are some alternative mutations of the fly ali-esterase that replace a bulky Trp residue at position 233 in the acyl binding pocket (a strongly hydrophobic pocket that accommodates the acyl moiety of bound substrate) with the smaller Leu, Ser or Gly [11,14,17,18]. Thus, by analogy, it is possible that the G119S substitution in the mosquitoesÕ AChE-1 causes a steric shift to enhance carbamate, and to a lesser extent, OP turnover. It is important to note that the results of the ÔinsensitivityÕ experiment of Weill et al. [1] could be consistent with either this scenario or with a blockage in the initial reaction of AChE-1 with carbamates in particular. The rates of turnover achieved by mutant esterases to confer protection against OPs are very slow [15] and there is no reason to think they would need to be faster for this mutant with the carbamates. Importantly also, a G119S substitution is not always associated with either resistance to, or the ability to degrade, carbamate or OP insecticides. Although Gly is usually found at position 119, Ser, Ala, and even Arg are all found at this site in naturally occurring carboxyl/cholinesterases [18]. The bacterial carboxylesterase, PCD, that was isolated because of its ability to degrade the carbamate herbicide, phenmedipham, has a Gly at position 119 [19] and is not inhibited by OPs (M. Williams and I. Horne, unpublished). A multiply resistant strain of the two-spotted spider mite,

Tetranychus urticae, has an AChE that is more sensitive to OPs and carbamates than the susceptible strain, and has Ser at position 119; the susceptible strain is polymorphic for both Ser and Gly [20]. So it is apparent that a Gly-Ser mutation per se at position 119 is not enough to render any carboxyl/cholinesterase insensitive to OPs and carbamates; the protein scaffold in which the mutation sits is obviously important as well. It may be that the mosquito AChE-1 G119S resistance mechanism is not available to all insect species. Conversely it may be possible that substitutions at position 119 to residues other than Ser can also confer Pattern I AChE insensitive resistance. Notably, Ordentlich et al. [21] replaced G119 in human AChE with Ala and created an enzyme with increased insensitivity to both OPs and carbamates. AChE is one of the most conserved enzymes of higher eukaryotes. It is widely known for its role in hydrolysing acetylcholine, but it also has another, much less studied role in the development, maturation, and maintenance of vertebrate and invertebrate nervous systems [22,23]. Futhermore, emerging sequence data [1,8,23] show that AChE has a dynamic evolutionary history. We can now identify four gene duplication events and at least one gene deletion event in the evolution of AChE from nematodes to humans (Fig. 1). The first gene duplication occurred before the split of nematodes, arthropods, and vertebrates giving rise to AChE-1 and AChE-2. The other three duplications all occurred in the nematode or vertebrate lineages. However, a gene loss occurred in insects, specifically in the higher Diptera, which have lost their version of the AChE-1 otherwise found from nematodes through to humans (Fig. 1). Intriguingly, there is growing evidence to suggest that AChE-1 is the molecule that processes acetylcholine in the majority of insect and arthropod lineages [24] (Fig. 1). So in higher Diptera these functions must now reside in their single AChE enzyme, which is actually derived from the ancestral ace-2 gene [25]. We have seen that three of the ten cases of Pattern II resistance in Table 1 involve the higher Diptera and the mutations underlying these changes have not been found to confer Pattern I or Pattern II resistance outside the higher

R.J. Russell et al. / Pesticide Biochemistry and Physiology 79 (2004) 84–93

Diptera. Interestingly also, no case of Pattern I resistance has yet been reported in the higher Diptera. It may be that the options for target site resistance in this lineage differ from those of other insects or mites because of the different evolutionary background and at least partly distinct suite of functions of its cholinergic AChE. Weill et al. [1] pointed out that there could be some major implications of any recurring patterns in AChE insensitivity mutations for the management of resistance, and the insecticides. Clearly however, there are some major questions to address before the nature of these implications is clear. One key proposition to test is our hypothesis of two major classes of target site resistance mechanisms, which can be tested by further molecular and bioassay work on some of the less well characterised cases of insensitive AChE listed in Table 1. Others obviously concern the biochemical mechanism underlying Pattern I resistance and the range of substitutions that could give rise to the phenotype. Then there is the issue of potential molecular, organismal and population level interactions between the two classes of resistance. Answering questions such as these will be crucial for the effective, ongoing use of two chemistries that still serve about half the world-wide insecticide market.

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