www.elsevier.com/locate/ibmb
Mini review
The molecular basis of two contrasting metabolic mechanisms of
insecticide resistance
Janet Hemingway
Cardiff School of Biosciences, Cardiff University, PO Box 915, Cardiff CF1 3TL, UK
Received 10 January 2000; received in revised form 28 March 2000; accepted 28 March 2000
Abstract
The esterase-based insecticide resistance mechanisms characterised to date predominantly involve elevation of activity through gene amplification allowing increased levels of insecticide sequestration, or point mutations within the esterase structural genes which change their substrate specificity. The amplified esterases are subject to various types of gene regulation in different insect species. In contrast, elevation of glutathione S-transferase activity involves upregulation of multiple enzymes belonging to one or more glutathione S-transferase classes or more rarely upregulation of a single enzyme. There is no evidence of insecticide resistance associated with gene amplification in this enzyme class. The biochemical and molecular basis of these two metabolically-based insecticide resistance mechanisms is reviewed. 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Esterase; Glutathione; S-transferase; DDT; Organophosphate; Mosquito
1. Introduction
Resistance to organochlorine, organophosphate and carbamate insecticides is conferred by a limited number of mechanisms in all insects analysed to date. These mechanisms predominantly involve either metabolic detoxification of the insecticide before it reaches its tar-get site, or changes in sensitivity of the tartar-get site so that it is no longer susceptible to insecticide inhibition. The most common metabolic resistance mechanisms involve esterases, glutathione S-transferases or monoox-ygenases (the latter has been the subject of a recent review by Scott et al., 1998). In most, but not all, instances of metabolic resistance, individual resistant insects can be detected through increased quantities of enzyme compared to their susceptible counterparts (Brown and Brogdon, 1987; Hemingway, 1989; Hem-ingway et al., 1995). Over the last decade the molecular basis of these resistance mechanisms has gradually been elucidated, opening up the exciting possibility of manipulation of these enzyme systems in the long term to restore insecticide susceptibility by manipulation of their expression patterns. The esterase and glutathione S-transferase (GST)-based insecticide resistance mech-anisms in a range of insects present a number of
con-0965-1748/00/$ - see front matter2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 0 0 ) 0 0 0 7 9 - 5
trasting ways in which metabolically-based resistance has been selected for at the molecular level.
2. Esterase-based resistance
Esterase-based resistance to organophosphorus and carbamate insecticides is common in a range of different insect pests (Field et al., 1988; Hemingway and Karunar-atne, 1998). The esterases either produce broad spectrum insecticide resistance through rapid-binding and slow turnover of insecticide, i.e. sequestration, or narrow spectrum resistance through metabolism of a very restricted range of insecticides containing a common ester bond (Herath et al., 1987; Karunaratne et al., 1995). The majority of esterases which function by seques-tration are elevated through gene amplification, (Vaughan and Hemingway, 1995; Mouches et al., 1986; Field et al., 1988). The one exception to this appears to be the elevated esta1 gene of Culex pipiens from France for which there is no evidence of amplification (Raymond et al., 1998). Esterase gene amplification is well documented in resistant strains of the aphid, Myzus
persicae, the mosquitoes Culex quinquefasciatus, C. pip-iens, C. tarsalis and C. tritaeniorhynchus and the brown
1998; Mouches et al., 1986; Field and Devonshire, 1998; Small and Hemingway, 2000b).
Esterases which produce resistance by increased metabolism are thought to occur by single point mutations in the structural genes, although few have been characterized at the nucleotide level. These mech-anisms often involve resistance to the organophosphorus insecticide malathion. Such point mutations can dramati-cally alter the substrate specificities of the enzyme, as seen in the E3 malathion carboxylesterase from the sheep blow fly Lucillia cuprina (Campbell et al., 1998) and the Musca domestica alpha E7 gene (Claudianos et al., 1999). Resistance to malathion is caused by a single (Trp251-Leu) substitution within the blow fly E3 esterase.
A second Gly139-Asp substitution in E3 confers broad
spectrum cross-resistance to a range of organophos-phates, excluding malathion (Campbell et al., 1998). This Gly-Asp substitution is also found in M. domestica (Claudianos et al., 1999). Malathion-specific esterase-based mechanisms occur commonly in Anopheles spec-ies where they are not associated with any increase in enzyme activity with general esterase substrates in resist-ant insects. The presumed point mutations in these ester-ases in Anopheles have yet to be characterized, although three malathion metabolizing esterases from malathion resistant An. stephensi have recently been biochemically purified and characterized kinetically (Hemingway et al., 1998). These esterases are standard “B” esterases on the classification of Aldridge (1953), but have little or no activity with the general naphthyl acetate enzyme sub-strates. Possible links of this “mutant ali-esterase” in
Musca to a general resistance loci controlling elevation
of monooxygenase and/or glutathione S-transferase up regulation are under active investigation (Feyereisen, 1999).
In aphids there are two common amplified esterase variants E4 and FE4, which appear to have had single independent origins (Devonshire et al., 1998). The amplicons containing each esterase variant are much larger than the esterase genes themselves, although only one gene has been characterized on each amplicon.
The E4 and FE4 enzymes both occur, in their non-amplified forms, in susceptible aphids. They are the result of a relatively recent duplication, differing only at their 39 ends through a mutation in the E4 stop codon resulting in a further 12 amino acids being added to the FE4 enzyme. The E4 esterase occurs at a single chromo-somal location, but there are multiple sites of insertion of the FE4 genes on different aphid chromosomes (Blackman et al., 1999).
Amplification of the E4 gene is in linkage disequilib-rium with a kdr-type pyrethroid resistance mechanism. This may reflect insecticide selection pressures favour-ing aphids with multiple resistance mechanisms, tight chromosomal linkage or the prominence of parthenogen-esis in this insect (Devonshire et al., 1998).
In contrast to the elevated esterases in other insects, the elevated esterase band in N. lugens occurs as a large diffuse band on polyacrylamide gels of planthoppers from a range of different continents, (Fig. 1). This band resolves into several enzyme variants on isoelectric focusing. The variants are caused by differential glycos-ylation and phosphorglycos-ylation of the same underlying esterase protein (Small and Hemingway, 2000a). The amplification of the esterase in N. lugens appears to have occurred only once and spread rapidly, as the amplified esterases are identical at the nucleotide level in insects from different continents, which is perhaps not surpris-ing, given the highly migratory nature of this insect (Small and Hemingway, unpublished data).
In C. quinquefasciatus the majority of esterase-based resistance involves two co-amplified esterases, esta21
and estb21 (Vaughan et al., 1997). Insects carrying this
esta21/ estb21 amplicon may have a significant fitness
advantage in the presence of insecticide over those with
Fig. 1. Polyacrylamide gel of Culex quinquefasciatus (Pel RR)
Nila-parvata lugens amplified esterase. Individual insects were
other amplified variants of the same esterase loci, as they occur in .80% of all characterized insecticide resistant strains. The local invasion of this amplicon into Culex populations in southern France is well documented, (Raymond et al., 1998). It was first found near Marseilles airport and spread within a few years to all surrounding organophosphorus (OP) treated areas, despite the earlier occurrence of other OP resistance mechanisms in this
Culex population (Raymond et al., 1998). The reason for
a selective advantage is not immediately apparent, as all the elevated esterases have similar affinities and turnover rates for the different insecticides (Karunaratne et al., 1993).
At least eight different esterase-containing amplicons have been recorded in Culex. One major difference between amplicons is the presence of an aldehyde oxi-dase (ao1) gene on the esta21/ estb21 amplicon. This is
expressed in insects with this amplicon, but is found only as a series of truncated 39 ao ends on the esta3/ estb1
amplicons in other Culex strains (Hemingway et al., 2000). The role of this amplified ao1 gene is not yet fully characterized, although it is elevated in activity assays in resistant compared to susceptible insects, and interacts with insecticides and herbicides containing aldehyde groups, hence a functional role is possible.
The esta and estbgenes are the result of an ancient gene duplication which appears to predate Culex speci-ation (Hemingway and Karunaratne, 1998). The two genes occur as single copies in a head to head arrange-ment 1.7 kb apart in the susceptible (PelSS) strain of C.
quinquefasciatus from Sri Lanka. In resistant insects
with the esta21/ estb21 amplicon the intergenic spacer
has been expanded to 2.7 kb with the insertion of two large and one small indels (Vaughan et al., 1997) com-pared to the susceptible PelSS spacer. The intergenic spacer in other susceptible strains is variable in size, (Guillemaud et al. 1996, 1999). The insertions in the resistant spacer introduce a number of possible zeste regulatory sequences into the intergenic spacer (Hemingway et al., 1998). These elements, which affect expression of multiple gene copies in Drosophila, may influence the levels of expression of the amplified ester-ases (Benson and Pirrotta, 1988). In contrast to the Culex amplified esterases, which are expressed in all life stages, the E4 esterase gene of aphids can be switched off completely in revertant insects by methylation of the gene. The pattern of methylation differs from many other organisms, where methylated genes are usually switched off. In aphids E4-related sequences are highly methyl-ated at Msp1 sites in all resistant aphid clones, but not in revertant clones, (Field et al., 1989). Although the
esta21 and estb21 genes are present in a 1:1
stoichi-ometry, there is up to four times more Estβ21produced
in the resistant insects, (Paton et al., 2000). This differ-ence in protein level is reflected in the expression pat-terns, although there is no direct link between activity
and amplification level in either resistant C.
quinquefas-ciatus or C. tritaeniorhydchus, (Paton et al., 2000).
Clon-ing of the intergenic spacer in both orientations upstream of a luciferase reporter gene has resulted in preliminary characterization of the estb21promoter, (Hemingway et
al., 1998).
The esta21 promoter is inoperative when inserted at
the same site. The difference in promoter strength may reflect differences in tissue specific expression of the esterases, as changing the relative position of the puta-tive esta21 promoter with respect to the luciferase
reporter gene does not influence expression (Hawkes and Hemingway, [in preparation]). The amplified esta21
gene is expressed at a high level only in the malpighian tubules, cuticle, gut and salivary glands, (Fig. 2), whilst the expression pattern of the estb21 gene is as yet
uncharacterized.
3. GST-based resistance
The glutathione S-transferases (GSTs) belong to a superfamily which currently has almost 100 sequences. There are at least 25 groups (families) of GST-like pro-teins, with one well supported large clade containing currently recognised mammalian, arthropod, helminth, nematode and mollusc GST classes (Snyder and Maddi-son, 1997). GSTs can produce resistance to a range of insecticides by conjugating reduced glutathione (GSH) to the insecticide or its primary toxic metabolic products. The majority of reports involve organophosphate resist-ance in houseflies (Clark et al., 1986; Motoyama and Dauterman 1977, 1975), however, recent work on recombinant Anopheles class I GST enzymes has shown that they recognize pyrethroids as either substrates or inhibitors, (Ranson et al., 1997; Prapanthadara et al., 1998), and there is now evidence that they are directly involved in pyrethroid resistance in the planthopper N.
lugens, (Vontas, Small and Hemingway, [unpublished
data]). A subset of GSTs are also able to dehydrochlorin-ate insecticide such as DDT, in a reaction where GSH acts as a co-factor rather than a conjugate (Clark and Shamaan, 1984). This is probably the most common DDT resistance mechanism in mosquitoes. Where conju-gation of primary metabolites occurs, the GST mech-anism often acts as a secondary resistance mechmech-anism in linkage disequilibrium with a monooxygenase or ester-ase-based resistance mechanism, as in An. subpictus (Hemingway et al., 1991).
The molecular basis of GST-based resistance is best understood in Musca domestica and the mosquitoes
Anopheles gambiae and Aedes aegypti. In all cases,
identify and characterize these regulators using a pos-itional cloning approach which has already identified crude chromosomal locations, which should contain these regulatory genes (Ranson et al., 1999).
Insect GSTs are currently classified into two groups, class I and class II GSTs. This classification is almost certainly over simplified. Class I GSTs are most closely
Fig. 2. High resolution electron micrograph (×25,000) of the esta21 expression visualized by gold labelling in: (A) the salivary gland of insecticide resistant 4th instar larval Culex quinquefasciatus, and, (B) the cuticle of resistant 4th instar larvae compared to the lack of staining in (C), the cuticle of insecticide susceptible larvae. Salivary glands were dissected out of individual larvae and sectioned. The cuticle was visualised in cross-sections of whole larvae. Fresh salivary gland was lightly fixed in a glutaraldehyde:formaldehyde mixture for 30 minutes at 4°C and was prepared using the Tokuyasu protocol for immuno-electronmicroscopy. After fixation, the material was infused with 2.3 M sucrose and then vitrified using liquid N2. Sections were cut at 2100°C using a Reichert Ultracut E fitted with a FC4 Cryochamber and were thawed onto 2 M sucrose solution. After treatment with pri-mary antibody (Ab2; diluted 1:200), and secondary rabbit IgG conju-gated to 10 nm gold particles, the sections were embedded on their grids using a methyl cellulose:uranyl acetate mix before examination in the electron microscope. For micrographs (B) and (C) after sec-tioning, the material was lightly fixed in a glutaraldehyde:paraformal-dehyde mixture for 30 minutes at 4°C, and after rapid dehydration in 70% alcohol was embedded in Hard Grade LR White resin. Sections nominally 60 nm thick were cut using a Reichert Ultracut E ultramicro-tome and placed onto copper EM grids. These were treated with pri-mary antibody Ab2 (made against purified Culex esterase at 1:200 dilution) and then with rabbit IgG conjugated to 10 nm colloidal gold. Immunolabelling was followed by routine uranyl acetate and lead cit-rate staining. Sections were examined and photographed using a JEOL 1210 TEM. Labelling shows as small intense black dots.
related at the amino acid level to mammalian theta class GSTs, while class II GSTs are related to the pi class (see Fig. 3), this relationship between insect and mammalian classes does not extend to their substrate specificities.
Fig. 3. Phylogenetic tree of the insect glutathione S-transferases with selected mammalian and parasite GST sequences. Bootstrap values are given for the insect GST relationships.
Fig. 4. Diagrammatic representation of the Anopheles gambiae Class I GST gene cluster. Six different GST transcripts are produced from the genes in both resistant and susceptible insects. Four of the transcripts originate from alternate splicing of the aggst1αgene.
The class I GSTs cloned to date still represent only a tiny subset of the GST variation seen biochemically in
An. gambiae which suggests that there are still further
insect GST classes for which we do not have molecular data. Screening of an An. gambiae BAC library has con-firmed this and details of new GSTs should be published shortly (Ranson and Collins [personal communication]). There are a number of possible regulatory elements upstream of the cloned GSTs, but the regulatory
mech-anism producing resistance still needs to be charac-terized. A positional cloning programme, using the An.
gambiae microsatellite markers, have identified a QTL
in which the probable trans-acting regulator of An.
gam-biae should reside. Further microsatellite loci are being
4. Conclusions
The last decade has seen large advances in our under-standing of the molecular basis of insecticide resistance. The structural genes coding for the enzymes, which are elevated in a number of insect species, have been cloned and characterized. Our understanding of how these genes are regulated will form another major advance in our understanding of such systems, moving us closer to the goal of manipulating pest insect species with the aim of restoring insecticide susceptibility.
Acknowledgements
The electron micrographs reported in this review would not have been possible without the expert techni-cal assistance of Mrs C. Winters at the Cardiff School of Biosciences.
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