Insect Biochemistry and Molecular Biology 30 2000 1009–1015 www.elsevier.comlocateibmb
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 target 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-
0965-174800 - see front matter
2000 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 est
a1 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
planthopper, Nilaparvata lugens Karunaratne et al.,
1010 J. Hemingway Insect Biochemistry and Molecular Biology 30 2000 1009–1015
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 Trp
251
-Leu substitution within the blow fly E3 esterase. A second Gly
139
-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 andor 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 3
9 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 phosphorylation 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, est
a2
1
and est b2
1
Vaughan et al., 1997. Insects carrying this est
a2
1
est b2
1
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 homogen- ised in 100
µ l phosphate buffer 0.02 M pH 7.5 and 25
µ l loaded
onto 7.5 PAGE. Gels were stained for esterase activity with 0.04 wv
α - and
β -naphrhyl acetate and 0.1 wv Fast Blue B in 100
mM phosphate buffer pH 7.4.
1011 J. Hemingway Insect Biochemistry and Molecular Biology 30 2000 1009–1015
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 est
a2
1
est b2
1
amplicon. This is expressed in insects with this amplicon, but is found only
as a series of truncated 3 9 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 est a and estb genes 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 est
a2
1
est b2
1
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 est
a2
1
and est b2
1
genes are present in a 1:1 stoichi- ometry, there is up to four times more Est
β 2
1
produced 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 est
b2
1
promoter, Hemingway et al., 1998.
The est a2
1
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 est a2
1
promoter with respect to the luciferase reporter gene does not influence expression Hawkes and
Hemingway, [in preparation]. The amplified est a2
1
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 est b2
1
gene is as yet uncharacterized.
3. GST-based resistance
