Insect Biochemistry and Molecular Biology 34 (2004) 653–665

www.elsevier.com/locate/ibmb

The molecular basis of insecticide resistance in mosquitoes

Janet Hemingway
, Nicola J. Hawkes, Lynn McCarroll, Hilary Ranson
Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK

Received 8 March 2004; accepted 18 March 2004

Abstract

Insecticide resistance is an inherited characteristic involving changes in one or more insect gene. The molecular basis of these
changes are only now being fully determined, aided by the availability of the Drosophila melanogaster and Anopheles gambiae
genome sequences. This paper reviews what is currently known about insecticide resistance conferred by metabolic or target site
changes in mosquitoes.
# 2004 Elsevier Ltd. All rights reserved.

Keywords: Esterase; Insecticide resistance; Molecular mechanism; Glutathione S-transferase (GST); GABA; rdl; Sodium channel; kdr;
Carboxylesterase; P450 monooxygenase; Acetylcholinesterase

1. Introduction they are used as indoor residual house sprays to

In this review we discuss the data currently available
regarding chemical insecticide resistance in mosquitoes.
Most resistance mechanisms can be divided into two
groups, metabolic (alterations in the levels or activities
of detoxification proteins), and target site (mutations in
the sodium channel, acetylcholinesterase and GABA
receptor genes). Alone or in combination these
mechanisms confer resistance, sometimes at an
extremely high level, to all of the available classes of
insecticides. In addition, many insecticides such as
DDT and permethrin also influence behavioural chan-
ges in the insect—for example, by reducing the rate of
mosquito entry into houses, increasing the rate of early
exit from houses and inducing a shift in biting times
(Lines et al., 1987; Miller et al., 1991; Mbogo et al.,
1996; Mathenge et al., 2001). Some mosquitoes have
also evolved thicker or altered cuticles, reducing pen-
etration of the insecticide (Stone and Brown, 1969;
Apperson and Georghiou, 1975).
Pyrethroids account for approximately 25% of the
world insecticide market. For the public health market,


Corresponding author. Tel.: +44-151-705-3261; fax: +44-151-707-
0155.
E-mail address: hemingway@liv.ac.uk (J. Hemingway).
0965-1748/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibmb.2004.03.018
impregnate bednets, curtains and screens, and in coils,
mats and aerosols. Pyrethroids are popular due to their
very low toxicity in humans, and rapid killing effect on
the insect, although this varies from product to product
and overall efficacy of treatment and knock-down
properties depend on irritancy and excito-repellancy
(Hougard et al., 2002). For example, bifenthrin has a
much lower irritancy effect than deltamethrin, thus the
mosquito will stay on bifenthrin-treated material for a
longer period of time and so obtain a higher dose of
insecticide. However, the well-documented cross-resist-
ance that occurs between pyrethroids means that as a
replacement for permethrin, bifenthrin may not be
effective for long (Chandre et al., 1999). Since pyre-
throids are the only option for treating bednets for the
control of malaria at present, it is important that the
efficacy of this class is maintained for as long as poss-
ible. Worryingly, resistance to pyrethroids may be
achieved through several of the mechanisms described
below. The use of insecticide mixtures or rotational use
of insecticides to delay the development and/or spread
of resistance is one strategy worth consideration
(Curtis et al., 1998) and is currently under investigation
on a large scale in Mexico (Penilla et al., 1998).
654 J. Hemingway et al. / Insect Biochemistry and Molecular Biology 34 (2004) 653–665
2. Metabolic resistance mechanisms
2.1. Carboxylesterases
2.1.1. Quantitative changes
Over-production of non-specific carboxylesterases as
an evolutionary response to organophosphorus and
carbamate insecticide selection pressure has been docu-
mented in numerous arthropod species including mos-
quitoes, cattle ticks, aphids and cockroaches. The most
widely studied mosquito species demonstrating this
resistance mechanism are members of the Culex genus,
including Culex pipiens pipiens, C. p. quinquefasciatus
and C. tritaeniorhynchus (reviewed in Hemingway and
Karunaratne, 1998). These enzymes are B esterases
with an active site serine residue (Aldridge, 1953). In
OP-susceptible insects, the active oxon analogues of the
insecticides act as esterase inhibitors, because they are
poor substrates with a high affinity for the enzymes.
Esterases from susceptible insects are significantly less
reactive with xenobiotics (including insecticides) than
their counterparts from resistant insects, which seques-
ter the oxon analogues and thus protect the acet-
ylcholinesterase target site (Karunaratne et al., 1995).
The amplified esterases may comprise up to 0.4% of
total protein, all with rapid sequestration and slow
insecticide hydrolysis rates (Karunaratne et al., 1993).
The predominant cause of this excessive enzyme syn-
thesis is amplification of the gene(s) within the genome
(Mouches et al., 1986; Vaughan and Hemingway, 1995;
Vaughan et al., 1995), although up-regulated transcrip-
tion without an underlying gene amplification event
has been reported (Rooker et al., 1996). There are
many esterase alleles associated with resistance. The
most common genotype is the co-amplification of two
esterase genes (esta21 and estb21) on an amplicon of
approximately 28 kbp, which is reiterated up to 80
times in the genomes of highly resistant C. p. quinque-
fasciatus mosquitoes (Vaughan et al., 1997; Paton et al.,
2000). This genotype is present in >90% of resistant C.
p. quinquefasciatus. Its selective advantage may arise
not only from the contributions of two insecticide-
sequestering esterases, but also from the presence of a
third gene with putative detoxification capacity on the
amplicon. A full-length aldehyde oxidase gene lies just
Fig. 1. Schematic of the amplicon from the organophosphate-resistant Culex quinquefasciatus strain PelRR, showing the relative proximities and
transcriptional orientation of three detoxification genes. Arrows indicate the direction of transcription.
downstream of esta21 (Fig. 1; Hemingway et al., 2000;
Coleman et al., 2002).
In C. p. pipiens, early estimates of gene copy number
elevated ~250-fold relative to susceptible mosquitoes
(Mouches et al., 1990) have subsequently been reduced
to ~20-fold amplification, although whether this reflects
technical inaccuracies or an actual lessening of the
resistance level within the laboratory population with
time is unknown (Guillemaud et al., 1997). In situ
hybridisations have revealed a tandem arrangement of
the amplified genes in both C. p. pipiens and in C. p.
molestus (Nance et al., 1990; Tomita et al., 1996).
In the OP-resistant C. p. quinquefasciatus laboratory
strain PelRR, the esta21 and estb21 genes are physically
very close, and oriented head-to-head (Vaughan et al.,
1997). They are thus divergently transcribed. This is also
true of the alleles in the OP-susceptible strain. Despite
linear (1:1) gene co-amplification within the genome,
estb21 transcript levels are 2–30-fold higher than esta21
transcript levels within individual adult mosquitoes
(Paton et al., 2000), and on average 3-fold more Estb21
protein is found in a mass homogenate of resistant
insects than Esta21 (Karunaratne, 1994). Functional
promoter analysis has shown that the b-esterase pro-
moters are active in vitro and that transcription from
the estb21 promoter requires an initiator sequence
approximately 125 bp upstream of the initiating meth-
ionine, but does not involve a TATA-box (Hemingway
et al., 1998; Hawkes and Hemingway, 2002). Rather, the
presence of a site, 28 bp downstream of the initiator,
perfectly matching the Drosophila downstream pro-
moter element (DPE) appears important (Burke and
Kadonaga, 1996; Hawkes, unpublished data).
Recent studies have found an inverse correlation
between esterase activity in C. p. quinquefasciatus and
filarial burden (McCarroll et al., 2000), suggesting that
high levels of esterase are adversely affecting the sur-
vival and/or development of Wuchereria bancrofti filar-
iae, but the overall effects on transmission are
unknown.
There are many reports of enhanced esterase activi-
ties in other mosquitoes, for example, in permethrin-
resistant An. gambiae (Vulule et al., 1999) and An.
albimanus (Brogdon and Barber, 1990), and in resistant
 J. Hemingway et al. / Insect Biochemistry and Molecular Biology 34 (2004) 653–665
Ae. aegypti (Mourya et al., 1993). However the gene(s)
involved are unknown. Whilst homologs of the co-
amplified Culex carboxylesterases have been identified
in An. gambiae in the same physical proximity and
orientation (Ranson et al., 2002a), it remains to be seen
whether they are involved in resistance, particularly
since the Culex esterases are ineffective against pyre-
throids (Karunaratne et al., 1993). The amplified car-
boxylesterases of some insects (the cattle tick
Boophilus microplus and the peach-potato aphid Myzus
persicae) do have activity against both pyrethroids and
OPs (Hernandez et al., 2002; Devonshire and Moores,
1982). However, there is as yet no evidence of this
mechanism in pyrethroid-resistant mosquitoes.
2.1.2. Qualitative changes
In some mosquito species with resistance, elevated
carboxylesterase activity involves rapid hydrolysis of
the insecticide, rather than increased sequestration.
This mechanism is almost always found in association
with malathion resistance, and gives a much narrower
cross-resistance spectrum (sometimes malathion-spe-
cific) than the amplified esterase-based mechanism.
Malathion carboxylesterase resistance has been found
in An. culicifacies, An. stephensi and An. arabiensis
(Herath et al., 1987; Hemingway, 1982a,b, 1983).
Although the genetic alteration(s) generating these
qualitative changes have not as yet been identified in
mosquito populations, data from other arthropods sug-
gest that only one or two amino acid mutations may be
responsible. Qualitative changes conferring resistance
to malathion have been reported in carboxylesterase
genes from Musca domestica, the sheep blowfly Lucilia
cuprina and the parasitoid wasp Anisopteromalus
calandrae (Zhu et al., 1999; Campbell et al., 1998;
Claudianos et al., 2002). In each case, the same trypto-
phan residue is mutated, to leucine in the blowfly and
housefly, and to glycine in the wasp, leading to the
speculation that a similar mutation in the orthologous
gene in the above Anopheles species may thus also be
mutated in malathion resistance.
The above carboxylesterase-based resistance
mechanisms are not mutually exclusive; both malathion
carboxylesterase and amplified carboxylesterase
mechanisms have been found in C. tarsalis (Ziegler
et al., 1987).
2.2. P450 monooxygenases
Cytochrome P450-dependent monooxygenases are an
important and diverse family of hydrophobic, heme-
containing enzymes involved in the metabolism of
numerous endogenous and exogenous compounds.
This generally results in the detoxification of the sub-
strate, although the activation of OP insecticides from
the phosphorothionate to the more toxic oxon form is
655
a notable exception. The majority of eukaryotic P450s
require the flavoprotein NADPH cytochrome P450
reductase, and sometimes cytochrome b5, for activity.
Diversity is conferred by the existence of multiple P450
isoforms, different expression patterns and wide sub-
strate spectra (for recent reviews see Scott and Wen,
2001; Feyereisen, 1999).
There are many reports demonstrating elevated P450
monooxygenase activities in insecticide-resistant mos-
quitoes, frequently in conjunction with altered activities
of other enzymes. For example, Vulule et al. (1999)
demonstrated elevated oxidase and esterase levels
in permethrin-resistant An. gambiae from Kenya.
Brogdon et al. (1999a,b) have reported oxidase-based
and esterase-based resistance mechanisms alone and in
combination in permethrin-resistant An. albimanus
from Guatemala. However, the identification of the
particular P450 gene(s) associated with resistance has
been far from easy in any insect.
In most cases where a link between insecticide resist-
ance and elevated P450 activity has been shown, the
Cyp gene belongs to the Cyp6 family. For example,
CYP6D1 is over-produced in pyrethroid-resistant M.
domestica due to up-regulated transcription (Kasai and
Scott, 2000) and CYP6A1 is similarly associated with
organophosphate resistance (Sabourault et al., 2001;
Andersen et al., 1994). Drosophila CYP6A2 expressed
in vitro is capable of metabolising diazinon and cyclo-
dienes (Dunkov et al., 1997). Amino acid substitutions
in the resistant Cyp6a2 allele confer increased activity
against DDT (Berge et al., 1998). Up-regulation of
Cyp6g1 orthologs are linked with DDT resistance in
field isolates of both Drosophila melanogaster and D.
simulans (Daborn et al., 2002). Laboratory selection of
D. melanogaster with DDT also results in up-regulation
of Cyp6g1 (sometimes in conjunction with Cyp12d1),
or Cyp6a8 (Brandt et al., 2002; Le Goff et al., 2003).
The presence of a transposable element at the 50 end of
Cyp6g1 in DDT-resistant alleles from multiple sites
worldwide suggests that this genetic event occurred
only once. Its selective advantage over other p450 up-
regulation genotypes may be due to the wider cross-
resistance (to imidacloprid, malathion and lufenuron)
conferred (Le Goff et al., 2003; Daborn et al., 2002).
Prior to the release of the An. gambiae genome (Holt
et al., 2002), Ranson et al. (2002b) had identified 34
P450 genes. Subsequent analysis of genome data
revealed a total of 111 P450 monooxygenases in An.
gambiae (Ranson et al., 2002a), arranged mostly in
clusters within the genome. Nikou et al. (2003) have
recently shown elevated transcript levels (not due to
gene amplification) of an adult-specific Cyp6 P450
gene, Cyp6z1, in a pyrethroid-resistant strain of An.
gambiae from East Africa. This gene lies within a clus-
ter of 17 P450 genes located within the boundaries of a
quantitative trait locus on chromosome arm 3R asso-
656 J. Hemingway et al. / Insect Biochemistry and Molecular Biology 34 (2004) 653–665
ciated with permethrin resistance in this strain (Ranson
et al., 2004). A second QTL on chromosome arm 2L
coincides with the para voltage-gated sodium channel
gene, and Ranson et al. (2000a,b) identified a leucine to
serine mutation within a codon frequently associated
with pyrethroid resistance in many arthropods.
A further Cyp6 gene implicated in insecticide resist-
ance is Cyp6f1 from the mosquito C. p. quinquefascia-
tus. Kasai et al. (2000) reported slightly elevated levels
(~2.5-fold) of Cyp6f1 transcript in a strain with perme-
thrin resistance. Deltamethrin resistance in a close rela-
tive, C. p. pallens, appears to involve up-regulation of
members of the Cyp4 family (Shen et al., 2003). Cyp4
gene over-expression has also been found in pyre-
throid-resistant Drosophila (Amichot et al., 1994) and
other species (Pridgeon et al., 2003; Pittendrigh et al.,
1997).
2.3. Glutathione S-transferases
Glutathione S-transferases (GSTs) are found ubiqui-
tously in aerobic organisms. Their central role in
detoxification and drug resistance pathways in mam-
mals has now been established but additional functions
are continually being attributed to this complex
enzyme family (see Ranson and Hemingway, 2004).
There are two distantly related classes of GSTs, classi-
fied according to their location within the cell: micro-
somal or cytosolic. There are only three distinct
microsomal GSTs transcribed in An. gambiae. Their
homology with vertebrate microsomal GSTs suggests a
similar trimeric structure, with a molecular mass of
approximately 50 kDa. Their tertiary structure in vivo
has not been resolved. This class will not be discussed
further.
The vast majority of GSTs are cytosolic dimeric pro-
teins comprising two subunits each around 24–28 kDa
in size. Each subunit has two binding sites, the G site
and the H site. The highly conserved G site binds the
tripeptide glutathione and is largely composed of
amino acid residues found in the N terminal of the
protein. The H site or substrate binding site is more
variable in structure and is largely formed from resi-
dues in the C-terminal (Mannervik, 1985). The active
site residue at the N termini of the protein interacts
with and activates the sulphydryl group of glutathione
to generate the catalytically active thiolate anion
(Armstrong, 1991). This nucleophilic thiolate anion is
then capable of attacking substrates bound to the H
site. The active site residue is generally conserved
within GST classes but differs between the classes
(Wilce and Parker, 1994; Agianian et al., 2003;
Sheehan et al., 2001).
Kinetic characteristics have proven a poor criterion
for classification of GSTs (Ranson and Hemingway,
2004). GST sequences that share over 40% sequence
identity are generally assigned to the same class, with
other properties such as phylogenetic relationships,
immunological properties, tertiary structure and the
ability to form heterodimers also employed. At least six
classes of insect GSTs have been identified in An.
gambiae (Ranson et al., 2002a), found in several large
clusters on all three chromosomes. The Delta and Epsi-
lon classes found exclusively in insects are the largest
classes of insect GSTs. Members of both these classes
have been implicated in resistance to all the major clas-
ses of insecticide. The other insect GSTs belong to the
Omega, Sigma, Theta and Zeta classes (Tang and Tu,
1994; Wang et al., 1991; Board et al., 1997, 2000;
Huang et al., 1998; Wei et al., 2001; Ranson et al.,
2002a; Vontas et al., 2002; Ortelli et al., 2003). Despite
the availability of full genome sequence (Holt et al.,
2002), the possibility that further insect GST classes
exist cannot be discounted, as at least three of the
An. gambiae GSTs could not be reliably assigned to
any of the pre-designated classes (Ding et al., 2003).
Individual GST subunits are assigned names indicat-
ing the species they were isolated from and the GST
class. They are also given a number that may either
reflect the order of discovery or the genomic organis-
ation. For example, AgGSTd12 is the 12th member of
the An. gambiae Delta class of GSTs to be identified.
The wide range of functions performed by GSTs is
aided by the extensive nature of the GST supergene
family in insects and the broad substrate specificities of
individual enzymes. Dramatic changes in substrate
specificity can be achieved by a small number of amino
acid substitutions (Ortelli et al., 2003). The primary
function of GSTs is generally considered to be the
detoxification of both endogenous and xenobiotic com-
pounds either directly or by catalysing the secondary
metabolism of a vast array of compounds oxidised by
the cytochrome P450 family. GSTs also play an impor-
tant role in stress physiology and have been implicated
in intracellular transport and various biosynthetic
pathways (Wilce and Parker, 1994).
Elevated GST activity has been implicated in resist-
ance to at least four classes of insecticides. Higher
enzyme activity is usually due to an increase in the
amount of one or more GST enzymes, either as a result
of gene amplification or more commonly through
increases in transcriptional rate, rather than qualitative
changes in individual enzymes (for recent review see
Ranson and Hemingway, 2004).
Resistance due to increases in GST activity was first
identified in organophosphate (OP) resistance, and
GSTs have now been implicated in OP resistance in
many insect species (Hayes and Wolf, 1988). Recombi-
nant GST enzymes from the diamondback moth and
housefly have verified the role of these enzymes in OP
metabolism (Huang et al., 1998; Wei et al., 2001).
Detoxification occurs via an O-dealkylation or O-dear-
 J. Hemingway et al. / Insect Biochemistry and Molecular Biology 34 (2004) 653–665
ylation reaction. In O-dealkylation, glutathione is con-
jugated with the alkyl portion of the insecticide (Oppe-
noorth et al., 1979), whereas the reaction of
glutathione with the leaving group (Chiang and Sun,
1993) is an O-dearylation reaction. GSTs can also cata-
lyse the secondary metabolism of OP insecticides. This
insecticide class is usually applied in its non-insecticidal
phosphorothionate form and activated to the insecti-
cidal form by the action of cytochrome P450s in the
insect. GSTs that can detoxify the active oxon ana-
logue have been described in mosquitoes and elevated
activity of these enzymes is a cause of OP resistance in
An. subpictus (Hemingway et al., 1991).
Dehydrochlorination of DDT is a major route of
detoxification in insects. The enzyme catalysing this
reaction was not initially recognised as a member of
the GST family, partly due to the inability to detect
even a transient GSH conjugate and also because of
the lack of DDT dehydrochlorinase activity in some
purified GSTs. Evidence that DDT dehydrochlorinase
is a GST was finally provided when three enzymes with
CDNB conjugating activity were isolated from a DDT-
resistant strain of housefly. Two of these were also
active with DDT as a substrate (Clark and Shamaan,
1984). The DDT dehydrochlorinase reaction proceeds
via a base abstraction of hydrogen, catalysed by the
thiolate anion generated in the active site of the GST,
leading to the elimination of chlorine from DDT, gen-
erating DDE. An increased rate of dehydrochlorination
confers resistance to DDT in Ae. aegypti (Grant et al.,
1991) and An. gambiae (Prapanthadara et al., 1993).
GSTs have no direct role in the metabolism of pyre-
throid insecticides but they play a very important role
in conferring resistance to this insecticide class by
detoxifying lipid peroxidation products induced by pyr-
ethroids (Vontas et al., 2001). GSTs may also protect
against pyrethroid toxicity in insects by sequestering
the insecticide (Kostaropoulos et al., 2001).
GSTs play a vital role in the inactivation of toxic
products of oxygen metabolism. Reactive oxygen spe-
cies (ROS), including hydrogen peroxide, superoxide
anions and hydroxyl radicals, are generated during
aerobic respiration. These ROS trigger a cascade of
reactions that can be highly damaging to the cells. The
generation of ROS initiates the conversion of poly-
unsaturated fatty acids to lipid hydroperoxides that are
toxic at high concentrations, but some of these have
important signalling functions affecting cell prolifer-
ation, apoptosis and differentiation at low concentra-
tions (Sawicki et al., 2003). Peroxidase activity has
been detected in insect GSTs from the Delta, Epsilon
and Sigma classes (Vontas et al., 2001; Singh et al.,
2001; Ortelli et al., 2003).
657
2.4. The role of Regulatory Factors
As described above, transcriptional up-regulation
underlies many instances of insecticide resistance, yet
the factors responsible for this are largely undeter-
mined. Amino acid alterations may be important in
some instances, e.g. CYP6A2 (Berge et al., 1998), but
cis- and trans-acting factors are also involved. In
houseflies, the expression of Cyp6a1 is influenced by a
trans-acting factor (Carino et al., 1994). Sabourault
et al. (2001) found a strong correlation between the
presence of an ali-esterase mutation (G137D: G119D
by Torpedo numbering) or of a deletion at this locus
and the over-production of CYP6A1, with concomitant
diazinon resistance. The authors suggested that
hydrolysis by the wild-type ali-esterase generates a pro-
duct that represses Cyp6a1 expression; absence of wild-
type esterase results in transcriptional up-regulation of
this OP-metabolising p450, and potentially other
detoxification genes also. Housefly Cyp6d1 expression
also involves cis- and trans-acting regulators (Liu and
Scott, 1997). In insecticide-resistant Drosophila strains,
mutation in a trans-acting repressor permits constitut-
ive over-expression of Cyp6a8 (Maitra et al., 1996,
2000, 2002).
Most studies of GSTs suggest that regulation occurs
at the transcriptional level. Several regulatory elements
have been identified in the promoter regions of GSTs
that may mediate their induction but the significance of
these findings is unclear in the absence of functional
studies. In Ae. aegypti, a mutation in a trans-acting
repressor element is the proposed mechanism for the
enhanced expression of a Delta class GST in a DDT-
resistant strain (Grant and Hammock, 1992). Genetic
mapping of the major genes controlling GST-based
DDT resistance in An. gambiae also provided tentative
evidence for a trans-acting regulator (Ranson et al.,
2000a,b) although in this species, mutations in pro-
moter elements of the Epsilon GST cluster are also
associated with resistance (Ranson et al., 2001).
The significance of alternative splicing in regulating
GST expression has not been fully investigated. Four
alternative transcripts of a Delta class gene and two
of a Sigma GST have been detected in An. gambiae (Ran-
son et al., 1998), and it is possible that different inducers
or stress treatments may affect the choice of splice site.
Maitra et al. (2002) have suggested that inefficient spli-
cing may generate different isoforms of CYP6A8.
The majority of individual insect GSTs whose
expression profile has been determined are expressed
constitutively in all life stages. Recent experiments
found that in An. gambiae, transcripts for all but one
of the GST supergene family were detectable in one-
day-old adult mosquitoes by RT-PCR (Ding et al.,
2003). It is apparent from several studies in other
insects that the levels of individual enzymes can fluctu-
658 J. Hemingway et al. / Insect Biochemistry and Molecular Biology 34 (2004) 653–665
ate widely during the life span of an insect and in dif-
ferent insect tissues.
In a number of studies insects have been exposed to
various xenobiotics to determine the effect of these che-
micals on GST expression levels (Ottea and Plapp,
1984; Yu, 1984, 1996). In the majority of cases, a
model substrate, such as 1-chloro-2,4-dinitrobenzene
(CDNB), is used to assay GST activity from crude
insect homogenates and hence the results are general
measurements of the activity of a large subset of GSTs
and do not reveal much about fluctuations in the level
of individual enzymes. A detailed account of the effect
of dietary compounds, insecticides and laboratory
inducers on GST expression is provided in two review
articles (Clark, 1989; Yu, 1996). Studies using GST
inducers have demonstrated that multiple mechanisms
are involved in the regulation of these enzymes. Up-
regulation of some p450s after exposure to insecticide
or to phenobarbital has been well documented (e.g.
Brandt et al., 2002; Sierra-Santoyo et al., 2000; Maitra
et al., 1996; Carino et al., 1994). Barbie box response
elements have been identified in Drosophila Cyp6a8 and
Cyp6a2 promoters (Maitra et al., 2002; Dombrowski
et al., 1998).
The simultaneous over-expression of multiple Cyp
genes has been reported in Drosophila and M. domes-
tica (Amichot et al., 1994; Brandt et al., 2002; Le Goff
et al., 2003; Carino et al., 1992). Ding et al. (2003)
identified differential up-regulation of specific An.
gambiae GST genes within a cluster. Le Goff et al.
(2003) reported up-regulation of GST and Cyp genes in
DDT-resistant D. simulans using a D. melanogaster
microarray. Microarrays of whole genomes or of
enzyme families (e.g. detoxification enzymes) will be a
powerful tool in dissecting resistance (Ranson, pers.
commun.; Le Goff et al., 2003). Vulule et al. (1999)
found a correlation between elevated p450 and carbox-
ylesterase activity levels in a resistant strain of An.
gambiae, suggesting co-regulation. The availability of
genome sequence for An. gambiae (Holt et al., 2002)
and D. melanogaster (Misra et al., 2002) may reveal
similarities within promoters of co-expressed genes that
will lead to the identification of their regulators, and
potentially to novel control methods.
Finally, it should be remembered that most studies
on gene regulation rely on the detection of steady state
mRNA levels. The role of differential stability of tran-
scripts is often overlooked.
3. Target site resistance
3.1. Insensitive acetylcholinesterase
Biochemical assays in several mosquito species have
identified insecticide resistance due to insensitive acet-
ylcholinesterase (AChE), the target site of OP and car-
bamate insecticides. AChE has a key role in the
nervous system, terminating nerve impulses by catalys-
ing the hydrolysis of the neurotransmitter acetylcho-
line. The insecticides inhibit enzyme activity by
covalently phosphorylating or carbamylating the serine
residue within the active site gorge (Corbett, 1974).
Studies on An. albimanus from Central America have
shown that an altered AChE is the most common OP/
carbamate resistance mechanism (Ayad and Geor-
ghiou, 1979; Hemingway and Georghiou, 1983), and
this is true also in Mexican populations of the same
mosquito (Penilla et al., 1998). Insensitive AChE has
been reported from C. p. pipiens, C. p. quinquefasciatus
and C. tritaeniorhynchus, An. nigerimus, An. atroparvus
and An. sacharovi (Villani and Hemingway, 1987;
Bisset et al., 1990; Hemingway et al., 1985, 1986;
Hemingway, 1982a,b) but in An. gambiae insensitive
AChE has only recently been described (N’Guessan
et al., 2003).
Across all insect species there are two very distinct
types of target site resistance, conferring high carba-
mate and low OP resistance, or high OP with either
equivalent or low carbamate resistance (Russell et al.,
2004). All the mosquito AChE target site resistances to
date fall into the former category.
In Drosophila there is only one gene encoding AChE,
ace (Fournier et al., 1989), and various point mutations
have been described in resistant strains (Mutero et al.,
1994). Two of these residues are also altered in the sin-
gle ace gene from resistant M. domestica (Walsh et al.,
2001); and one is found in conjunction with a novel
mutation in OP-resistant olive fruit fly, Bactrocera
oleae (Vontas et al., 2002). Individual mutations confer
a low level of insensitivity without dramatically affect-
ing the rate of neurotransmitter hydrolysis; combina-
tions of mutations generate enhanced resistance. These
mutations constrict the neck of the active site gorge,
limiting insecticide access to the catalytic residues loca-
ted at the base of the gorge.
In C. p. pipiens and in C. tritaeniorhynchus, the pres-
ence of at least two AChE genes was inferred from the
findings that AChE-based resistance mapped to chro-
mosome II, whereas the cloned AChE gene (ace-2) is
sex-linked (Bourguet et al., 1996; Malcolm et al., 1998;
Mori et al., 2001). Recently, the sequences of two
AChE genes (ace-1 and -2) were identified from the An.
gambiae genome (Holt et al., 2002); they show only
53% identity. A partial fragment of ace-1 was amplified
by degenerate PCR from C. p. pipiens (Weill et al.,
2002), and tight linkage between this gene and pro-
poxur resistance was demonstrated using an RFLP
within the coding region. However, sequence analysis
of this fragment did not show any likely resistance-
associated mutations. Homologous ace-1 fragments
were also isolated from several other mosquito species,
including An. gambiae and Ae. aegypti. Subsequently,
 J. Hemingway et al. / Insect Biochemistry and Molecular Biology 34 (2004) 653–665 659

the entire ace-1 coding regions from susceptible and
resistant C. p. pipiens strains were compared. A single
amino acid substitution (G119S) was identified, and
found also in other insensitive AchE strains of C. p.
pipiens, C. p. quinquefasciatus and An. gambiae (Weill
et al., 2003). This glycine residue lies at the base of the
active site gorge, close to the oxyanion hole.
Intriguingly, a different mutation (G119D) at the
oxyanion hole of related carboxylesterases is respon-
sible for OP-specific resistance in L. cuprina and
M. domestica (Newcomb et al., 1997; Campbell et al.,
1998; Claudianos et al., 1999), and linked with p450
over-expression (Sabourault et al., 2001), whilst mala-
thion-specific resistance is conferred by mutations at a
tryptophan residue as discussed earlier.
A mutation in the ace-1 ortholog from Japanese C.
tritaeniorhynchus has recently been described, associa-
ted with high levels of OP resistance (Nabeshima et al.,
2004). The mutation (F455W, corresponding to F331
in Torpedo) involves a different amino acid to that
described by Weill et al. (2003), located close to the
catalytic His within the acyl pocket of the enzyme.
Replacements at this position have also been described
in carbamate-resistant Myzus persicae (Nabeshima
et al., 2003) and OP-resistant two-spotted spider mite
Tetranychus urticae (Anazawa et al., 2003).
Fig. 2 summarises the known AChE/carboxylester-
ase genotype/phenotype relationships; for a compre-
hensive discussion of structure–function correlations,
see Russell et al. (2004).
3.2. The GABA receptor mutation
The target site of cyclodiene insecticides such as diel-
drin is the type A receptor for the neurotransmitter c-
aminobutyric acid (GABA). Binding of GABA to the
receptor elicits rapid gating of an integral chloride-
selective ion channel. GABA receptors comprise five
subunits arranged around the central ion channel. They
have a wide distribution in both vertebrates and inver-
tebrates. Insect resistance to cyclodienes is widespread,
and the cause of this insensitivity has been most exten-
sively studied in D. melanogaster (reviewed in Hosie
et al., 1997). Mutations at a single codon in the Rdl
(resistance to dieldrin) gene (encoding one receptor
subunit), from an alanine residue to a serine or more
rarely to a glycine, have been documented in all diel-
drin-resistant insect species to date (ffrench-Constant
et al., 1998), although the only mosquito studied thus
far is Ae. aegypti (Thompson et al., 1993). This
mutation lies within the second transmembrane region
of the RDL subunit, presumed to be the principal con-
stituent of the ion-channel lining, and appears to con-
fer both insensitivity to the insecticide and a decreased
rate of desensitisation.
The phenyl pyrazole insecticide fipronil also targets
the GABA receptor, and so problems of resistance due
to mutation may arise, should fipronil be employed in
vector control programmes (Kolaczinski and Curtis,
2001). The appearance of such resistance in areas pre-
viously treated with cyclodienes may be much quicker

Fig. 2. Genotype–phenotype correlations between carboxy/cholinesterase mutations and organophosphate/carbamate resistance. Torpedo AchE
numbering is used; the catalytic triad residues are denoted by solid circles. Solid lines indicate AchE mutations; diamonds denote ali-esterase
mutations. Based on data from Mutero et al. (1994), Walsh et al. (2001), Vontas et al. (2002), Weill et al. (2002, 2003), Nabeshima et al. (2003,
2004), Anazawa et al. (2003), Newcomb et al. (1997), Campbell et al. (1998), Claudianos et al. (1999, 2002), Zhu et al. (1999), Russell et al. (2004).
660 J. Hemingway et al. / Insect Biochemistry and Molecular Biology 34 (2004) 653–665
than anticipated, since the alanine!serine mutation
can persist in Drosophila populations long after insecti-
cide selection has been withdrawn, presumably due to
the lack of a selective disadvantage in resistant flies
(Hosie et al., 1997)
3.3. Mutations in the voltage-gated sodium channel
Pyrethroid insecticides have a rapid ‘knock-down’
effect. However, the intensive use of DDT and pyre-
throids has led to the development of knock-down
resistance (kdr) in many insect species (Liu et al., 2000;
Soderlund and Knipple, 2003) including Anopheles and
Aedes. The use of both DDT and pyrethroids in the
control of rice and cotton pests is likely to have con-
tributed significantly to the development of resistance
in An. gambiae from West Africa (Elissa et al., 1993;
Martinez-Torres et al., 1998). The molecular mech-
anism of kdr can be distinguished from metabolic
resistance by the use of synergists (Enayati et al., 2003)
and is well characterised.
Pyrethroids and DDT both target the voltage-gated
sodium channel, which comprises four domains (I–IV),
each consisting of six transmembrane helices (S1–S6).
Action potentials are generated across the membrane
in order to conduct electrical information throughout
the nervous system. In an inactive state, when the
membrane is at the resting potential, the sodium chan-
nel is closed. However, upon channel activation, the
membrane becomes depolarised. This causes the
sodium channel to open by generating a transient
sodium current. After approximately 1 ms, inactivation
occurs due to a conformational change in the sodium
channel that blocks the passage of ions across the
membrane. When the membrane potential returns to
the resting level, the channel closes (Vais et al., 2001).
Pyrethroids modify the gating kinetics of voltage-
sensitive sodium channels (Lund and Narahashi, 1983)
by slowing both the activation and inactivation of the
channel. The pyrethroid class of insecticides can be
subdivided into two groups, each of which has a differ-
ent effect. Type I pyrethroids (e.g. permethrin) lack a
cyano group. Type II pyrethroids (e.g. deltamethrin)
contain a cyano group in the a-benzylic position. Type
II pyrethroids prolong the sodium current during an
action potential more than Type I pyrethroids. The
half activation potential (the membrane potential at
which 50% of the available channels are open) is more
negative when using Type I pyrethroids (Vais et al.,
2001). This results in the channel opening at the resting
potential, with consequent depolarisation of the neuro-
nal membrane and the initiation of repetitive dis-
charges in motor and sensory axons that causes
paralysis and death. All pyrethroids have a similar
mode of action, but neurophysiological studies on C. p.
quinquefasciatus from Saudi Arabia indicated that kdr-
type nerve insensitivity might not confer similar levels
of resistance to all pyrethroids (Amin and Hemingway,
1989).
Cross-resistance studies and single channel studies in
the housefly confirmed that the molecular mechanism
of pyrethroid resistance is due to a functional change
in the sodium channel (Chinn and Narahashi, 1986).
Using genetic mapping techniques, Ranson et al.
(2000a,b) demonstrated that the sodium channel map-
ped to a location in the genome corresponding to a
major quantitative trait locus determining resistance to
permethrin, and that a nucleotide substitution in the
sodium channel was linked to the inheritance of perme-
thrin resistance in a Kenyan strain of An. gambiae. kdr-
like mutations in this gene have been linked to changes
in target sensitivity in a range of insects. A comprehen-
sive review of the molecular biology of knock-down
resistance to pyrethroids in other insect species has
recently been published (Soderlund and Knipple, 2003).
kdr occurs due to a change in affinity between the
insecticide and its binding site on the sodium channel,
caused by single or multiple substitutions in the sodium
channel gene. It is possible that a limited number of
substitutions in the target site can lead to nerve insensi-
tivity (Martinez-Torres et al., 1998). In several insect
species, the most common kdr mutation is a leucine to
phenylalanine (Leu!Phe) substitution in the S6 hydro-
phobic segment of domain II in the sodium channel
gene, such as that found in pyrethroid-resistant
West African An. gambiae (Williamson et al., 1996;
Martinez-Torres et al., 1998) and An. stephensi
(Enayati et al., 2003). However, a second substitution
at the same position (Leu!Ser) has been found in East
African An. gambiae (Ranson et al., 2000a,b). Both
mutations have been reported in An. sacharovi Favre in
Turkey (Lüleyap et al., 2002).
Cross-resistance and neurophysiological studies
inferred target site resistance in Ae. aegypti
(Hemingway et al., 1989), and a recent molecular study
has now confirmed this (Brengues et al., 2003). Analy-
sis of the cDNA sequence of the S6 hydrophobic
domain II of six pyrethroid-resistant Aedes strains
showed that the common Leu!Phe substitution found
in An. gambiae was not found in these strains of Aedes.
This may be because Aedes codon usage does not easily
allow for this substitution without a simultaneous dou-
ble nucleotide substitution. However, all insects from
a Vietnamese pyrethroid-resistant Aedes strain were
homozygous for a double nucleotide change, resulting
in a Leu!Trp mutation, slightly upstream of the com-
monly mutated codon. This double mutation includes a
common silent polymorphism found in a range of
Aedes strains. All individuals from a Brazilian strain of
pyrethroid-resistant Aedes were either homozygous or
heterozygous for both of two, non-adjacent, amino
acid alterations (isoleucine!methionine and glyci-
 J. Hemingway et al. / Insect Biochemistry and Molecular Biology 34 (2004) 653–665
ne!valine), each of which occur as a result of a single
nucleotide mutation. These alterations were also found
in mosquitoes from two other Central American coun-
tries, suggesting geographical dispersal from a single
originating location. Aedes samples from Indonesia and
Thailand had a mutation of valine !glycine as a result
of a single nucleotide change.
In total, over 20 unique sodium channel amino acid
sequence polymorphisms have been identified in associ-
ation with pyrethroid resistance. All mutations so far
identified in mosquito vectors have been found in
domain II of the sodium channel. Super-kdr mutations
found in other insects (Soderland and Knipple, 2003)
that are highly resistant to pyrethroids have not yet
been identified in mosquitoes.
Direct DNA sequence analysis can be used to estab-
lish the presence of mutations in the voltage-gated
sodium channel. However, this is not practical for
large-scale population studies. A simple diagnostic
PCR assay has been developed (Martinez-Torres et al.,
1998) that enables the rapid diagnosis of the common
Leu!Phe substitution in resistant mosquitoes, and spe-
cific assays for other mutations have been developed
(Ranson et al., 2000a,b; Enayati et al., 2003). The
sodium channel gene has been used as a molecular tool
to look at An. gambiae population structure in Mali
and Burkina Faso (Fanello et al., 2003a,b; Diabate
et al., 2003).
4. Conclusions
Resistance to insecticides is one of the most well
documented, and rapid, cases of evolutionary adap-
tation to environmental changes. Although only a lim-
ited number of resistance mechanisms have been
implicated to date, the diversity within those enzyme
families involved in metabolic resistance is likely to
contribute substantially to resistance to many insecti-
cide classes. The advent of post-genomic technology is
likely to reveal the contributions of hitherto unsuspec-
ted enzymes. The ability to compare the transcriptome
or proteome of a resistant mosquito strain with a sus-
ceptible equivalent will substantially enhance our
understanding of resistance and its complexity. In the
long term this can only benefit those who suffer from
mosquito-borne disease.
Acknowledgements
We would like to acknowledge anonymous reviewers
for their constructive comments.
661
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