Medical and Veterinary Entomology (1998) 12, 112

Mosquito carboxylesterases: a review

of the molecular biology and biochemistry
of a major insecticide resistance mechanism
J . H E M I N G WA Y and S . H . P. P. K A R U N A R AT N E 1
School of Pure and Applied Biology, University of Wales, Cardiff, U.K. and 1Department of Zoology, University of Peradeniya,
Peradeniya, Sri Lanka

Abstract. The major mechanism of organophosphorus insecticide resistance in Culex

mosquitoes involves the elevation of one or more esterases. The general mechanism
underlying this resistance is the amplification of the structural genes. This review
covers the classification of the mosquito esterases in the context of classical esterase
nomenclature. The function of the amplified esterases and the structure of the amplified
DNA on which they occur are also described. Implications of information on the
esterase amplicons are discussed in relation to the evolution and migration of
insecticide resistance in Culex.
Key words. Culex, Culicidae, mosquitoes, insecticide resistance, esterase,
carboxylesterase, carbamate, organophosphorus insecticides.

Introduction
Major mechanisms of insecticide resistance involve either
mutation within the target site of the insecticide, or an alteration
in the rate of insecticide detoxification. The enzymes involved
in insecticide detoxification may be qualitatively and/or
quantitatively changed to give resistance. There are three
major groups of such enzymes; glutathione-S-transferases,
monooxygenases and carboxylesterases. The carboxylesterasebased resistance mechanism has been reported from more than
thirty different medical, veterinary and agricultural insect pests.
In mosquitoes (Diptera: Culicidae) it is the primary mechanism
for organophosphorus insecticide (OP) resistance as well as
a secondary mechanism for carbamate resistance (Peiris &
Hemingway, 1993), whereas in some other insect species,
pyrethroid resistance is also conferred (Devonshire &
Moores, 1982).
The target site of the OP and carbamate insecticides is
acetylcholinesterase (AChE), a serine esterase, which
hydrolyses the neurotransmitter acetylcholine. Once AChE is
inhibited by these insecticides, paralysis or death of the insect
occurs. The majority of OPs are esters of phosphoric acid
and can be hydrolysed by carboxylesterases. Most OPs are
administered in the phosphorothionate form which is activated
to the organophosphate by monooxygenases within the insect.
Correspondence: Professor Janet Hemingway, School of Pure and
Applied Biology, University of Wales, PO Box 915, Cardiff CF1 3TL,
U.K. E-mail: Hemingway@cardiff.ac.uk

1998 Blackwell Science Ltd

These oxons are more neurotoxic than their thionate analogues,

for example malaoxon is . 2000-fold better than malathion at
inhibiting Culex tarsalis AChE (Matsumura & Brown, 1961).

Classification of carboxylesterases
Carboxylesterase or esterase is a collective term for the enzymes
which hydrolyse carboxylic esters. Classification of these
enzymes is difficult because of their overlapping substrate
specificity (Heymann & Jakoby, 1980). However, the esterase
classification of Aldridge (1953a) is generally recognized.
According to that classification, esterases inhibited by paraoxon
in a progressive and temperature-dependent manner are called
B esterases and those which are not inhibited are A esterases
(Aldridge, 1953a,b, 1993). Some A esterases can hydrolyse
OPs, through an acylated cysteine in their active site, and are
termed phosphoric triester hydrolases (EC 3.1.8) (Reiner, 1993;
Walker, 1993). The term carboxylesterase (EC 3.1.1.1) is now
mainly attributed to B esterases (Reiner, 1993; Walker, 1993).
These enzymes have an active site serine residue, hence the
terms B esterase and serine hydrolase are synonymous.
Apart from this general classification, different
nomenclatures have been adopted to describe the esterases
present in a particular species or a group of closely related
species. Mentlein et al. (1984, 1985) used the most prominent
natural substrate to classify rat liver microsomal esterases.
However, the most commonly used criterion in this type of
classification is the different mobilities apparent after native

J. Hemingway and S. H. P. P. Karunaratne

electrophoresis. For example, aphid esterases E1

E7 (Devonshire, 1975), Drosophila esterases Est.2, Est.5, etc.
(Zouros et al., 1982), German cockroach esterases E-1, E-2,
etc. (Prabhakaran & Kamble, 1993).
For Culex mosquitoes, an alternative esterase nomenclature
was developed on the combined criteria of their preference for
hydrolysing the synthetic esters - and -naphthyl acetate and
electrophoretic mobility (Georghiou et al., 1980; Raymond
et al., 1991). The esterases which preferentially hydrolysed and -naphthyl esters were called A and B esterases,
respectively. Numerical subscripts indicated mobility, starting
from the slowest running esterase. However, as more esterases
were described at the electrophoretic and molecular level, the
mobility criterion became impractical and subscripts were
allocated on a historical basis. Also, unlike the terminology
used for other taxonomic groups of organisms, the esterase
series from different Culex species were mixed: the majority
of the esterase series occur in Culex quinquefasciatus (Table 1),
but the A3 and B3 esterases have been described only in Culex
tarsalis (Prabhakar et al., 1987). To overcome these and other
difficulties with the previous classification, a new but
conventional nomenclature of Culex esterases was introduced
by Vaughan & Hemingway (1995). This, in keeping with the
rat and mouse esterase nomenclature (Zutphen, 1983, 1987;
Bender et al.,1984; Simon et al., 1985), refers to enzymes
specific for - and -naphthyl acetate as Est and Est,
respectively, with the genes italicised. Numbers for
electrophoretically distinct enzymes are allocated historically,

hence esterases B1 and B2 become Est1 and Est2. Once

characterized at the nucleotide level, the esterases are
additionally allocated superscripts, for example the B1 from
Cx quinquefasciatus TEMR strain becomes Est11, whereas
the MRes B1 which differs from that of TEMR at the
nucleotide but not the electrophoretic level becomes
Est12 (Vaughan et al., 1995). With the earlier nomenclature
some esterases need to be completely renamed as work on
them progresses. For example, the esterase in the MRes
strain of Cx quinquefasciatus, referred to in several papers as
B1 (Bisset et al., 1990, 1995; Rodriguez et al., 1993), would
become B8 on the basis of its nucleotide sequence (Rooker
et al., 1996). The new system has the advantage that esterases
can be accurately classified at either the electrophoretic or
molecular level initially, avoiding changes of nomenclature in
the literature for the same esterase. With this conventional
classification, esterases from other Culex species are
differentiated from those of Cx quinquefasciatus by addition
of an identifying leader, as is common for mammalian and
Drosophila enzymes, for example, the amplified Est in Cx
tritaeniorhynchus becomes CtrEst11 (Karunaratne et al.,
1997). Use of and in the nomenclature also avoids clashes
with the Aldridge classification, on which all the Culex esterases
to date are B esterases. Hence the earlier Culex A and B terms
are misleading, as both these groups are B or serine esterases
(Aldridge, 1953a,b, 1993). Table 1 gives the nominal
equivalents of Culex esterases identified under both
classification systems in the context of conventional enzyme
nomenclature (IUBMB, 1992).

Table 1. Esterase nomenclature for Culex mosquitoes:

synonymy of previous terminology with conventional nomenclature developed here.
Esterase terminology (original reference)
Species and Strains

Conventional

Previous

Est series
Culex quinquefasciatus (Say)
S54
PelRR 1 numerous
PelSS
COLOMBIA
Western Mediterranean
Eastern Mediterranean
China
Culex tarsalis (Coquillett)

Est1
Est21
Est3
Est31
Est4
Est5
Est6
CtaEst1

Current
(Vaughan & Hemingway, 1995)
(DeSilva et al., 1997)
(DeSilva et al., 1997)
Current
Current
Current
Current

A1
A2
None
None
A4
A5
A6
A3

Est Series
Culex quinquefasciatus (Say)
TEMR

Est11

(Vaughan et al., 1995)

B1

MRES

Est12

(Vaughan et al., 1995)

B 1 & B8

PelSS
PelRR 1 numerous
Western Mediterranean
Eastern Mediterranean
Culex tritaeniorhynchus (Giles)
Culex tarsalis (Coquillett)

Est13
Est21
Est4
Est5
CtrEst11
CtaEst1

(Vaughan et al., 1995)

(Vaughan et al., 1995)
Current
Current
(Karunaratne et al., 1997)
Current

None
B2
B4
B5
None
B3

(Pasteur & Georghiou, 1981)

(Peiris & Hemingway, 1993)

(Poire et al., 1992)

(Severini et al., 1997)
(Rooker et al., 1996)
(Prabhaker et al., 1987)

(Pasteur et al., 1981b;

Raymond et al., 1987)
(Bisset et al., 1995;
Guillemaud et al., 1996)
(Peiris & Hemingway, 1993)
(Poire et al., 1992)
(Severini et al., 1997)
(Prabhaker et al., 1987)

1998 Blackwell Science Ltd, Medical and Veterinary Entomology, 12, 112

Mosquito carboxylesterases
Detection of esterase-based resistance
Early work implicated esterases in insecticide resistance by the
use of synergists such as DEF (S,S,S-tributyl phosphorothioate),
TPP (triphenyl phosphate) and IBP (S-benzyl O,O-diisopropyl
phosphorothionate) (Apperson & Georghiou, 1975; Georghiou
et al., 1980; Hemingway, 1982, 1983a; Magnin et al., 1988;
Hemingway et al., 1989; Wirth et al., 1990; Bisset et al.,
1995). Insecticide metabolism studies were used to implicate
carboxylesterase involvement in resistance, through increased
accumulation of hydrolytic products in insecticide resistant
compared to susceptible insect strains (Matsumura & Brown,
1961; Hemingway, 1982, 1989; Herath et al., 1987; Zeigler
et al., 1987). Esterases can also act by sequestering the
insecticide, i.e. rapidly binding and slowly releasing the
insecticide metabolites (Karunaratne et al., 1993). This latter
type of resistance requires the presence of increased quantities
of esterase due to the 1:1 stoichiometry of the reaction and the
slow metabolism times. Such a mechanism is not easily detected
by classical metabolism studies. However, as sequestering
esterases occur in increased quantities in resistant insects, their
presence can be detected in resistant insects on native PAGE
gels or by simple biochemical microplate or filter paper assays,
usually with - and/or -naphthyl acetate as the substrates
(Georghiou & Pasteur, 1978; Pasteur et al., 1981a, 1992;
Devonshire et al., 1986a; Dary et al., 1990; Ketterman et al.,
1992). Immunoassays have been developed to show the elevated
esterase levels in resistant insects (Devonshire et al., 1986a;
Beysat-Arnaouty et al., 1989; Devonshire & Field, 1991).
These assays demonstrate the increased level of esterase protein,
compared to the biochemical assays which measure only
enzyme activity levels.
Physico-chemical characterization
To date, the carboxylesterase Est1, Est21, Est3, Est11,
Est12, Est13, Est21 and CtrEst11 from Culex pipiens, Cx
quinquefasciatus or Cx tritaeniorhynchus have been partially
or fully purified and characterized. All the amplified forms of
these enzymes are involved in insecticide resistance
(Jayawardena et al., 1994; Karunaratne et al., 1995a;
Karunaratne & Hemingway, 1996; Ketterman et al., 1992,
1993; Karunaratne et al., 1997; Small et al., 1998). Molecular
weights of the Culex esterases are between 60 and 67 kDa,
within the range of the carboxylesterases of other organisms
(Heymann & Jakoby, 1980). All the purified esterases are
monomers, with the exception of Est1, which is reported as
a homodimer (Fournier et al., 1987), but this may be due to a
temporary association between monomers in the partially
purified preparation.
Iso-electric points (pI) of the mosquito carboxylesterases are
between 5 and 6 (Fournier et al., 1987; Ketterman et al., 1992;
Jayawardena et al., 1994; Karunaratne et al., 1993), which is
similar to other esterases, for example insecticide-resistant
houseflies have two esterases with pI values of 5.1 and
5.3 (Kao et al., 1985) and twenty-two soluble esterases of
D. melanogaster have a pI range of 3.86.2 (Healy et al., 1991).
From a range of physiological substrates, medium chain
length mono- and di-acylglycerols are hydrolysed preferentially

by Est21 and Est21 (Jayawardena, 1992; Ketterman et al.,

1992). The Est esterases are more reactive than Est esterases
towards the xenobiotic substrates (Ketterman et al., 1993;
Karunaratne et al., 1993) and the Est3 and Est13 esterases
from susceptible mosquitoes are significantly less reactive with
xenobiotics, including insecticides, than their counterparts from
resistant mosquitoes (Karunaratne et al., 1995a). In contrast,
in aphids the esterases from both susceptible and resistant
insects have similar kinetics with the xenobiotic substrates
(Devonshire & Moores, 1982).
Mechanism of insecticide resistance
The majority of serine hydrolases contain a serinehistidine
glutamic acid catalytic triad, but some (e.g. cholesterol esterase)
have aspartic acid as the acidic member of the triad (Cygler
et al., 1993). OPs and carbamates inhibit B esterases by rapid
esterification of the serine residue in the active site. This
reaction is often followed by a slow hydrolysis of the new
ester bond. Therefore, these insecticides can be considered as
inhibitors of the esterases, because they are poor substrates
which have a high affinity for the enzymes. The generally
accepted reaction mechanism is:
k1
k2
k3
E 1 I EI EI9 1 P1 E 1 P2
k-1
H2O
where E is the enzyme, I the inhibitor, EI the Michaelis
complex, EI9 the acylated enzyme, P1 the first metabolite
(alcohol) and P2 the second metabolite (acid).
The reaction from E 1 I EI9 (i.e. the formation of the
acylated enzyme) is measured by the kinetic constant ka, the
bimolecular rate constant.
For substrates, the whole reaction is very fast, with rapid
hydrolysis of the substrate and regeneration of the free enzyme.
For inhibitors, the acylated enzyme is formed quickly (ka is
high) but it is either stable or its rate of hydrolysis (k3) is very
slow, becoming a rate limiting step. Hence B esterases, which
are inhibited by paraoxon, have a very high affinity and a very
low capacity for the organophosphates and carbamates because
of the irreversible 1:1 stoichiometry of the reaction in the
active site. The presence of large amounts of these enzymes
produces resistance as the activated insecticides are rapidly
sequestered before they reach the insects AChE.
Esterases cause insecticide resistance by sequestration in the
ten different elevated insect carboxylesterases studied. The
kinetic constants for enzymeinsecticide interactions have been
determined only for aphid and Culex esterases (Devonshire &
Moores, 1982; Karunaratne et al., 1993, 1995a). The rate of
interaction of these esterases with organophosphates is very
fast, for example, the bimolecular rate constant for the reaction
between Est21 and chlorpyrifos-oxon is 1.5 3 108 M1 min1
(Karunaratne et al., 1993). The deacylation rate is very slow,
i.e. in paraoxon-inhibited mosquito Est21 and Est21, k3 is
0.031 h1, so to hydrolyse a paraoxon molecule completely,
one molecule of enzyme takes about 32 h (Karunaratne et al.,
1993). The deacylation rates are significantly higher for aphid
paraoxon inhibited esterase E4 where one molecule is
completely hydrolysed in 3 h (Devonshire & Moores, 1982).

1998 Blackwell Science Ltd, Medical and Veterinary Entomology, 12, 112

J. Hemingway and S. H. P. P. Karunaratne

Determination of several kinetic constants for mosquito

carboxylesterases has shown that ka is the most important,
correlating directly with the cross-resistance spectrum of the
strain. These kinetic data indicate that the insect
carboxylesterases protect the target site, AChE, by interacting
with the oxons more readily than does the target site itself. The
rates of interaction of Est and Est with the organophosphorus
insecticides were insignificant compared to the oxon analogues.
The Culex resistance mechanism is ineffective with pyrethroids
and weakly effective with carbamates (Karunaratne et al.,
1993). In contrast, the aphid E4 hydrolyses the (1S)transenantiomer of the pyrethroid permethrin (Devonshire &
Moores, 1982).
Approximately 7.7 pmol of elevated esterases, accounting
for about 0.4% of the total soluble protein, can be found in the
OP-resistant PelRR strain of Cx quinquefasciatus (Karunaratne,
1994). However, in the most resistant variants of aphids, the
amplified esterases comprise about 3% of their total protein
(Devonshire & Moores, 1982). The lower percentage content
of these esterases in PelRR homogenate is evident as a
significant band at the level of Est21, and Est21 is not
observed on SDS-PAGE gels stained for proteins (Karunaratne
et al., 1993). In contrast, E4 protein can be identified as a
prominent band in SDS electrophoresis of resistant aphid
homogenates (Devonshire & Moores, 1982).
Antisera raised against Est11 from the OP-resistant TEMR
strain of Cx quinquefasciatus* and Est1 of the OP-resistant
S54 strain of Cx pipiens* have been used to show that esterases
are overproduced by at least 500-fold and 70-fold, respectively,
as compared with the corresponding susceptible strains. The
Est11 antiserum cross-reacted with other Ests, but not with
Ests (Mouche`s et al., 1987). Similarly, the Est1 antiserum
reacted with other Ests, but not with Ests. Proteins
immunologically related to Ests were detected in Aedes
aegypti, Anopheles albimanus, An. stephensi, Musca domestica,
Myzus persicae and Cx tarsalis strains (Beyssat-Arnaouty et al.,
1989; Field et al., 1989). With the advent of more sensitive
detection methods and antiserum raised to native Est21 and
Est21, it was shown that the Est and Est enzymes do share
common epitopes (Karunaratne et al.,1995b, 1997). The Est21
antiserum also cross-reacts with many insect AChEs and with
some of the esterases and cholinesterases belonging to distant
animal groups such as mammals (Karunaratne et al., 1995b).
In contrast, the Est21 antiserum does not cross-react with
AChE (Karunaratne et al., 1997).
Non-elevated esterases also cause OP resistance. In humans,
hens and rabbits, OP insensitivity is mainly due to the presence
of serum paraoxonases, these are A esterases which can
hydrolyse the insecticides rapidly (Du et al., 1993; Furlong
et al., 1993; Li et al., 1993b). The overproduction of these
enzymes is not necessary, as they do not act by sequestration.
A similar type of mechanism occurs in some malathionresistant strains of Anopheles arabiensis, An. culicifacies, An.
stephensi and Cx tarsalis mosquitoes (Hemingway, 1982,
1983a; Herath et al., 1987; Ziegler et al., 1987; Malcolm &

*see Editorial Postscript on the Culex pipiens complex.

Boddington, 1989). In contrast to the Culex esterases, these

enzymes metabolise malathion not malaoxon (Hemingway,
1985). The esterases involved in insecticide resistance do not
show elevated expression in native PAGE gels and appear
to be qualitatively different from the susceptible enzymes
(Hemingway, 1982, 1983a). Resistant An. stephensi esterases
hydrolyse malathion, but are inhibited by paraoxon
(Boddington, 1992). It is still not known whether these
malathion-specific esterases are A or B esterases. They are
stage specific: An. arabiensis from Sudan has adult, but not
larval, malathion resistance (Hemingway, 1983a), whereas
resistance peaks in one-day old adult An. stephensi, then
decreases with the age of the adult (Rowland & Hemingway,
1987). This contrasts with amplified esterases in Culex which
are found in all life stages. Unlike the amplified Culex B
esterases, these malathion-specific esterases confer a narrow
cross-resistance spectrum (Hemingway, 1982; Herath et al.,
1987). Therefore, an important factor favouring the selection
of the amplified esterase-based mechanism may be its ability
to sequester a wide range of insecticides, giving the insect
greater protection. The two types of esterases can coexist in
the same strain: for example, a highly malathion-resistant strain
of Cx tarsalis has both elevated and non-elevated types of
esterase resistance mechanisms (Ziegler et al., 1987).
Molecular biology and classical genetics of
mosquito esterases
Formal genetic studies of the carboxylesterase-based
resistance in Anopheles has focused on mortalities in crosses
after malathion exposure. All these resistances are inherited as
single autosomal semi-dominant characteristics (Hemingway,
1983b; Lines et al., 1984). Studies on the Culex esterases are
based on polymorphism on native gels and mortalities after
insecticide exposure (Guillemaud et al., 1996). Linkage
relationships have been analysed from the esterase banding
patterns or esterase activity of the progeny from mass crosses
between resistant and susceptible strains, followed by backcrosses to the susceptible parents (De Stordeur, 1976; Pasteur
& Georghiou, 1981; Pasteur et al., 1981b; Peiris & Hemingway,
1983; Villani et al., 1983; Prabhaker et al., 1987; Takahashi &
Yasutomi, 1987; Georghiou & Pasteur, 1989). Resistance is
autosomal, although in Cx quinquefasciatus a minor maternal
effect on resistance occurs, with back-crosses involving
resistant (F1) females giving significantly lower mortalities
than those involving resistant (F1) males (De Stordeur, 1976;
Georghiou & Pasteur, 1989; Peiris & Hemingway, 1993). Most
mosquitoes have three pairs of chromosomes (White, 1981;
Besansky et al., 1992). The pattern of inheritance with recessive
morphological markers for each linkage group has indicated
that the resistant gene(s) are in linkage group II of Cx
tritaeniorhynchus (Takahashi & Yasutomi, 1987) and linkage
group III of the Cx pipiens complex (Pasteur et al., 1981b),
although recent molecular data (Nance et al., 1990) and
polytene chromosome mapping (Heyse et al., 1996) indicate
that Est11 is on chromosome arm 2L (Tewfik & Barr, 1974).
Est and Est have been attributed to two closely linked
gene loci Est-3 and Est-2, now designated Est and Est,

1998 Blackwell Science Ltd, Medical and Veterinary Entomology, 12, 112

Mosquito carboxylesterases
respectively (Pasteur & Georghiou, 1981; Pasteur et al., 1981b).
Some progeny of a cross between OP-resistant TEMR and a
susceptible strain had higher esterase activity than in any of
their parents, suggesting an enhanced expression of the
amplified Est11 genes when they are present in only one of
the two homologous chromosomes (Ferrari & Georghiou,
1990). The same phenomenon has also been shown with Sri
Lankan Cx quinquefasciatus for Est21 and Est21 (Peiris &
Hemingway, 1993). The early values for linkage distances
between the two esterase genes in Cx quinquefasciatus (Wirth
et al., 1990) have since been shown to be incorrect by molecular
mapping (Vaughan et al., 1995). Est4 and Est4, recently
described for a Cx pipiens strain from France, are reported to
be 0.8 centimorgans apart (Poire et al., 1992) but, given the
demonstrable inaccuracy of the Est21 and Est21 data, this
value may also be questionable.
The molecular mechanism of esterase elevation
The development of resistance to toxins by amplification of
genes involved in their detoxication is common in several
organisms (Devonshire et al., 1986b). The resistant TEMR
strain of Cx quinquefasciatus with Est11 was originally
estimated to have up to 250-fold more copies of this gene per
cell than the susceptible strain (Mouche`s et al., 1990), although
recent estimates of gene copy number have been 10-fold lower
than this (Guillemaud et al., 1996). An Est11 cDNA probe
hybridizes with other Est genes, but not with Est genes
(Raymond et al., 1987, 1989). In situ hybridization showed
that the Est11 genes are clustered between the centromere
and the apex of chromosome II, in a tandem arrangement,
making this chromosome longer in resistant insects (Nance
et al., 1990). Hence, the amplified genes are inherited as
clusters in a pseudo-monofactorial manner (Wirth et al., 1990).
Est11 spans 540 amino acid residues, with an active site
polypeptide sequence similar to those of eukaryotic serine
esterases (Mouche`s et al., 1992). Several Est cDNAs
(including one from a susceptible strain), the Est21 gene
and an Est21 cDNA and gene have been sequenced and
characterized (Vaughan et al., 1995, 1997; Vaughan &
Hemingway, 1995). cDNA sequences of PelRR Est21, MRes
Est12 and the partial PelSS Est13 (Vaughan et al., 1995)
have been compared with the sequence of TEMR Est11 and
a previously sequenced partial Est21 (Mouche`s et al., 1992).
They share between 95.2% and 98.8% identity (see Table 2).
Deduced amino acid identity between the esterases ranges from
95.2% to 98.6% (Vaughan & Hemingway, 1995). The high
identities at the molecular level suggest that the Ests may be
an allelic series from a single locus, although PCR data from
the susceptible PelSS strain of Cx quinquefasciatus suggest
that there are probably two non-amplified Est loci, one closely
linked with an Est locus (Vaughan et al., 1995), the other
sitting alone (Hemingway & Vaughan, unpublished data). The
Est21 has 47% deduced amino acid sequence homology
with all the Ests, but the predicted secondary structure of the
Est21 and Est21 proteins is nearly identical (Vaughan &
Hemingway, 1995). The CtrEst11 cDNA from Cx
tritaeniorhynchus has 88% homology with the Ests from

Cx quinquefasciatus. Immunological data suggest that this

species also has expressed Est alleles, but there is no evidence
that these are amplified alongside the CtrEst11 (Karunaratne
et al., 1997). Fig. 1 shows an unrooted evolutionary tree of the
Culex esterases and their relationship to other insect esterases,
including AChE.
The Est11 gene is 2773 base pairs long with three introns.
The Est21 gene has an identical number of introns and the
boundaries of these between the two Est genes are conserved.
The Est21 gene has six introns and three of these introns
have conserved boundaries with those of the Est genes, the
other three introns are novel and split Est21 exon 3 into exons
3, 4 and 5 and Est21 exon 4 into exons 6 and 7 (Vaughan
et al., 1995, 1997). The TEMR amplified section of DNA or
amplicon, which includes the Est11 gene, contains a highly
conserved 25 kb core sequence within an amplified sequence
of at least 30 kb (Mouche`s et al., 1992) on which there is no
amplified Est gene (Vaughan et al., 1995; Rooker et al.,
1996). In the susceptible PelSS strain the nonamplified Est3
and Est1 loci are arranged head-to-head, separated by 1.8 kb
of non-coding DNA (Vaughan et al., 1997). These nonamplified
genes are also present in strains with the amplified Est11, e.g.
strain TEMR, or Est12 (e.g. strain MRes), even when these
strains have been highly selected for elevated esterase over
many generations (Georghiou et al., 1978; Georghiou &
Pasteur, 1989). The amplified Est21 and Est21 loci, on an
amplicon of similar size to that of Est11, are also in a headto-head orientation, but the non-coding region separating them
is 2.7 kb. It is not possible to determine whether the nonamplified genes are still present in the amplified Est21 and
Est21 strains, because the larger amplification product from
the amplicon swamps the PCR reaction (Vaughan et al., 1997).
The Est and Est loci probably evolved from an initial
tandem duplication of a common ancestral locus; different
Est and Est genes and alleles could have arisen secondarily
from each of these loci forming a multigene family, a situation
resembling the organization of the mammalian and Drosophila
esterase genes (Peters, 1982; Zutphen & Bieman, 1984; Ronai
et al., 1985; Hedrich & Deimling., 1987).
Gene expression
The elevated esterases of Cx quinquefasciatus and Cx
tritaeniorhynchus appear to be constitutively expressed,

Fig. 1. Unrooted evolutionary tree of the Culex esterases and their

relationship to other B esterases including acetylcholinesterase.
Sequence alignments were done through the Megalign programme of
DNAStar using the Clustal method.

1998 Blackwell Science Ltd, Medical and Veterinary Entomology, 12, 112

J. Hemingway and S. H. P. P. Karunaratne

although there is indirect evidence from crosses to suggest that

there is variability in expression levels (see above). The Est21
and Est21 genes occur in a 1:1 ratio through their tight
linkage, but approximately 3-fold more Est21 than Est21
appears to be produced (Karunaratne, 1994). The non-coding
region between the Est and Est genes must contain the
promoter region and possibly associated cis and\or trans acting
factor binding sites. The difference between the size of noncoding DNA between the non-amplified and amplified Est
and Est genes is accounted for by two inserts, both of
500 bp (Vaughan et al., 1997). There is also a short section
of DNA in the non-amplified intergenic region at the site of
the second insertion that appears to have been deleted in the
resistant insects amplicon (see Fig. 2). The inserts are of
interest, as they contain a number of elements with high
homology to the 15 bp conserved sequence of the BARBIE
box, and have the common conserved AAAG core of other
regulatory signal sequences such as the ARE elements which
influence induction (Vaughan et al., 1997).
There is no evidence as yet in Culex that the esterase loci
can be present in the genome without being expressed, but
non-expression occurs in the aphid Myzus persicae, with
revertant insects containing high numbers of non-expressed E4
genes. The amplified aphid E4-related sequences are highly
methylated at HpaII/MSP1 restriction enzyme sites in all
resistant strains apart from insects where resistance has reverted.
This suggests that loss of methylation has resulted in the loss
of transcriptional expression of these aphid genes, giving an
apparently susceptible phenotype (Field et al., 1988). This is
the reverse of the mammalian methylation system, in which
methylated genes are transcriptionally silent and, as yet, its
mechanism is poorly understood in aphids.

Generation of different electromorphs

Specific glycosylation may explain some of the mobility
differences of the different Est and Est electromorphs. There
are five possible N-linked glycosylation sites amongst all the
Cx quinquefasciatus Est cDNAs, only one of these is common
to all four sequences and none of the esterases have all five
sites (Mouche`s et al., 1992; Vaughan & Hemingway, 1995),
whereas the CtrEst11 has four glycosylation sites including
the common one. As the esterases have different potential
glycosylation sites, allele specific glycosylation leading to
mobility differences on native PAGE is possible. The predicted
molecular weight of Est11 from the cDNA is 59 kDa, which
is 8 kDa less than its native form (Fournier et al., 1987;
Mouche`s et al., 1992). The glycosylated nature of esterase E4
of Myzus persicae has been shown by its high affinity for lectin
(Con-A Sepharose) and by non-denaturing electrophoresis
gels, stained for sugars. Differences in glycosylation may be
responsible for 68 kDa difference between the native esterases,
E4 and FE4, and their respective nascent forms (Devonshire
& Moores, 1982; Mouche`s et al., 1992). However, unlike the
aphid E4 gene, the Cx quinquefasciatus esterases lack the
initial signal sequence necessary for the secretion of the protein,
during which process glycosylation is likely to take place
(Mouche`s et al., 1992; Vaughan & Hemingway, 1995). Also,
the PelRR Est21 does not bind to Con-A Sepharose, indicating
that the participation of glucose and mannose residues in
glycosylation is insignificant in this enzyme (Karunaratne
et al., 1993).
Evolution and the spread of esterase genes
On the basis of identical restriction digest patterns of the
flanking regions of the elevated Est21 genes from different

Fig. 2. Diagrammatic representation of the intergenic spacer region between the Est21 and Est21 genes of the insecticide resistant PelRR and
susceptible PelSS strains of Culex quinquefasciatus. The two esterases are in a head-to-head orientation.

1998 Blackwell Science Ltd, Medical and Veterinary Entomology, 12, 112

Mosquito carboxylesterases
populations, it was proposed that amplification of the Est21
had occurred only once and spread worldwide (Raymond et al.,
1991). Est21 is in complete linkage disequilibrium with
Est21, as both esterases sit on the same amplicon (Rooker
et al., 1996; Vaughan et al., 1997). Therefore, Est21 has
spread concurrently with Est21. Using the amplified esterase
restriction maps and male genitalia as morphological markers
to discriminate tropical, temperate and hybrid forms of Cx
quinquefasciatus and Cx pipiens within the Cx pipiens complex,
the recent migration of the Est21 and Est21 amplicon into
Cx pipiens from France has been shown (Rivert et al., 1993).
Multiple amplified Ests and Ests can occur within a single
population of Cx quinquefasciatus. For example, in Cuba,
the field population with a high frequency of Est12 was
subsequently invaded by the Est21 and Est21 amplicon,
which is now found at a higher frequency than the Est12
amplicon (Karunaratne, 1994).
Migration has undoubtedly played an important role in the
spread of these amplicons in the presence of the positive
selection pressure of the insecticides. However, the current
evidence suggests that mutation and amplification of the Est
and Est loci must have occurred on several occasions. Hence
one interesting question is: what gives the very common
Est21\Est21 its evident selective advantage over the other
amplified esterase phenotypes? It may just be the presence of
two expressed esterases on a single amplicon. Another possible
explanation may be the presence of other genes on some, but
not all, of the amplicons. A third gene has already been
discovered on the Est21\Est21 amplicon and, if this gene is
actively transcribed, it may contribute to the fitness of
individuals carrying this amplicon.
Variability of biochemical and molecular data
Significant kinetic differences exist among Est21 and Est21
esterases purified from strains originating in different
geographical areas, with differences being greater between
Est21s than Est21s (Hemingway et al., 1993; Ketterman
et al., 1993; Karunaratne et al., 1995a). However, these strains
have identical restriction digest patterns for both the Est21
and Est21 genes (Karunaratne, submitted). There could be
several explanations for these apparently contradictory
biochemical and molecular biological data. Perhaps there are
minor differences in the gene sequences, not detectable by
restriction digestion, which result in slight kinetic differences
between the esterases. A related example is the single amino
acid substitution, in a position distinct from the active site,
which causes kinetic differences between two human serum
paraoxonases (Humbert et al., 1993). This explanation appeared
plausible with the discovery of three different deduced amino
acid residues between the reported partial Est2 sequence from
a French Cx quinquefasciatus strain (Mouche`s et al., 1992)
and the corresponding region of the PelRR Est21 (Vaughan
et al., 1995). However, extensive sequencing of genomic clones
from many strains suggests that the genes from different strains
are in fact identical; hence the differences between the earlier
readings are due to inaccuracies in the sequencing of the
partial Est2 (Vaughan et al., 1995; Vaughan & Hemingway,

unpublished data). Another possible explanation is that there are

post-transcriptional or post-translational modifications which
slightly modify the catalytic activity of the esterases. On the
basis of current literature, the existence of such a mechanism
seems unlikely. Sex-, tissue- and age-specific regulation of the
genes of esterase 6 and esterase S in Drosophila do not produce
catalytic differences (Karotam & Oakeshott, 1993; Ludwig
et al., 1993; Sergeev et al., 1993). Extensive polymorphism of
the gene product after mRNA processing and subsequent
post-translational modifications has been reported for AChE
(Chatonnet & Lockeridge, 1989; Taylor, 1991; Li et al., 1993a).
Post-translational proteolytic processing and core-glycosylation
have been studied in vitro in rat liver esterases (Robbi &
Beaufay, 1986, 1987, 1988). Two of the forms, one the result
of proteolytic processing of the other, were similar to the pI
6.0 and 6.4 esterases which had different substrate preferences
(Mentlein et al., 1984). However, it is uncertain whether such
proteolytic processing can occur in vivo (Mentlein et al., 1984;
Robbi & Beaufay, 1988).
Alternatively, as many non-amplified forms of both Est
and Est occur, it is possible that some of these were copurified
with the Est21 and Est21. This is supported by the variability
of sequence which resulted from N-terminal analysis of the
purified Est21 (Karunaratne, 1994), suggesting a mixture of
proteins. However, the extensive purification work carried out
on the non-amplified PelSS enzymes show that the bulk of the
non-amplified esterases, as detected using the antisera described
earlier, would not have been co-purified with the amplified
forms. Hence contamination should have been very minor.
Does the genomic locality of the Est and Est
genes represent an amplification hot spot?
It has been suggested on the basis of the similarity of the
amplified Est2 and Est2 restriction digest patterns
worldwide, that amplification is a rare or unique event. We
now know that, within Cx quinquefasciatus, amplification of
one or both of these esterase loci has occurred independently
at least five times for Est and at least three times for Est.
In addition to the Est2\Est2 amplicon, two co-elevated Ests
and Ests have been found in Cx pipiens in France and
Cyprus with different restriction patterns (Poire et al., 1992).
Amplification of the Est11 gene alone has occurred in the
TEMR strain of Cx quinquefasciatus. Similar restriction digest
patterns occur for Est11 in strains of the Cx pipiens complex
from France, Venezuela, Puerto Rico, U.S.A. and China (Qiao
& Raymond, 1995), whereas the distinct Est12 occurs in Cx
quinquefasciatus from Cuba (Vaughan et al., 1995).
Additionally, amplicons containing amplified Est3s, distinct
from Est21, occur in three strains of Cx quinquefasciatus,
two of which also contain the Est11 amplicon, whereas the
third has the Est12 amplicon (De Silva et al., 1997).
Further evidence that this chromosomal region represents an
amplification hot spot comes from other Culex species. In Cx
tritaeniorhynchus the CtrEst1 gene has been amplified. The
cDNA sequence for this esterase has demonstrated that this is
the heterologous gene to Est in Cx quinquefasciatus and,
unlike between Cx pipiens and Cx quinquefasciatus, there is

1998 Blackwell Science Ltd, Medical and Veterinary Entomology, 12, 112

J. Hemingway and S. H. P. P. Karunaratne

no possibility of this amplification having occurred through

gene flow from the Cx pipiens complex (Karunaratne et al.,
1997). Additionally, elevation of esterase activity associated
with insecticide resistance has been reported in another Culex
species group, that of Cx tarsalis (Ziegler et al.,1987).
The mechanism of amplification
Transposable elements or long interspersed repetitive elements
(LINEs) which are capable of accelerating the frequency of
gene mutation and gene amplification have been found in
association with the mosquito carboxylesterase genes. LINEs,
designated as Juan-C, are closely associated with the amplified
Est11 in the TEMR strain of Cx quinquefasciatus (Mouche`s
et al., 1992). Transposition can be influenced by environmental
factors such as insecticidal pressure which may play an
important role in adaptation (Wilson, 1993). Many full-length
copies of Juan-C LINEs have been described recently from
the genomes of different strains of Cx quinquefasciatus that
originated from different continents (Mouche`s et al., 1986;
Agarwal et al., 1993). However, the different levels of
amplification of the amplicon core and extremities make
it unlikely that active transposition has been involved in
amplification (Besansky et al., 1992). It has been shown that
amplification is a rare event and its frequency in cultured
mammalian cells is 106104 (Schimke, 1984). Point mutation
is also a rare event, but the changes that have occurred between
Est alleles are all due to such single nucleotide substitutions
rather than chromosomal rearrangements, as the nucleotide
changes are scattered throughout the coding sequence (Vaughan
et al., 1995).
Knowledge of kinetic and catalytic properties, and molecular
structure of the amplified carboxylesterases should lead to their
manipulation to facilitate insect control. The different esterase
genes are now being expressed in a baculovirus system and
site-directed mutagenesis of these enzymes and the subsequent
characterization of the proteins will allow further elucidation
of the gene-protein structural and functional relationships.
Understanding the possible events/factors which can influence
the mutation and amplification of these esterase genes
(Guillemaud et al., 1997) should help towards developing new
strategies for the prevention and management of resistance.
Accession numbers: The Culex nucleotide sequences reported
in this paper have been submitted to GenBankTM\EMBL Data
Bank with accession numbers Z32694, Z32696 and Z47988.
References
Agarwal, M.L., Bensaadi, N., Salvado, J.C., Campbell, K. & Mouche`s,
C. (1993) Characterization and genetic organization of full-length
copies of a LINE retroposon family dispersed in the genome of Culex
pipiens mosquitoes. Insect Biochemistry and Molecular Biology, 23,
621629.
Aldridge, W.N. (1953a) Serum esterases. 1. Two types of esterases (A
and B) hydrolysing p-nitrophenyl acetate, propionate and butyrate, and
a method for their determination. Biochemical Journal, 53, 110117.
Aldridge, W.N. (1953b) Serum esterases. 2. An enzyme hydrolysing
diethyl p-nitrophenyl phosphate (E600) and its identity with the Aesterase of mammalian sera. Biochemical Journal, 53, 117124.

Aldridge, W.N. (1993) The esterases perspectives and problems.

ChemicoBiological Interactions, 87, 513.
Apperson, C.S. & Georghiou, G.P. (1975) Mechanisms of resistance to
organophosphorus insecticides in Culex tarsalis. Journal of Economic
Entomology, 68, 153157.
Bender, K., Adams, M., Baverstock, P. et al. (1984) Biochemical markers
in inbred strains of the rat (Rattus norvegicus). Immunogenetics, 19,
257266.
Besansky, N.J., Finnerty, V. & Collins, F.H. (1992) Molecular
perspectives on the genetics of mosquitoes. Advances in Genetics, 30,
123184.
Beyssat-Arnaouty, V., Mouche`s, C., Georghiou, G.P. & Pasteur, N. (1989)
Detection of organophosphate detoxifying esterases by dot-blot
immunoassay in Culex mosquitoes. Journal of the American Mosquito
Control Association, 5, 196200.
Bisset, J.A., Ortiz, E., Rodriguez, M. & Hemingway, J. (1995)
Comparison of microtitre plate and filter paper assays of elevated
esterase-based resistance frequencies in field and laboratory
populations of the mosquito Culex quinquefasciatus from Cuba.
Medical and Veterinary Entomology, 9, 9497.
Bisset, J.A., Rodriguez, M.M., Diaz, C., Ortiz, E., Marquetti, M.C.
& Hemingway, J. (1990) The mechanisms of organophosphate and
carbamate resistance in Culex quinquefasciatus (Diptera: Culicidae)
from Cuba. Bulletin of Entomological Research, 80, 245250.
Boddington, R.G. (1992) Characterisation of malathion
carboxylesterases and non-specific esterases in the malaria mosquito
Anopheles stephensi. PhD thesis. University of London.
Chatonnet, A. & Lockridge, O. (1989) Comparison of
butyrylcholinesterase and acetylcholinesterase. Biochemical Journal,
260, 625634.
Cygler, M., Schrag, J.D., Sussman, J.L., Harel, M., Silman, I., Gentry,
M.K. & Doctor, B.P. (1993) Relationship between sequence
conservation and three-dimensional structure in a large family of
esterases, lipases and related proteins. Protein Science, 2, 366382.
Dary, O., Georghiou, G.P., Parsons, E. & Pasteur, N. (1990) Microplate
adaptation of Gomoris assay for quantitative determination of general
esterase activity in single insects. Journal of Economic Entomology,
83, 21872192.
De Silva, D., Hemingway, J., Ranson, H. & Vaughan, A. (1997) Est3,
a novel amplified esterase associated with Est1s from insecticide
resistant strains of the mosquito Culex quinquefasciatus. Experimental
Parasitology, in press.
De Stordeur, E. (1976) Esterases in the mosquito Culex pipiens L.: formal
genetics and polymorphism of adult esterases. Biochemical Genetics,
14, 481493.
Devonshire, A.L. (1975) Studies of the carboxylesterases of Myzus
persicae resistant and susceptible to organophosphorus insecticides.
Proceedings of the 8th British Insecticide Fungicide Conference,
6773.
Devonshire, A.L., Devine, G.J. & Moores, G.D. (1992) Comparison of
microplate esterase assays and immunoassay for identifying
insecticide resistant variants of Myzus persicae (Homoptera:
Aphidae). Bulletin of Entomological Research, 82, 459464.
Devonshire, A.L. & Field, L.M. (1991) Gene amplification and
insecticide resistance. Annual Review of Entomology, 36, 123.
Devonshire, A.L. & Moores, G.D. (1982) A carboxylesterase with broad
substrate specificity causes organophosphorus, carbamate and
pyrethroid resistance in peach-potato aphids (Myzus persicae).
Pesticide Biochemistry and Physiology, 18, 235246.
Devonshire, A.L., Moores, G.D. & ffrench-Constant, R.H. (1986a)
Detection of insecticide resistance by immunological estimation of
carboxylesterase activity in Myzus persicae (Sulzer) and cross reaction
of the antiserum with Phorodon humuli (Shrank) (Hemiptera:
Aphididae). Bulletin of Entomological Research, 76, 97107.

1998 Blackwell Science Ltd, Medical and Veterinary Entomology, 12, 112

Mosquito carboxylesterases
Devonshire, A.L., Searle, L.M. & Moores, G.D. (1986b) Quantitative
and qualitative variation in the messenger-RNA for carboxylesterases
in insecticide-susceptible and resistant Myzus persicae (Sulz). Insect
Biochemistry, 16, 659665.
Du, B.N.L., Adkins, S., Kuo, C.X. & Lipsig, D. (1993) Studies on human
serum paraoxonase/arylesterase. ChemicoBiological Interactions,
87, 2534.
Ferrari, J.A. & Georghiou, G.P. (1990) Esterase B1 activity variation
within and among insecticide resistant, susceptible and heterozygous
strains of Culex quinquefasciatus (Diptera, Culicidae). Journal of
Economic Entomology, 83, 17041710.
Field, L.M., Devonshire, A.L., ffrench-Constant, R.H. & Forde, B.G.
(1989) Changes in DNA methylation are associated with loss of
insecticide resistance in the peach-potato aphid Myzus persicae (Sulz).
FEBS Letters, 243, 323327.
Field, L.M., Devonshire, A.L. & Forde, B.G. (1988) Molecular evidence
that insecticide resistance in peach-potato aphids (Myzus persicae
Sulz.) results from amplification of an esterase gene. Biochemical
Journal, 251, 309312.
Fournier, D., Bride, J.-M., Mouche`s, C., Raymond, M., Magnin, M.,
Berge, J.-B., Pasteur, N. & Georghiou, G.P. (1987) Biochemical
characterisation of the esterases A1 and B1 associated with
organophosphate resistance in the Culex pipiens L. complex. Pesticide
Biochemistry and Physiology, 27, 211217.
Furlong, C.E., Costa, L.G., Hassett, C. et al. (1993) Human and rabbit
paraoxonases: purification, cloning, sequencing, mapping and role of
polymorphism in organophosphate detoxification. ChemicoBiological Interaction, 87, 3548.
Georghiou, G.P., Ariaratnam, V., Pasternak, M.E. & Lin, S.C. (1978)
Organophosphorus multi-resistance in Culex pipiens quinquefasciatus
in California. Journal of Economic Entomology, 68, 461467.
Georghiou, G.P. & Pasteur, N. (1978) Electrophoretic esterase patterns in
insecticide resistant and susceptible mosquitoes. Journal of Economic
Entomology, 71, 201205.
Georghiou, G.P. & Pasteur, N. (1989) Novel tests for organophosphate
insecticide resistance in single mosquitoes: an overview of recent
progress and outline of filter paper test. 57th Annual Conference of the
California Mosquito and Vector Control Association, 174178.
Georghiou, G.P., Pasteur, N. & Hawley, M.K. (1980) Linkage
relationship between organophosphate resistance and a highly active
esterase-B in Culex quinquefasciatus from California. Journal of
Economic Entomology, 73, 301305.
Guillemaud, T., Makate, N., Raymond, M., Hirst, B. & Callaghan, A.
(1997) Esterase gene amplification in Culex pipiens. Insect Molecular
Biology, 6, 319327.
Guillemaud, T., Rooker, S., Pasteur, N. & Raymond, M. (1996) Testing
the unique amplification event and the world-wide migration
hypothesis of insecticide resistance genes with sequence data.
Heredity, 7, 535543.
Healy, M.J., Dumancic, M.M. & Oakeshott, J.G. (1991) Biochemical
and physiological studies of soluble esterases from Drosophila
melanogaster. Biochemical Genetics, 29, 365388.
Hedrich, H.J. & Deimling, O. (1987) Re-evaluation of LGV of the rat
and assignment of 12 carboxylesterases to two gene clusters. The
Journal of Heredity, 78, 9296.
Hemingway, J. (1982) The biochemical nature of malathion resistance
in Anopheles stephensi from Pakistan. Pesticide Biochemistry and
Physiology, 17, 149155.
Hemingway, J. (1983a) Biochemical studies on malathion resistance in
Anopheles arabiensis from Sudan. Transactions of the Royal Society
of Tropical Medicine and Hygiene, 77, 477480.
Hemingway, J. (1983b) The genetics of malathion resistance in
Anopheles stephensi from Pakistan. Transactions of the Royal Society
of Tropical Medicine and Hygiene, 77, 106108.

Hemingway, J. (1985) Malathion carboxylesterase enzymes in Anopheles

arabiensis from Sudan. Pesticide Biochemistry and Physiology, 23,
309313.
Hemingway, J. (1989) A note on simple biochemical methods for
resistance detection and their field application in Sri Lanka. Pesticide
Science, 27, 281285.
Hemingway, J., Callaghan, A. & Kurtak, D.C. (1989) Temephos
resistance in Simulium damnosum Theobald (Diptera: Simulidae): a
comparative study between larvae and adults of the forest and savanna
strains of this species complex. Bulletin of Entomological Research,
79, 659669.
Hemingway, J., Ketterman, A.J., Karunaratne, S.H.P.P., Jayawardena,
K.G.I., & Vaughan, A. (1993) Amplified esterases A2 and B2: has
resistance occurred once or several times? Proceedings of the 1st
International Congress of Insect Pests in the Urban Environment, 1,
319328.
Herath, P.R.J., Hemingway, J., Weerasinghe, I.S. & Jayawardena, K.G.I.
(1987) The detection and characterization of malathion resistance in
field populations of Anopheles culicifacies B in Sri Lanka. Pesticide
Biochemistry and Physiology, 29, 157162.
Heymann, E. & Jakoby, W.B. (1980) Carboxylesterases and amidases.
Enzymatic basis of detoxication, Volume II. (ed. by E. Heymann and
W. B. Jokoby) pp. 291323. Academic Press, New York.
Heyse, D., Catalan, J., Nance, E., Britton-Davidian, J. & Pasteur, N.
(1996) Unconventional organization of amplified esterase B gene in
insecticide-resistant mosquitoes of the Culex pipiens complex. Journal
of the American Mosquito Control Association, 13, 199205.
Humbert, R., Adler, D.A., Disteche, C.M., Hassett, C., Omiecinski, C.J.
& Furlong, C.E. (1993) The molecular basis of the human serum
paraoxonase activity polymorphism. Nature Genetics, 3, 7376.
IUBMB (1992) Enzyme Nomenclature: Recommendations 1992.
International Union of Biochemistry and Molecular Biology,
Nomenclature Committee. Academic Press, San Diego, U.S.A.
Jayawardena, K.G.I. (1992) Purification and characterisation of two
carboxylesterases from organophosphate resistant Culex
quinquefasciatus from Sri Lanka. PhD thesis. University of London.
Jayawardena, K.G.I., Karunaratne, S.H.P.P., Ketterman, A.J. &
Hemingway, J. (1994) Determination of the role of elevated B2
esterase in insecticide resistance in Culex quinquefasciatus (Diptera:
Culicidae) from studies on the purified enzyme. Bulletin of
Entomological Research, 84, 3944.
Kao, L.R., Motoyama, N. & Dauterman, W.C. (1985) The purification
and characterisation of esterases from insecticide resistant and
susceptible houseflies. Pesticide Biochemistry and Physiology, 23,
228239.
Karotam, J. & Oakeshott, J.G. (1993) Regulatory aspects of esterase-6
activity variation in sibling Drosophila species. Heredity, 71, 4150.
Karunaratne, S.H.P.P. (1994) Characterisation of multiple variants of
carboxylesterases which are involved in insecticide resistance in the
mosquito Culex quinquefasciatus. PhD thesis, University of London.
Karunaratne, S.H.P.P. & Hemingway, J. (1996) Different insecticides
select multiple carboxylesterase isoenzymes and different resistance
levels from a single population of Culex quinquefasciatus. Pesticide
Biochemistry and Physiology, 54, 411.
Karunaratne, S.H.P.P., Hemingway, J., Jayawardena, K.G.I.,
Dassanayaka, V. & Vaughan, A. (1995a) Kinetic and molecular
differences in the amplified and non-amplified esterases from
insecticide resistant and susceptible Culex quinquefasciatus
mosquitoes. Journal of Biological Chemistry, 270, 3112431128.
Karunaratne, S.H.P.P., Jayawardena, K.G.I. & Hemingway, J. (1995b)
The cross-reactivity spectrum of a polyclonal antiserum raised against
the A2 esterase involved in insecticide resistance. Pesticide
Biochemistry and Physiology, 53, 7583.

1998 Blackwell Science Ltd, Medical and Veterinary Entomology, 12, 112

10

J. Hemingway and S. H. P. P. Karunaratne

Karunaratne, S.H.P.P., Jayawardena, K.G.I., Hemingway, J. &

Ketterman, A.J. (1993) Characterisation of a B-type esterase involved
in insecticide resistance from the mosquito Culex quinquefasciatus.
Biochemical Journal, 294, 575579.
Karunaratne, S.H.P.P., Vaughan, A., Paton, M.G. & Hemingway, J. (1998)
Amplification of a serine esterase is involved in insecticide resistance
in Sri Lankan Culex tritaeniorhynchus. Insect Molecular Biology,
in press.
Ketterman, A.J., Jayawardena, K.G.I. & Hemingway, J. (1992)
Purification and characterisation of a carboxylesterase involved in
insecticide resistance from the mosquito Culex quinquefasciatus.
Biochemical Journal, 287, 355360.
Ketterman, A.J., Karunaratne, S.H.P.P., Jayawardena, K.G.I. &
Hemingway, J. (1993) Qualitative differences between populations of
Culex quinquefasciatus in both the esterases A2 and B2 which are
involved in insecticide resistance. Pesticide Biochemistry and
Physiology, 47, 142148.
Li, Y., Camp, S. & Taylor, P. (1993a) Tissue specific expression and
alternative mRNA processing of the mammalian acetylcholinesterase
gene. Journal of Biological Chemistry, 268, 57905797.
Li, W.F., Costa, L.G. & Furlong, C.E. (1993b) Serum paraoxonase status
a major factor in determining resistance to organophosphates. Journal
of Toxicology and Environmental Health, 40, 337346.
Lines, J.D., Ahmed, M.A.E. & Curtis, C.F. (1984) Genetic studies of
malathion resistance in Anopheles arabiensis Patton (Diptera:
Culicidae). Bulletin of Entomological Research, 74, 317325.
Ludwig, M.Z., Tamarina, N.A. & Richmond, R.C. (1993) Localisation
of sequences controlling the spatial, temporal and sex-specific
expression of the esterase 6 locus in Drosophila melanogaster adults.
Proceedings of the National Academy of Sciences of the United States
of America, 90, 62336237.
Magnin, M., Marboutin, E. & Pasteur, N. (1988) Insecticide resistance
in Culex quinquefasciatus (Diptera: Culicidae) in West-Africa.
Journal of Medical Entomology, 25, 99104.
Malcolm, C.A. & Boddington, R.G. (1989) Malathion resistance
conferred by a carboxylesterase in Anopheles culicifacies Giles
(Species B) (Diptera: Culicidae). Bulletin of Entomological Research,
79, 193199.
Matsumara, F. & Brown, A.W.A. (1961) Biochemistry of malathion
resistance in Culex tarsalis. Journal of Economic Entomology, 54,
11761185.
Mentlein, R., Berge, R.K. & Heymann, E. (1985) Identity of purified
monoacylglycerol lipase, palmitoyl-CoA hydrolase and aspirinmetabolizing carboxylesterase from rat-liver microsomal fractions
a comparative study with enzymes purified in different laboratories.
Biochemical Journal, 232, 479483.
Mentlein, R., Suttorp, M. & Heymann, E. (1984) Specificity of purified
monoacylglycerol lipase, palmitoyl-coA hydrolase, palmitoylcarnitine hydrolase and nonspecific carboxylesterase from rat liver
microsomes. Archives of Biochemistry and Biophysics, 228, 230246.
Mouche`s, C., Bensaadi, N. & Salvado, J. (1992) Characterization of a
LINE retroposon dispersed in the genome of three non-sibling Aedes
mosquito species. Gene, 120, 183190.
Mouche`s, C., Magnin, M., Berge, J.B., De Silvestri, M., Beyssat, V.,
Pasteur, N. & Georghiou, G.P. (1987) Overproduction of detoxifying
esterases in organophosphate-resistant Culex mosquitoes and their
presence in other insects. Proceedings of the National Academy of
Sciences of the United States of America, 84, 21132116.
Mouche`s, C., Pasteur, N., Berge, J.B., Hyrien, O., Raymond, M., De
Saint Vincent, B.R., De Silvestri, M. & Georghiou, G.P. (1986)
Amplification of an esterase gene is responsible for insecticide
resistance in a Californian Culex mosquito. Science, 233, 778780.
Mouche`s, C., Pauplin, Y., Agarwal, M. et al. (1990) Characterisation of

amplification core and esterase B1 gene responsible for insecticide

resistance in Culex. Proceedings of the National Academy of Sciences
of the United States of America, 87, 25742578.
Nance, E., Heyse, D., Britton-Davidian, J. & Pasteur, N. (1990)
Chromosomal organisation of the amplified esterase B1 gene in
organophosphate-resistant Culex pipiens qinquefasciatus Say
(Diptera:Culicidae). Genome, 33, 148152.
Pasteur, N. & Georghiou, G.P. (1981) Filter paper test for rapid
determination of phenotypes with high esterase activity in
organophosphate resistant mosquitoes. Mosquito News, 41, 181183.
Pasteur, N., Iseki, A. & Georghiou, G.P. (1981a) Genetic and biochemical
studies of the highly active esterases A and B associated with
organophosphate resistance in mosquitoes of the Culex pipiens
complex. Biochemical Genetics, 19, 909919.
Pasteur, N., Sine`gre, G. & Gabinaud, A. (1981b) Est-2 and Est-3
polymorphisms in Culex pipiens L. from Southern France in relation
to organophosphate resistance. Biochemical Genetics, 19, 499508.
Peiris, H.T.R. & Hemingway, J. (1993) Characterisation and inheritance
of elevated esterases in organophosphorus and carbamate insecticide
resistant Culex quinquefasciatus (Diptera: Culicidae) from Sri Lanka.
Bulletin of Entomological Research, 83, 127132.
Peters, J. (1982) Non-specific esterases of Mus musculus. Biochemical
Genetics, 20, 585606.
Poirie, M., Raymond, M. & Pasteur, N. (1992) Identification of two
distinct amplifications of the esterase B locus in Culex pipiens (L.)
mosquitoes from Mediterranean countries. Biochemical Genetics, 30,
1326.
Prabhakaran, S.K. & Kamble, S.T. (1993) Activity and electrophoretic
characterisation of esterases in insecticide-resistant and susceptible
strains of German-cockroach (Dictyoptera: Blattellidae). Journal of
Economic Entomology, 86, 10091013.
Prabhaker, N., Georghiou, G.P. & Pasteur, N. (1987) Genetic association
between highly active esterases and organophosphate resistance in
Culex tarsalis. Journal of the American Mosquito Control Association,
3, 473475.
Qiao, C.-L. & Raymond, M. (1995) The same esterase B1 haplotype is
amplified in insecticide resistant mosquitoes of the Culex pipiens
complex from the Americas and China. Heredity, 74, 339345.
Raymond, M., Beyssat-Arnaouty, V., Sivasubramanian, N., Mouche`s,
C., Georghiou, G.P. & Pasteur, N. (1989) Amplification of various
Esterase Bs responsible for organophosphate resistance in Culex
mosquitoes. Biochemical Genetics, 27, 417423.
Raymond, M., Callaghan, A., Fort, P. & Pasteur, N. (1991) World-wide
migration of amplified insecticide resistance genes in mosquitoes.
Nature, 350, 151153.
Raymond, M., Pasteur, N., Georghiou, G.P., Mellon, R.B., Wirth, M.C.
& Hawley, M. (1987) Detoxification esterases new to California,
USA, in organophosphate resistant Culex quinquefasciatus (Diptera:
Culicidae). Journal of Medical Entomology, 24, 2427.
Reiner, E. (1993) Recommendations of the IUBMB nomenclature
committee comments concerning classification and nomenclature
of esterases hydrolysing organophosphorus compounds. Chemico
Biological Interactions, 87, 1516.
Rivert, Y., Marquine, M. & Raymond, M. (1993) French mosquito
populations invaded by A2-B2 esterases causing insecticide resistance.
Biological Journal of the Linnean Society, 49, 249255.
Robbi, M. & Beaufay, H. (1986) Biosynthesis of rat liver pI 5.0 esterases
in cell-free systems and in cultured-hepatocytes. European Journal of
Biochemistry, 158, 187194.
Robbi, M. & Beaufay, H. (1987) Biosynthesis of rat-liver pI 6.1 esterase,
a carboxylesterase of the cisternal space of the endoplasmic-reticulum.
Biochemical Journal, 248, 545550.
Robbi, M. & Beaufay, H. (1988) Immunochemical characterisation and

1998 Blackwell Science Ltd, Medical and Veterinary Entomology, 12, 112

Mosquito carboxylesterases

11

biosynthesis of pI 6.4 esterase, a carboxylesterase of rat liver

microsomal extracts. Biochemical Journal, 254, 5157.
Rodriguez, M., Ortiz, E., Bisset, J.A., Hemingway, J. & Saledo, E. (1993)
Changes in malathion and pyrethroid resistance after cypermethrin
selection of Culex quinquefasciatus field populations of Cuba. Medical
and Veterinary Entomology, 7, 117121.
Ronai, A., Wassmer, B., Looze, S.D. & Deimling, O.V. (1985)
Immunochemical interrelationships between carboxylesterase
isozymes (EC-3.1.1.1) of the house mouse, Mus musculus.
Comparative Biochemistry and Physiology B-Comparative
Biochemistry, 82, 347355.
Rooker, S., Guillemaud, T., Berge, J., Pasteur, N. & Raymond, M. (1996)
Co-amplification of esterase A and B genes as a single unit in Culex
pipiens mosquitoes. Heredity, 77, 555561.
Rowland, M. & Hemingway, J. (1987) Changes in malathion resistance
with age in Anopheles stephensi from Pakistan. Pesticide Biochemistry
and Physiology, 28, 239247.
Schimke, R.T. (1984) Gene amplification in cultured animal cells. Cell,
37, 705713.
Sergeev, P.V., Yenikolopov, G.N., Peunova, N.I., Kuzin, B.A.,
Khechumian, R.A., Korochkin, L.I. & Georgiev, G.P. (1993)
Regulation of tissue-specific expression of the esterase-s gene in
Drosophila virilis. Nucleic Acids Research, 21, 35453551.
Severini, C., Romi, R., Marinucci, M., Guillemaud, T. & Raymond, M.
(1997) Esterases A5B5 in organophosphate resistant Culex pipiens
from Italy. Medical & Veterinary Entomology, 11, 123126.
Simon, B., Looze, S., Ronai, A. & Deimling, O. (1985) Identification of
rat liver carboxylesterase isozymes (EC 3.1.1.1.) using
polyacrylamide gel electrophoresis and iso-electric focusing.
Electrophoresis, 6, 575582.
Small, G.J., Karunaratne, S.H.P.P. & Hemingway, J. (1998)
Characterisation of amplified Est1 associated with organophosphate
resistance in a multi-resistant population of the mosquito Culex
quinquefasciatus (Diptera: Culicidae) from Cuba. Medical and
Veterinary Entomology, 12, in press.
Takahashi, M. & Yasutomi, K. (1987) Insecticidal resistance of Culex
tritaeniorhynchus (Diptera: Culicidae) in Japan: Genetics and
mechanisms of resistance to organophosphorus insecticides. Journal
of Medical Entomology, 24, 595603.
Taylor, P. (1991) The cholinesterases. Journal of Biological Chemistry,
266, 40254028.
Tewfik, H.R. & Barr, A.R. (1974) The salivary gland chromosomes of
Culex pipiens L. Mosquito News, 34, 4754.
Vaughan, A., Hawkes, N. & Hemingway, J. (1997) Co-amplification

explains linkage disequilibrium of two mosquito esterase genes in

insecticide resistant Culex quinquefasciatus. Biochemical Journal,
325, 359365.
Vaughan, A. & Hemingway, J. (1995) Mosquito carboxylesterase Est21
(A2). Cloning and sequence of the full length cDNA for a major
insecticide resistance gene world-wide in the mosquito Culex
quinquefasciatus. Journal of Biological Chemistry, 270, 17044
17049.
Vaughan, A., Rodriguez, M. & Hemingway, J. (1995) The independent
gene amplification of indistinguishable esterase B electromorphs from
the insecticide resistant mosquito Culex quinquefasciatus.
Biochemical Journal, 305, 651658.
Villani, F., White, G.B., Curtis, C.F. & Miles, S.J. (1983) Inheritance and
activity of some esterases associated with organophosphate resistance
in mosquitoes of the complex of Culex pipiens L. (Diptera: Culicidae).
Bulletin of Entomological Research, 73, 153170.
Walker, C.H. (1993) The classification of esterases which hydrolyse
organophosphates recent developments. ChemicoBiological
Interactions, 87, 1724.
White, G.B. (1981) Academic and applied aspects of mosquito
cytogenetics. Insect Cytogenetics (ed. by R. L. Blackman et al.)
Symposia of the Royal Entomological Society, 10, 245274.
Wilson, T.G. (1993) Transposable elements as initiators of insecticide
resistance. Journal of Economic Entomology, 86, 645651.
Wirth, M.C., Marquine, M., Georghiou, G.P. & Pasteur, N. (1990)
Esterases A2 and B2 in Culex quinquefasciatus (Diptera: Culicidae):
role in organophosphate resistance and linkage. Journal of Medical
Entomology, 27, 202206.
Ziegler, R., Whyard, S., Downe, A.E.R., Wyatt, G.R. & Walker, V.K.
(1987) General esterase, malathion carboxylesterase and malathion
resistance in Culex tarsalis. Pesticide Biochemistry and Physiology,
28, 279285.
Zouros, E., Delden, W., Odense, R. & Dijk, H. (1982) An esterase
duplication in Drosophila: differences in expression of duplicate loci
within and among related species. Biochemical Genetics, 20, 929942.
Zutphen, L.F.M.V. (1983) Revision of the genetic nomenclature of
esterase loci in the rat (Rattus norvegicus). Transplantation
Proceedings, 15, 16871688.
Zutphen, L.F.M. & Bieman, M.G.C.W. (1984) Rat esterases: an update
of linkage group V. Journal of Hereditary, 75, 313.
Zutphen, L.F.M., Bieman, M.G.C.W., Deimling, O. & Fox, R.R. (1987)
Genetics of a tissue esterase polymorphism (Est-6) in the rabbit
(Oryctolagus cuniculus). Biochemical Genetics, 25, 335344.

Editorial Postscript. The mosquitoes Culex quinquefasciatus

Say 1823 and Cx pipiens Linnaeus 1758 are generally regarded
as a pair of sister species or subspecies, the main members of the
cosmopolitan Culex (Culex) pipiens group or species complex,
collectively known as Cx pipiens sensu lato (s.l.). Until the 1980s
Cx quinquefasciatus was often called Cx fatigans Wiedemann
1828, a junior synonym (Belkin, 1977). Culex quinquefasciatus
(redescribed by Sirivanakarn & White, 1978) is an urban pest in
hotter parts of the world, breeding perennially in polluted water
and transmitting pathogens causing human diseases (arboviruses
and filariasis), hence the importance of its control. Culex pipiens
(redescribed by Harbach et al., 1985) inhabits temperate latitudes
and cooler tropical highlands, usually overwintering by female
hibernation. Culex pipiens is the type-species of the genus Culex
Linnaeus, from which the family name Culicidae is derived
(Edwards, 1932). Typical Cx pipiens sensu stricto (s.s.) is not
anthropophilic, but the biotype Cx pipiens form molestus Forskal

1775 (redescribed by Harbach et al., 1984) is a widespread manbiting nuisance characterized by autogeny and stenogamy
without diapause. Intergradation between pipiens and
quinquefascatus occurs in some hybrid zones, e.g. around the
Madagascan plateau (Urbanelli et al., 1995) and in North
America between latitudes 36 and 39 North (Barr, 1957), but
these two taxa remain generally distinct worldwide. Populations
of Cx pipiens s.l. with intermediate pipiens/quinquefasciatus
characteristics predominate in Japan (Tanaka et al., 1979), where
they are known as Cx pipiens pallens Coquillett 1898. See
Mattingly et al. (1951), Jupp (1978), White (1979), Subra (1981),
Barr (1982), Cheng et al., 1982), Miles (1982), Urbanelli et al.
(1985, 1997), Cranston et al. (1987), Pryor & Daly (1991), Ishii
(1991), Vinogradova (1992), Tongyan & Baolin (1995) and
Debrunner-Vossbrinck et al, 1996) for biosystematics of the Cx
pipiens complex relevant to population genetics of their
esterases. Crabtree et al. (1997) identified a pipiens-specific

Accepted 3 August 1997

1998 Blackwell Science Ltd, Medical and Veterinary Entomology, 12, 112

12

J. Hemingway and S. H. P. P. Karunaratne

DNA sequence of 600 bp and developed a PCR assay to

distinguish pipiens from quinquefasciatus.
Two more Culex species with esterases classified by
Hemingway & Karunaratne (1998) belong to other species
groups within subgenus Culex of the genus Culex Linnaeus.
Culex tarsalis Coquillett 1896 is the nominotypical member of an
important species group of American arbovirus vectors (Darsie &
Ward, 1981). Culex tritaeniorhynchus belongs to the
Palaeotropical Vishnui subgroup of the Sitiens group
(Sirivanakarn, 1976). Culex tritaeniorhynchus is the major vector
of Japanese Encephalitis in the Oriental Region and the range of
this species extends to the Afrotropical Region. G. B. White
References
Barr, A.R. (1957) The distribution of Culex pipiens pipiens and Culex
pipiens quinquefasciatus in North America. American Journal of
Tropical Medicine and Hygiene, 6, 153165.
Barr, A.R. (1982) The Culex pipiens complex. Recent Developments in
the Genetics of Insect Disease Vectors (ed. by W. W. M. Steiner et al.),
pp. 551572. Stiles Publishing Co., Champaign, Illinois, U.S.A.
Belkin, J.N. (1977) Quinquefasciatus or fatigans for the tropical
(southern) house mosquito (Diptera: Culicidae). Proceedings of the
Entomological Society of Washington, 79, 4552.
Cheng, M.L., Hacker, C.S., Pryor, S.C., Ferrell, R.E. & Kitto, G.B.
(1982) The ecological genetics of the Culex pipiens complex in North
America. Recent Developments in the Genetics of Insect Disease
Vectors (ed. by W. W. M. Steiner et al.), pp. 581627. Stiles Publishing
Co., Champaign, Illinois, U.S.A.
Coquillett, D.W. (1896) New Culicidae from North America. Canadian
Entomologist, 28, 4344.
Coquillett, D.W. (1898) Report on a collection of Japanese Diptera,
presented to the U.S. National Museum by the Imperial University
of Tokyo. Proceedings of the United States National Museum, 21,
301340.
Crabtree, M.B., Savage, H.M. & Miller, B.R. (1997) Development of a
polymerase chain reaction assay for differentiation between Culex
pipiens pipiens and Cx p. quinquefasciatus (Diptera: Culicidae) in
North America based on genomic differences identified by subtractive
hybridization. Journal of Medical Entomology, 34, 532537.
Cranston, P.S., Ramsdale, C.D., Snow, K.R. & White, G.B. (1987) Keys
to the Adults, Male Hypopygia, Fourth Instar Larvae and Pupae of
the British Mosquitoes (Culicidae) with Notes on their Ecology and
Medical Importance. Freshwater Biological Association. Scientific
Publication No. 48 152 pp.
Darsie, R.F. & Ward, R.A. (1981) Identification and geographical
distribution of the mosquitoes of North America, north of Mexico.
Mosquito Systematics. Supplement 1.
Debrunner-Vossbrinck, B.A., Vossbrinck, C.R., Vodkin, M.H. & Novak,
R.J. (1996) Restriction analysis of the ribosomal DNA internal
transcribed spacer region of Culex restuans and mosquitoes in the
Culex pipiens complex. Journal of the American Mosquito Control
Association, 12, 477482.
Edwards, F.W. (1932) Diptera Fam. Culicidae. Genera Insectorum (P.
Wytsman), Quatre-Bras, Tervueren, Belgium. Fascicle 194.
Forskal, P. (1775) Descriptiones Animalium, Avium, Amphibiorum,
Piscium, Insectorum, Vermium, quae in itinere orientali observavit,
post mortem auctoris edidit Carsten Niebuhr. Havniae, Moeller.
Harbach, R.E., Dahl, C. & White, G.B. (1985) Culex (Culex) pipiens
Linnaeus (Diptera: Culicidae): concepts, type designations, and
description. Proceedings of the Entomological Society of Washington,
87, 124.

Harbach, R.E., Harrison, B. & Gad, A.M. (1984) Culex (Culex) molestus
Forskal (Diptera: Culicidae). Neotype designation, description,
variation and taxonomic status. Proceedings of the Entomological
Society of Washington, 86, 521542.
Hemingway, J. & Karunaratne, S.H.P.P. (1998) Mosquito
carboxylesterases: a review of the molecular biology and biochemistry
of a major insecticide resistance mechanism. Medical and Veterinary
Entomology, 12, 111.
Ishii, T. (1991) Integrated study on the Culex pipiens complex species
diversion in the Culex pipiens complex. Akaieka Newsletter, 14, 540.
Jupp, P.G. (1978) Culex (Culex) pipiens pipiens Linnaeus and Culex
(Culex) pipiens quinquefasciatus Say in South Africa: morphological
and reproductive isolation in favour of their status as two species.
Mosquito Systematics, 10, 461473.
Linnaeus, C. (1758) Systema Naturae per Regna Tria Naturae. 10th edn.
Holmiae. Vol. 1.
Mattingly, P.F., Rozeboom, L.E., Knight, K.L., Laven, H., Drummond,
F.M., Christophers, S.R. & Shute, P.G. (1951) The Culex pipiens
complex. Transactions of the Royal Entomological Society of London,
102, 33138.
Miles, S.J. (1982) Genetic relationships among members of the Culex
pipiens group of taxa: the practical implications. Recent Developments
in the Genetics of Insect Disease Vectors. (ed. by W. W. M. Steiner
et al.) pp. 573580. Stiles Publishing Co., Champaign, Illinois, U.S.A.
Pryor, S.C. & Daly, J. (1991) Temporal variation in morphological and
genetic characteristics within a hybrid population of Culex pipiens
(Diptera: Culicidae). Journal of Medical Entomology, 28, 481486.
Say, T. (1823) Descriptions of dipterous insects of the United States.
Journal of the Academy of Natural Sciences of Philadelphia, 3, 954.
Sirivanakarn, S. (1976) A revision of the subgenus Culex in the Oriental
Region (Diptera: Culicidae). Medical Entomology Studies III.
Contributions of the American Entomological Institute, 12, iii 1 272
pp.
Sirivanakarn, S. & White, G.B. (1978) Neotype designation of Culex
quinquefasciatus Say (Diptera: Culicidae). Proceedings of the
Entomological Society of Washington, 80, 360372.
Subra, R. (1981) Biology and control of Culex pipiens quinquefasciatus
Say 1823 (Diptera, Culicidae) with special reference to Africa. Insect
Science and its Applications, 1, 319338.
Tanaka, K., Mizusawa, K. & Saugstad, E.S. (1979) A revision of the
adult and larval mosquitoes of Japan etc. Contributions of the American
Entomological Institute, 16.
Tongyan Zhao & Baolin Lu (1995) Biosystematics of Culex pipiens
complex in China. Entomologia Sinica, 2, 18.
Urbanelli, S., Silvestrini, F., Reisen, W.K., De Vito, E. & Bullini, L.
(1997) Californian hybrid zone between Culex pipiens pipiens and
Cx.p.quinquefasciatus revisited. Journal of Medical Entomology, 34,
116127.
Urbanelli, S., Silvestrini, F., Sabatinelli, G., Raveloarifera, F., Petrarca,
V. & Bullini, L. (1995) Characterization of the Culex pipiens complex
(Diptera: Culicidae) in Madagascar. Journal of Medical Entomology,
32, 778786.
Urbanelli, S., Villani, F. & Bullini, L. (1985) Electrophoretic studies on
Culex p. quinquefasciatus Say from Africa: genetic variability and
divergence from Culex p. pipiens L. Bulletin of Entomological
Research, 75, 291304.
Vinogradova, E.B. (1992) Morphology, ecology and control of the Culex
pipiens complex in the USSR. Akaieka Newletter, 15, 110.
White, G.B. (1979) Problems in the identification of mosquitoes as
vectors of malaria and filariasis. Symposia of the British Society for
Parasitology, 16, 103143.
Wiedemann, C.R.G. (1828) Aussereuropaische Zweiflugelige Insekten.
Hamm. Vol. 1, 608 pp.

1998 Blackwell Science Ltd, Medical and Veterinary Entomology, 12, 112