From the
Organophosphorus insecticide resistance in Culex mosquitoes is commonly caused by increased activity of one or more
esterases. The commonest phenotype involves elevation of the esterases
Est
Insecticide resistance is a significant problem in many insect
pests. Elevation of carboxylesterase activity is the major mechanism of
resistance to the organophosphorus insecticides in a wide range of
insect
species(1, 2, 3, 4, 5, 6, 7) .
However, the resistance mechanism has been studied in depth at the
biochemical and/or molecular level in only a few species. In the
mosquito Culex quinquefasciatus and the aphid Myzus
persicae, amplification of at least one esterase gene underlies
the increase in esterase activity, with up to 250 copies of an esterase
gene per genome being recorded in Culex (8-10). The
esterases involved in both species are standard B or serine esterases
according to the classification of Aldridge(11) . This is a
widely distributed family of enzymes, which hydrolyze carboxylester,
amide, and thioester bonds in a variety of compounds.
The mosquito C. quinquefasciatus is a major biting nuisance insect
worldwide, and a vector of filarial and viral disease on at least two
continents. Esterase banding patterns after polyacrylamide or starch
gel electrophoresis of crude homogenates of this mosquito can be
complex(12, 13) . Esterases can be classified on the
basis of their biochemical, molecular, immunological, and
electrophoretic characteristics, although their broad substrate
specificities and multi-allelic nature make classification difficult. A
classification system based on the esterase's preference for the
general esterase substrates
We here report the original
cloning and cDNA sequence of the A
The Culex esterase nomenclature is currently out of
line with that used for other organisms, and there is confusion between
the earlier general esterase classification (11) and that
subsequently proposed for mosquitoes(14) . Karunaratne (22) has proposed that the Culex esterases should be
renamed using
A heterogeneous population (Pel) of mosquito was collected
from Peliyagoda, Sri Lanka, in 1986. It was selected to give an
insecticide-susceptible strain, Pel SS, and a resistant strain, Pel RR
(23, 24). Pel RR was 31-fold more resistant to the organophosphorus
insecticide temephos than Pel SS(25) .
The Pel SS strain was
obtained by multiple single family selection from the Pel
strain(23) . The Pel RR strain was obtained by mass selection
with temephos(24) . Since then, insecticide resistance in this
strain has been maintained by exposing fourth instar larvae every third
generation to the LD
A polyclonal antiserum was prepared by injecting a New
Zealand White rabbit with purified nondenatured Est
Four positive plaques were obtained from the initial
screening of the 200,000 recombinant clones of the unamplified Pel RR
cDNA library. The number of positives obtained from this screening
suggested that the target sequence was initially present at a higher
frequency than that expected for a single copy gene. The four plaques
were purified, in vivo excised, and partially sequenced with
M13 forward and reverse primers. The sequence for all four clones over
400 nucleotides was identical. The insert from one of the plasmids
(pBlueAV.A2) was completely sequenced in both directions.
The cDNA
sequence had an open reading frame at its 5`-end, and terminated in a
stop codon, 3`-untranslated region, and a poly(A) tail. There was no
start methionine codon (AUG), so a modified 5`-RACE procedure (17) was used to isolate the 5`-end of the cDNA. The sequence of
the two subcloned 5`-RACE PCR products analyzed was identical in both
directions and also overlapped exactly with the previously sequenced
partial length cDNA clone. The full-length cDNA (made up of the insert
from pBlueAV.A2 and the 5`-RACE PCR product) had an open reading frame
of 1623 base pairs complete with an AUG start codon and coded for a
protein of 540 amino acids. This is in the expected range, since the
purified monomeric protein has an estimated molecular mass of
58-67 kDa estimated by SDS-PAGE, native PAGE, and Sephacryl S200
chromatography(29) . Fig. 1shows the full-length est
Possible N-linked glycosylation sites,
conforming to the sequence NXT or NXS, where X is not proline are underlined in Fig. 2. There are
only two possible glycosylation sites in est
The predicted secondary structure of Est
In this paper a new Culex esterase nomenclature
system (17) has been adopted, as the molecular data now accumulating is
making the old system unworkable. For example, a nonamplified B
esterase from a susceptible Culex strain, which has an
identical electrophoretic mobility to B
The est
The different RFLP pattern for est
The high level of amino
acid identity of Est
In many soluble carboxylesterases that are
retained by the endoplasmic reticulum, the tetrapeptide KDEL
(Lys-Asp-Glu-Leu) occurs at the carboxyl terminus(41) . Variants
of the KDEL sequence that direct intracellular retention of proteins
have since been identified, although it appears that the Glu-Leu is a
major requirement(42) . The four Culex est
Twenty-three amino acids
are conserved through a series of 29 related proteins, and it was
argued that these amino acids are essential for the structure (salt
bridges, packing, and disulfide bridges) and function (active site) of
the proteins(30) . Only 21 of these are conserved in the
Est
Having shown that the mechanism of elevation of the Est
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
2 (A
) and Est
2 (B
). A cDNA
encoding the Est
2 esterase has now been isolated from a Sri Lankan
insecticide-resistant mosquito (Culex quinquefasciatus, Say)
expression library. In line with a recently suggested nomenclature
system (Karunaratne, S. H. P. P.(1994) Characterization of Multiple
Variants of Carboxylesterases Which Are Involved in Insecticide
Resistance in the Mosquito Culex quinquefasciatus. Ph.D. thesis,
University of London), as the first sequenced variant of this esterase,
it is now referred to as Est
2
. The full-length cDNA of est
2
codes for a 540-amino acid protein,
which has high homology with other esterases and lipases and belongs to
the serine or B-esterase enzyme family. The predicted secondary
structure of Est
2
is similar to the consensus
secondary structure of proteins within the esterase/lipase family where
the secondary and tertiary structures have been resolved. The level of
identity (
47% at the amino acid level) between the est
2
and the various Culex est
(B
and B
) cDNA alleles that have been cloned
and sequenced suggests that the two esterase loci are closely related
and arose originally from duplication of a common ancestral gene. The
lack of a distinct hydrophobic signal sequence for Est
2
and two possible N-linked glycosylation sites, both
situated close to the active site serine, suggest that it is a
nonglycosylated protein that is not exported from the cell. Southern
and dot blot analysis of genomic DNA from various insecticide-resistant
and susceptible mosquito strains show that the est
2
gene, like est
2
, is amplified in
resistant strains. The restriction fragment length polymorphism
patterns, after probing Southern blots of EcoRI-digested
genomic DNA with est
2
cDNA, show that the
amplified and nonamplified est
alleles differ in the
resistant and susceptible Sri Lankan mosquitoes.
- or
-naphthyl acetate and their
relative electrophoretic mobilities has been suggested(14) . On
the basis of this classification, two predominant elevated esterase
phenotypes, B
and co-elevated A
/B
,
which cause insecticide resistance, can be distinguished
electrophoretically in the mosquito. The A
/B
phenotype is by far the most common, occurring in all continents
where C. quinquefasciatus is found. The elevated A
and B
esterases are in complete linkage
disequilibrium in mosquito populations, i.e. the two esterases
are co-elevated in all mosquitoes. Amplification of the B
esterase was first shown in the Californian Tem-R strain of
mosquito(9) . Immunological and molecular studies have since
shown that B
and B
were originally alleles of
the same locus and that the B
gene is also amplified in
resistant insects(10, 15, 16, 17) .
Initial reports suggested that there was no cross-reaction between the
A and B esterases at either an immunological or molecular level, and it
was concluded that if these genes had arisen from duplication of a
common ancestral gene, this must undoubtedly have occurred a long time
ago(15, 18) . However, we have recently shown that a
polyclonal antiserum raised to the nondenatured A
esterase
does cross-react with the B
, although the specificity is
50-fold lower for B
than A
, suggesting
that the two enzymes share some common
epitopes(19, 20) .
mosquito esterase from
an insecticide-resistant strain of C. quinquefasciatus from
Peliyagoda, Sri Lanka(21) . The cDNA sequence is compared with
the other mosquito and aphid esterases that are involved in insecticide
resistance. We also show that the underlying mechanism of A
elevation in the Pel RR insecticide-resistant strain is gene
amplification.
Mosquito Esterase Nomenclature
and
rather than the A and B of Raymond et
al.(14) , with numerical superscripts denoting sequence
variants with the same electrophoretic mobility. Hence the two
amplified B
s, which differ in their inferred amino acid
sequence but are electrophoretically identical, become Est
1
and Est
1
, from the Californian and Cuban strains
of Culex, respectively, and the nonelevated B from the Sri
Lankan Pel SS strain, again with the same electrophoretic mobility,
becomes Est
1
. This system ensures that esterases can
still be named initially on the basis of their electrophoretic
mobilities, but distinct DNA sequence variants can also be indicated as
this level of data becomes available. As this nomenclature system will
be adopted in this paper, a summary of the past and proposed
nomenclature for mosquito strains where esterase DNA sequence data is
available is given in . On the basis of this
classification(22) , the Pel RR A
esterase now
becomes Est
2
, as the first available sequence of this
esterase.
Mosquito Strains
concentration of temephos. The only
organophosphate resistance mechanism in this strain is co-elevation of
the Est
2
and Est
2
esterases.
Production of a Polyclonal Antiserum
2
from Pel RR. The esterase was administered after mixing with
reconstituted Ribi adjuvant to give a final enzyme concentration of 200
µg/ml. Four injections of 1 ml of antigen were given at 2-week
intervals. Each injection was split between intramuscular, intradermal,
and subcutaneous sites(26) . The resultant antiserum had a high
level of specificity to Est
2 and much lower (
50-fold) levels
of sensitivity to the Est
2 and Est
1 esterases and the
organophosphate target site
acetylcholinesterase(19, 20) . This antiserum was used
to screen a Pel RR cDNA expression library.
Isolation of a cDNA Clone for Esterase Est
2
Pel RR cDNA Library Construction
The cDNA for
library construction was synthesized from 5 µg of mRNA from fourth
instar Pel RR larvae by a method previously used(17) . The cDNA
was blunted and size-selected (>400 base pairs) on a Sephacryl S-400
column (Pharmacia Biotech Inc.). After EcoRI linker addition
(Promega) and subsequent phosphorylation, the cDNA was ligated into
Lambda ZAP II (Stratagene). This vector allows plasmid containing cDNAs
to be directly excised from the phage library. The bacteriophage arms
were supplied digested with EcoRI and dephosphorylated. 5
µg of arms were ligated to 125 ng of cDNA and packaged with
Packagene extracts (Promega). Approximately 200,000 recombinant clones
were obtained.
Immunoscreening
This was carried out with the
Protoblot Immunoscreening System (Promega). The 200,000 unamplified
recombinant clones were screened with the polyclonal
anti-Est2
esterase antisera at a dilution of 1:10,000.
In Vivo Excision and Sequencing
Positive clones
were purified by successive rounds of screening and in vivo excised to give recombinant pBluescript plasmids. Sequencing was
carried out with Sequenase Version 2.0 (Amersham Corp.) using universal
primers complemented with the ExoIII/Mung Bean Nuclease deletion method
(Stratagene) and with primers specific to the est2
clone. This allowed the sequencing of both strands of the insert
cDNA from the plasmid.
Modified 5`-Rapid Amplification of cDNA Ends
(RACE)
A modified 5`-RACE procedure as performed
previously (17) was used to isolate the full-length est2
cDNA. The reverse primer used in the PCR
was 5`-ACCGTACATCTCCACTCC-3` and was close to the 5`-end of the cDNA
isolated from the cDNA library. The PCR product was subcloned into the
pBluescript T vector(17, 27) . Two separate PCR products
were sequenced in both directions.
Genomic DNA Studies
A Pel RR est2
cDNA fragment was used as a probe to
determine the haplotype of the Est
esterases from the Pel RR and
Pel SS strains. Genomic DNA was isolated from fourth instar larvae as
described previously(17) . 10 µg of genomic DNA was digested
to completion with EcoRI and separated by gel electrophoresis
through 0.8% (w/v) agarose. The DNA was transferred to charged nylon
membranes (Amersham Corp.) and hybridized with a
P-labeled
Est
2
cDNA probe (specific activity > 2
10
cpm/µg) at 65 °C for 16 h in hybridization
buffer (5
Denhardt's solution, 6
SSC, 0.1% (w/v)
SDS, 0.1% (w/v) sodium pyrophosphate, 5% (w/v) PEG 8000, and 100
µg/ml boiled, sheared herring sperm DNA). The final washes were at
65 °C in 0.1
SSC and 0.1% (w/v) SDS for 20 min.
Sequence Analysis
Similarity searches of protein
data bases with the the est2
cDNA sequence
were undertaken using the B17049 and MPsearch programs available
through NCBI (National Institutes of Health) and EMBL (Heidelberg),
respectively. Family structure determination was undertaken using the
Prosite section of the Motif finder program through the Motif E-mail
server on Genome. Prediction of secondary structure was undertaken
using the PHD program(28) . Sequence alignments were undertaken
using the MegAlign program of the LASERGENE package (DNASTAR).
Similarity indexes were calculated using the FASTA algorithm, via the
GeneMan program of LASERGENE.
2
cDNA nucleotide sequence and the
proposed amino acid sequence. Fig. 2shows the predicted amino
acid sequence of Est
2
and its alignment with the Culex Est
2
(Pel RR B
) and aphid
E4 amplified esterases. The Est
2
sequence, like the
Est
2
, contained nine cysteine residues, but only three
of these are conserved between the two esterases. The triad of
precisely located active site amino acids, Ser, Glu, and His, of the
serine esterase family were present in the Est
2
sequence at positions 190, 324, and 445, respectively.
Figure 1:
The nucleotide and predicted amino acid
sequence of the cDNA for carboxylesterase Est2
(A
) from Pel RR, an OP-resistant strain of Culex
quinquefasciatus. The cDNA was isolated from a cDNA expression
library by immunoscreening with antibody against Est
2 and by a
modified RACE procedure. The nucleotide and amino acid sequences are
numbered from the ATG start codon. An in-frame stop codon (TGA)
upstream of the ATG start codon is underlined. The start (ATG,
amino acid M) and stop (TAG, represented by X) codons are underlined, as is the putative polyadenylation signal (AATAAA)
beginning at nucleotide 1727. The amino acid residues thought to make
up the active site triad (Ser
, Glu
, and
His
) are doubleunderlined, and the
start of the poly(A)
tail is represented by
$.
Figure 2:
Alignment
of the amino acid sequence of Culex mosquito serine
Est2
and Est
2
esterases with the
aphid E4 esterase involved in insecticide resistance. Possible
glycosylation sites are underlined. Conserved amino acids in a
large range of esterases (30) are in boldface.
The est2
had a similarity index of 49.2 with est
2
, which was the highest similarity in the
protein database. The top 30 alignments of est
2
undertaken with both the B17049 and MPsearch programs were all
esterases or lipases. The protein was also unambiguously assigned to
the carboxylesterase B-1 family of B type serine esterases using the
Motif program.
2
; both are very close to the active site
serine and are not shared by other esterases in the alignment. Since
the est
2
is not proceeded by a signal
sequence, and as the purified mature protein is not retained by Con A
chromatography(22) , it is probable that the protein is not
glycosylated.
2
obtained using the PHD program (28) is given in Fig. 3compared with the known consensus secondary structure of
the Torpedo californica acetylcholinesterase and Geotrichum candidum lipase(30) . The predicted
secondary structures of the Est
2
and Est
2
proteins, determined independently, were identical with the
exception of the final
-helix, which was not predicted in
Est
2
. The predicted secondary structure is remarkably
similar to that of the known consensus sequence for serine esterases
and lipases, differing only in three
-helices. One of these, the
helix between
-sheets 8 and 9 is also present in the G.
candidum lipase structure but is not present in T. californica acetylcholinesterase (30).
Figure 3:
Predicted secondary structure of
Est2
compared with the known consensus secondary
structure of T. californica acetylcholinesterase and G.
candidum lipase. The predicted structure of Est
2
differs only in the final
-helix. Arrows indicate
-sheets, and boxes indicate
-helices. A,
consensus secondary structure of T. californica acetylcholinesterase and G. candidum lipase. B,
predicted secondary structure of the Est
2
Culex esterase.
The est2
cDNA was used as a probe for Southern blot analysis of EcoRI restriction digests of equal amounts of genomic DNA from
the insecticide-susceptible (Pel SS) and resistant (Pel RR) mosquito
strains. After hybridization and high stringency washing, the probe
bound to a single 7.4-kb band of Pel SS DNA (Fig. 4), which
demonstrates the existence of an est
gene with high
homology to est
2
in this strain. A 7.5-kb
band was found in the resistant strain at an equally low intensity,
suggesting that the resistant strain still carries a nonamplified est
allele. The Pel RR strain contained a 5.8-kb band
with a high signal intensity, which was not present in the Pel SS
strain. The higher signal intensity in the resistant strain compared
with the susceptible, implies that gene amplification is the underlying
mechanism of the Est
2
-associated resistance. Further
proof of this was obtained by undertaking dot blots with the est
2
cDNA probe, using genomic DNA from four
other resistant strains of C. quinquefasciatus from different
geographical locations with elevated Est
2 activity. All the
resistant strains gave a much higher intensity of signal than the
susceptible strain (Pel SS), and the signals obtained were similar when
the blots were reprobed with an est
2
cDNA.
This suggests that the amplification levels of the est
2 and est
2 genes are similar in the resistant strains.
Figure 4:
Southern blot of EcoRI
restriction digests of equal amounts of Pel RR (lane 1) and
Pel SS (lane 2) genomic DNA hybridized with a Pel RR
Est2
cDNA probe.
, has been shown to
be distinct from the B
s from TemR and MRES, which are in
turn distinct from each other, and from esterase B
at both
a kinetic and amino acid level(17) .
(
)Thus on the basis of restriction fragment length
polymorphism (RFLP) patterns, DNA sequences, inferred amino acid
sequences, and kinetic interactions of these enzymes at least one
nonamplified and two amplified ``B
'' esterases
occur(17) .
(
)This level of variability of
electrophoretically identical esterases makes application of the
earlier classification difficult, and the problem has been compounded
by electrophoretically similar esterases being given different
numerical values on the basis of their distinct RFLP
patterns(31) . The nomenclature system we have used allows
preliminary assignment of the esterase by electrophoresis, with an
extension when sequence data is available, while the old system means
esterases may need complete reclassification once sequence or RFLP data
is available. With the new classification the esterase that we have now
cloned, which was previously referred to as A
, is
classified as Est
2
on the basis of its electrophoretic
mobility and sequence.
2
esterase
cDNA from the insecticide-resistant Pel RR strain of the C.
quinquefasciatus mosquito has now been cloned and sequenced. The
high number of positive clones obtained from the initial screening of
the cDNA library suggested either that there was increased
transcription of the gene or that gene amplification was the underlying
mechanism of increased esterase activity. Southern blot analysis showed
that the resistant strain had a unique, amplified 5.8-kb EcoRI
RFLP when compared with the susceptible strain, which had a single
unamplified band at 7.4 kb. Amplification of the est
2 gene was also shown in other C. quinquefasciatus strains
with elevated Est
2 and Est
2 activity, using dot blot analysis
of genomic DNA. Since amplification of est
2 is already
well documented(17) , the underlying genetic mechanism for
organophosphorus insecticide resistance in mosquitoes with the elevated
Est
2/Est
2 activity phenotype is therefore amplification of
both esterase genes.
in the Pel RR and Pel SS strains demonstrates the existence of two
distinct est
alleles. The amplified and nonamplified
Est
and Est
esterases have been purified and characterized
physically and kinetically from a range of insecticide-resistant
strains and a susceptible strain of
mosquito(28, 32, 33, 34) .
The Est
enzymes from the susceptible (Pel SS) and resistant
(Pel RR) strains are kinetically and electrophoretically distinct from
each other,
suggesting that they are different alleles,
which is supported by the different RFLP patterns seen in the two
strains. The amplified Est
2
, Est
1
,
and Est
2
esterases all have similar sizes (
60
kDa) and PI values(29, 33) .
We now know
that all three esterases are coded for by cDNAs with the same length
open reading frame. The amplified esterases E4 and FE4 in
insecticide-resistant aphids are comparable in function and size with
the mosquito esterases, but unlike the Culex esterases these
enzymes confer resistance to both organophosphates and
pyrethroids(35, 36, 37) . The E4 esterase and
the Est
2
and various Est
Culex esterases
all produce resistance to the organophosphates by sequestration,
although the aphid and mosquito Est
and Est
esterases share
only 21.8 and 22.9% similarity, respectively, while the similarity of
the mosquito esterases is 49.2%. Hence the same kinetic properties can
clearly be conferred by a large number of different sequences. This is
perhaps not surprising when the predicted secondary structures of these
esterases are considered. The predicted secondary structures for the
Est
2
and Est
2
esterases differ only
in the final element, and the structure bears a striking resemblance to
actual secondary structures already resolved for two members of the
serine esterase/lipase family(30) .
2
and Est
2
suggests that they originated from a common ancestor. The two
genes probably arose through gene duplication and subsequently
diversified. The Culex esterase cDNAs each code for proteins
of 540 amino acids, and they have a higher sequence homology with each
other than for any other sequence within the data banks. The two
esterases also share a number of common features. For example, many
carboxylesterases contain a short hydrophobic leader sequence, which
initially directs protein sorting down a secretory pathway. All
sequenced human liver carboxylesterases and the aphid E4
carboxylesterase contain signal sequences of between 17 and 23 amino
acids(38, 39) . The mosquito est
2
sequence and the est
2
sequence are
relatively unusual in that they contain no signal sequence, suggesting
that neither esterase is exported from the cell. Neither of these
purified esterases bind to Con A chromatography columns(22) ,
which suggests that the esterases are not glycosylated, in contrast to
the aphid and some human esterases(39, 40) . This is
further supported by the sequence data, since there are only two
possible N-linked glycosylation sites on the Est
2
esterase, which are situated only 4 and 31 amino acids from the
active site serine.
cDNAs
sequenced to date all end in the sequence NDELF. However, the carboxyl
terminus of Est
2
is KDKLY.
2
. A cysteine is one of the nonconserved residues,
and it is notable that both the Est
2
and
Est
2
have nine cysteine residues, the majority of
which are not conserved in any other serine esterase. The missing
cysteine, at position 65 (Cys
and Cys
in T. californica AChE and G. candidum lipase,
respectively) in the Pel RR Est
2
sequence,
forms a disulfide bridge in T. californica acetylcholinesterase and G. candidum lipase with a
cysteine at position 84 (Cys
and Cys
), which
is a serine in the Est
2
and various Est
Culex esterases. In the alignment of Cygler et al.(30) ,
the only esterase in which the latter cysteine was not conserved was
the Culex TemR Est
1
. The reason for the large
number of cysteines in the Culex esterases is unknown, but it
has been suggested that five of these may not be involved in disulfide
bond formation, leaving them free to oxidize. This results in the
development of evenly spaced satellite bands after native
polyacrylamide gel electrophoresis of purified native esterases under
buffer conditions that do not protect the thiol groups(43) .
2
esterase is gene amplification, we now intend to determine the
genomic structure of this esterase. The almost complete linkage
disequilibrium in which these two elevated esterases occur, coupled
with their sequence similarities, indicating that they arose through
gene duplication, may also suggest that the two esterases are situated
on the same amplification unit. This has, however, been contradicted by
some classical genetic data on the inheritance patterns of these
esterases,(12, 44) , and the final determination of the
physical location of the two esterases in relation to each other awaits
further genomic studies.
Table: Suggested nomenclatures for the esterases of the
mosquito Culex quinquefasciatus
/EMBL Data Bank with accession number(s)
Z47988.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.