Báo cáo Y họcMolecular characterization of the amplified aldehyde oxidase from insecticide resistant Culex quinquefasciatus docx

Molecular characterization of the amplified aldehyde oxidase
from insecticide resistant
Culex quinquefasciatus
Michael Coleman, John G. Vontas and Janet Hemingway
Liverpool School of Tropical Medicine, UK
Primary structural information including the complete
nucleotide sequence of the first insect aldehyde oxidase (AO)
was obtained from the common house mosquito Culex
quinquefasciat us (Say) through cloning and sequencing o f
both g enomic DNA and cDNA. The d educed amino-acid
sequence encodes a 150- kDa p rotein of 1266 amino-acid
residues, which is consistent with the expected monomeric
subunit size of AO. The Culex AO sequence contains a
molybdopterin cofactor binding domain and two iron–sul-
fur centres. A comparison of the partial sequen ces of AO
from insecticide resistant and susceptible strains of C. quin-
quefasciatu s shows two distinct alleles of this enzyme, one of
which is amplified in the insecticide resistant strain on a
30-kb DNA amplicon alongside two resistance-associated
esterases. The amplified AO gene results in elevated AO
activity in all life stages, but activity is highest in 3rd instar
larvae. The elevated enzyme can b e seen as a separate band
on polyacrylamide gel electrophoresis. The role of AO in
xenobiotic oxidation in mammals and t he partial inhibition
of elevated AO activity by a range of insecticides in Culex ,
suggest that this AO may play a role in insecticide resistance.
Keywords: aldehyde oxidase; mosquito; insecticide resis-
tance.
Culex mosquitoes are major vectors of filariasis and
Japanese encephalitis as well as a general biting nuisance.
Amplification of nonspecific esterases accounts for > 90%

of known insecticide resistance in Culex populations. In the
C. pipiens complex, distribution of amplified esterase alleles
is geographically restricted, except for esta2
1
and estb2
1
,
which are coamplified on a single DNA amplicon and occur
world-wide. The rapid spread of this amplicon through
Culex populations already containing alternative esterase
alleles on resistance-associated amplicons suggests that the
esta2
1
/estb2
1
amplicon has a very strong selective advantage
over other esterase-based resistance mechanisms [1]. A
comparison of esterases from resistant C. quinquefasciatus
strains with different amplified esterases suggests no s elec-
tive advantage of esta2
1
and estb2
1
over other resistant
strains based on their enzyme activity alone [2]. The selective
advantage observed for strains, such as PelRR, with this
amplicon, must therefore be due to some other factor.
Recently, w e have reported that the PelRR amplicon
contains a third complete gene, putatively aldehyde o xidase
(AO) [3]. It is possible that either the AO or esterases on the

amplicon affect mosquito viability in the presence of filarial
parasites, as we have shown that parasite survival and
insecticide resistance status are highly negative ly correlated
[4], which may contribute to the lack of correlation between
mosquito biting rates, prevalence of microfilaraemia and
disease which has been noted in several studies [5].
Alternatively, the AO m ay have a direct role in insecticide
resistance.
Cytochrome P450s have traditionally been thought of as
the sole enzyme system associated with increased l evels of
insecticide oxidation. However, in mammals oxidation of
xenobiotics by the molybdopterin family of enzymes is now
well documented [6,7]. AO is a molybdenum-containing
enzyme belonging to this family. The tissue localization and
physiological roles of this enzyme are still not fully
understood. The broad substrate specificity of AO makes
it a useful mammalian prodrug activator [8,9], and AO
functions in herbivores to protect them against plant toxins
[6]. The amplification of AO in insecticide-resistant insects
may therefore have a functional significance, which h as been
overlooked to date.
There is no known sequence data for AO in insects,
although t he enzyme has a number of diverse physiological
functions [10–12] and its distribution patterns in Drosophila
[13,14] and Musca domestica have been studied histochem-
ically [15]. Here we report the first genomic, cDNA and
deduced protein sequences of AO from the mosquito Culex
quinquefasciatus, demonstrate that the allele present on the
amplicon is expressed in insecticide-resistant insects and that
it interacts with insecticides.

EXPERIMENTAL PROCEDURES
Mosquito strains
C. quinquefasciatus larvae were collected from Peliyagoda,
Sri Lanka in 1984. Th is population, which had been under
fenthion selection pressure for several years, was selected i n
the laboratory to produce two strains: an insecticide
susceptible strain, PelSS, with the nonamplified esterases
esta3 and estb1
2
, and an organophosphorus insecticide-
resistant strain, PelRR, with the two coamplified esterases
esta2
1
/estb2
1
[16,17]. The C. quinquefasciatus str ain TemR
Correspondence to J. Hemingway, Liverpool School of Tropical
Medicine, Pembroke Place, Liverpool L3 5QA, UK.
Fax: + 44 151 7088733, Tel.: + 44 151 7089393,
E-mail:
Abbreviations: AO, aldehyde oxidase; ALDH, aldehyde dehydrogen-
ase; XDH, xanthine dehydrogenase.
(Received 18 July 2001, revised 14 November 2001, accepted 16
November 2001)
Eur. J. Biochem. 269, 768–779 (2002) Ó FEBS 2002
was obtained from G. Georghiou, University of Califor-
nia, Riverside, USA. TemR is resistant to organophos-
phates due to the amplification of a single esterase gene,
estb1
1

[18].
Genomic DNA sequence
A genomic library of PelRR fourth-instar larvae was
constructed in the kGEM-11 vector (Promega) and probed
with a partial esta2
1
cDNA as previously described [19,20].
The sequence downstream of the esta2
1
gene from the
resultant positive bacteriophage clone (A2) suggested a third
ORF with high homology to the molybdenum containing
enzymes xanthine dehydrogenase (XDH) and AO [3].
Bacteriophage A2 was produced for analysis by inoculating
400 mL of Luria–Bertani broth [0.1% (w/v) bacto-tryptone,
0.05% (w/v) bacto yeast extract, 0.1% (w/v) NaCl] with
6mLofEscherichia c oli LE392 culture, grown overnight in
Luria–Bertani broth + 0.2% maltose and incubated for
20 min, 37 °C, 225 r.p.m. The culture was inoculated with
10
9
plaque forming units of bacteriophage A2. After
allowing the mixture to stand for 20 min at 37 °C, the
culture was grown at 37 °C, 225 r.p.m. for 6 h. Chloroform
(2 mL) w as added to lyse any remaining cells. Ten grams
NaCl, RNase 1 m gÆmL
)1
and DNase 1 mgÆmL
)1
were

added and the mixture incubated for 1 h at room
temperature. The cell d ebris was removed by centrifugation
at 12 000 g,4°C for 10 min 10% (w/v) of poly(ethylen e
glycol) 6000 was gently dissolved into the s upernatant, and
the mixture incubated at 4 °C for 10 min and resuspended
in 5 mL S M [0.58% ( w/v) NaCl, 0.2% (w/v) M gSO
4
,5%
1
M
Tris/HCl pH 7.5, 0.01% gelatin].
Chloroform extraction was carried out and the superna-
tant removed. CsCl
2
(0.75 g per mL) was dissolved into the
supernatant a nd the m ixture centrif uged at 100 000 g,
10 °C for 24 h. The DNA band was dialysed overnight
against 10 m
M
NaCl, 50 m
M
Tris/HCl pH 8.0, 10 m
M
MgCl
2
EDTA. After dialysis, proteinase K (50 mgÆmL
)1
)
and SDS (0.5%) were added and the mixture incubated at
65 °C for 1 h . Phenol and chloroform extractions were

carried out before precipitating the DNA in ethanol and
resuspending in Tris/HCl pH 8.0.
Restriction digests and subcloning of the A2 insert was
undertaken to analyse the AO sequence. A2 was digested
with BamHI and SacI and run o n a 1% agarose gel (Bio-
Rad). The three resultant bands were extracted from the gel
with the Wizard DNA Clean-up System (Promega) and
subcloned into pBluescript (Stratagene). The ligation prod-
ucts were used to transform E. coli XL-1Blue (Stratagene)
and recombinant plasmids were isolated from a mplified
bacterial colonies using a standard miniprep method
(Qiagen).
PCR was used to produce sequence spanning across the
three subcloned fragments. P rimer X6 (5¢-GGTGTACA
ACGTGCAGGA-3¢)andY4(5¢-GAGCGAGAACGAG
CCGGAAC-3¢)wereusedtoPCRbetweenplasmids
AO1 and AO2. Primer Y6 (5¢-GCCGAAATGTGATTAT
TTG-3¢)andA1(5¢-TTAGCCC GAACCGCGGCC-3¢)
were used to PCR across plasmids AO2 and AO3. These
PCR products were ligated into p GEMT-easy (Promega)
and positive colonies were s elected and prepared as a bove.
A contig of the complete bacteriophage insert was made b y
combining these sequence data.
Synthesis of cDNA and sequencing of AO
Total RNA was isolated from 1 g of fourth instar larval
PelRR using TRI reagent (Sigma) according to the man-
ufacturer’s instructions. Reverse transcription of first strand
cDNA from mRNA was accomplished with SuperScript
TM
RT (Gibco BRL) according to the manufacturers instruc-

tions with an oligo(dT) adaptor primer [5¢-GACTCG
AGTCGATCGA-(dT)
17
-3¢].
Primers were designed to the putative 5¢,F1(5¢-A TG
GAAGTCATATTTACGAT-3¢)and3¢,F2(5¢-TTG
TAGTTTAAACTGTTC-3¢) ends of AO based on the
genomic sequence. The 50-lL PCR reaction contained
20 ng of first strand cDNA, 150 ng o f each primer, 0.5 m
M
dNTPs, 2.5 m
M
MgCl
2
,1.25UPfu DNA polymerase
(Stratagene) 5 U of Amplitaq Gold DNA polymerase
(PerkinElmer) and Taq DNA polymerase buffer. After one
cycle of 95 °C for 10 min to activate the Amplitaq,35cycles
of amplification were carried out as follows: 95 °C, 45 s;
50 °C, 45 s; 72 °C, 7.5 min.
3¢ RACE
RACEwasusedtoobtainthe3¢ UTR of AO. Primer A20
(5¢-CCGAGAACTTGATCTACAG-3¢)designedtothe3¢
end o f t he cDNA was used in conjunction with an adaptor
primer (5¢-GACTCGAAGTCGACATCGA-3¢)inaPCR
reaction. The PCR reaction was carried out as above,
without Pfu DNA polymerase and with an extension time
of 2 min.
5¢ PCR
The p artial 5¢ UTR was obtained using a primer designed

from the genomic DNA 102 base pairs upstream of the
transcription start codon, UTR5 (5¢-GCACTGTTTAACT
CAGTTCG-3¢) and a primer (X7) designed to t he 5¢ end of
the cDNA (5¢-TCCTGCACGTTGTACACC-3¢). The PCR
reaction was carried out as for the 3¢ RACE.
PCR products were isolated using a Wizard DNA Clean-
up System (Promega) and subcloned into pGEMT-Easy
(Promega) for sequencing.
DNA sequencing
Initially the inserts were sequenced using universal M13
forward and reverse primers complimentary to the plas-
mids. Internal primers were synthesized based on this initial
sequence data. Sufficient primers were synthesized to allow
sequencing of both strands of the AO gene at least twice.
Sequencing was carried out with an ABI Automatic
Sequencer (PerkinElmer). Sequence data were analysed
with the
DNASTAR
(Lasergene) program.
Genomic Southern blots
Genomic DNA was extracted by the method of Vaughan
et al. [21]. Ten milligrams of PelRR, PelSS and TemR DNA
was digested with Eco RV restriction enzyme a nd the
products separated by electrophoresis on a 0 .8% agarose
gel. The DNA was denatured, neutralized [22] and trans-
ferred to a nylon membrane (Amersham) using the Hybaid
Vacu-Aid. The DNA was fixed to the membrane with UV
Ó FEBS 2002 AO from C. quinquefasciatus (Eur. J. Biochem. 269) 769
light. Membranes were prehybridized at 65 °Cfor1hin
hybridization buffer (6 · NaCl/Cit, 0.1% (w/v) SDS, 0.1%

(w/v) sodium pyrophosphate, 5% poly(ethylene glycol),
5 · Denhardt’s solution). The genomic clone AO1 was
digested with HincII and PstI. Products were separated by
electrophoresis and the 0.65-kb band with high homology to
AO was extracted from the agarose using a Wizard
TM
kit
(Promega). It was labelled with
32
P (specific activities >
2 · 10
6
c.p.m.Æmg
)1
) by random priming with a Pharmacia
oligonucleotide labeling kit and used a s a probe. The probe
was hybridized to t he phage DNA overnight at 60 °Cin
hybridization buffer. Final washes were in 0.1 · NaCl/Cit,
0.1% (w/v) SDS at 60 °C for 45 min.
Aldehyde oxidase assay
Individual mosquitoes were assayed for AO activity by a
method adapted from Moura & Barata [23] and Mira et al.
[24]. Briefly, individual larvae were homogenized in 40 lL
potassium p hosphate buffer pH 7.8 with 1 m
M
EDTA.
Two replicates of 10 lL were transferred to a microplate
and 200 lL of reaction mixture containing 0.1 mgÆmL
)1
phenazine methosulfate, 0.1 mgÆmL

)1
2,6-dichloroindophe-
nol, 50 l
M
allopurinol, and 0.1 m
M
of a ‘neat’ m ixture of
aldehyde substrates (1 : 1, v/v, acetaldehyde/benzaldehyde)
was added. AO a ctivity was determined by measuring the
rate of 2,6-dichloroindophenol reduction at 600 nm
(e ¼ 21 m
M
)1
Æcm
)1
) as aldehyde is enzymatically oxi-
dized. Kinetics were read immediately, by following the
decrease in absorbance at 650 nm for 5 min. Specific
activities are given in UÆmg
)1
protein where a unit corre-
sponds to 1 lmol o f 2,6-dichloroindophenol reduced per
min, under the assay conditions used. All assays were
compared to controls of id entical composition lacking
substrate (aldehydes) or homogenate.
Measurement of aldehyde dehydrogenase (ALDH)
activity
The ALDH assay was performed by the method of Tasayco
& Prestwich [25], under anaerobic conditions. A 1-mL
aliquot of potassium phosphate buffer pH 7.8 saturated

with nitrogen, containing 1 m
M
NAD
+
and 10–15 mg
protein of pooled crude homogenate was added to a 2-mL
cuvette. Four millilite rs of 10 m
M
aldehyde in ethanol was
added anaerobically and the appearance of NADH
(e ¼ 6.22 m
M
)1
Æcm
)1
) was recorded continuously for
10 min at 340 n
M
. Specific activities are given in UÆmg
protein
)1
. A unit corresponds to the production of 1 nmol
NADH in 1 min.
Protein assay
Protein content was determined b y the method of Bradford
[26]. Protein values in mg ÆmL
)1
were calculated from a
standard curve o f a bsorbance of known concentrations o f
bovine serum albumin.

Inhibition of AO activity by pesticides and inhibitors
Crude homogenates for the pesticide and effector experi-
ments were prepared in ice-cold 0.1
M
phosphate buffer
(pH 7 .8) with 5% (v/v) glycerol. S topped time inhibition
assays were performed. Solutions of AO inhibitors and
various pesticides were prepared in either phosphate buffer
or acetonitrile depending on their solubility (acetonitrile
concentration of the medium never exceeded 1%, v/v). Each
effector was preincubated with the crude homogenate for
15 min at 20 °C. AO or ALDH residual activity were then
measured as described above in t he presence of each
effector, except that phenazine methosulfate was omitted
from the AO activity reaction mixture.
Electrophoresis
Electrophoresis of native protein samples was performed in
a Phastsystem (Pharmacia). Crude homogenates were
prepared as described above. Two microliters of each
sample ( 5 lg p rotein) w as applied to a 8–25% gradie nt
Phastgel (Pharmacia) with standard molecular mass mark-
ers (Sigma) and subjected to native PAGE Phastsystem
(400 V, 10 mA, 2.5 w, 10 °C, 390 Vh).
Gels were divided to v isualize standard proteins by
Comassie Blue R250 staining and AO activity bands using a
formazan staining solution, prepared according to Tasayco
& Prestwich [25]. Briefly, 20 mg Nitro Blue tetrazolium,
0.8 m g phenazine methosulfate, 50 mg allopurinol (to
inhibit xanthine oxidase and XDH) and 1 mL of aldehyde
substrates (acetaldehyde, benzaldehyde, Dimethyl-amino-

benzaldehyde or heptaldehyde), were added in 50 mL 0.1
M
Tris/borate buffer, pH 8.0 with 1 m
M
EDTA. All native
gels were compared to controls of identical composition
with aldehyde omitted.
RESULTS
A complete ORF (ORF) coding for a putative AO enzyme
was obtained f rom genomic DNA of the C. quinquefascia-
tus mosquito strain PelRR (Fig. 1). The computer-based
analysis of the 5¢ flanking region of the AO gene identified
several potential transcription factor-binding sites. Amongst
them were three Barbie boxes located at )1137 to )1122,
) 666 to )652 and )539 to )525 to this ORF. These
sequences have been previously found in the promoter
regions of most GSTs with the core sequence o f AAAG
common in all of them [27,28]. This element might be
responsible for the induction of the GST genes by the drug
phenobarbital and may play a role in drug resistance during
cancer treatment. The common arthropod initiator
sequence, TCAGT, occurs at both positions 13–18 (AI1)
and 25–29 (AI2), with a possible TATA box located at )12
to )3, relative to the +1 of the 5¢ UTR, suggesting that this
ORF codes for a functional gene.
Primers designed to the 5¢ and 3¢ ends of the AO genomic
DNA sequence were used to obtain a full-length cDNA of
3798 nucleotides from fourth instar Culex larvae. This
cDNA sequence was completely homologous to the
predicted exon regions of the genomic DNA sequence and

the structure is superimposed on the genomic sequence in
Fig. 1. A presumptive polyadenylation site, AATAA, is
located at 5884–5889 (7540 on genomic sequence). The
cDNA ORF pred icts a protein of 1266 amino-acid residues
and an molecular mass o f 150 kDa. This is consistent with
the expected monomeric subunit size for AO. 3¢ RACE was
used to obtain a 3¢ UTR of 67 nucleotides that includes the
presumptive polyadenylation site. A 102-bp 5¢ UTR w as
770 M. Coleman et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Ó FEBS 2002 AO from C. quinquefasciatus (Eur. J. Biochem. 269) 771
772 M. Coleman et al. (Eur. J. Biochem. 269) Ó FEBS 2002
obtained by PCR usin g primers designed to the genomic
DNA sequence.
The cDNA encodes a protein with two predicted iron–
sulfur [2Fe)2S] centres between amino-acid residues 33–62
and 138–164. There is a highly conserved molybdopterin
cofactor binding site between residues 706–752 (Fig. 2 ), and
a region with high homology to the NADH binding site of
various Drosophila enzymes [29] is located at 343–515. This
region of the PelRR AO sequence has little homology with
the related molybdopterin enzyme XDH and there is no
putative NAD
+
binding domain in the mosquito sequence,
as would be expected if this ORF coded for XDH. The
PelRR AO has a high d egree of homology to XDH from
Drosophila melanogaster (51%), Bombyx mori (51%) and
D. subobscura (50% ) which is expected, as AO and XDH
are closely related enzymes. Assigning this gene as an AO is
supported by the three conserved active site centers and the

high homology to bovine AO (51%), human AO (51%) and
Arabidopsis AO (50%) (Fig. 2).
A Southern blot of genomic DNA from various
mosquito strains (Fig. 3), shows that this AO is amplified
in PelRR, in contrast to PelSS and the insecticide resistant
strain TemR, which has an amplicon containing estb1
1
.The
complete AO ORF in PelRR is located on the same
insecticide resistance-associated amplicon as the esterase
genes esta2
1
/estb2
1
. The coding regions of the genomic AO
and esta2
1
DNA sequences overlap at their extreme 3¢ ends
in the PelRR amplicon. There are five introns within the
ORF. All are small introns of < 200 bp with the exception
of intron 3 which is  1700 bp in length (Fig. 1). A partial
sequence of an AO from PelSS DNA is conserved at the
3¢ end of the gene (329/330 nucleotides) and has lower
conservation in t he mid r egion (263/276 nucleotides) when
PelRR and PelSS sequences are c ompared, suggesting that
the two strains carry different alleles of this gene. The 3¢ end
of the gene contains the conserved active site centers hence
the lack of variability in this region i s expected.
On native PAGE gels it is difficult to distinguish between
ALDH and AO [30]. Allopurinol was used to inhibit

xanthine oxidase and XDH bands which will stain with AO
substrates. Figure 4 indicates that the upper band is
xanthine oxidase and the two lower bands in PelRR are
either AO or ALDH. Three bands occurred in PelRR adults
in contrast to two in P elSS when equal amounts of protein
were loaded (Fig. 5). Size estimation of the novel AO band
was obtained under native conditions using its relative
mobility and the linear regression equation for the molec-
ular mass markers. The apparent molecular m ass of the
amplified AO was estimated in its native form as approx-
imately 302 kDa, which is in line with t he predicted
molecular mass from the translated amino-acid sequence
for a homodimeric AO, as recorded in other insects and
mammals. The novel AO band in PelRR is stage specific
(Fig. 6), with highest activity during t he larval stages,
peaking in the third and fourth instars and decreasing to low
levels in the pupal and adult stages.
The specific AO activity of PelRR larval crude homo-
genate using this assay was approximately fourfold higher
than that in PelSS crude homogenates (Fig. 7). However,
this fourfold elevation in the aldehyde oxidizing activity
can not be readily compared to the degree of the AO
genomic amplification (which is estimated as being much
higher), as a many fold AO upregulation may only
increase the overall activity a few fold. Adult PelRR and
PelSS had similar levels of AO activity (data not shown),
due to the lower expression of the amplified AO in this life
stage, in comparison with other aldehyde oxidizing
enzymes (such as ALDH).
The identification of the novel PelRR enzyme as an AO

was confirmed by measuring AO substrate oxidation
anaerobically. These conditions suppress AO activity and
any remaining enzymatic activity is due to ALDH. PelRR
and PelSS had similar ALDH specific activities (Table 1).
Any differences between the activity of the two strains with
Fig. 1. Nucleotide sequence of C. quinquefasciatus PelRR AO genomic sequence, deduced after complete sequencing of both DNA strands (Accession
no. AF 202953). A PelRR AO cDNA was a lso sequenced in both directions and e xactly matched the underlined exon sequences of the genomic
DNA. The potential arthropod initiator sequences and poly A site are indicated as AI1, AI2 and PA, respectively. Some of t he p rimers used in PCR
have been indicated. The 5¢ UTR and 3¢ UTR sequences are also shown.
Ó FEBS 2002 AO from C. quinquefasciatus (Eur. J. Biochem. 269) 773
774 M. Coleman et al. (Eur. J. Biochem. 269) Ó FEBS 2002
AO substrates is therefore due to differences in AO activity.
The novel PelRR AO had a high affinity for the substrate
heptaldehyde, which is a good AO, but poor ALDH
substrate, supportin g our earlier suggestion from anaerobic
assays that this novel band is not an ALDH. Both bands
were prominent with acetaldehyde.
Because t he true kinetic parameters (V
max
or K
m
)and
inhibition constants are not readily obtainable for a mixed
Fig. 3. Southern blot of genomic DNA from the PelRR, PelSS and
TemR strains of Culex quinquefasciatus dige sted with EcoRV demon-
strating genomic amplification of AO in resistant PelRR, but not in
resistant TemR.
Fig. 4. Specific staining and inhibition of the xanthine oxidase (XO)
stained band. Equal crude h omogen ate protein samples from C. quin-
quefasciatus PelRR adults were loaded to each well of a gradient

PhastGel (8–25%) and subjected to native PAGE P hastsystem. The
formazan staining solution was prepared as described in materials and
methods, with acetaldehyde/benzaldehyde substrates and the xanthin e
oxidase inhibitor allopurinol (lane 1) and the specific xanthine oxidase
substrate hypoxanthine (lane 2). The positions of the molecular mass
markers are indicated on the left.
Fig. 5. The amplified AO stained band in the insecticide resistant
C. quinquefasciatus PelRR strain. (A)NativePAGEoftheinsecticide
susceptible PelSS (lane 1) and the insecticide resistant PelRR (lane 2)
Culex quinque fasciat us adults. Equal amounts of crude homogenate of
pooled samples were loaded to each well o f a gradien t P hastGel (8–
25%) and a neat mixture of aldehyde substrates [1 : 1 (v/v) a cetalde-
hyde/benzaldehyde] was used a s substrate. (B) The molecular mass was
estimated by bilogarithmic plotting of molecular masses of the stan-
dards against T%, which was the total polyacrylamide concentration
reached by each protein afte r electroph oresis. The stan dard markers
were as follows: (a) urease (hexamer, M
r
545 0 00); (b) urease (trimer,
M
r
272 000); (c) albumin bovine s erum (dimer, M
r
132 000);
(d) albumin bo vine seru m (mono mer, M
r
66 000); (e) albumin chicken
egg (M
r
45 000).

Fig. 2. Comparison of the putative AO f rom PelRR with the amino-acid
sequences of bovine, human and maize AO with XDH from Drosophila
melanogaster. Common residues between sequences are boxed.
Ó FEBS 2002 AO from C. quinquefasciatus (Eur. J. Biochem. 269) 775
enzyme system a simple inhibition study with various AO
inhibitors and pesticides w as performed. Table 2 shows the
percentage inhibition for each chemical tested on t he
standard AO activity assay. Methadone, a potent inhibitor
of rat AO, was the most potent inhibitor of PelRR AO
activity. All four pesticides at concentrations of 0.05–
0.1 m
M
produced partial inhibition of AO activity, as did
two commonly used herbicides a t concentrations of 1 m
M
.
Only the triazine h erbicides had an inhibitory effect on
anaerobically measured ALDH activity (data not shown),
suggesting that inhibition is due to interaction with the AO
enzyme.
DISCUSSION
Cytochrome P450s have traditionally been considered as the
only enzymes to oxidize insecticides. Other oxidizing
enzymes, such as AO and XDH have only recently been
recognized as important in the oxidation of many drugs and
xenobiotics [8]. A O is capable of utilizing a wide r ange of
substrates such as, N-heterocyclics, aldehydes (which
includes a number of drugs), azo dyes and N-oxides.
Hepatic AO in humans mediates the oxidation of a large
number of such compounds [31]. Bovine AO is expressed at

high levels in the liver and lungs and is implicated in the
detoxification of environmental pollutants [32]. The pres-
ence of an amplified AO on the insecticide resistance-
associated amplicon of C. quinquefasciatus opens up the
possibility that this enzyme, may play a role in insecticide
Table 2. Influence of pesticides and inhibitors on the AO activity of
pooled C. quinquefasciatus PelRR larvae. AO activity was measured
with a reaction mixture containing 2,6,d ichloroindo phen ol and 1 : 1
acetaldehyde/benzaldeh yde (0.1 m
M
). The data are means ± SD of
three separate experiments each of which was performed in duplicate.
Pesticides
Inhibitor
concentration (m
M
)
Remaining
activity (%)
Herbicides
S-Triazine 1 73.9 ± 2.0
Atrazine 1 67.4 ± 3.2
Insecticides
Thiabendazole 0.05 76.2 ± 4.5
Methidathione 0.5 82.5 ± 3.5
Diazinon 0.1 85.7 ± 8.4
Parathion 0.1 89.7 ± 5.6
AO inhibitors
Menadione 1.0 56.6 ± 4.2
Methadone 0.1 32.1 ± 2.5

SKF-525A (Profidane) 1 64.0 ± 3.8
Table 1 . ALDH specific activities in insecticide resistant PelRR and
insecticide susceptible PelSS C. quinquefasciatus larvae. Assays were
performed anaerobically and activities were estimated for pooled
C. qu inque fasciatus PelRR and PelSS fourth instar larvae.
Strain
ALDH activity
(nmolÆmin
)1
Æmg protein
)1
)
PelRR 1.24 ± 0.2
PelSS 1.15 ± 0.2
Fig. 6. AO enzymatic activity in different life stages of insecticide
resistant C. quinquefasciatus PelRR. Upper panel: AO specific activity
was measured in pooled crude homogenates of isogenic lines. Activity
means were d etermin ed f or each of three indepe ndent i sogenic s amples
at each time point. Results are means ± SD. Lower panel: native AO
stained bands from equal loading of crude homogenate proteins of
different life stages of an isogenic C. qu inqu efasc iatus PelRR line.
Fig. 7. Ranges of AO activity from individual PelRR and PelSS larvae.
Ranges of AO specific activity from individual C. quinque fasciat us
PelRR and PelSS fourth instar larvae, respectively (n is the number of
individuals tested).
776 M. Coleman et al. (Eur. J. Biochem. 269) Ó FEBS 2002
resistance. This may account for the selective advantage of
insects carrying the esta2
1
/estb2

1
amplicon over those with
other esterase containing amplicons.
An AO gene has been cloned from the resistance-
associated amplicon of the PelRR strain of C. quinquefas-
ciatus. It contains the t hree conserved active site centres
expected of an AO enzyme. This is the first reported AO
sequence from any insect, hence absolute identification of
the enzyme through DNA sequence homology alone is
difficult [33]. However, the Culex enzyme clearly differs
from XDH, a similar molybdenum containing enzyme in
this family, as it lacks the NAD
+
domain which is essential
for XDH activity. The predicted amino-acid sequence of the
Culex ORF e ncodes fo r two [ 2Fe)2S] centres, a n NADH
binding site and a molybdenum binding domain, which is
consistent with the primary structure of AO from a range of
species. There are several complete AO sequences on the
database; within these there is a high degree of homology
between human and bovine AO sequence, but little
homology between these and Arabidopsis AO [9,32]. The
Culex sequence has similar levels of homology with all three
AOs.
The lack of structural c onservation between AO of
different species is suggested by Southern blots where
bovine AO cDNA probes did not cross hybridize with
Drosophila or to ad DNA, but did cross hybridize with
lizard, chicken, mouse, rat and h uman DNA. AO may be
less conserved than XDH, as a Southern blot using bovine

XDH cross-hybridized to DNA from all the organisms
above [9].
Identification of this enzyme as an AO is further
supported by the gene structure. Three introns are con-
served in all 31 insect XDHs recorded to date and a fourth is
conserved in all but one insect species. There are three
narrowly distributed novel introns, one in the medfly and
two in the Willistroni group of Drosophila, one of which is
shared by a second Drosophila group [35]. All of these
introns, along with numerous others, are found in the
genomic DNA encoding mammalian X DHs and AOs. Of
the five in trons in the Culex AO genomic DNA, introns
1 and 5 occur in all known XDH and AO sequences. Introns
3 and 4 occur in all mammalian AO and XDH sequences
but in no insect XDH sequence, and intron 2 is novel to this
Culex AO sequence (Fig. 8).
TheamplifiedAOoccursonthecommonesta2
1
/estb2
1
amplicon, but does not occur on either of the two estb1
amplicons in the TemR or COL Culex strains [3]. We have
previously shown a ladder of truncated AO bands in the
COL strain [3], and the current study shows TemR has no
amplified AO sequence, further confirming the differences
in the amplicons of these two strains despite both having an
amplified estb
1
[18,36].
To influence fitness of mosquitoes carrying the amplicon,

and p lay a role in insecticide detoxification, the amplified AO
needs to be expressed. Multiple allelic variants of the esterase
genes occur in Culex, making it easy to identify transcripts
from the esterases on the insecticide re sistance-associated
amplicons. PCR analysis was undertaken to see whether a
similar level of allelic variation occurred in the AO locus.
Comparison of the AO from resistant PelRR and susceptible
PelSS strains of Culex show that there is allelic variation at
this locus. The amplified AO diverges significantly from its
nonamplified counterparts a t its 5¢ end, although all allelic
variants are highly conserved at their 3¢ ends in line with
other known AO sequences [32,34]. The variability be tween
the nonamplified and a mplified alleles of AO from PelSS and
PelRR, coupled with the complete homology between the
PelRR genomic exons and the cDNA sequence from PelRR,
suggests that the AO cDNA cloned from PelRR was
transcribed from the amplicon and not from an un-amplified
AO elsewhere in the genome. The strength of PCR product
in PelRR also suggests that the rates of AO expression are
higher in this strain than in PelSS.
The normal physiological role of AO in mosquitoes is, a s
yet, unknown, hence it i s difficult to p redict what effect if
any the over-expression of this enzyme w ould have on the
fitness of the mosquito carrying the AO containing ampli-
con. Histochemical studies on the patterns of AO content in
imaginal wing discs in Minute mutants o f hybrids of
D. melanogaster [33] an d hybrids of M. domestica [15]
suggest that it plays a role in larval development. In male
moths AO assists in the catabolism of pheromones for
location of female moths [10,12]. In some f emale moths their

response to aldehydes in plant material is mediated by AO
[12].
To further characterize the effe cts of the amplified AO,
we analysed AO activity in resistant PelRR and s usceptible
PelSS m osquitoes. The active site of AO includes an
extended lipophilic active site [37], which can accommodate
the diphenyl ring systems of methadone and SKF-525A.
SKF-525A (Profidane) and its related analogues and
methadone are potent inhibitors of rat liver cytsolic AO
[38]. Methadone was a potent inhibitor o f mosquito AO
activity, whilst SKF-525A was a poorer inhibitor.
The absence of clear trends in structural requirements for
substrates of AO, has been attributed to the flexibility of its
substrate binding sites and its multiple productive orienta-
tion [39]. This makes AO an effective detoxifying enzyme
for a broad range of substrates in higher vertebrates. The
broad substrate specificity of AO, including the oxidative or
reductive metabolism of a wide variety of nitrogen or sulfur
containing heterocyclic xenobiotics has been well docu-
mented [6,7]. This suggests that insecticides and their
metabolites may make good substrates for AO. AO from
higher vertebrates is involved in the oxidative metabolism of
neurotoxins [40], substituted quinazolines and pthalazines
[41], chinhona antimalarials [42], purines and their ana-
logues [39], quinoloinium cations a nd quinines [43,44],
Fig. 8. Schematic diagram of the intron positions of PelRR AOscom-
pared to AOs and XDHs from a range of species. Introns at positions A
andGarecommontoPelRRAOandinsectXDHs. Intron* is novel to
the Culex AO, while the four remaining introns are at positions in
common with mammalian AO and XDH .

Ó FEBS 2002 AO from C. quinquefasciatus (Eur. J. Biochem. 269) 777
benzothiazole [45], and certain drugs, such as zonisamide
[46]. The role of AO in the o xidative metabolism of these
nitrogen or sulfur containing heterocyclic compounds is the
catalysis of the oxidation of a ring methine group usually
vicinal to a ring nitrogen or sulfur. Such a nu cleophilic
attack at an electron deficient carbon atom, adjacent to a
ring nitrogen or sulfur atom, occurs in the metabolic
pathways of thiocarbamate, phosphorothioate, phosphora-
mide and other heterocyclic pesticides. However, the role of
oxidative enzymes, such as AO, in the metabolism o f these
insecticides has not been investigated to date. Inhibition
studies on PelRR larvae suggested that many of these
pesticides may interact with mosquito AO.
AO is involved in the reductive metabolism of a broad
variety of nitro or sulfur compounds that can a ct as
electron acceptors, such as N-heterocyclic and aromatic
nitro c ompounds, nitrosamines, hydroxamic acids, azo
dyes [32], and certain drugs s uch as the anticancer agent
DACA [47]. However AO activity with sulfoxides [48,49],
and i n the reduction of N-oxide compounds to their
corresponding amino derivatives [48,50–52], is of greater
significance with regard to insecticide resistance. It is
possible that the novel mosquito AO may be involved in
the nitroreductase activity based metabolic pathways of
major insecticides, such as the conversion of parathion to
aminoparathion, which is 100–300-fold less toxic than t he
parent compound [53]. The partial inhibition of Culex AO
activity by a range of insecticides, including 0.1 m
M

parathion, suggests that it may recognize this insecticide
as a s ubstrate.
The ability of AO to oxidize a range o f xenobiotics may
give mosquitoes with the esta2
1
/estb2
1
amplicon, a selective
advantage over oth er resistance-associated amplicons, par-
ticularly in the presenc e of insecticides. As AO is capable of
interacting with e nvironmental pollutants, in particu lar
pesticides or their by-products, then it is feasible that the
amplified AO fo und on the esta2
1
/estb2
1
amplicon has a
functional significance in resistant insects.
The low level of amplified AO activity in adult
compared to larval PelRR mosquitoes, s uggests that the
coamplified esterases, rather than the AO, are primarily
responsible for the strong negative correlation between this
insecticide resistance mechanism and filariasis vectorial
capacity [4].
ACKNOWLEDGEMENTS
M. C. was supported by a Wellcome Trust Prize Studentship. J. G. V.
was supported by an European Union Marie Curie TMR grant
(MCFI-1999–002 59).
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