Transactions of the Royal Society of Tropical Medicine and Hygiene (2009) 103, 1139—1145

available at www.sciencedirect.com

journal homepage: http://www.elsevier.com/locate/trstmh

Evidence of multiple pyrethroid resistance

mechanisms in the malaria vector Anopheles
gambiae sensu stricto from Nigeria
T.S. Awololaa,∗ , O.A. Oduolaa, C. Strodeb, L.L. Koekemoerc,d,
B. Brookec,d, H. Ransonb

a Nigerian Institute of Medical Research, Yaba, Lagos, Nigeria b

Liverpool School of Tropical Medicine, Liverpool, UK
c Vector Control Reference Unit, National Institute for Communicable Diseases, Johannesburg, South Africa
d Division of Virology and Communicable Disease Surveillance, School of Pathology of the National Health Laboratory Service and the

University of the Witwatersrand, Johannesburg, South Africa

Received 17 April 2008; received in revised form 15 August 2008; accepted 15 August 2008
Available online 30 September 2008

KEYWORDS Summary Pyrethroid insecticide resistance in Anopheles gambiae sensu stricto is a major con-
Anopheles; cern to malaria vector control programmes. Resistance is mainly due to target-site insensitivity
Malaria; arising from a single point mutation, often referred to as knockdown resistance (kdr). Metabolic-
Metabolism; based resistance mechanisms have also been implicated in pyrethroid resistance in East Africa
Pyrethroid; and are currently being investigated in West Africa. Here we report the co-occurrence of both
Insecticide resistance mechanisms in a population of An. gambiae s.s. from Nigeria. Bioassay, synergist
resistance; and biochemical analysis carried out on resistant and susceptible strains of An. gambiae s.s.
Nigeria from the same geographical area revealed >50% of the West African kdr mutation in the resis-
tant mosquitoes but <3% in the susceptible mosquitoes. Resistant mosquitoes synergized using
pyperonyl butoxide before permethrin exposure showed a significant increase in mortality com-
pared with the non-synergized. Biochemical assays showed an increased level of monooxygenase
but not glutathione-S-transferase or esterase activities in the resistant mosquitoes. Microarray
analysis using the An. gambiae detox-chip for expression of detoxifying genes showed five over-
expressed genes in the resistant strain when compared with the susceptible one. Two of these,
CPLC8 and CPLC#, are cuticular genes not implicated in pyrethroid metabolism in An. gambiae
s.s, and could constitute a novel set of candidate genes that warrant further investigation.
© 2008 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights
reserved.

∗ Corresponding author. Present address: Medical Research Council Laboratories, The Gambia, P.O. Box 273, Banjul, The Gambia.

Tel.: +220 702 2832; fax: +220 449 7952.

E-mail addresses: awololas@hotmail.com, tsawo2001@yahoo.co.uk (T.S. Awolola).

0035-9203/$ — see front matter © 2008 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.trstmh.2008.08.021
1140
1. Introduction
Pyrethroid resistance in the major Afrotropical malaria vec-
tor Anopheles gambiae sensu stricto is common in West
Africa and presents a major challenge facing malaria vec-
tor control. Two broad classes of resistance mechanism
play an important role in mosquito resistance to insecti-
cide: target-site insensitivity and metabolic enzyme-based
resistance.1 Target-site insensitivity to pyrethroids and DDT
in An. gambiae is associated with single point mutations,
commonly referred to as knockdown resistance (kdr). These
mutations lead to modification of the voltage-gated sodium
channel protein, making it less susceptible to the bind-
ing of pyrethroids and DDT. 2 Two different kdr mutations
have been identified in resistant An. gambiae s.s. from
West and East Africa. West African kdr is characterized by
a leucine—phenylalanine substitution at position 1014 of
the voltage-gated sodium channel gene.2 East African kdr
involves a leucine—serine substitution at the same position.3
Both kdr mutations have recently been reported in Central
Africa.4 The kdr resistance mutations in An. gambiae have
previously been used as markers for monitoring introgression
between the molecular M and S forms of An. gambiae s.s.5,6
Metabolic-based resistance mechanisms, although equally
important in terms of conferring resistance in insect species,
have been under-investigated in many African countries,
including Nigeria.7
Metabolic enzyme-based resistance is principally asso-
ciated with three enzyme families: the cytochrome P450
monooxygenases (P450s), carboxylesterases (COEs) and
glutathione-S-transferases (GSTs).1,8 Each of these families
contains large numbers of genes with various roles in insect
metabolism, but only a small number are thought to be
directly involved in insecticide detoxification. Techniques
designed to determine the role of metabolic enzymes, par-
ticularly monooxygenases in pyrethroid resistance, include
the use of the synergist piperonyl butoxide, an inhibitor
of mixed function oxidase activity.8 Biochemical techniques
have also been designed to quantify levels of detoxifying
enzymes in resistant and susceptible mosquitoes. 9 These,
however, do not provide information on the specific gene(s)
involved. This gap can be filled using gene expression pro-
filing based on microarray technology: a powerful tool for
examining the relationship between global gene expression
profiles and various physiological states.10
Following the launch of the Roll Back Malaria initiative
in 2000, there has been an increase in the distribution
of insecticide-treated nets (ITNs) in Nigeria. This effort
is being complemented by pilot indoor residual spraying
(IRS) programmes (C.N. Amajoh, personal communication).
The sustainability of these insecticide-based interventions
relies on the continuing susceptibility of Anopheles vec-
tors to the few available insecticides. Historically, the first
report of insecticide (DDT and dieldrin) resistance in African
Anopheles came from Nigeria. 11 The numbers of reports
of Anopheles resistance to insecticide have since then
increased, but insecticide resistance monitoring is consid-
ered a low priority in the mainstream of the national malaria
control programme.
Pyrethroid resistance has been reported in An. gam-
biae from different localities in Nigeria, with frequencies
of the West African kdr genes varying from 50 to 75% in
T.S. Awolola et al.
the resistant mosquitoes.12—15 Here we present the results
of bioassay, synergist, biochemical and microarray analyses
and reveal the co-occurrence of kdr metabolic and poten-
tially cuticular resistance mechanisms in An. gambiae s.s.
2. Materials and methods
2.1. Field collections
Anopheles gambiae were collected at Ipokia (03◦24′E,
06◦27′N), where pyrethroid resistance in An. gambiae has
been reported, and Alakia (03◦54′E, 07◦26′N), where the
mosquitoes are susceptible to pyrethroids. 12 Both collec-
tions were made in October 2005. The study sites, method of
mosquito collection and identification have been described
elsewhere.13 Anopheles gambiae s.s. populations at both
sites consist of approximately 30 and 70% of the molecu-
lar M and S forms, respectively. Wild-caught females were
induced to lay eggs in the laboratory insectaries, and their F1
progeny, henceforth referred to as ‘Ipokia’ and ‘Agib’, were
used in subsequent studies. The Ipokia strain was maintained
under routine permethrin selection pressure.
2.2. Synergist studies
The synergist piperonyl butoxide (PBO), an inhibitor
of monooxygenases, was used in conjunction with the
pyrethroids permethrin and deltamethrin in a subset of
bioassays. For each test, a cohort of Ipokia mosquitoes
was divided into two equal samples. One sample was pre-
exposed to 4% piperonyl butoxide-impregnated filter paper
for 1 h and then both samples were immediately exposed to
0.75% permethrin or 0.05% deltamethrin for 1 h according to
standard WHO specifications using WHO insecticide test kits
and treated papers.16 Final mortalities 24 h post-exposure
were compared between the synergized and unsynergized
samples. The Agib samples were not synergized but were
exposed to permethrin- and deltamethrin-treated papers.
2.3. Identification of the knockdown resistance
mechanism
Following synergist and insecticide susceptibility tests, the
kdr genotypes were determined for all specimens. Both sus-
ceptible and resistant specimens were assayed using an
allele-specific PCR diagnostic test designed for the West
African leucine—phenylalanine mutation.2 Owing to recent
reports of the presence of the East African kdr mutation in
An. gambiae in neighbouring countries,4 the presence of this
mutation was also investigated in the samples assayed here.
2.4. Biochemical analysis
Biochemical assays were used to quantify levels of monooxy-
genase and activity of non-specific esterases and GST in
individual mosquitoes from the Ipokia strain using batches
of 47 two-day-old unfed female mosquitoes per microtitre
plate.9 Each plate also contained 10 specimens of two-
day-old female mosquitoes from the susceptible Agib strain
for comparative purposes. Analyses were carried out on an
Pyrethroid resistance mechanisms in Anopheles gambiae s.s.
Anthos spectrophotometer (Anthos 2010, Anthos Labtech,
GmbH, 5071 Wals Salzburg, Austria). Mean enzymatic
level/activities were compared between the Agib and Ipokia
strains using two-sample Student’s t tests.
2.5. Microarray analysis
The microarray analysis was carried out using the Anophe-
les detox chip: a highly specific microarray developed for
studying metabolic-based insecticide resistance in malaria
vectors.10
2.5.1. Target preparation and microarray hybridizations
RNA extractions, cRNA synthesis and labelling reactions
were performed independently for each biological replicate.
Total RNA was extracted from batches of 10 three-day-old
adult female mosquitoes using a PicoPure RNA isolation kit
(Arcturus; Biocomp Inc., San Francisco, CA, USA) accord-
ing to the manufacturer’s instructions. Total RNA quantity
and quality was assessed using a NanoDrop spectrophotome-
ter (NanoDrop Technologies, Thermo Fisher Scientific Inc.,
Waltham, MA, USA) before further use. RNA was amplified
using a RiboAmp RNA amplification kit (Arcturus) accord-
ing to the manufacturer’s instructions. Amplified RNAs were
checked for quantity and quality by spectrophotometry and
agarose gel electrophoresis. Amplified RNA was reverse tran-
scribed into labelled cDNA and hybridized to the array as
previously described.10 Each comparison was repeated three
times with different biological samples. For each biological
replicate, two hybridizations were performed, in which the
Cy3 and Cy5 labels were swapped between samples so a total
of six hybridizations were performed for each comparison.
2.5.2. Microarray data analysis
Spots that failed to meet any of the following criteria in
either channel were rejected: (1) an intensity value of
>300; (2) signal-to-noise ratio of >3; and (3) greater than
60% pixel intensity superior to the median of the local
background ± 2 SD. Normalization and statistical anal-
yses of the data were performed using the Limma 1.9
software package for R 2.3.1, available from the CRAN
repository.17 Background corrected intensities from the red
(R, Cy5) and the green (G, Cy3) channels were transformed
to intensity log-ratios, M = log R/G, and their correspond-
ing geometrical means, A = (log R + log G)/2. Within each
array, M-values were normalized as a function of A using
the locally weighted scatterplot smoothing (LOWESS) func-
tion and scaled to equalize the median absolute value across
all arrays to account for technical biases between replicate
Table 1 Bioassay results comparing pyperonyl butoxide synergized and unsynergized Ipokia mosquitoes and the susceptible
Agib strain
Mosquitoes No. exposed (24 h % mortality)a
1% permethrin 4% PBO + 1% permethrin
Ipokia 200 (58.0) 200 (87.0)
Agib 200 (100) — 200 (100)
PBO: piperonyl butoxide.
a Figures in parentheses denote % mortality of the mosquitoes exposed.
1141
Figure 1 Mean level of monooxygenase in the pyrethroid-
resistant Ipokia and the susceptible Agib strain.
hybridizations.18 Mean expression ratios were submitted to
a one-sample Student’s t test against the baseline value of 1
(equal gene expression in both samples) with a multiple test-
ing correction.19 Genes showing both t test P-values <0.001
and ≥2-fold over- or under-expression were considered dif-
ferentially expressed between comparisons. The expression
data from these microarray experiments can be accessed at
VectorBase.20
3. Results
3.1. Synergist and biochemical analysis
Table 1 shows the results of the pyrethroid susceptibility
test and the synergist study. The Agib strain is 100% sus-
ceptible to pyrethroids, whereas the 24 h post-exposure
mortality of the Ipokia strain was 58 and 72% in perme-
thrin (0.75%) and deltamethrin (0.05%), respectively. The
difference in mortality 24 h post-exposure between syn-
ergized and unsynergized samples from Ipokia exposed to
permethrin was highly significant (P < 0.001). Synergized and
unsynergized samples exposed to deltamethrin gave sim-
ilar results (P < 0.001). Even then, the synergized Ipokia
mosquitoes exposed to permethrin or delatamethrin still
showed much lower mortality when compared with Agib
(Table 1). More than 90% of synergized mosquitoes that
survived pyrethroid exposure were of the S form, whereas
those unsynergized mosquitoes that survived the same insec-
ticide exposure included equal proportions of the M and
S forms (data not shown). Biochemical analysis revealed
a significantly increased (P = 0.017) level of monooxyge-
nase in the Ipokia samples compared with the susceptible
Agib (Figure 1), suggesting monooxygenase involvement in
0.05% deltamethrin PBO + 0.05% deltamethrin
200 (72.0) 200 (92.0)
—
1142 T.S. Awolola et al.

Table 2 Final mortalities 24 h post-exposure of Anopheles gambiae (Ipokia and Agib) strains exposed to 1% permethrin for 1 h
and the corresponding as knockdown resistance (kdr) allele frequencies

Mosquitoes Genotype and frequency of the kdr alleles (%)a

No. assayed 24 h % mortality RR RS SS F (R)

Ipokia 1040 58.4 50.2 4.6 45.2 54.8

Agib 1040 100 0 2.5 97.5 2.5
a kdr genotypes: RR: resistant; RS: heterozygous susceptible; SS: homozygous susceptible.

pyrethroid metabolism in the Ipokia population. The dif-

ference in the mean GST activity between both strains
was not significant (P = 0.52). The difference in esterase
activity in both strains was not significant using either
para-nitrophenyl acetate (P = 0.59) or -naphtyl acetate as
substrate (P = 0.61).

3.2. Frequency of the kdr mutations

The leucine—phenylalanine kdr mutation was detected

in the molecular S form but not in the M form. The
leucine—serine mutation was not detected in any of the sam-
ples assayed. The frequency of the kdr allele (R) was >50% in
those Ipokia mosquitoes that survived 24 h post-exposure but
<5% in the cohort that died (Table 2). Although there was a
significant difference in mortality between synergized and
unsynergized Ipokia mosquitoes, the frequency of the kdr
alleles was similar in both synergized and unsynergized sub- Figure 2 Microarray analysis comparing constitutive detoxifi-
samples (data not shown). The Agib strain presented as fully cation gene expression of the pyrethroid-resistant Ipokia strain
susceptible to permethrin and deltamethrin. Nevertheless, with the susceptible Agib colony. The solid horizontal line repre-
the kdr allele was also found in these samples, although at sents a threshold P-value of <0.001 and the dashed vertical lines
a very much lower frequency and only in the heterozygotic represent a fold over- or under-expression in either direction.
phase (Table 2). Those genes showing both a P-value of <0.001 and a ± 2-fold
expression are named on the graph.

3.3. Microarray analysis

Figure 2 shows the microarray analysis comparing constitu-

tive expression of detoxification genes in the pyrethroid-
resistant Ipokia strain with the susceptible Agib. Of the
233 gene probes on the An. gambiae detox chip, 36
were differentially expressed. The cut-off value (± 2-
fold differential expression) and threshold P-value (<0.001)
established to select biologically relevant candidate genes
among those showing over-expression revealed six genes
with a significant difference in expression. The genes that
were significantly over-expressed in the resistant Ipokia
strain include two cuticular genes (CPLC8 and CPLC#), a
cytochrome P450 (CYP325A3), a sigma class GST (GSTS1-
2) and a thioredoxin peroxidase (TPX4) gene. Of these,
TPX4 had the highest fold expression (3.29). The two
cuticle genes, CPLC# and CPLC8, were over-expressed
2.37- and 2.86-fold, respectively, while CYP325A3 and Figure 3 Microarray analysis comparing detoxification gene
GSTS1-2 had 2.44- and 2.68-fold expression. TPX2 was expression of the pyrethroid-resistant Ipokia strain (1 h per-
the only gene that showed significantly higher expression methrin exposure) with the susceptible Agib colony. The solid
(2.6-fold) in the susceptible Agib (Figure 2). The Ipokia horizontal line represents a threshold P-value of <0.001 and the
mosquitoes assayed after 1 h exposure to permethrin showed dashed vertical lines represent a fold over- or under-expression
much higher expression levels of CPLC8, CPLC# and TPX4 in either direction. Those genes showing both a P-value of
(Figure 3). <0.001 and a ±2-fold expression are named on the graph.
Pyrethroid resistance mechanisms in Anopheles gambiae s.s.
4. DISKUSI
hasil yang disajikan di sini menunjukkan bahwa piretroid
resis-bantuan di An. gambiae dari Afrika Barat multifaktor
dan mencakup mekanisme metabolik yang belum
sepenuhnya dijelaskan. Banyaknya leusin — fenilalanin kdr
mutasi telah disorot di An. gambiae dari Africa6 Barat, 13,
21-24 dan baru-baru ini di pusat Africa4, 25 dan mungkin
lebih luas daripada yang diperkirakan sebelumnya. Tidak
adanya alel kdr di tahan nyamuk bentuk M molecu-lar,
bagaimanapun, mengusulkan detoksifikasi metabolik yang
account untuk produksi fenotipe perlawanan. Ini adalah
paling jelas berdasarkan hasil dari studi Syn-inti, di mana
mereka sampel Ipokia yang saling bersinergi menggunakan
pyperonyl butoxide sebelum paparan permetrin
menunjukkan peningkatan yang signifikan dalam mortalitas
dibandingkan dengan non - sampel sinergis. Analisis
biokimia mengungkapkan tingkat peningkatan
monooxygenase kegiatan Ipokia piretroid-tahan strain,
memberikan bukti yang mendukung berbasis metabolik
perlawanan. Peningkatan kadar monooxygenase juga telah
terlibat dalam perlawanan piretroid. Funes-tus dari
temuan serupa Africa.26,27 Selatan telah dilaporkan di An.
gambiae dari tengah dan Afrika Timur, 25, 28, 29
menyarankan distribusi ini perlawanan mecha-nism.
Sementara tes biokimia instruktif dalam mengidentifikasi
kelas enzim detoksifikasi, detoksifikasi chip pergi bulu-sana
oleh mengidentifikasi gen orang putatively terlibat dalam
perlawanan dan sangat mempercepat pemahaman kita
tentang metabolik perlawanan. Gen enam yang
constitutively over dinyatakan dalam Ipokia tahan, tiga
(CYP325A3, GSTS1-2 dan TPX4) sebelumnya telah
ditunjukkan untuk terlibat dalam metabolisme pyrethroid,
khususnya di Afrika Timur ketegangan An. gambiae.10...
Temuan ini dapat dijelaskan berdasarkan fakta bahwa
gen ini juga sangat dinyatakan dalam penerbangan
dipteran muscles.35 GSTS1-2 baru-baru ini telah
ditunjukkan untuk menjadi lebih-dinyatakan dalam
piretroid Tahan regangan, Odumasy, dari Ghana.7
Selatan peran GSTS1-2 Penganugerahan perlawanan
insecti-cide saat ini belum diketahui; Namun, sigma
GSTs melindungi terhadap stres oksidatif yang
dihasilkan dalam penerbangan muscles.36 Akibatnya,
itu mungkin memiliki peran sekunder dalam
manajemen stres oksidatif yang disebabkan oleh
tindakan pyrethroids. Penelitian ini juga
mengungkapkan tingkat over ekspresi gen cuticular
dua (CPLC8, CPLC #). Tingkat signifikan lebih tinggi
ekspresi gen ini bersama-sama dengan TPX4 setelah
eksposur permetrin ketegangan Ipokia menunjukkan
fungsi metabolisme potensi mereka. Selain itu, CPLC8
dan CPLC # tidak sebelumnya telah terlibat dalam
metabolisme piretroid dalam An. gambiae dan bisa
1143
merupakan seperangkat novel calon gen yang
menjamin lebih lanjut investigati...
Mengikuti 1 h paparan permetrin, hanya tiga (TPX4, CPLC8
dan CPLC #) dari lima sebelumnya diamati
selama-menyatakan gen yang signifikan berlebihan
mengungkapkan di Tahan regangan. Satu jam mungkin
terlalu cepat untuk mengamati efek induksi. Sementara ada
literatur yang menggambarkan gen induksi, 43-46 induksi di
Aedes aegypti yang kurang tahan regangan dibandingkan
dengan suscepti-ble ketegangan. Gen induksi di rentan
nyamuk adalah sangat penting di 3h Post-Exposure (WC
hitam, komunikasi pribadi). Demikian pula, setelah paparan
dosis sublethal permetrin dan DDT, efek induksi tidak diamati
pada resistan terhadap An. gambiae (G. Warr, komunikasi
pribadi). Meskipun dampak operasional piretroid
perlawanan pada efektivitas ITNs tidak jelas, laporan
terbaru telah menggambarkan khasiat yang
mengurangi ITNs dan IRS tempat
piretroid-perlawanan di Republik Benin, mana
frekuensi kdr adalah high.23,24 ini menekankan
perlunya insektisida perlawanan pemantauan dalam
program-program pengendalian malaria yang
mengandalkan ITNs dan IRS intervensi di seluruh
Afrika. Fakta bahwa studi ini menunjukkan bahwa
populasi tahan mungkin mengandung berbagai
mekanisme tahan mengkhawatirkan. Aplikasi
insektisida alternatif dan rotasi penggunaan
insektisida telah menganjurkan untuk perlawanan
management.1,47 Unfor-tunately, ada beberapa
alternatif insektisida yang tersedia untuk
pengendalian vektor malaria. Sama seperti yang
mendukung penggunaan DDT pengendalian penyakit
malaria, 48 sana adalah dire perlu menemukan
insektisida alterna-tive untuk meminimalkan
penyebaran cross-resistance antara DDT dan
pyrethroids.
Authors’ contributions: TSA conceived the study and
drafted the manuscript; OAO carried out field and laboratory
analysis; CS and HR designed the microarray experiment and
analysis; LLK and BB carried out molecular and biochemical
analysis and revised the manuscript. All authors read and
approved the final paper. TSA is guarantor of the paper.
Penghargaan: Kami terima kantor regional WHO untuk Afrika
(WHO-Afro) untuk menyediakan makalah diperlakukan insektisida dan
Patricia Okoye untuk membantu dalam tes biokimia. Dana:
Penyelidikan ini menerima dukungan keuangan dari inisiatif
Multilateral Malaria (MIM) proyek melalui Bank
UNICEF/UNDP/dunia/WHO program khusus penelitian dan pelatihan di
tropis penyakit (TDR) melalui pemberian nomor A60039 TSA. Konflik
kepentingan: tidak ada yang menyatakan.
 1144
Persetujuan etis: penelitian telah disetujui
oleh Komite etika penelitian dari Nigeria
Institute of Medical Research dan etika Komite
Review dari WHO - MIM/TDR, Jenewa,
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