Ahoua Alou et al. Malaria Journal 2010, 9:167
Distribution of ace-1R and resistance to carbamates
and organophosphates in Anopheles gambiae s.s.
populations from Côte d'Ivoire
© 2010 Alou et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons At-
tribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Ludovic P Ahoua Alou1,2, Alphonsine A Koffi*1, Maurice A Adja1,2, Emmanuel Tia3, Philippe K Kouassi2, Moussa Koné1
and Fabrice Chandre4
Background: The spread of pyrethroid resistance in Anopheles gambiae s.s. is a critical issue for malaria vector control
based on the use of insecticide-treated nets. Carbamates and organophosphates insecticides are regarded as
alternatives or supplements to pyrethroids used in nets treatment. It is, therefore, essential to investigate on the
susceptibility of pyrethroid resistant populations of An. gambiae s.s. to these alternative products.
Methods: In September 2004, a cross sectional survey was conducted in six localities in Côte d'Ivoire: Toumbokro,
Yamoussoukro, Toumodi in the Southern Guinea savannah, Tiassalé in semi-deciduous forest, then Nieky and Abidjan
in evergreen forest area. An. gambiae populations from these localities were previously reported to be highly resistant
to pyrethroids insecticides. Anopheline larvae were collected from the field and reared to adults. Resistance/
susceptibility to carbamates (0.4% carbosulfan, 0.1% propoxur) and organophosphates (0.4% chlorpyrifos-methyl, 1%
fenitrothion) was assessed using WHO bioassay test kits for adult mosquitoes. Then, PCR assays were run to determine
the molecular forms (M) and (S), as well as phenotypes for insensitive acetylcholinesterase (AChE1) due to G119S
Results: Bioassays showed carbamates (carbosulfan and propoxur) resistance in all tested populations of An. gambiae
s.s. In addition, two out of the six tested populations (Toumodi and Tiassalé) were also resistant to organophosphates
(mortality rates ranged from 29.5% to 93.3%). The M-form was predominant in tested samples (91.8%). M and S
molecular forms were sympatric at two localities but no M/S hybrids were detected. The highest proportion of S-form
(7.9% of An. gambiae identified) was in sample from Toumbokro, in the southern Guinea savannah. The G119S
mutation was found in both M and S molecular forms with frequency from 30.9 to 35.2%.
Conclusion: This study revealed a wide distribution of insensitive acetylcholinesterase due to the G119S mutation in
both M and S molecular forms of the populations of An. gambiae s.s. tested. The low cross-resistance between
carbamates and organophosphates highly suggests involvement of other resistance mechanisms such as metabolic
detoxification or F290V mutation.
Malaria vectors control mainly relies on the use of insec-
ticide-treated nets (ITN) and indoor residual spraying
(IRS). Pyrethroids are the only group of insecticides cur-
rently recommended for net treatment . Although
pyrethroid resistance in the most important malaria vec-
tor Anopheles gambiae s.s. has become widespread in sev-
eral African countries [2-5], field studies in experimental
huts and at community level using malaria indicators
have shown that pyrethroid-treated bed nets remain usu-
ally effective against pyrethroid resistant mosquitoes [6-
9]. However, the evolution of pyrethroid resistance in An.
gambiae s.s. represent a threat for malaria control. To
prevent any significant decline of the efficiency of pyre-
throids, harmful to the malaria control, management
strategies of pyrethroid resistance are envisaged through
* Correspondence: email@example.com
1 Institut Pierre Richet (IPR), BP 47 Abidjan, Côte d'Ivoire
Full list of author information is available at the end of the article
Ahoua Alou et al. Malaria Journal 2010, 9:167
Page 2 of 7
the exploration of news tools or combination of existing
One of these strategies, used in agriculture as well as in
public health, consists to associate in the same treatment,
several molecules having different modes of action.
Although developed initially for agricultural use and for
indoor residual spraying, carbamates and organophos-
phates constitute a new prospect to circumvent pyre-
throid resistance in An. gambiae s.s.
In area of high prevalence of kdr in Côte d'Ivoire, exper-
imental hut trials of carbamates or organophosphates
alone and in combination with pyrethroids on mosquito
nets showed very promising results [8,10-13]. However,
little is known about the susceptibility status of pyre-
throid resistance populations of An. gambiae to organo-
phosphates and carbamates in Côte d'Ivoire, as well as
potential resistance mechanisms.
Acetylcholinesterase (AChE) is a common target for
carbamates and organophosphates. These insecticides
blocks transmission of nerve impulses by irreversible
inhibition of AChE at cholinergic synapses, causing
insect death. Cross-resistance to carbamates and organo-
phosphates can arise by insensitive AChE mechanism
due to the glycine to serine substitution (G119S muta-
tion) resulting from a single point mutation in the ace-1
gene . The G119S mutation was selected indepen-
dently in several mosquitoes species including An. gam-
biae s.s., the major malaria vector in Africa [11,14-17].
This mutation was found in both M and S molecular
forms of An. gambiae from Côte d'Ivoire [16,17].
In the current study, the geographic extent of insensi-
tive AChE mechanism in An. gambiae s.s. populations
from Côte d'Ivoire according to molecular forms, as well
as their susceptibility status to carbamates and organo-
phosphates were investigated.
Mosquito populations and sampling sites
The study sites form a north-to-south transect across the
Southern Guinea savannah, the semi-deciduous forest
and the evergreen forest areas in Côte d'Ivoire. The last
two zones are characterized by intensive human activities
and agricultural land-degraded forest mosaic. Mosqui-
toes were collected during the rainy season from six
localities: Toumbokro (7°N; 5°35' W), Yamoussoukro
(6°82' N; 5°28' W) and Toumodi (6°55' N; 5°03' W) located
in the Southern Guinea savannah, Tiassalé (5°88' N; 4°38'
W) in a semi-deciduous forest area, then Nieky (5°20'N;
4°10'W) and Abidjan (5°33'N; 4°03' W) in a evergreen for-
est area (Figure 1). Samples were collected from coffee
and cocoa industrial plantations in Toumbokro, banana
cultivation fields in Nieky and in urban areas in Yamous-
soukro, Toumodi, Tiassalé and Abidjan. Mosquitoes were
collected at larval stage, brought to the laboratory and
reared until for emergence of adults. A reference labora-
tory strain of An. gambiae s.s. named "Kisumu", native
from Kenya and susceptible to all insecticides was used as
Bioassays were carried out using WHO test kits for adults
mosquitoes  with four insecticides of technical grade
quality: two carbamates (0.4% carbosulfan, 0.1%
propoxur) and two organophosphates (0.4% chlorpyrifos-
methyl and 1% fenitrothion). Filter papers were impreg-
nated according to WHO specifications by the Institut
Pierre Richet de Bouaké. Papers were stored at 4°C and
were not used more than three times.
Tests were performed with batches of 25 unfed females
of An. gambiae s.s., 2-5 days old, four replicates per insec-
ticide. Mosquitoes were exposed to the insecticide
treated papers for 60 min at 27 ± 1°C and 80% relative
humidity. After exposure, mosquitoes were kept in obser-
vation tubes, supplied with 10% honey solution and held
for 24 h before scoring mortality. Batches exposed to
untreated papers were used as control.
M/S taxon determination
According to previous studies, An. gambiae complex in
Côte d'Ivoire was only represented by An. melas on the
Atlantic littoral area and An. gambiae s.s., the most wide-
spread all over the country [19-21]. So the PCR analysis
in this study was carried directly on the molecular forms
of An. gambiae s.s.. Genomic DNA was extracted from
individual mosquitoes according to Collins et al  and
used for PCR analysis to determine M/S taxon according
to Favia et al . The PCR conditions were 10 min at
94°C as initial step, followed by 29 cycles (94°C for 30 sec-
onds, 63°C for 30 seconds and 72°C for 30 seconds). After
the last cycle the products were finally extended for 7 min
at 72°C. Primers used in the PCR were: R5 5'GCCAAT
CCGAGCTGATAGCGC3', R3 5'CGAATTCTAGGGAG
CTCCAG3', Mopint 5'GCCCCTTCCTCGATGGCAT3',
B/S 5'ACCAAGATGGTTCGTTGC3'. Amplified frag-
ments were analysed on a 1.5% agarose gel.
DNA diagnostic test for insensitive acetylcholinesterase
Genomic DNA extracted from the field samples and used
for PCR was also used to determine the phenotypes for
insensitive AChE G119S mutation according to Weill et
al . The DNA was PCR amplified with the primers
Ex3Agrev 5'AGGATGGCCCGCTGGAACAG3' for an
initial denaturation step of 3 min at 94°C, followed by
thirty-five cycles (94°C for 30 seconds, 62°C for 30 sec-
onds and 72°C for 20 seconds). After the final cycle the
products were extended for 5 min at 72°C. The PCR frag-
ments were then digested with Alu I restriction enzyme
Ahoua Alou et al. Malaria Journal 2010, 9:167
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Figure 1 Map of Côte d'Ivoire showing the localities in the different ecological zones where anopheline mosquitoes were collected
Ahoua Alou et al. Malaria Journal 2010, 9:167
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and fractionated on a 2% agarose gel. The two primers
produced a 403 bp fragment, which is undigested by AluI
for susceptible homozygous mosquitoes (SS), and cut into
two fragments (253 bp and 150 bp) for homozygous resis-
tant (RR). Heterozygous individuals (RS) display a com-
Mortality data were analysed according to WHO . To
compare the status of insecticide resistance, Fisher's exact
test was performed to determine if there was any signifi-
cant difference between mortality rates of two given pop-
ulations of An. gambiae s.s. using Statistica 6.0. Allelic
frequencies of G119S mutation were analysed using the
version 3.2a of Genepop . To assess if the mutation
frequencies was identical across populations, the test of
genotypic differentiation was performed .
Susceptibility to carbamates and organophosphates
Mortality rates of the Kisumu reference strain to all insec-
ticides was 100% (Table 1). Conversely, all the field sam-
ples were resistant to carbamates, with mortalities rates
less than 83%. Susceptibility to chlorpyrifos-methyl was
assessed on five populations except on the Yamoussoukro
population. Chlorpyrifos-methyl resistance was detected
in Toumodi and Tiassalé, with 82-94% mortality rates,
while it was suspected in Toumbokro with 97% mortality
rate. The two other populations were fully susceptible to
this organophosphate. Fenitrothion resistance was
observed in five out of the six populations tested (Toum-
bokro, Toumodi, Tiassalé, Nieky, Abidjan). Only the
Yamoussoukro population was fully susceptible to this
insecticide. Overall the two populations from Toumodi
and Tiassalé were resistant to all insecticides used. Tias-
salé sample was the most resistant to carbamates and
organophosphates with mortality rates of 3% and 12% for
carbosulfan and propoxur and 83% and 30% for chlorpy-
rifos-methyl and fenitrothion, respectively.
Molecular forms and frequencies of the G119S mutation
All PCR analysis to determine M/S molecular forms real-
ized in this study were positive, showing either the form
M or the form S. So it was not necessary to make the PCR
analysis for species identification .
Three hundred twenty-eight mosquitoes were identi-
fied to molecular forms and analyzed for the G119S
mutation; results are shown in Table 2. The M and S
molecular forms of An. gambiae s.s. occurred in sympatry
in two of the six localities, namely Toumbokro and Tou-
modi in the savannah area. However, the M-form was
predominant in the six areas, representing 91.8% of the
whole sample (n = 328). In sympatric areas, the frequen-
cies of the S-form were 41.9% (n = 62) and 1.3% (n = 76)
respectively in Toumbokro and Toumodi. However, no
M/S heterozygote was found.
The G119S mutation was detected in all the six popula-
tions tested, but only at heterozygote state, either in the
M or in the S form. The highest mutation frequency was
observed in the M form from the Tiassalé urban area
located in semi-deciduous forest (50%) and the lowest in
the M form from the Nieky banana cultivation fields in
evergreen forest (12%). No significant difference was seen
between G119S mutation frequencies in M and S forms
from Toumbokro (p = 0.9153).
Table 1: Mortality of a susceptible strain (Kisumu) and wild populations of Anopheles gambiae s.s. exposed to diagnostic
doses of technical material of insecticides
Carbosulfan (0.4%)Propoxur (0.1%) Chlorpyrifos-methyl (0.4%)Fenitrothion (1%)
LocalitiesMort StatusMort StatusMort StatusMort Status
Kisumu 100 (101)S 100 (104)S 100 (104)S 100 (102)S
R 96.9 (97)S
R NT 99.0 (98)S
Number of tested mosquitoes in parentheses; NT: no tested; Mort: Mortality rate 24 h post exposure; S: indicates susceptibility; R: suggests
NB: Numbers in the same column with the same superscript do not differ significantly by Fisher's exact test (p > 0.05)
Ahoua Alou et al. Malaria Journal 2010, 9:167
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The distribution of M and S molecular forms of An. gam-
biae s.s. in the study agrees with previous findings that
reported both M and S forms in Guinea savannah areas
and only the M form in the forest areas [27-30]. This geo-
graphic distribution seems to follow more the global
environment than the breeding sites nature. Both forms
are involved in carbamate and organophosphate resis-
tance, although at different level according to insecti-
cides. Indeed, in this study, An. gambiae s.s. displayed
large variations in resistance level to carbamates and
organophosphates. Although the wild populations were
all resistant to carbamates, resistance was less marked to
propoxur than to carbosulfan at WHO diagnostic con-
All these populations were as resistant to carbosulfan as
the population of Yaokoffikro in surrounding area of
Bouaké . The resistance reported in Bouaké was
attributed to agricultural or domestic hygiene or public
health use of carbamates. In Burkina-Faso, Diabaté et al
 attributed An. gambiae s.s. pyrethroid resistance in
cotton field areas to their use in agriculture.
The observed cross-resistance to organophosphates
and carbamates in Tiassalé and Toumodi highlights
implication of their common target site: the AChE-1.
Although the mutation ace-1 G119S provided cross-
resistance to organophosphates and carbamates, the
resistance level greatly varied between both insecticide
families. This difference observed in resistance level
could be the consequence of their difference observed in
dominance level relied on insecticide specificity. Accord-
ing to Djogbénou et al , dominance status of ace-1
G119S varied between semirecessivity with fenitrothion
and chlorpyrifos methyl to semidominance with
propoxur and carbamates. The fact that low cross-resis-
tance was observed in the other populations, suggests
and confirms potential involvement of metabolic resis-
tance mechanisms and/or alternative mutation associ-
ated to G119S. This may explain why mortality rates to
organophosphates among samples from Nieky, Abidjan
and Yamoussoukro were so strong despite confirmed
resistance level to carbamates in bioassays.
Such result could also be explained by possible cross-
resistance between organophosphates and pyrethroids
based on an increased detoxification mechanism were as
suggested for other anopheline species selected for pyre-
throid resistance .
Moreover an alternative mutation in ace-1 gene was
described in the Culex pipiens strain originating from
Cyprus. This mutation is F290V substitution and it con-
fers cross-resistance to OP and carbamate insecticides
. Because C. pipiens and An. gambiae s.s. share
G119S, it is possible that they share also this other muta-
tion. Asidi et al  had noted that G119S mutation did
not confer effective resistance to chlorpyrifos-methyl.
Yet, the G119S mutation involved certainly a high resis-
tance to carbamate but could enhance organophosphate
hydrolysis. Similar mutations in a homologous position
to G119S are known to alter substrate specificity in Dros-
ophila melanogaster and enhanced hydrolysis of some
The presence of G119S mutation in both M and S
forms of An. gambiae s.s. has already been reported by
Weill et al  and Djogbénou et al  and was sug-
gested to result from introgression between forms. The
wide distribution of ace-1R reported here could result
Table 2: Acetylcholinesterase phenotypes and frequency of G119S mutation in the molecular M and S forms of Anopheles
LocalityM FormS Form
S RSR F(G119S)%(%)*S RSR F(G119S)%(%)*
*: Percentage of each molecular form in the population tested
a,b Values sharing a superscript letter are not significantly different at the 5% level for G119S mutation distribution
Ahoua Alou et al. Malaria Journal 2010, 9:167
Page 6 of 7
from an unique event that then spread as reported in C.
pipiens amplified esterase B2 genes through the world
The absence of homozygous resistant individuals might
be related to high fitness cost of the G119S mutation,
resulting on death of the homozygous resistant
[13,16,17]. Indeed, greater mortality of resistant individu-
als during pupation relative to their sensitive counter-
parts was reported. There was also evidence for costs to
adult fitness as resistant individuals were smaller than
sensitive adults . Consequently, in area where the
resistant allele ace-1R is present, resistant mosquitoes will
mainly at heterozygote state (ace-1RS). Because of this fit-
ness cost, at least one duplication combining resistant
and susceptible alleles of the ace-1 locus has recently
appeared, started to spread and replace ace-1R in treated
areas [17,38-41]. Duplications lead to an excess of
heterozygotes in natural populations because that
heterozygotes involving either ace-1S or ace-1R alleles do
not exhibit deleterious side effects. To date, no specific
test is available for detecting specifically ace-1 duplica-
tions as mosquitoes carrying duplications appear as
heterozygous for ace-1R mutation.
Further investigation is needed to tackle the origin of
the difference of resistance between carbamates and
Data from this study complemented resistance to car-
bamates and organophosphates in An. gambiae s.s. popu-
lations from Côte d'Ivoire and the wide distribution of
G119S mutation in both molecular forms. The low cross-
resistance between carbamates and organophosphates
through susceptibility tests in most of the populations
suggests the involvement of other resistance mecha-
nisms, probably a metabolic detoxification or an alterna-
tive mutation such as the F290V substitution. These
results must be carefully considered while elaborating
malaria control programs in Côte d'Ivoire.
The authors declare that they have no competing interests.
LPAA, AAK designed the study, conducted the field work, genotyping, summa-
rized the data and drafted the manuscript. MAA, ET jointly carried out PCR
assays, and interpreted the results. PKK and MK supervised LPAA and AAK and
contributed to the manuscript. FC contributed to design of the study and
manuscript drafting. All authors read and approved the final manuscript.
We wish to thank all the staff at the Institut Pierre Richet, Bouaké, Côte d'Ivoire
for their hard work during the field and laboratory process and for their con-
tinuing commitment despite extremely difficult conditions during the out-
break of civil war. Special thanks to Mahama Touré, Aboubacar Koné and JB
Assamoi. We are also grateful to Nestor Konan for his technical assistance and
Dr. Josiane Etang for her helpful comments on the manuscript.
1Institut Pierre Richet (IPR), BP 47 Abidjan, Côte d'Ivoire, 2Laboratoire de
Zoologie et Biologie Animale, Université de Cocody, 22 BP 582 Abidjan 22, Côte
d'Ivoire, 3Centre d'Entomologie Médicale et Vétérinaire (CEMV), Université de
Bouake, 27 BP 529 Abidjan 27, Côte d'Ivoire and 4Institut de Recherche pour le
Développement (IRD)/Laboratoire de Lutte Contre les Insectes Nuisibles (LIN)
LIN, IRD/UR 016, 911 Ave Agropolis, 34394 Montpellier Cedex 5, France
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Received: 1 March 2010 Accepted: 16 June 2010
Published: 16 June 2010
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Cite this article as: Ahoua Alou et al., Distribution of ace-1R and resistance to
carbamates and organophosphates in Anopheles gambiae s.s. populations
from Côte d'Ivoire Malaria Journal 2010, 9:167