Tropical Medicine and International Health

doi:10.1111/j.1365-3156.2008.02010.x

volume 13 no 3 pp 430433 march 2008

Short Communication

Knockdown resistance mutations (kdr) and insecticide

susceptibility to DDT and pyrethroids in Anopheles gambiae
from Equatorial Guinea
M. Moreno1, J. L. Vicente2, J. Cano1,3, P. J. Berzosa1, A. de Lucio1, S. Nzambo3, L. Bobuakasi3, J. N. Buatiche3,
M. Ondo3, F. Micha3, V. E. Do Rosario2, J. Pinto2 and A. Benito1
1 Centro Nacional de Medicina Tropical. Instituto de Salud Carlos III, Madrid, Spain
2 Centro de Referencia para el Control de Endemias, Centro Nacional de Medicina Tropical, Instituto de Salud Carlos III, Bata, Guinea
Ecuatorial
3 Centro de Malaria e outras Doencas Tropicais, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Portugal

Summary

objectives To determine the frequency of knockdown resistance (kdr) mutations in the malaria vector
Anopheles gambiae s.s. from continental Equatorial Guinea; and to relate kdr genotypes with
susceptibility to DDT and pyrethroid insecticides in this vector.
methods Female mosquitoes were collected in two villages, Miyobo and Ngonamanga, of mainland
Equatorial Guinea. Insecticide susceptibility tests were performed following WHO procedures. Anopheles
gambiae complex specimens were identified to species and molecular form by PCR. Genotyping of the
kdr locus was performed by allele-specific PCR and direct sequencing in a subset of samples.
results Both M and S molecular forms of A. gambiae were found in Ngonamanga whereas only the
S-form was identified in Miyobo. The two kdr mutations were detected in S-form samples of both
villages, with a higher frequency of the kdr-e (Leu-1014-Ser) allele (Miyobo: 16%; Ngonamanga: 40%).
The kdr-w (Leu-1014-Phe) mutation was also detected in 3% of the M-form. All individuals tested for
pyrethroids were susceptible. A mortality rate of 86% was obtained for DDT. An overall kdr allele
frequency (i.e. kdr-e + kdr-w) of 22% was detected in DDT resistant individuals, whereas susceptible
individuals had a kdr frequency of 6%.
conclusion The co-occurrence of both kdr mutations and reduced susceptibility to DDT found in
A. gambiae highlights the importance of implementing efficient surveillance of insecticide resistance in
Equatorial Guinea.
keywords Anopheles gambiae s.s., DDT, pyrethroids, kdr mutations, Equatorial Guinea

Resistance to pyrethroids in anopheline vectors may pose a

serious threat for the sustainability of malaria control
programmes. Pyrethroids are the insecticides of choice for
the impregnation of mosquito bednets, due to their great
effectiveness and low toxicity in mammals (WHO UNICEF RBM 2005). More recently, the exceptional use of
DDT for malaria vector control in sub-Saharan Africa has
been considered by the World Health Organization
(WHO) (Mandavilli 2006).
Two main mechanisms of resistance to DDT and
pyrethroid insecticides have been described in the Afrotropical malaria vector Anopheles gambiae s.s.: metabolic
resistance has been implicated, mainly through altered
430

expression of cytochrome P450 and glutathione S-transferase detoxifying enzymes (David et al. 2005). Knockdown resistance (kdr) is a type of target-site resistance
arising from point mutations in sodium channel genes of
the insect nervous system and conferring cross-resistance
to DDT and pyrethroids (Soderlund & Knipple 2003).
In A. gambiae, two kdr mutations have been described.
One involves the substitution of a leucine amino acid
(TTA: wild-type allele hereafter termed kds) by a phenylalanine (TTT: hereafter termed as kdr-w) at position
1014 and is widely distributed in West Africa (MartinezTorres et al. 1998; Gentile et al. 2004). The other was
described in East Africa and involves the substitution of a

2008 Blackwell Publishing Ltd

Tropical Medicine and International Health

volume 13 no 3 pp 430433 march 2008

M. Moreno et al. Knockdown resistance in Anopheles gambiae from Equatorial Guinea

leucine (TTA) by a serine (TCA: hereafter termed as

kdr-e) at the same position, conferring high resistance
to DDT (Ranson et al. 2000). More recently, the
co-occurrence of both mutations has been reported in the
neighbouring countries of continental Equatorial Guinea
(i.e. Gabon and Cameroon) and in Uganda (Etang et al.
2006; Pinto et al. 2006; Verhaeghen et al. 2006). The
phenotypic outcome of the presence of both kdr mutations at the heterozygous state remains unclear. The
distribution of kdr mutations also differs among the two
molecular forms recognized in A. gambiae s.s., denoted M
and S (della et al. 2005). kdr-w alleles are generally much
more frequent in the S-form than in the M-form, even
when both forms occur in sympatry (see della et al. 2005,
and references therein). No record of kdr-e alleles has
been reported in the M-form so far.
Information on insecticide resistance in malaria vectors
from Equatorial Guinea is scarce and mainly focused on
the analysis of the kdr mutations for the island of Bioko.
Reimer et al. (2005) detected an unusual frequency of the
kdr-w allele in M-form individuals from Malabo, the
capital of the country. No studies have yet been performed
on the continental part of Equatorial Guinea. Here we
present the first data on the frequency of kdr mutations in
both molecular forms of A. gambiae from mainland
Equatorial Guinea. We also attempt to correlate kdr
genotypes with resistance phenotypes to DDT and
pyrethroids.
The study was conducted in two localities from continental Equatorial Guinea, Central Africa. Miyobo
(145 N 1010 E) is an inland rural village located in the
south margin of river Wele, approximately 48 km east of
the main city Bata. Ngonamanga (209 N 946 E) is a
coastal fishery village, located approximately 33 km north
of Bata and 63 km northwest of Miyobo. In both localities,
indoor resting mosquitoes were collected early in the
morning in June 2004 and June 2005. After morphological
identification (Gillies & Coetzee 1987), individual female
A. gambiae s.l. were kept in silica gel until DNA extraction
(Collins et al. 1988). Anopheles gambiae complex species
and molecular form identification was performed by PCR
(Scott et al. 1993; Favia et al. 2001). Genotyping of the
kdr locus was performed by two PCR assays to distinguish
both kdr-w and kdr-e mutations (Martinez-Torres et al.
1998; Ranson et al. 2000). The kdr genotype of a subset of
the samples was confirmed by direct sequencing (Pinto
et al. 2006).
We analysed 289 mosquitoes. In Miyobo, all 95 specimens belonged to the S-form of A. gambiae s.s. (Table 1).
Both M- and S-forms were present in the sample from
Ngonamanga, but with a much higher frequency of the
M-form (89.7%). No M S hybrids were detected.

2008 Blackwell Publishing Ltd

Table 1 Knockdown resistance allele and genotype frequencies,

according to molecular forms of Anopheles gambiae and collection
sites

n
kds
kdr-w
kdr-e
kds kds
kdr-w kdr-w
kdr-e kdr-e
kds kdr-w
kds kdr-e
kdr-w kdr-e

Miyobo

Ngonamanga

S-form

M-form

S-form

95
0.75
0.09
0.16
0.61

0.07
0.16
0.14
0.02

174
0.97
0.03

0.95
0.01

0.04

20
0.32
0.28
0.40
0.10
0.05
0.15
0.25
0.25
0.20

n, sample size; , not found.

Sequencing of the kdr locus confirmed the genotypes

obtained by PCR in a subset of 24 specimens analysed. In
both localities surveyed, genotypic frequencies at the kdr
locus met HardyWeinberg proportions within each
molecular form (data not shown). As in previous studies
from neighbouring countries (Etang et al. 2006; Pinto
et al. 2006), the two kdr mutations were detected in the
S-form samples of both localities, with kdr-e being the
more frequent allele (Table 1). In addition, the kdr-w
allele was also detected in eight M-form individuals, two
homozygous and six kds kdr-w heterozygous. Although
the occurrence of kdr-w alleles in the M-form of
A. gambiae is uncommon, this finding agrees with
previous reports of this exceptional case on the island of
Bioko and in neighbouring Cameroon (Reimer et al.
2005; Etang et al. 2006).
In order to relate kdr genotypes with their phenotypic
outcome, susceptibility assays were performed in a
subsample of females collected from both localities in June
2005, using WHO kits and procedures (WHO 1998). The
test papers used were DDT (4%), permethrin (0.75%),
deltamethrin (0.05%) and respective controls, in 60 min
exposures. Mortality was recorded at 24 h post-exposure.
We obtained 100% mortality in the 39 mosquitoes tested
with pyrethroids. No kdr-e allele was present in this sample
but kdr-w was found in 5% of both homozygous and
heterozygous (with the kds allele) dead individuals. For
DDT, a mortality rate of 85.9% was recorded, suggesting a
degree of reduced susceptibility to this insecticide
(Table 2). Both kdr mutations were detected in this sample
but no homozygous individuals for any of the kdr alleles
were found. Nevertheless, kdr alleles were more frequent in
resistant individuals (kdr-w: 16.7%; kdr-e: 5.6%) than in
susceptible ones (kdr-w: 4.6%; kdr-e: 1.8%). Considering
431

Tropical Medicine and International Health

volume 13 no 3 pp 430433 march 2008

M. Moreno et al. Knockdown resistance in Anopheles gambiae from Equatorial Guinea

Table 2 Distribution of kdr genotypes according to susceptible

and resistant groups obtained by susceptibility assays to DDT
(WHO, 1998)
Miyobo

kds kds
kdr-w kdr-w
kdr-e kdr-e
kds kdr-w
kds kdr-e
kdr-w kdr-e
Total

Ngonamanga

Susceptible

Resistant

Susceptible

Resistant

19
0
0
1
1
0
21

1
0
0
3
1
0
5

30 (30 0)
0
0
3 (2 1)
0
1 (0 1)
34 (32 2)

4 (4 0)
0
0
0
0
0
4 (4 0)

For Ngonamanga, values in parenthesis represent the number of

M-form S-form A. gambiae individuals in each cell (only S-form in
Miyobo). , not done.

that kdr is a recessive trait (Martinez-Torres et al. 1998;

Ranson et al. 2000), the absence of kdr homozygotes in the
analysed sample could not allow assessing association
between kdr and the resistance phenotype. On the other
hand, the presence of kds kds and kds kdr-w genotypes in
resistant individuals suggests involvement of resistance
mechanisms other than kdr, such as metabolic resistance.
Our results on the correlation between kdr genotypes
and the resistance phenotype should be interpreted with
caution. Mosquito samples used in susceptibility assays
were blood-fed females collected at the adult stage and the
sample sizes never reached the required 100 2448 h postemergence non-fed females per test recommended by the
WHO (1998). Therefore, these results should only be
considered as preliminary. The phenotypic implications of
the co-occurrence of both kdr mutations, particularly when
found at a heterozygous state, need to be further examined
with appropriate biological material, adequate sample sizes
and including synergists in bioassays to ascertain the
possible role of metabolic resistance mechanisms.
The presence of both kdr mutations described for A.
gambiae, the occurrence of the kdr-w allele in the M-form,
the possible association between these mutations and the
resistance phenotype, and the suspicion of non-target site
resistance mechanisms, highlight the importance of more
detailed surveys and continued monitoring of insecticide
resistance levels in Equatorial Guinea. An effective monitoring system, combining both the analysis of kdr mutations and susceptibility assays, will provide crucial
information for the rational management of insecticides
application overtime. This will be of particular relevance
for the sustainability of the insecticide-based vector control
efforts that are currently being implemented in this and
other countries of the West-Central African region.
432

Acknowledgements
We thank the National Malaria Control Program,
Republic of Equatorial Guineas Ministry of Health and
Social Welfare, for technical support. This study received
financial support from the Spanish International Cooperation Agency, the Institute of Health Carlos III within the
Network of Tropical Diseases Research Centers and from
the Foundation for Science and Technology, Portugal. J.P.
was funded by Foundation of Science and Technology,
Portugal.
References
Collins FH, Finnerty V & Petrarca V (1988) Ribosomal DNAprobes differentiate five cryptic species in the Anopheles gambiae complex. Parassitologia 30, 231240.
David JP, Strode C, Vontas J et al. (2005) The Anopheles gambiae
detoxification chip: a highly specific microarray to study metabolic-based insecticide resistance in malaria vectors. Proceedings
of the National Academy of Sciences of the United States of
America 102, 40804084.
della Torre A, Tu Z & Petrarca V (2005) On the distribution
and genetic differentiation of Anopheles gambiae s.s. molecular forms. Insect Biochemistry and Molecular Biology 35,
755769.
Etang J, Fondjo E, Chandre F et al. (2006) First report of knockdown mutations in the malaria vector Anopheles gambiae from
Cameroon. The American Journal of Tropical Medicine and
Hygiene 74, 795797.
Favia G, Lanfracotti A, Spanos L, Siden K & Louis C (2001)
Molecular characterization of ribosomal DNA polymorphisms
discriminating among chromosomal forms of Anopheles gambiae s.s. Insect Molecular Biology 10, 1923.
Gentile G, Santolamazza F, Fanello C et al. (2004) Variation in
an intron sequence of the voltage-gated sodium channel gene
correlates with genetic differentiation between Anopheles
gambiae s.s. molecular forms. Insect Molecular Biology 13,
371377.
Gillies MT & Coetzee MT (1987) A supplement to the Anophelinae of Africa south of the Sahara (Ethiopian zoogeographical
region). The South African Institute for Medical Research 2, 55.
Mandavilli A (2006) Health agency backs use of DDT against
malaria. Nature 443, 250251.
Martinez-Torres D, Chandre F, Williamson MS et al. (1998)
Molecular characterization of pyrethoid knockdown resistance
(kdr) in the major malaria vector Anopheles gambiae s.s. Insect
Molecular Biology 7, 179184.
Pinto J, Lynd A, Elissa N et al. (2006) Co-occurrence of East and
West African kdr mutations suggests high levels of resistance to
pyrethroid insecticides in Anopheles gambiae from Libreville,
Gabon. Medical and Veterinary Entomology 20, 2732.
Ranson H, Jensen B, Vulule JM et al. (2000) Identification of a
point mutation in the voltage-gated sodium channel gene of
Kenyan Anopheles gambiae associated with resistance to DDT
and pyrethroids. Insect Molecular Biology 9, 491497.

2008 Blackwell Publishing Ltd

Tropical Medicine and International Health

volume 13 no 3 pp 430433 march 2008

M. Moreno et al. Knockdown resistance in Anopheles gambiae from Equatorial Guinea

Reimer LJ, Tripet F, Slotman M et al. (2005) An unusual distribution of the kdr gene among populations of Anopheles gambiae on the island of Bioko, Equatorial Guinea. Insect Molecular
Biology 14, 683688.
Scott JA, Brogdon WG & Collins FH (1993) Identification of
single specimens of the Anopheles gambiae grouby polymerase
chain reaction. The American Journal of Tropical Medicine and
Hygiene 49, 520529.
Soderlund DM & Knipple DC (2003) The molecular biology of
knockdown resistance to pyrethroid insecticides. Insect Biochemistry and Molecular Biology 33, 563577.

Verhaeghen K, Van Bortel W, Roelants P, Backeljau T & Coosemans M (2006) Detection of the East and West African kdr
mutation in Anopheles gambiae and Anopheles arabiensis from
Uganda using a new assay based on FRET Melt Curve analysis.
Malaria Journal 5, 16.
WHO (1998) Test Procedures for Insecticide Resistance
Monitoring in Malaria Vectors, Bio-Efficacy and Persistence of Insecticides on Treated Surfaces. WHO CPC
MAL 98.12
WHO UNICEF RBM (2005) World Malaria Report. http://
www.rbm.who.int/wmr2005/index.html

Corresponding Author M Moreno, Centro Nacional de Medicina Tropical, Instituto de Salud Carlos III, c Sinesio Delgado, Madrid,
Espana. Tel.: +34 91 822 22 23; Fax: +34 387 77 56; E-mail: martamor@isciii.es

2008 Blackwell Publishing Ltd

433