Insect Molecular Biology (2005)
© 2005 The Royal Entomological Society
Blackwell Publishing, Ltd.
An unusual distribution of the
of Bioko, Equatorial Guinea
on the island
L. J. Reimer*, F. Tripet*, M. Slotman*, A. Spielman†,
E. Fondjo‡ and G. C. Lanzaro*
Diseases, Harvard University, Boston, MA, USA; and
‡National Malaria Control Program, National Ministry
of Public Health, Yaoundé, Cameroon
Department of Entomology, University of California, Davis,
†Department of Immunology and Infectious
In West Africa,
subpopulations known as the M and S molecular forms.
Although these forms occur in sympatry, pyrethroid
knock-down resistance (
with the S molecular form. On the island of Bioko,
Equatorial Guinea we found high frequencies of the
mutation in M form individuals (55.8%) and a
complete absence of
in the S form. We also report
the absence of the
allele in M and S specimens
from the harbour town of Tiko in Cameroon, represent-
ing the nearest continental population to Bioko. The
allele had previously been reported as absent in
on Bioko. Contrary to earlier
reports, sequencing of intron-1 of this sodium channel
gene revealed no fixed differences between M form
resistant and susceptible individuals. The mutation may
have recently arisen independently in the M form on
Bioko due to recent and intensive pyrethroid application.
exists in discrete
) is strongly associated
, Bioko, molecular
Pyrethroid insecticides are the preferred choice for impreg-
nating bed nets because of their high efficacy, rapid rate of
knockdown, residual and strong mosquito excito-repellent
properties and low mammalian toxicity (Diabate
2002). The use of insecticide-treated bed nets (ITN) offers
both individual and community protection against malaria,
sometimes reducing morbidity by as much as 50% and
global mortality by 20–30% (Binka
spread of pyrethroid resistance in
ulations across Africa is becoming a hindrance to malaria
control programs based on ITNs using pyrethroids
(Elissa et al., 1993). Knockdown resistance is primarily
conferred by the knock down resistance (
dre et al., 1999), a single point mutation in domain II of the
voltage-gated sodium channel gene – located on the left
arm of chromosome 2 in An. gambiae
1996; Ranson et al., 2000a). This mutation involves the
replacement of leucine by phenylalanine. The addition of
the large aromatic side chain of phenylalanine may impose
steric hindrance interfering with insecticide binding to the
sodium channel thus rendering the individual mosquito
resistant (Ranson et al., 2000b).
Anopheles gambiae s.s is classified into two molecular
forms, Mopti (M) and Savannah (S). The forms exhibit
strong assortative mating and are considered by many
to be incipient species (Lanzaro & Tripet, 2003). Pyrethroid
kdr occurs in many populations of
the species range: in West Africa,
with the S molecular form of An. gambiae
was first identified in the S molecular form of
and until recently had yet to be detected in the M form, even
at sites where the two are sympatric.
Resistance in the S form has been reported from the Ivory
Coast (Weill et al., 2000; della Torre
(della Torre et al., 2001), Nigeria (Awolola
(Fanello et al., 2003) and Burkina Faso (Diabate
Resistance in the M form has only been found in Benin
(Weill et al., 2000; della Torre
(Diabate et al ., 2004) and at very low frequency in Ghana
(Yawson et al., 2004). This suggested that the
reached the M form only recently through introgression from
S form populations (Weill et al
been detected in either form in the Central African Republic
et al., 1996). The
Anopheles gambiae pop-
kdr) gene (Chan-
(Williamson et al.,
kdr is strongly associated
et al., 2001), Ghana
et al., 2003), Mali
et al., 2003).
et al ., 2001), Burkina Faso
., 2000). Resistance has not
Received 23 April 2005; accepted after revision 15 July 2005. Correspond-
ence: Gregory C. Lanzaro, Department of Entomology, University of Califor-
nia, One Shields Avenue, Davis, California CA 95616, USA. Tel.: +1 5307
525 652; fax: +1 5307 521 537; e-mail: email@example.com
684L. J. Reimer et al.
© 2005 The Royal Entomological Society, Insect Molecular Biology ,
The volcanic islands of Equatorial Guinea are tropical,
with a hot and humid climate year-round. Bioko Island lies
40 km off the coast of Cameroon in the Bight of Biafra. The
island rises steeply to an altitude of 2850 m, with two main
peaks, one in the north and one in the south. The southern
area is rugged and inaccessible. Recent research on Bioko
has included seroparasitological studies of malaria, the
vitro responses of Plasmodium falciparum
mosquito taxonomic surveys and mosquito species distribu-
tions. An. gambiae and Anopheles funestus
the main malaria vectors in Bioko. Malaria is hyperendemic
on Bioko and is one of three main causes of morbidity and
mortality on the island (Berzosa
Malaria exhibits a stable year round pattern of transmission
with seasonal fluctuations. In a study of the
P. falciparum to antimalarial drugs, resistant isolates
were found to exhibit interregional differences (Benito
1995). Prevalence of malaria infection is 29.8% in Bioko,
parasitic prevalence (malaria index) was 26.6%, and the
splenic index was 57.0% (Roche
indicate that Bioko is an area of stable hyperendemic malaria.
Populations of An. gambiae
the kdr allele between 1998 and 2001, despite the use of
pyrethroid-impregnated bed nets (Berzosa
The fact that the local population was found to be suscep-
tible justified the implementation of a larger scale ITN
program through the Malaria Control Program of Equatorial
Guinea. In addition, in 2003 Marathon Oil Corporation and
Medical Care Development International joint launched a
five year Bioko Island Malaria Control Project that includes
indoor residual spraying with deltamethrin, a synthetic
pyrethroid, in 25 000 homes in Malabo, Bioko Island (MCDI
Newsletter, March, 2004).
., 2003; Gentile
., 2000), Senegal, The Gambia, Guinea, Angola
., 2004) or Cameroon (Weill
et al., 2004).
et al ., 2000; Etang
et al., 2002).
in vitro response
et al .,
et al ., 1991). These results
on Bioko were found to lack
et al., 2002).
We surveyed mosquito populations from six villages near
Malabo for the presence of the
diagnostic (Martinez-Torres et al
resistance allele in the M molecular form in Malabo. Previous
studies have shown polymorphism in intron 1 and fixed differ-
ences between the M and S populations from different locales
on the mainland (Weill
et al., 2000). Weill
et al. (2004) reported that the patterns of polymor-
phism in the intron suggest introgression of the
from the S to the M form. In order to determine whether the
allele arose through introgression from mainland mosquitoes
or independently through continuous pyrethroid exposure
we examined these polymorphic sites in M and S molecular
form samples from Bioko and nearby mainland Cameroon.
kdr gene using a PCR-based
., 1998). We found the kdr
et al . (2000) and
Results and discussion
Species and molecular form composition
Mosquitoes were collected from 6 villages within 8 km of
Malabo, the capital of Bioko and from the village of Tiko in
Cameroon. M and S molecular form diagnostics were
performed on 75 An. gambiae from Malabo and 68 from Tiko.
In Malabo 27.7% of mosquitoes collected were
(n = 33) and 72.3% were An. gambiae
(36%) were S form and 48 (64%) were M form. In Tiko 100%
(n = 68) of mosquitoes collected were
(21%) were S form and 54 (79%) were M form. Although M
and S form individuals were collected from the same sites
no M/S hybrids were observed in either Malabo or Tiko. This
suggests that if hybrids are generated, it is at a very low fre-
quency. This observation is consistent with what has been
reported throughout West Africa (Lanzaro & Tripet, 2003).
= 86); of these 27 (n
An. gambiae , 14
Genetic differentiation and gene flow
between one island and three mainland populations, as
values, based on eight microsatellite loci,
Table 1. Matrices of pair-wise estimates of genetic divergence (FST) and significance levels of exact tests of genetic differentiation between and among
molecular forms on Bioko Island and three locales in Cameroon – Malantouen, Mutanguene and Tiko
Location and molecular form
0.1085** 0.0383** 0.1299**0
0.0766** 0.00740.0891**0.0575** 0.0914**0
*P < 0.01; **P < 0.001.
© 2005 The Royal Entomological Society, Insect Molecular Biology,
well as between molecular forms of
calculated (Table 1). All between form
indicating that gene flow between molecular forms is
limited at every location sampled, even where the two
occur in sympatry.
estimates between Tiko, mainland
Cameroon and Bioko Island were high and significant, both
between and within forms. Tiko is a harbour town, 75 km
across the Bight of Biafra on mainland Cameroon. Although
trade and transportation are frequent between Bioko Island,
Cameroon and Equatorial Guinea, Tiko represents the nearest
continental population to the island. In order to determine
whether the Bight of Biafra poses a physical barrier to gene
flow, we also included in our analysis two additional loca-
tions in Cameroon; Mutanguene (
(n = 27). The distance from each village to Tiko is 85 km
and 345 km, respectively. F
estimates within form among
mainland populations were all low, suggesting the Bight of
Biafra is a strong physical barrier to gene flow between
populations on Bioko Island and mainland Cameroon.
s were significant,
n = 29) and Malantouen
Frequency and distribution of kdr allele
Presence of the
described by Martinez-Torres
kdr allele was not detected in any
from Bioko collected between 1998 and 2001 (Berzosa
et al ., 2002), we found the kdr allele at high frequency in our
M form samples from Bioko. Near Malabo, the
was present in 55.8% of the M molecular form (5 R/R, 12
R/S and 2 R/?) but was absent in the S molecular form. The
kdr allele was also absent in all
Tiko (n = 68). The Tiko samples belong to the forest chro-
mosomal form and consist of both the M and S molecular
form. A recent study by Berzosa
that all An. gambiae s.s. on Bioko Island belong to the forest
kdr allele was determined by PCR as
et al. (1998). Although the
An. gambiae samples
An. gambiae sampled from
et al . (2002) demonstrated
Origin of kdr in An. gambiae on Bioko
We analysed a 526 bp region including the kdr mutation
and a portion of the intron upstream of the kdr mutation
(Fig. 1). We also sequenced the same region in four mos-
quitoes collected from Tiko. Weill et al. (2000) identified
two polymorphic sites at nucleotide positions 702 and 896
(following the notation by Weill et al., 2000) in which M form
susceptible individuals carry C-A or C-C combinations
and M form resistant along with most S form carry a T-C
combination. A recent study by Gentile et al. (2004) has
shown a correlation between the substitution at site 702
and molecular form, whereby M form resistant individuals
and all S form individuals exhibit the thymine substitution
and M form susceptible individuals exhibit cytosine at this
site. The few exceptions to this trend were thought to have
arisen from past introgression events between M and S
(Gentile et al., 2004). In this study we found the expected
T-C combination at sites 702 and 896, respectively, in M
form resistant mosquitoes from Bioko. However, the T-C
combination, thought to be characteristic of all S form
individuals, but only in M form individuals carrying the kdr
gene, was also found in M form susceptible mosquitoes on
Bioko (Table 2). Therefore we found no differences between
susceptible and resistant M form individuals outside of the
Figure 1. Partial sequence of the sodium channel
gene showing the Leu-Phe kdr mutation that is
associated with pyrethroid knockdown resistance
(shaded box), a portion of the 920 bp intron 1 that
precedes it and the 57 bp intron 2 (bold). Two
positions in intron 1 are known to be polymorphic in
Anopheles gambiae s.s. (702 and 896), though in
this study polymorphism was only found at site 702.
Forward and reverse primers are also shown
Table 2. Polymorphisms in Anopheles gambiae s.s. molecular forms
collected on the island of Bioko and the port city of Tiko in Cameroon. Unlike
previous data (Weill et al., 2000; Diabate et al., 2004), no polymorphism was
observed at site 896 and the polymorphisms at site 702 were not associated
with molecular form or resistance. Pyrethroid knockdown resistance (r) and
susceptibility (s) determined by sequencing
Location and molecular form Sample size702 896kdr
686L. J. Reimer et al.
© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 683–688
single kdr point mutation. The absence of the T-C combina-
tion in susceptible M form in West Africa has been used to
argue for the introgression of the kdr allele from the S form
into the M form (Weill et al., 2000). Our observations, that
the T-C combination is present in susceptible M form indi-
viduals on Bioko and the absence of the kdr gene in the
nearest mainland population in Cameroon, suggests that
the emergence of kdr resistance in the M population of
An. gambiae on Bioko occurred as an independent evolu-
The T-C combination in the M form in Bioko and Cameroon
most likely represents an ancestral shared polymorphism.
The molecular forms are thought to have diverged only very
recently and differentiation between them is limited to a few
genomic regions. The alternative scenario that the M form
acquired the T-C combination through introgression from a
susceptible S form cannot be ruled out. However, the kdr
allele is located in one of the few areas of the genome that
appears to resist introgression between forms (Chandre
et al., 1999; Turner et al., 2005). In addition, the scenario of
an independent origin of the kdr mutation on Bioko is sup-
ported by the great physical distance between Bioko Island
and the nearest source of kdr carrying M form individu-
als from the mainland, in Benin. Furthermore, our esti-
mates of gene flow between Bioko and the nearest
mainland population Tiko, suggest that An. gambiae popu-
lations on Bioko are to a large extent isolated from main-
land populations. Finally, an independent origin of the kdr
mutation is not without precedent. Based on intron poly-
morphisms, Diabate et al. (2004) concluded that the muta-
tion arose independently in Anopheles arabiensis. A
separate leucine-serine substitution at the kdr site, associ-
ated with DDT and pyrethroid resistance, was found in An.
gambiae from Kenya (Ranson et al., 2000a).
It is also important to note that the kdr gene was not
present on the island prior to 2001, before any large scale
application of insecticide (Berzosa et al., 2002). In 2001
antimalaria efforts in Malabo increased, with frequent and
intense application of pyrethroid aerosols from trucks and
by hand (A. Spielman, personal communication, May 11,
2005). By 2002, when our samples were collected, the
frequency of the kdr gene within the M form population
around Malabo was 55.8%, a staggering increase. Other
studies have shown a strong increase in the frequency of
the kdr gene immediately following the implementation
of an impregnated bed net program (Ranson et al., 2000a;
Stump et al., 2004), although these increases were not as
dramatic as we report here. The unusual distribution of the
kdr mutation in the M form may have arisen independently
and recently on the island of Bioko in response to this
intensive series of pyrethroid applications. Although the
individual and community protection offered by treated bed
nets continue to have a powerful impact on malaria trans-
mission (Curtis et al., 2003), the potential of wide spread
pyrethroid resistance in An. gambiae could be disastrous to
Mosquitoes for the kdr analyses were collected from two locations:
from urban and peri-urban settings in the city of Malabo, Bioko
Island, Equatorial Guinea (3°45′N, 8°47′E) and from the rural
areas surrounding the harbour town of Tiko, Cameroon (4°04′N,
9°22′E). Collections were also made from the villages of Malan-
touen (5°44′N, 12°00′E) and Mutanguene (4°50′N, 9°17′E) in
Cameroon for estimates of gene flow based on microsatellite allele
frequencies. In Bioko, adult female mosquitoes were collected by
hand using aspirators and human bait catches in August and
September, 2002. The mosquitoes were sorted to genus using
morphological identification and genomic DNA of anophelines was
extracted using a DNeasy extraction kit (Qiagen, Valencia, CA,
USA). In Cameroon, adult resting females were collected indoors
using aspirators in September 2003. DNA was extracted using a
standard extraction protocol (Post et al., 1992).
Species and form diagnostic
The PCR assay described by Scott et al. (1993) was used to
identify members of the An. gambiae species complex. An additional
diagnostic described by Koekemoer et al. (2002) was used to
identify An. funestus from Bioko Island. Molecular identification of
forms within An. gambiae s.s. was based on the method described
by Fanello et al. (2002).
Estimation of gene flow
We estimated the amount of genetic differentiation between island
and mainland populations using eight 3rd chromosome microsat-
ellite loci of GT repeats that were isolated and mapped by Zheng
et al. (1996). The loci were PCR amplified using fluorescent primers
and an MJ Research PTC-200 thermal cycler (MJ Research,
Watertown, MA). PCR products were mixed with a Genescan
(Perkin-Elmer, Norwalk, CT) size standard and run on an ABI 3100
capillary sequencer (Perkin Elmer). The gels were analysed using
the ABI PRISM Genescan Analysis Software and Genotyper DNA
Fragment Analysis Software (Perkin-Elmer). Arlequin version 2.001
(Schneider et al., 2001) available at http://lgb.unige.ch/arlequin
was used to calculate FST values between all pairs of populations.
Detection of the Leu-Phe kdr mutation was based on the PCR
diagnostic test developed by Martinez-Torres et al. (1998) with
modifications. 2 µl of extracted genomic DNA was combined with
primers Agd1, Agd2, Agd3 and Agd4 to a total reaction volume of
25 µl. PCR conditions included an initial 13 min at 95 °C, 1 minute
at 95 °C, 30 s at 48 °C, 30 s at 72 °C for 60 cycles and a final
extension step at 72 °C for 10 min. Amplified fragments were
analysed by electrophoresis using 1.5% agarose gel.
Based on results of the PCR diagnostic, 24 M and S form, knock-
down resistant and susceptible individuals were selected (19 from
© 2005 The Royal Entomological Society, Insect Molecular Biology, 14, 683–688
Malabo, 5 from Tiko) for sequence determination. A 602 bp genomic
region containing the kdr mutation site and a portion of intron
1 was PCR amplified using primers Agd8 (5′-CACAACAAGTA-
CAAAATGTCTCGC-3′) and kdr-rev (5′-GCAAGGCTAAGAAAAG-
GTTAAGCA-3′). The region was trimmed to 526 bp for analysis.
PCR fragments were purified using QIAquick PCR purification kit
(Qiagen) and sequences were analysed using an ABI 3100 Genetic
Analyser (Foster City, CA, USA). The sequences were aligned
using the Clustal-V method (Megalign program, DNASTAR, Inc.,
Madison, WI, USA).
We thank Michael Reddy for helpful comments on the
manuscript. This work was supported by grant no. AI 40308
from the National Institutes of Health to G.C.L.
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