# Evidence of increasing Leu-Phe knockdown resistance mutation in Anopheles gambiae from Niger following a nationwide long-lasting insecticide-treated nets implementation

Cyrille Czeher, Rabiou Labbo, Ibrahim Arzika, Jean-Bernard Duchemin

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Cyrille Czeher, Rabiou Labbo, Ibrahim Arzika, Jean-Bernard Duchemin. Evidence of increasing Leu-Phe knockdown resistance mutation in Anopheles gambiae from Niger following a nationwide long-lasting insecticide-treated nets implementation. Malaria Journal, 2008, 7 (1), pp.189. 10.1186/1475-2875-7-189. doi:10.1186/1475-2875-7-189

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# Evidence of increasing Leu-Phe knockdown resistance mutation in *Anopheles gambiae* from Niger following a nationwide long-lasting insecticide-treated nets implementation

Cyrille Czeher*, Rabiou Labbo, Ibrahim Arzika and Jean-Bernard Duchemin

Address: Parasitology Unit, CERMES (Centre de Recherche Médicale et Sanitaire), Institut Pasteur International Network, Niamey, Niger

Email: Cyrille Czeher* - cyril_czeher@yahoo.fr; Rabiou Labbo - rabiou@cermes.org; Ibrahim Arzika - ibrarzika@yahoo.fr; Jean-Bernard Duchemin - jbduchemin@yahoo.fr

Published: 25 September 2008

Malaria Journal 2008, 7:189 doi:10.1186/1475-2875-7-189

This article is available from: http://www.malariajournal.com/content/7/1/189

© 2008 Czeher et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution 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.

Received: 22 May 2008

Accepted: 25 September 2008

## Abstract

**Background:** At the end of 2005, a nationwide long-lasting insecticide-treated net (LLIN) distribution targeting the most vulnerable populations was implemented throughout Niger. A large number of studies in Africa have reported the existence of anopheline populations resistant to various insecticides, partly due to knockdown resistance (kdr) mutations, but few operational wide-scale control programmes were coupled with the monitoring of such mutations. The distribution of the kdr-west (kdr-w) Leu-Phe mutation was studied in *Anopheles gambiae* s.l. populations from Niger and temporal variations were monitored following the nationwide LLIN implementation.

**Methods:** Mosquitoes were collected from 14 localities during the wet seasons of 2005, 2006 and 2007 with additional sampling in the capital city, Niamey. After morphological identification of *Anopheles gambiae* s.l. specimens, DNA extracts were used for the determination of species and molecular forms of the *Anopheles gambiae* complex and for the detection of the *kdr-w* mutation.

**Results:** Around 1,500 specimens collected in the three consecutive years were analysed. All *Anopheles arabiensis* specimens analysed were homozygous susceptible, whereas the few *Anopheles gambiae* S forms exhibited a high overall *kdr-w* frequency. The M form samples exhibited a low overall *kdr-w* frequency before the LLIN distribution, that increased significantly in the two wet season collections following the LLIN distribution. Higher *kdr* frequencies were repeatedly noticed within host-seeking females compared to resting ones in indoor collections. In addition, preliminary results in M form urban populations from Niamey showed far higher *kdr* frequencies than in all of the rural sites studied.

**Discussion:** This study describes the first case of *kdr* mutation in *Anopheles gambiae* populations from Niger. It is suspected that the LLIN have caused the important temporal increase of *kdr-w* mutation observed during this study. While the *kdr* mutation is still found at a low level, this rapid increase could potentially lead to high *kdr* frequencies within a few years.

**Conclusion:** These results are of prime importance in the effort to document multiple effects of operational control programmes on mosquito vectors, and to conceive sustainable control strategies for future malaria control programmes.

<footnote>* Corresponding author</footnote>
**Background**

*Plasmodium falciparum*, one of the deadliest pathogens cir-
culating in Niger and many other sub-Saharan countries,
is contributing significantly to the high estimated child
mortality rate (United Nations Children's Fund 2005).
The major vectors responsible for *P. falciparum* transmis-
sion in Niger are *Anopheles gambiae* sensu lato (s.l.) and
*Anopheles funestus*, the former being far more widespread
over the country whereas the last is found in more limited
areas [1]. Of the sibling species and forms constituting the
*Anopheles gambiae* complex, *Anopheles arabiensis* and both
molecular M and S forms of *An. gambiae* sensu stricto (s.s.)
have been found to date in Niger [2,3].

At the end of 2005, a nationwide long-lasting insecticide-
treated net (LLIN) distribution targeting the most vulner-
able members of the population was organized in con-
junction with an integrated poliomyelitis vaccination
campaign [4]. This LLIN coverage aimed to reduce mor-
bidity and mortality caused by malaria clinical cases. Such
a reduction can be observed when the bed net usage rate
in the population is high enough [5-9]. Although bed nets
were already quite commonly used by the population,
principally for protection against biting nuisance mosqu-
toes, this campaign strongly increased insecticide-treated
bed net usage in households all over the country [10].

The effectiveness of bed nets impregnated with pyrethroid
insecticides derives from several mechanisms that
decrease the probability of a host-seeking female mos-
quito to succeed in taking a blood-meal. Besides the phys-
ical barrier constituted by the net, a deterrent effect limits
unfed mosquitoes entering houses where a treated net is
present, and in case of tarsal contact with the treated net,
the insecticide compound could repel, hurt, or kill the
mosquito. These properties have led to substantial reduc-
tions in indoor mosquito densities, and biting, feeding
and survival rates in field trials [11-14] and experimental
huts evaluations [15-18]. Some of these studies have also
demonstrated a reduction in *P. falciparum* transmission
[11,13,14].

However, a number of studies throughout Africa have
reported the existence of anopheline populations resistant
to various insecticides [15,19-23] involving two major
types of resistance mechanisms : insecticide target-site
insensitivity due to single nucleotide polymorphisms
(SNP), and increased enzymatic metabolization of insect-
ticidal compounds [24]. Two SNP at amino-acid position
1014 in the voltage-gated sodium channel gene have been
described in *An. gambiae* [25,26], leading to amino-acid
substitutions involved in reducing the affinity of DDT and
pyrethroid insecticides for their target site in the insect
nervous system. These SNP, named *knock-down resistance
(kdr)* mutations in relation to their phenotypic expres-

sion, were found in many field populations of *An. gambiae*
s.s. and *An. arabiensis* covering West, Central and East
Africa [27-50]. In West and West-Central Africa, it was
shown that the *kdr-w* (or L1014F) allele was far more fre-
quent and widespread in the S molecular form of *An. gam-
biae* [23,27-29,32-38,50], whereas only few M form
populations from a limited area near the Gulf of Guinea
presented *kdr-w* alleles at low frequencies [23,31,32,36-
38,50], except in few urban and peri-urban coastal areas
where it reached high frequencies [20,35,40-45]. How-
ever, to date there have been almost no studies on *kdr*
mutations in sahelian *An. gambiae* populations except one
in Burkina Faso that included two sites in the sahelian
zone of the country [32]. No *kdr* M forms were found
whereas one village exhibited *kdr-w* S forms.

The monitoring of insecticide resistance in malaria vectors
is of prime importance especially where control pro-
grammes are planned or already running, in order to
assess potential selection effects of insecticidal com-
pounds on vector populations, and to take appropriate
measures such as switching to other classes of com-
pounds. For this goal, the presence and frequency of the
*kdr* mutations constitute a valuable and useful resistance
marker for two main reasons. First, it provides an early
warning of resistance development as the mutation arises
well before any effect on phenotype can be detected in a
population [51]. Indeed, the expression of the 24 h-sur-
vival diagnostic phenotype [52] appear to be recessive
[45,53]. Therefore, a population presenting a low *kdr* fre-
quency mainly in heterozygous state is likely to show a
high mortality rate during bioassays. As further evidences
supporting the advantage of *kdr* genotyping over bio-
assays for emergent resistance detection, Chandre *et al.*
[53] found a significant mortality reduction only when
heterozygous females proportion reached 60%, and a sig-
nificant increase of Knockdown time (KdT) only with
40% heterozygous females. Secondly, the *kdr* mutations
seem to be well correlated with resistance phenotype
[25,38,45,53] in both *An. gambiae* molecular forms, even
if metabolic resistance mechanisms could also be
involved in increased tolerance to pyrethroids [27].

Several authors have studied the effect of insecticide
treated nets (ITNs) with pyrethroids on *An. gambiae* pop-
ulations and the possible selection of *kdr* alleles either in
experimental huts trials [18,45,53,54] or laboratory
experiments [45,53], helping in the understanding of
advantages conferred by the mutations on survival and
blood-feeding in areas of ITN use. However, few data are
available on long-term and/or large-scale ITN coverage
effect on resistance and *kdr* mutations in natural settings.
In East Africa, no selection effect of long-term ITNs use on
phenotypic resistance was noticed [55,56], whereas
Stump *et al.* [57] reported a significant increase of *kdr-east*
mutation frequency in *An. gambiae* S form populations
from Kenya after four years of ITNs community use. In
West-Central Africa, a rapid increase of *kdr-w* mutation
was observed in M forms from the island of Bioko follow-
ing a large-scale insecticide residual spraying (IRS) pro-
gramme [41,58].

The distribution of the *kdr-w* mutation was studied in *An. gambiae* s.l. populations from the sahelian area of Niger during three consecutive wet seasons, allowing to monitor temporal variations following the nationwide LLIN coverage implemented after the first collection event. This study reports the first documented case of *kdr* mutation in *An. gambiae* populations of Niger and provides crucial information about potential effects of wide-scale LLIN coverage on *kdr* mutation selection.

**Methods**

Study area

Niger is a West-African country spanning three biocli-
matic zones, with a marked aridity gradient from the
southern sahelo-sudanian zone (Niger rainfalls per
year) to the northern pre-Saharan zone (less rainfalls per
year). Located between these zones, the sahelian
zone represents the northern limit of malaria endemicity
in the region, and has relatively high human population
densities compared to the north of the country, where
malaria is more marginal and epidemic-prone and where
*Anopheles* species population densities are very low, highly
variable and heterogenous (in prep.).

Apart from describing the distribution of the *kdr-w* muta-
tion in *An. gambiae* s.l. populations in Niger, we moni-
tored its potential variations in frequency and spatial
distribution following the countrywide LLIN coverage
implemented by the end of 2005 that targeted children
under five years of age. The nationwide extent of the cam-
paign prevented us from monitoring control sites without
LLIN usage, and the only available methodology was to
study the temporal evolution of the *kdr-w* mutation in
selected sentinel sites before and after the campaign.

Study design and mosquito collections

Mosquitoes were collected from 14 localities situated in
the sahelian zone during the wet season (between July
and September) of 2005, 2006 and 2007 (Figure 1). The
2007 collections were made in eight of the 14 initial study
sites because of logistical constraints. This sampling
scheme allowed us to collect specimens from West to East
over a distance of around 1,700 km. In each village, land-
ing catches on human adult male volunteers and indoor
spray catches with pyrethroids (Mobil insecticide, Mobil
Oil Nigeria, Lagos, Nigeria) were employed following
standardized procedures and with agreement of the
National Ethics Committee. The 2005 collections were

made before the implementation of the nationwide LLIN
coverage to provide baseline *kdr* mutation frequency data,
whereas the 2006 and 2007 collections were made
approximately 7-8 months and 19-20 months after the
LLIN distribution, respectively. Additionally, some larval
samples collected in Niamey (the capital city) during the
2003 and 2007 wet seasons were included in the study.

**Laboratory processing and data analysis**

Mosquito species were morphologically identified in the
field and put in 96-well microplates with dessicant, and
stored between -20 to -28°C in the laboratory before
processing. Some specimens identified as *An. gambiae* s.l.
were processed for DNA extraction from four to six legs
using Chelex-100 resin (Sigma-Aldrich, St. Louis, MO,
USA), while whole bodies were kept for transmission
studies. DNA extracts were used for the determination of
species and molecular forms of the *An. gambiae* complex
by Polymerase Chain Reaction – Restriction Fragment
Length Polymorphism (PCR-RFLP) assay [59], allowing
discrimination of *An. arabiensis*, *An. gambiae* S form, and
*An. gambiae* M form. These DNA extracts were then sub-
jected to PCR assay for detection of the *kdr-w* mutation
[25] with some modifications. As already done [49] and
to get a more reliable genotype determination, we per-
formed two different PCRs, one with primers Agd1 and
Agd3 for the detection of the mutant allele, and the other
one with primers Agd2 and Agd4 to detect the wild-type
allele. Genotypes of every specimen harbouring at least
one *kdr-w* allele were confirmed in a second run of both
PCR, and a third run was done if the two first results were
discordant. The *kdr-east* (Leu-Ser) mutation was not
searched for as it is very rare in *An. gambiae* M forms
[15,37,38,50]. The conformance of genotypic frequencies
with Hardy-Weinberg equilibrium expectations was deter-
mined by exact test [60] as computed in Arlequin version
3.1.1 [61]. All other statistical analysis were performed
online with OpenEpi version 2.2.1 [62]. One-tail p-values
were employed for Chi square and Fisher exact tests con-
sidering the hypothesis of *kdr* frequency increase over the
study period. Also, it should be noted that allelic fre-
quency of the *kdr-w* mutation (number of chromosomes
presenting the mutation divided by total number of chro-
mosomes analysed) is always presented rather than fre-
quency of *kdr* specimens (presenting one or two copies of
the mutation).

**Results**

Global results

One thousand five hundred and seventeen specimens col-
lected in the three consecutive years and different sites
were analysed for the species/forms and the *kdr-w* muta-
tion. 54 individuals (3.6%) could not be genotyped for
the *kdr-w* mutation after two to three attempts, and were
excluded from further analysis. *An. arabiensis* and *An. gam-
<figure><img src="image_2.png" /><figcaption>**Figure 1**
Map of the study area and collection sites.</figcaption></figure>

*biae* M form were found in sympatry in all 14 collection sites and all years except in the village of Bosso where only *An. arabiensis* was found. Also, very few *An. gambiae* M forms were repeatedly collected in Guidimouni. Consequently, detailed *kdr* results for *An. gambiae* M forms will be presented for 12 villages and Niamey. However, important variations of abundances and relative proportions of these two species were observed throughout the country (in prep.), leading to the analysis of small sample sizes in some locations or years for either one or both species. The reliability of the method employed to distinguish *kdr-w* genotypes was good, with 83% of concordance between first and second PCR runs considering only *kdr* M forms from rural areas (n = 45). In addition, on 26 *kdr* M forms from Niamey (12 homozygotes and 13 heterozygotes), all gave concordant results between the two runs except one (*kdr/kds* in the first assay, *kdr/kdr* in the second one) giving 96% of concordance.

All 456 *An. arabiensis* specimens analysed were homozygous susceptible *kds/kds* (n = 186 in 2005; n = 139 in 2006; n = 131 in 2007).

Only 19 *An. gambiae* S form specimens were found clus-
tered in four sites (Garey, Banizoumbou, Zindarou and
Fararatt), exhibiting an overall *kdr-w* frequency of 50.0%,
with five homozygous *kdr/kdr* genotypes, nine hetero-
zygous *kdr/kds* genotypes, and five homozygous *kds/kds*
genotypes. The proportion of these genotypes conformed
to Hardy-Weinberg equilibrium expectations. Only this
global result is given because the low sample size pre-
cluded any spatial or temporal variation analysis.

Dynamics of kdr frequency in An. gambiae M forms

Of the 986 *An. gambiae* M form specimens analysed, 42
specimens harboured one copy of the *kdr-w* mutation,
whereas only three specimens were homozygous for the
mutant allele, giving an overall *kdr-w* frequency of 2.4%. However, the mutation was not evenly distributed in space and time, and the proportion of sites where at least one *kdr*-harbouring mosquito was found within sufficient sample sizes varied from 5/11 sites in 2005 (45.5%) to 6/8 sites in 2006 (75%), and reached 6/6 sites in 2007 (100%). The genotype distribution was in accordance with Hardy-Weinberg proportions for each year's collection as a whole (all sites).

In the collection of 2005, corresponding to the pre-LLIN period, the mean *kdr-w* mutation frequency was 0.5% (n = 537) (Table 1). In each of the five sites where the mutation was detected, only one heterozygous *kdr-w* carrier was found, giving frequency estimates ranging from 0.6% (n = 86) to 1.6% (n = 32). The higher *kdr-w* frequency (8.3%) found in Damagaram Takaya was calculated for six individuals, and may not be representative of the population due to the small sample size.

The mean *kdr-w* mutation frequency significantly increased (X² = 8.935, p = 0.0014, df = 1) in the 2006 collection

(eight months after the LLIN distribution), as 2.0% of all M form specimens tested (n = 272) (Table 1) harboured the *kdr-w* allele in the heterozygous state only. Within the six locations where *kdr-w* carriers were found, the frequency of the mutation varied from 0.7% (n = 71) to 7.9% (n = 19). The site that had shown the highest *kdr* frequency within six individuals in 2005 gave similar results in 2006 (n = 5), indicating that the observed *kdr* frequency within only a few individuals might correctly estimate its real occurrence in that local population. Also, the 50.0% *kdr* frequency found in Guidimouni must be interpreted with caution as the M form sample represents only one heterozygous female.

In the 2007 wet season, the mean *kdr* frequency increased again in M form populations reaching 9.0% (X² = 23.16, p << 0.001, df = 1) (Table 1), and was found in the resistant homozygous state for the first time, in the village of Guidimouni. The number of M form specimens was again low in this locality (n = 4), and all were *kdr* carriers with three homozygous *kdr/kdr* females, giving the highest *kdr* frequency found during this study (87.5%). All five other

<table><caption>Table 1: *kdr-w* allelic frequency in An. gambiae M forms by site and year.</caption><thead><tr><th rowspan="2">Sites</th><th colspan="2">2005</th><th colspan="2">2006</th><th colspan="2">2007</th><th colspan="2">overall</th></tr><tr><th>n</th><th>kdr freq (%)<br>[95% CI]</th><th>n</th><th>kdr freq (%)<br>[95% CI]</th><th>n</th><th>kdr freq (%)<br>[95% CI]</th><th>n</th><th>kdr freq (%)<br>[95% CI]</th></tr></thead><tbody><tr><td>Firgoune</td><td>47</td><td>0.00<br>[0.0-3.8]</td><td>17</td><td>0.00<br>[0.0-10.3]</td><td>29</td><td>10.34<br>[3.9-21.2]</td><td>93</td><td>3.23<br>[1.2-6.9]</td></tr><tr><td>Garey</td><td>54</td><td>0.00<br>[0.0-3.4]</td><td>35</td><td>0.00<br>[0.0-5.1]</td><td>-</td><td>-</td><td>89</td><td>0.00<br>[0.0-2.1]</td></tr><tr><td>Banizoumbou</td><td>55</td><td>0.00<br>[0.0-3.3]</td><td>52</td><td>2.88<br>[0.6-8.2]</td><td>26</td><td>7.69<br>[2.1-18.5]</td><td>133</td><td>2.63<br>[1.1-5.3]</td></tr><tr><td>Zindarou</td><td>49</td><td>1.02<br>[0.0-5.6]</td><td>71</td><td>0.70<br>[0.0-3.9]</td><td>65</td><td>4.62<br>[1.7-9.8]</td><td>185</td><td>2.16<br>[0.9-4.2]</td></tr><tr><td>Maichilmi</td><td>77</td><td>0.00<br>[0.0-2.4]</td><td>10</td><td>5.00<br>[0.1-24.9]</td><td>-</td><td>-</td><td>87</td><td>0.57<br>[0.0-3.2]</td></tr><tr><td>Maikogo</td><td>86</td><td>0.58<br>[0.0-3.2]</td><td>19</td><td>7.89<br>[1.7-21.4]</td><td>34</td><td>8.82<br>[3.3-18.2]</td><td>139</td><td>3.60<br>[1.7-6.5]</td></tr><tr><td>Fararatt</td><td>71</td><td>0.70<br>[0.0-3.9]</td><td>53</td><td>0.94<br>[0.0-5.1]</td><td>19</td><td>7.89<br>[1.7-21.4]</td><td>143</td><td>1.75<br>[0.6-4.0]</td></tr><tr><td>Bermo</td><td>42</td><td>0.00<br>[0.0-4.3]</td><td>4</td><td>0.00<br>[0.0-36.9]</td><td>-</td><td>-</td><td>46</td><td>0.00<br>[0.0-3.9]</td></tr><tr><td>Tchake</td><td>32</td><td>1.56<br>[0.0-8.4]</td><td>3</td><td>0.00<br>[0.0-45.9]</td><td>-</td><td>-</td><td>35</td><td>1.43<br>[0.0-7.7]</td></tr><tr><td>Falenko</td><td>13</td><td>0.00<br>[0.0-13.2]</td><td>-</td><td>-</td><td>-</td><td>-</td><td>13</td><td>0.00<br>[0.0-13.2]</td></tr><tr><td>Damagaram</td><td>6</td><td>8.33<br>[0.2-38.5]</td><td>5</td><td>10.00<br>[0.3-44.5]</td><td>-</td><td>-</td><td>11</td><td>9.09<br>[1.1-29.2]</td></tr><tr><td>Guidimouni</td><td>3</td><td>0.00<br>[0.0-15.9]</td><td>1</td><td>50.00<br>[1.3-98.7]</td><td>4</td><td>87.50<br>[47.3-99.7]</td><td>8</td><td>50.00<br>[24.6-75.3]</td></tr></tbody><tfoot><tr><td>Total</td><td>537</td><td>0.47<br>[0.2-1.1]</td><td>272</td><td>2.02<br>[1.0-3.6]</td><td>177</td><td>9.04<br>[6.3-12.5]</td><td>986</td><td>2.43<br>[1.8-3.2]</td></tr></tfoot></table>

Six out of 12 villages were analysed in 2007. The study sites are presented following their longitudinal position, from West to East. Two sites (Goudoumaria and Bosso) where too few An. gambiae s.s. were found are not shown. n: specimens sample size. Kdr freq: *kdr-w* allelic frequency (percentage). 95% CI: 95% Confidence Interval
sites presented *kdr* carriers, with allelic frequencies ranging from 4.6% (n = 65) to 10.3% (n = 29).

Eight of the fourteen initial villages where sampled in 2007. Five of these presented enough sufficient M form sample sizes. The global temporal *kdr-w* frequency variation was also studied considering only those five sites. The results were quite similar, and with all five sites taken together, a highly significant increase of *kdr-w* frequency was detected (X² = 40.98, p << 0.001, df = 2), from 0.5% in 2005 to 1.9% in 2006 and 7.2% in 2007. Considering each site individually, only Maikogo presented a significant *kdr-w* frequency increase in the first wet season after the campaign (Fisher exact test, p = 0.019). In the second wet season after the campaign, *kdr* frequency increased significantly in Zindarou (Fisher exact test, p = 0.047) whereas significance was almost reached in Fararatt and Firgoune (Fisher exact test, p = 0.056 and 0.057, respectively). When comparing 2005 versus 2007 collections, all five sites except Zindarou presented a significant increase of *kdr* allelic frequency.

## Kdr frequency analysis by collection methods

The above results were presented without differentiation of collection methods, however they are meaningful because the proportion of tested individuals collected by each method was homogenous for the different years of collection (X² = 1.439, p = 0.487, df = 2 for five sites from Table 2; X² = 5.377, p = 0.0679, df = 2 for all sites). However, when analysing separately sub-samples of M forms collected by different methods, we found different *kdr-w*

frequencies, with a trend towards higher values for host-seeking mosquitoes collected by landing catches inside dwellings compared to indoor resting mosquitoes collected by spray catches (Figure 2). This trend was seen for each year but the difference was statistically significant only for 2007 (X² = 3.232, p = 0.036, df = 1) and for global results over all years (X² = 7.392, p = 0.003, df = 1). In addition, we observed quite constantly around two-fold higher *kdr-w* frequencies in host-seeking females.

By focusing again on the five villages used for temporal variation analysis, but considering only host-seeking females collected inside, the global increase of *kdr-w* frequency from 2005 to 2007 was also highly significant (X² = 25.35, p << 0.001, df = 2) (Table 2), however it was not the case when considering only resting females, giving only an increasing trend between 2005 and 2006 (Fisher exact test, p = 0.060).

## Preliminary results for urban M form populations

Thirty four *An. gambiae* M form females sampled as larvae in Niamey during 2003 and 53 during 2007 were also analysed. Several sites were sampled in 2003, whereas only one site was sampled in 2007, near a small stream called Gountou Yena (Figure 3). *Kdr* frequency was 32.4% in 2003 and reached 71.7% in 2007, constituting a highly significant increase (X² = 26.07, p << 0.001, df = 1). When analysing only the larval samples from the Gountou Yena stream, *kdr* frequency was 43.3% in 2003 (n = 15) and the 2003–2007 increase was still significant (X² = 8.318, p = 0.002, df = 1). In addition, considering the 2003 collec-

<table><caption>Table 2: *kdr-w* allelic frequency in *An. gambiae* M forms by site, year and method of collection.</caption><thead><tr><th rowspan="2">Sites</th><th rowspan="2">Collection</th><th colspan="2">2005</th><th colspan="2">2006</th><th colspan="2">2007</th><th colspan="2">overall</th></tr><tr><th>n</th><th>freq kdr (%)</th><th>n</th><th>freq kdr (%)</th><th>n</th><th>freq kdr (%)</th><th>n</th><th>freq kdr (%)</th></tr></thead><tbody><tr><td rowspan="3">Firgoune</td><td>ILC</td><td>24</td><td>0.00</td><td>5</td><td>0.00</td><td>21</td><td>14.29</td><td>50</td><td>6.00</td></tr><tr><td>IRC</td><td>23</td><td>0.00</td><td>8</td><td>0.00</td><td>8</td><td>0.00</td><td>39</td><td>0.00</td></tr><tr><td>total indoor</td><td>47</td><td>0.00</td><td>13</td><td>0.00</td><td>29</td><td>10.34</td><td>89</td><td>3.37</td></tr><tr><td rowspan="3">Banizoumbou</td><td>ILC</td><td>12</td><td>0.00</td><td>18</td><td>5.56</td><td>-</td><td>-</td><td>30</td><td>3.33</td></tr><tr><td>IRC</td><td>35</td><td>0.00</td><td>24</td><td>2.08</td><td>26</td><td>7.69</td><td>85</td><td>2.94</td></tr><tr><td>total indoor</td><td>47</td><td>0.00</td><td>42</td><td>3.57</td><td>26</td><td>7.69</td><td>115</td><td>3.04</td></tr><tr><td rowspan="3">Zindarou</td><td>ILC</td><td>22</td><td>0.00</td><td>52</td><td>0.96</td><td>50</td><td>5.00</td><td>124</td><td>2.42</td></tr><tr><td>IRC</td><td>6</td><td>0.00</td><td>2</td><td>0.00</td><td>15</td><td>3.33</td><td>23</td><td>2.17</td></tr><tr><td>total indoor</td><td>28</td><td>0.00</td><td>54</td><td>0.93</td><td>65</td><td>4.62</td><td>147</td><td>2.38</td></tr><tr><td rowspan="3">Maikogo</td><td>ILC</td><td>45</td><td>1.11</td><td>8</td><td>12.50</td><td>27</td><td>11.11</td><td>80</td><td>5.63</td></tr><tr><td>IRC</td><td>15</td><td>0.00</td><td>7</td><td>7.14</td><td>7</td><td>0.00</td><td>29</td><td>1.72</td></tr><tr><td>total indoor</td><td>60</td><td>0.83</td><td>15</td><td>10.00</td><td>34</td><td>8.82</td><td>109</td><td>4.59</td></tr><tr><td rowspan="3">Fararatt</td><td>ILC</td><td>35</td><td>0.00</td><td>15</td><td>0.00</td><td>7</td><td>14.29</td><td>57</td><td>1.75</td></tr><tr><td>IRC</td><td>32</td><td>0.00</td><td>31</td><td>1.61</td><td>10</td><td>5.00</td><td>73</td><td>1.37</td></tr><tr><td>total indoor</td><td>67</td><td>0.00</td><td>46</td><td>1.09</td><td>17</td><td>8.82</td><td>130</td><td>1.54</td></tr><tr><td rowspan="3">total</td><td>ILC</td><td>140</td><td>0.36</td><td>99</td><td>3.03</td><td>105</td><td>9.05</td><td>344</td><td>3.67</td></tr><tr><td>IRC</td><td>111</td><td>0.00</td><td>72</td><td>2.08</td><td>40</td><td>2.50</td><td>223</td><td>1.81</td></tr><tr><td>total indoor</td><td>251</td><td>0.20</td><td>171</td><td>2.63</td><td>145</td><td>7.24</td><td>567</td><td>2.88</td></tr></tbody></table>

The table presents data from the five sites analysed for the three years and with sufficient sample size. n: sample size. freq kdr: *kdr-w* allelic frequency (percentage). ILC: Indoor Landing Collections. IRC: Indoor Resting Collections
<figure><img src="image_3.png" /><figcaption>**Figure 2**
kdr-w allelic frequency in An. gambiae M forms by year and method of collection. Averaged kdr frequency over all rural sites (13 villages in 2005 and 2006, 6 villages in 2007). ILC: Indoor Landing Collections. IRC: Indoor Resting Collections</figcaption></figure>

tions, this *kdr* frequency in the Gountou Yena area was sig-
nificantly higher compared to other breeding sites in
Niamey (X² = 2.957, p = 0.043, df = 1) where the mean fre-
quency was 23.7%.

**Discussion**

This study describes the first case of *kdr* mutation-har-
bouring *An. gambiae* populations from Niger, to contrib-
ute to the description of the spatial distribution of *kdr-w*
mutation across West Africa, especially in the northern
limits of *An. gambiae* distribution area where such data are
very rare. Fully susceptible genotypes were found in every
*An. arabiensis* sample. This result is consistent with other
studies from neighbouring countries where *An. arabiensis*
exhibited very few if any *kdr* alleles [21,29,32]. Despite the
fact that our S form sample was too limited to describe
spatial and temporal dynamics of *kdr-w* mutation, its high
prevalence is nevertheless interesting. The cause of muta-
tion maintenance without significant insecticide pressure
is unknown, as the mutation frequency was actually
already 39% (n = 13) before the LLIN distribution.

The collected specimens allowed a more detailed study of
M form populations throughout the country. It suggests
that *kdr-w* mutation was already present at a low level in
M form populations before the nationwide LLIN distribu-
tion, in various localities distributed all over the sahelian
zone, at longitudes ranging approximately from 1°E to

10°E and latitudes below 16°N. This finding greatly
 extends the area of known *kdr*-carrying M form popula-
tions, that was to date limited to more humid areas in
Côte d'Ivoire [43], Ghana [36], Burkina Faso [31,32],
Benin [19,50], Nigeria [22,50], Cameroon [37,50], Equa-
torial Guinea [41,46] and Angola [47]. The mutation fre-
quencies encountered are consistent with studies from
neighbouring countries where *kdr-w* frequency was usu-
ally low in M molecular form : 6% in Malanville, North-
ern Benin, near the Nigerien border [19], 0.7% in Koubri
[36] and 2% in VK7 [32], southern Burkina Faso.

Based on strong supporting results, several authors
[19,21,29,33,34,36,48] hypothesized that past and cur-
rent agricultural use of pyrethroids and DDT for crop pro-
tection led to the selection of resistant individuals by
challenging larval stages with residual insecticide prod-
ucts accumulating in water bodies around cultivated
areas. This hypothesis was recently supported [44] by
showing indirectly the presence of pesticide residues in
soil and water from vegetable gardens and farms in Benin
that limited the emergence rate of challenged larvae. We
could relate our pre-LLIN low global kdr frequency to the
presumed limited environmental selection pressure on
An. gambiae populations, as the rural areas studied are
usually surrounded by seasonal millet and/or sorghum
subsistence cultivation for local consumption. These
farming practices employ negligible amounts of pesti-
<figure><img src="image_4.png" /><figcaption>**Figure 3**
**View of the Gountou Yena stream.** Picture taken in Niamey during early wet season (June 2008), showing the small stream surrounded by small-scale gardening areas (top-left) where kdr frequency within An. gambiae M form larvae was particularly high</figcaption></figure>

cides. Unreliable precipitation and limited commercial
demand tend to keep the use of inputs such as chemical
fertilizer, pesticides and hired labour to a minimum [64].
However, several localized mosquito populations might
experience more pyrethroid exposure, especially in irriga-
tion and gardening zones where agricultural production
demands and allows financially insecticide use. Urban
domestic use of pyrethroids for personal protection was
also suggested to favor the emergence of resistant individ-
uals within mosquito populations [29,39]. Indeed, the
highest *kdr* frequencies ever detected in M forms were
found around important cities in Côte d'Ivoire [43] and
Benin [19,35,44], corroborating the hypothesis of high
insecticide pressure within urban environnements. In
addition, it seems that M form populations breeding
inside the city of Niamey also present high *kdr-w* frequen-
cies, far greater than any analysed rural site. Some of the
sampled larval populations were breeding near a small
stream that crosses the city and is surrounded by year-
round vegetable cultivation areas. We therefore cannot

attribute potential selection effects to gardening and/or
domestic insecticide use, but strongly suspect far higher
insecticide exposure due to crop protection treatments.
This hypothesis is sustained by the higher *kdr* frequency
found near the cultivation areas compared to other larval
habitats in Niamey. We conducted a basic interview in the
cultivation areas, all 24 people reported repeated insecti-
cide use along the year, bought in local market places.
These multiple potential sources of pyrethroid pressure
are common in many sub-Saharan urban and peri-urban
areas [19,35,43,44], and are primarily due to the presence
of cultivated zones in the outskirts of cities. These local sit-
uations prevent a clear identification of factors responsi-
ble for the high resistance levels detected.

Compared to the pre-LLIN 2005 collections, a significant four-fold increase of global *kdr-w* mutation frequency was observed in the 2006 wet season, around 7-8 months after the nationwide LLIN distribution, again followed by a similar four-fold increase between 2006 and 2007 wet seasons. Although suspected, the selective pressure exerted by the nationwide LLIN coverage causing the *kdr* increase within mosquito populations cannot be demonstrated (mainly due to absence of control zones). Even though it is at a quite low level, this fast and linear increase could potentially lead to high *kdr* frequencies within a few years. A similar trend was reported in *An. gambiae* S form populations from West Kenya [57] after four years of ITNs trials in a 200 km² area whereas no variation was observed in control zones without ITNs. Sharp *et al.* [58] also reported an increasing *kdr-w* frequency in M forms from Bioko Island (Equatorial Guinea) in response to an IRS programme. On the same island, Reimer *et al.* [41] found a high allelic frequency in urban and peri-urban areas one year after the beginning of residual pyrethroids spraying, that they compare to the absence of *kdr* alleles reported before the IRS programme [65]. However, it should be noted that Berzosa *et al.* [65] analysed only ten larvae at each collection site, resulting in a high probability of missing *kdr* alleles present at low to moderate frequency. Indeed, a null *kdr* frequency within 10 individuals gives a 95% Confidence Interval upper bound of 16.8%, that means a 95% probability to find no *kdr* allele in the sample although present at a frequency below 16.8% in the population. A similar rapid *kdr* frequency increase was reported in Abidjan [25,28,43] from no *kdr* alleles around 1998 (n = 30) to 39% around 1999 (n = 27) and 70% in 2004, with the last sample collected in an outdoor deltamethrin-spraying area (n = 103). Unfortunately the two first studies pre-dated the description of *An. gambiae* molecular forms so we cannot exclude an effect of variation in molecular form proportions on *kdr* frequency. These studies and our results suggest a selection effect of large-scale insecticide-based control programmes on *kdr* mosquitoes. However, a recent study in Mali [34]
showed increasing *kdr* frequencies in S forms from three villages between 1993 and 2004 that were attributed to agricultural and domestic pyrethroid use in absence of any wide-scale control programme. Also, under the hypothesis of a recent introgression of the *kdr-w* allele from S to M form in southern forest areas, we cannot rule out the possibility that the global increase we described is simply due to current spatial expansion of the mutation in West Africa.

Marked seasonality and a short rainy season condition the annual expansion of mosquito populations and the start of a new malaria transmission season. Each new malaria transmission season's increased mosquito abundance stimulates the Sahel inhabitants to use their impregnated bed nets. Indeed, the nationwide LLINs distribution campaign took place during the beginning of the dry season when usually very low mosquito densities are found throughout the sahelian zone. Our personal observations of low LLIN usage before the wet season (R. Labbo, unpublished) were confirmed by the recently published coverage and usage study [10] and are consistent with this low biting nuisance. This fact could have important implications in terms of potential selective pressure exerted by the LLIN, that would consequently be highly seasonal and co-occurrent with the fast increase of mosquito population size soon after the beginning of the rainy season. The long-term effect of such explosive patterns of mosquito populations dynamics and LLIN usage on insecticide resistance is unknown, but could potentially differ from more humid zones where populations are more stable along the year. Also, this delay between bed nets distribution and maximal usage rate (around six months after) could also have delayed the start of the resistance increase, and could explain the low kdr frequency in 2006 only a few months after the nets began to be used largely.

Concerning our finding of lower *kdr* frequency within resting compared to host-seeking mosquitoes, the most likely hypothesis is directly linked to the collection method. The resting collections used pyrethroids (0.25% tetramethrin/0.05% cyphenothrin/0.04% prallethrin) and could therefore have preferentially killed susceptible *kds/kds* females whereas at least a portion of *kdr* females could have escaped or stayed on walls and ceilings without being knocked down by the spraying. If this hypothesis was true, one should consider the collection method employed when comparing published studies or planning field collections as it could bias the *kdr* frequency values. In addition, It seems unlikely that the observed variation between collection methods is due to different sub-populations within the domestic environnement. Resting females may have taken a blood-meal one or two days before and constitute the same population that blood-seeking females collected by indoor landing catches.

Once *kdr* mutations are found in a population, one important issue would be to determine to what extent it could decrease the benefits of control programmes. The relationship between *kdr* genotypes and resistance phenotypes was partly unraveled by laboratory studies [25,28,38,45,53] and experimental huts trials [18,45], but we are far from fully understanding the role of *kdr* mutations on mosquito survival in the field. Therefore, we can only speculate on the *kdr* frequency required to measure a significant effect on insecticide resistance. Some studies give however interesting indications about the personal and/or community protection provided by insecticide materials or treatments in areas of *kdr* populations. Sharp et al. [58] reported a reduced infection rate after an IRS programme with pyrethroids on An. gambiae M form populations exhibiting around 40% *kdr* frequency, however the abundances were only decreased after a shift towards carbamate spraying. The recent study of N'guessan et al. [15], although conducted in semi-field conditions with experimental huts and artificially holed bed nets, gives interesting clues because the same experiments were run in two contrasting environments of *kdr* frequency in M form populations. The personal protection provided by the treated holed nets was good when *kdr* frequency was 6%, but was much decreased when *kdr* frequency was around 80%. In addition, in a peri-urban area of Abidjan, Côte d'Ivoire, with M form populations with a *kdr* frequency of 70% [43], a spatial spraying programme of deltamethrin and fenitrothion in conjunction with deltamethrin-treated bed net usage in a french military camp did not significantly reduce the mosquito biting rates.

Therefore, as the present results indicate still low *kdr* frequencies mainly in the heterozygous state, it is unlikely that the current spread of the mutation has to date any significant effect on resistance phenotype and resulting efficacy of LLIN. However, it is feared that this continuous spread of *kdr-w* mutation could rapidly impede current efforts to reduce malaria transmission by implementing large-scale pyrethroid-treated nets coverage.

## Conclusion

These results are of prime importance in our effort to document multiple effects of operational control programmes on mosquito vectors, and to conceive sustainable control strategies for the future. As an increasing number of African countries plan to develop and scale up malaria control strategies including large vector control implementation, continued monitoring for insecticide resistance will be of utmost importance in a context of increasing extensive pyrethroid exposure. The documentation of the factors contributing to resistance selection within those populations is also highly important. Nevertheless, the long-term sustainability of such pro-
grammes will not be achieved without the availability of
alternative insecticidal compounds that could replace
pyrethroids for bed net impregnation where they would
appear inefficient due to *kdr* mutations and/or other
resistance mechanisms.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

CC carried out the molecular processing, the analysis and
interpretation of data, and contributed to the drafting of
the manuscript; RL participated to the conception of the
study and the field samples collection and identification;
IA participated to the collection and identification of the
samples and the molecular processing; JBD conceived and
coordinated the study, and contributed to the interpreta-
tion of data and the drafting of the manuscript. All
authors read and approved the final manuscript.

Acknowledgements

We thank the CERMES Parasitology unit staff for logistical and technical support during field and laboratory work. We acknowledge Fabrice Chan-dre and Jocelyn Ratovonjato for kindly providing positive controls for *kdr* mosquitoes and S form specimens, respectively. We are grateful to Arne Bomblies, Pr Marcel Hommel and the two anonymous reviewers for useful comments and corrections on the draft. We thank villagers from all studied sites for their collaboration. The study and CC's doctoral fellowship were funded by the IVth round of the Global Fund attributed to Niger through the International Federation of Red Cross and Red Crescent Societies for the two first years, and by the World Health Organization (APW AF/07/ 266865) for the third year of the project. Mosquito collections in Banizoumbou and Zindarou were funded by the European Union through AMMA (African Monsoon Multidisciplinary Analyses) project (reference CS 4089). Based on a French initiative, AMMA was built by an international scientific group and is currently funded by a large number of agencies, especially from France, UK, US and Africa. It has been the beneficiary of a major financial contribution from the European Community's Sixth Framework Research Programme. Detailed information on scientific coordination and funding is available on the AMMA International web site : http://www.amma-interna-tional.org.

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