# Culicidae diversity, malaria transmission and insecticide resistance alleles in malaria vectors in Ouidah-Kpomasse-Tori district from Benin (West Africa): A pre-intervention study

Armel Djènontin, Sahabi Bio-Bangana, Nicolas Moiroux, Marie-Claire Henry, Olayidé Bousari, Joseph Chabi, Razaki Ossè, Sébastien Koudénoukpo, Vincent Corbel, Martin Akogbéto, et al.

► To cite this version:

Armel Djènontin, Sahabi Bio-Bangana, Nicolas Moiroux, Marie-Claire Henry, Olayidé Bousari, et al. Culicidae diversity, malaria transmission and insecticide resistance alleles in malaria vectors in Ouidah-Kpomasse-Tori district from Benin (West Africa): A pre-intervention study. Parasites & Vectors, 2010, 3 (1), pp.83. 10.1186/1756-3305-3-83. doi:10.29657/pc.2010.3

HAL Id: hal-02965713

https://hal.science/hal-02965713

Submitted on 12 Jan 2022

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
*Culicidae* diversity, malaria transmission and insecticide resistance alleles in malaria vectors in Ouidah-Kpomasse-Tori district from Benin (West Africa): A pre-intervention study

Armel Djènontin¹,²*, Sahabi Bio-Bangana¹, Nicolas Moiroux², Marie-Claire Henry²,⁴, Olayidé Bousari², Joseph Chabi², Razaki Ossè¹, Sébastien Koudénoukpo¹, Vincent Corbel², Martin Akogbéto¹, Fabrice Chandre³

**Abstract**

**Background:** To implement an Insecticide Resistance Management (IRM) strategy through a randomized controlled trial (phase III), 28 villages were selected in southern Benin. No recent entomological data being available in these villages, entomological surveys were performed between October 2007 and May 2008, before vector control strategies implementation, to establish baseline data.

**Methods:** Mosquitoes were sampled by human landing collection (16 person-nights per village per survey per village) during 5 surveys. Mosquitoes were identified morphologically and by molecular methods. The *Plasmodium falciparum* circumsporozoïte indexes were measured by ELISA, and the entomological inoculation rates (EIRs) were calculated. Molecular detection of pyrethroid knock down resistance (*Kdr*) and of insensitive acetylcholinesterase were performed.

**Results:** 44,693 mosquitoes belonging to 28 different species were caught from October 2007 to May 2008. Among mosquitoes caught, 318 were *An. gambiae s.s.*, 2 were *An. nili*, 568 were *An. funestus s.s.*, and one individual was *An. leesoni*. EIR was 2.05 ± 1.28 infective bites per human per 100 nights on average, of which 0.67 ± 0.60 were from *An. funestus* and 1.38 ± 0.94 infective bites were from *An. gambiae*. Important variations were noted between villages considering mosquito density and malaria transmission indicating a spatial heterogeneity in the study area. The *kdr* allelic frequency was 28.86% in *An. gambiae s.s.* on average and significantly increases from October 2007 (10.26%) to May 2008 (33.87%) in M molecular form of *An. gambiae s.s.* *Ace 1* mutation was found in 5 molecular of *An. gambiae s.s.* at a low frequency (< 1%).

**Conclusion:** This study updates information on mosquito diversity and malaria risk in rural villages from south Benin. It showed a high spatial heterogeneity in mosquito distribution and malaria transmission and underlines the need of further investigations of biological, ecological, and behavioral traits of malaria vectors species and forms. This study is a necessary prerequisite to cartography malaria risk and to improve vector control operations in southern Benin.

**Background**

Malaria remains a major cause of morbidity and mortal-
ity in sub-Saharan Africa and represents one of the most
critical public health challenges for Africa. In 2008, 243
million cases of malaria was estimated worldwide leading
to 863 000 deaths of which 89% were in the African

Region [1]. Treatment with Artemisinin Combination
Therapy (ACT), the use of Indoor Residual Spraying
(IRS) and Insecticide Treated Nets (ITNs) represent the
main approaches of malaria control [1]. Household ITNs
ownership reached more than 50% in several high burden
African countries [1]. Pyrethroids are the only insecti-
cides used for net impregnation because of their strong
efficacy, their fast acting effect at low doses and their low
toxicity for mammals [2]. Unfortunately, pyrethroids
resistance in malaria vectors has spread across Africa and

<footnote>* Correspondence: armeldj@yahoo.fr
¹Centre de Recherche Entomologique de Cotonou (CREC), 06 BP 2604
Cotonou, Bénin
Full list of author information is available at the end of the article</footnote>
is now present in most of countries where national
malaria control programmes (NMCP) are implementing
large scale distribution of Long Lasting Nets to popula-
tions at risk, i.e. children under five and pregnant women
[3]. Up to now, there is no evidence that pyrethroids
resistance reduce the effectiveness of ITNs for malaria
control at operational scale [4]. However, a small scale
field trial carried out in an area of resistance in southern
Benin (Ladji) and Burkina Faso (Kou Valley) showed a
reduction of personal protection and overall insecticidal
effect of ITNs in experimental huts [5-7].

It is then urgent to find ways to manage this resis-
tance in malaria vectors. In this context, malaria vec-
tors control and insecticide resistance management
tools based on the use (alone or in combination with
pyrethroid-treated mosquito nets) of alternative classes
of insecticides with different mode of action than pyre-
throids were developed and have already been evalu-
ated in experimental huts, with good results against
wild populations of strongly resistant *An. gambiae*
[6,7].

To validate these strategies, their impact on malaria
transmission and insecticide resistance dynamic in
malaria vectors must be accessed through randomized
controlled trials under phase III. For that purpose, 28
villages were selected in Ouidah-Kpomasse-Tori
(OKT) health district in southern Benin. No entomolo-
gical data from OKT health district was available.
Recent data relative to *Culicidae* fauna of Benin go
back up to 1950s [8,9]. Thus, in order to collect base-
line data relative to mosquito's diversity and abun-
dance, malaria transmission and the prevalence of
insecticide resistance alleles in malaria vectors, ento-
mological surveys were performed between October
2007 and May 2008 in the study area before imple-
menting the vector control strategies. This paper
reports these baseline data.

**Methods**

Study area

The study was carried out in OKT health district. Twenty
eight villages were selected considering a size between
250-500 inhabitants, a distance between two villages
higher than two kilometers and the absence of a local
health center. The OKT health district is one of 36 health
districts in Benin. This district has essentially a sub-equa-
torial climate, with two dry seasons (August-September
and December-March), and two rainy seasons (April-July
and October-November). The average annual rainfall is
around 1200 mm, of which 700-800 mm come in the
first rainy season and 400-500 mm come in the second
rainy season. The average monthly temperatures vary
between 27 and 31°C. The northern part of the health
district is made of a plateau that drops into the Couffo

valley and the Allada depression. The southern is watered
by several ramification arms of Toho Lake (Fig 1). The
study zone is totally cleared of the original equatorial for-
est. Currently, the vegetation is characterized by bushes
and isolated trees, associated with areas with more or less
densely populated areas with oil palm trees.

Mosquito's collection and identification

Sampling of mosquitoes was done in 28 villages by
human landing catches using tubes that were plugged
with cotton. Mosquitoes collection was carried out dur-
ing 5 surveys from October 2007 to May 2008 (2 in the
beginning of rainy periods and 3 in dry periods) every
6 weeks both indoors and outdoors at 4 sites per village
from 10 p.m. to 6 a.m. and for two consecutive nights
per survey (i.e. 16 person-nights per village per survey).
Teams of collectors were rotated among the collection
points on different collection nights to minimize sam-
pling bias. Ethical clearance was given for the study by
the National Ethical Committee in Benin (Comité
National Provisoire d'Ethique pour la Recherche en
Santé) and IRD ethical committee (Comité Consultatif
de Déontologie et d'Ethique). Collectors gave prior
informed consent and they were vaccinated against yel-
low fever. Since study was done in area where malaria is
endemic, adult collectors that already acquired immu-
nity against malaria parasites, did not received chemo-
prophylaxis, but were medically supervised by local
physicians in case of illness.

Mosquitoes were identified on the field to species level
using morphological criteria according to the identifica-
tion keys [10-12]. All mosquitoes belonging to the
*An. gambiae* complex or *An. funestus* group were stored
in individual tubes with silica gel and preserved at -20°C
in the laboratory.

**Determination of EIRs**

Heads and thoraces of anopheline females were tested by
Enzyme-Linked Immunosorbent Assay (ELISA) for detec-
tion of *P. falciparum* circumsporozoite protein (CSP), as
previously described [13]. Samples having an optical den-
sity higher than 3 times the average of the optical density
of negative control were considered as positive. Those
having an optical density between 2 and 3 times this aver-
age were considered as doubtful and were retested.

The CSP index was calculated as the proportion of
mosquitoes found to be positive for CSP. Entomological
inoculation rates (EIRs) were estimated as the number
of infective bites per human per 100 nights.

**Molecular analysis**

All mosquitoes belonging to *An. gambiae* complex and *Anopheles funestus* group were identified by polymerase chain reaction (PCR) at the species levels [14,15].
<figure><img src="image_2.png" /><figcaption>Figure 1 Map of the study area.</figcaption></figure>

Molecular forms in *Anopheles gambiae s.s.* were also identified [16].

Molecular characterizations of the *Kdr* (west-African Leu-Phe mutation) and *Ace1* mutations were carried out on all mosquitoes belonging to *An. gambiae* complex as previously described [17,18]

## Statistical analysis

Chi-square tests were done to determine the odds of association of the CSP index with malaria vectors *An. gambiae* and *An. funestus*.

Data relative to mosquito's density and EIRs according to villages were analyzed using a Linear Mixed Effects (LME) model implemented in R software and written as follow:

$$ \text{Log}(x) = \beta_0 + \beta_{1i} + a_i, \text{ where:} $$

x = means of mosquitoes number (or EIRs) per village per survey

$\beta_0$ = estimated value of coefficient associated to the reference village

$\beta_{1i}$ = estimated value of coefficient associated to a village i

$a_i$ = intra-village variance

i = village index

Villages were grouped according to their homogeneity for the entomological indicator considered taking into account $\beta_i$ and its P value. In each group, the average of the number of mosquitoes caught per village per survey (or the number of infective bites per human per 100 nights) was calculated with its 95% confidence interval.

The genotypic differentiation of *kdr* and *Ace1* loci was tested using the Fischer exact test implemented in Gen-Pop software [19].

## Results

### Species diversity and density

A total of 44,693 mosquitoes belonging to 28 species were caught (table 1). *Mansonia Africana* (44.84%), *Culex gr. decens* (29.91%), *Culex quinquefasciatus* (9.81%), and *Culex nebulosis* (6.22%) were the most
<table><caption>**Table 1** Mosquitoes species caught from October 2007 to May 2008 in the study area</caption><thead><tr><th>Species</th><th>Indoor</th><th>Outdoor</th><th>Total</th><th>%</th></tr></thead><tbody><tr><td>Aedes aegypti</td><td>388</td><td>318</td><td>706</td><td>1.58</td></tr><tr><td>Aedes gr. palpalis</td><td>12</td><td>28</td><td>40</td><td>0.09</td></tr><tr><td>Aedes gr. tarsalis</td><td>4</td><td>4</td><td>8</td><td>0.02</td></tr><tr><td>Aedes luteocephalus</td><td>21</td><td>41</td><td>62</td><td>0.14</td></tr><tr><td>Aedes sp</td><td>13</td><td>37</td><td>50</td><td>0.11</td></tr><tr><td>Aedes vittatus</td><td>199</td><td>396</td><td>595</td><td>1.33</td></tr><tr><td>Anopheles brohieri</td><td>1</td><td>0</td><td>1</td><td>0.00</td></tr><tr><td>Anopheles coustani</td><td>3</td><td>5</td><td>8</td><td>0.02</td></tr><tr><td>Anopheles funestus</td><td>314</td><td>265</td><td>579</td><td>1.30</td></tr><tr><td>Anopheles gambiae</td><td>149</td><td>173</td><td>322</td><td>0.72</td></tr><tr><td>Anopheles nili</td><td>1</td><td>1</td><td>2</td><td>0.00</td></tr><tr><td>Anopheles pharoensis</td><td>60</td><td>135</td><td>195</td><td>0.44</td></tr><tr><td>Anopheles ziemanni</td><td>12</td><td>20</td><td>32</td><td>0.07</td></tr><tr><td>Culex annulioris</td><td>185</td><td>428</td><td>613</td><td>1.37</td></tr><tr><td>Culex duttoni</td><td>36</td><td>18</td><td>54</td><td>0.12</td></tr><tr><td>Culex fatigans</td><td>270</td><td>436</td><td>706</td><td>1.58</td></tr><tr><td>Culex gr. decens</td><td>4720</td><td>8649</td><td>13369</td><td>29.91</td></tr><tr><td>Culex nebulosis</td><td>1040</td><td>1738</td><td>2778</td><td>6.22</td></tr><tr><td>Culex perfuscus</td><td>2</td><td>0</td><td>2</td><td>0.00</td></tr><tr><td>Culex poicilipes</td><td>2</td><td>14</td><td>16</td><td>0.04</td></tr><tr><td>Culex quinquefasciatus</td><td>1170</td><td>3213</td><td>4383</td><td>9.81</td></tr><tr><td>Culex tigripes</td><td>17</td><td>38</td><td>55</td><td>0.12</td></tr><tr><td>Cx sp</td><td>17</td><td>17</td><td>34</td><td>0.08</td></tr><tr><td>Cx thalassius</td><td>10</td><td>16</td><td>26</td><td>0.06</td></tr><tr><td>Eretmapodites gr. quinquevittatus</td><td>0</td><td>3</td><td>3</td><td>0.01</td></tr><tr><td>Mansonia africana</td><td>7652</td><td>12390</td><td>20042</td><td>44.84</td></tr><tr><td>Mansonia uniformis</td><td>7</td><td>4</td><td>11</td><td>0.02</td></tr><tr><td>Uranotaenia gr. bilineata</td><td>1</td><td>0</td><td>1</td><td>0.00</td></tr><tr><td>Total</td><td>16306</td><td>28387</td><td>44693</td><td></td></tr></tbody></table>

abundant species caught. The proportion of malaria vectors (*Anopheles gambiae* and *Anopheles funestus*) was very low (~2%).

All mosquitoes belonging to *Anopheles gambiae s.l.*, were *An. gambiae s.s.* (217 from S molecular form and 101 from M form). Concerning *Anopheles funestus* group all mosquitoes were identified by PCR as *Anopheles funestus s.s.* except one individual which was *An. leesoni*, caught in Tokoli-Vidjinangnimon village.

Large variations were observed between villages considering mosquito's density. An average of 212 ± 46 *Culicidae* per village per survey and 531 ± 139 *Culicidae* per village per survey were caught in two different homogenous villages groups (Table 2). Concerning *An. gambiae s.l.* on average 0.4 ± 0.2; 1.8 ± 0.6 and 7.4 ± 3.5 individuals were caught per village per survey in three different homogenous villages groups (Table 3). In villages close to a arm of the Toho Lake, 13.48 ± 5.9 *An. funestus s.l.* were caught on average per village per

<table><caption>**Table 2** Average of *Culicidae* number caught per survey per village from October 2007 to May 2008 with 95% confidence intervals</caption><thead><tr><th>Villages groups</th><th>Average Culicidea number per survey per village with 95% confidence intervals</th></tr></thead><tbody><tr><td><strong>Group 1:</strong> Agouako; Assogbenoudaho; Guezohoue; Hla; Todo; Zoume; Adjahassa; Satre; Agadon; Aidjedo; Ayidohoue; Dokanme; Tokoli Vidjinangnimon; Hekandji; Hounkponouhoue; Dekponhoue; Soko; Wanho; Tokoli; Tanto</td><td>212 ± 46</td></tr><tr><td><strong>Group 2:</strong> Lokohoue; Tokoli; Vidjinangnimon; Manguevier; Adjame-Allagbede; Amoulehoue; Kindjitokpa; Abenihoue; Hinmandou</td><td>531 ± 139</td></tr></tbody></table>

survey, in contrast with 0.29 ± 0.1 in the others villages (Table 4).

## Vectors infection to CSP and malaria transmission risk

The CSP positivity rate was 9.63 ± 3.2% in *An. gambiae s.s.* (5.94 ± 0.4% in M molecular form and 11.52 ± 0.2% in S form) and 2.64 ± 1.3% in *An. funestus s.l.* *An. gambiae s.s.* was more infected than *An. funestus s.l.* (OR = 4.00 (95%IC 2.13-7.54), P < 0.001). The average of EIR from October 2007 to May 2008 was 2.05 ± 1.28 infective bites per human per 100 nights of which 0.67 ± 0.60 infective bites of *An. funestus* per human per 100 nights and 1.38 ± 0.94 infective bites of *An. gambiae* per human per 100 nights. EIR was very variable according to villages. Two homogenous villages groups with respectively 0.7 ± 0.4 and 6.1 ± 3.4 infective bites per human per 100 nights were observed (Table 5).

<table><caption>**Table 3** Average of *An. gambiae s.l.* number caught per survey per village from October 2007 to May 2008 with 95% confidence intervals</caption><thead><tr><th>Villages groups</th><th>Average An. gambiae s.l.<br>number per survey per village<br>with 95% confidence intervals</th></tr></thead><tbody><tr><td><strong>Group 1:</strong> Adjame-Allagbede; Agouako; Assogbenoudaho; Kindjitokpa; Guezohoue; Hinmandou; Hla; Abenihoue; Todo; Zoume</td><td>0.4 ± 0.2</td></tr><tr><td><strong>Group 2:</strong> Adjahassa; Satre; Lokohoue; Agadon; Aidjedo; Ayidohoue; Dokanme; Tokoli; Vidjinangnimon; Hekandji; Agokon; Hounkponouhoue; Dekponhoue; Manguevier</td><td>1.8 ± 0.6</td></tr><tr><td><strong>Group 3:</strong> Amoulehoue; Soko; Tokoli; Tanto; Wanho</td><td>7.4 ± 3.5</td></tr></tbody></table>
<table><caption>**Table 4 Average of An. funestus s.l. number caught per survey per village from October 2007 to May 2008 with 95% confidence intervals**</caption><thead><tr><th>Villages groups</th><th>Average An. funestus s.l. number per survey per village with 95% confidence intervals</th></tr></thead><tbody><tr><td><strong>Group 1:</strong> Adjame-Allagbede;<br>Agouako; Assogbenoudaho;<br>Guezohoue; Hinmandou; Hla;<br>Abenihoue; Todo; Zoume;<br>Adjahassa; Satre; Agadon; Agokon;<br>Aidjedo; Ayidohoue; Dokanme;<br>Hekandji; Hounkponouhoue;<br>Dekponhoue; Soko</td><td>0.29 ± 0.1</td></tr><tr><td><strong>Group 2:</strong> Amoulehoue; Tokoli;<br>Tanto; Lokohoue; Manguevier;<br>Kindjitokpa; Tokoli Vidjinangnimon;<br>Wanho</td><td>13.48 ± 5.9</td></tr></tbody></table>

### Kdr resistance gene status in *An. gambiae*.

The average of *kdr* allelic frequency from October 2007 to May 2008 was 28.86% in *An. gambiae s.s.* At the beginning of the study (From October to December 2007), *kdr* allelic frequency was significantly higher in S molecular form of *An. gambiae s.s.* (28.08%) compared to the M molecular form (10.26%) (P = 0.001). This allelic frequency significantly increased in the M molecular form and has reached 33.87% (P < 0.001) from January to May 2008 (Table 6).

### Insensitive acetylcholinesterase gene status in *An. gambiae s.l*

The allelic frequency of this gene was less than 1%. Only two heterozygous individuals was found in the S molecular form of *An. gambiae s.s.* at Tanto and Hekandji during October 2007.

### Discussion

The present study provides entomological baseline data on OKT health district. Twenty eight different species

<table><caption>**Table 5 Number of infective bites per human per 100 nights from October 2007 to May 2008 with 95% confidence intervals**</caption><thead><tr><th>Villages groups</th><th>Number of infective bites per human per 100 nights with 95% confidence intervals</th></tr></thead><tbody><tr><td><strong>Group 1</strong> : Adjame; Agouako;<br>Aidjedo; Asogbenoudaho;<br>Ayidohoue; Adjahassa; Dokanme;<br>Kindjitokpa; Guezohoue;<br>Hinmandou; Hla; Hounkponouhoue;<br>Abenihoue; Dekponhoue;<br>Manguevier; Satre; Todo; Tokoli;<br>Wanho; Agadon; Zoume</td><td>0.7 ± 0.4</td></tr><tr><td><strong>Group 2</strong> : Amoulehoue; Tokoli;<br>Vidjinangnimon; Hekandji; Agokon;<br>Soko; Lokohoue; Tanto</td><td>6.1 ± 3.4</td></tr></tbody></table>

<table><caption>**Table 6 kdr in M and S molecular forms of *An. gambiae* s.s. from October 2007 to May 2008 in the study area**</caption><thead><tr><th rowspan="2"></th><th colspan="2">October-December 2007</th><th colspan="2">January-May 2008</th></tr><tr><th>M form</th><th>S form</th><th>M form</th><th>S form</th></tr></thead><tbody><tr><td>RR</td><td>0</td><td>10</td><td>13</td><td>16</td></tr><tr><td>RS</td><td>8</td><td>62</td><td>16</td><td>20</td></tr><tr><td>SS</td><td>31</td><td>74</td><td>33</td><td>35</td></tr><tr><td>Total</td><td>39</td><td>146</td><td>62</td><td>71</td></tr><tr><td>Allelic frequency (%)</td><td><strong>10.26<sup>a</sup></strong></td><td><strong>28.08<sup>b</sup></strong></td><td><strong>33.87<sup>b</sup></strong></td><td><strong>36.62<sup>b</sup></strong></td></tr></tbody></table>

*Kdr* allelic frequency values carrying the same letter was not significantly different (P > 0.05)

were caught during surveys whereas Huttel (1950) and Hamon (1954) had collected respectively 13 species (adult's collection in the dwellings) and 45 species (adult and larval collection) in the south-east of Benin (Cotonou and Porto-Novo) [8,9]. If the *Culicidae* diversity is lower in our study, we have identified 3 *Anopheles* species that was not mentioned in Hamon study: *An. brohieri*, *An. coustani* and *An. nili*. These 3 species were present at very low densities, explaining why they were not reported previously. More unexpected is the predominance in our study of *Mansonia africana* and the presence of *Cx. quinquefasciatus* that were not reported by Hamon. The relative abundance of *Cx. quinquefasciatus* is particularly surprising for a species which is adapted to polluted larval breeding sites and usually more familiar with urban environment than traditional villages from this study. Results showed a high heterogeneity of the study area in terms of *Culicidae* and especially malaria vectors abundance. It could be due to the geographic pattern of this area that is characterized by a northern part made of a plateau and the southern watered by several ramification arms of Toho Lake. *An. funestus* density was higher in villages close to arms of Toho Lake (< 2 km), since some of its larval breeding sites are the bank of lake or river with vegetation. In most villages of the study area, the soil is made of a thick bed of a mixture of sand and clay allowing a rapid water infiltration after rain. This impedes the formation of *An. gambiae* larval breeding sites that could explain the low density of malaria vectors. However, on the 5 surveys conducted in each village, only two were conducted during a rainy period (in the beginning of the rainy periods). This could explain the very low number of anopheles vectors caught during the study. Further studies conducted on a long period and taking into account environmental factors are required to better understand mosquito's distribution determinants in this area.

*Anopheles gambiae s.s.* and *An. funestus s.s.* were shown to be the main vectors in the study area, confirming previous studies in West Africa [20-22]. The
relative abundance of *An. funestus* in the study area and its CSP positivity rate (2.64 ± 1.3) indicate that this mosquito is involved in malaria transmission in southern Benin. No data relative to resistance status and mechanisms of this vector being available in Benin, further studies about *An. funestus* populations and their resistance mechanisms are required. The EIR, 2.05 ± 1.28 infective bites per human per 100 nights on average, indicates that in the study area, malaria is mesoendemic. These data agree with parasitological and clinical data collected in the same way in these villages indicating an annual prevalence rate of 21.8% (19.1-24.4) in young asymptomatic children and a clinical incidence of *Plasmodium falciparum* malaria of 1.5 (1.2-1.9) per child per year (Damien *et al.* 2010, unpublished data).

The *kdr-w* resistance allele was identified in *An. gambiae* populations examined in this study and have significantly increased from October 2007 to May 2008 in M molecular form in contrast with S form, indicating probably that M and S molecular forms of this vector undergo different selection pressures in the study area and underlines the need of further studies taking into account ecological and others factors. The presence of the *kdr-w* allele in *An. gambiae s.s.* and the increase of its frequency are worrying because this gene and metabolic resistance mechanisms in southern Benin appears to have a significant impact on LLIN efficacy as demonstrated by recent experimental hut trial [23]. Many other studies have showed a strong decrease of Pyrethroid-Treated Nets performance against pyrethroid-resistant malaria vectors in Benin [5,6], Burkina Faso [7], and Côte d'Ivoire [24].

Regarding carbamate resistance, we confirmed the low allelic frequency of the ace-1<sup>R</sup> allele (< 1%) in *An. gambiae s.s.* in West Africa [25], showing that there is no (or very low) selection pressure on this allele in the study area. However, a monitoring of this resistance gene is required considering the large scale implementation of Indoor Residual Spraying using bendiocarb by President's Malaria Initiative program in south Benin that will inevitably increase the selection pressure on vectors populations.

## Conclusion

This study updates information on mosquito's distribution and their role in malaria transmission in southern Benin. It showed a high spatial heterogeneity in mosquitoes distribution and malaria transmission and then underlines the need of further investigations of biological, ecological, and behavioral traits of malaria vectors species and forms. That will allow to cartography

malaria risk and then to improve vector control interventions in Benin.

## Acknowledgements

We thank the Ministère Français des Affaires Etrangères for project REFS N° 2006-22 and the Institut de Recherche pour le Développement, which financially and materially supported the work. We thank populations of the study area, OKT health district, for their collaboration. We also thank Seth Irish for his linguistic assistance while drafting this manuscript.

## Author details

¹Centre de Recherche Entomologique de Cotonou (CREC), 06 BP 2604 Cotonou, Bénin. ²Institut de Recherche pour le Développement (IRD/UR016), 01 BP 4414 Cotonou, Bénin. ³Laboratoire de lutte contre les Insectes Nuisibles (LIN/IRD), 911 Ave Agropolis BP 64501, 34394 Montpellier Cedex 5, France. ⁴Service de Coopération française, Ambassade de France, Cotonou, Bénin.

## Authors' contributions

FC and M-CH conceived of the study. AD, JC and FC have participated in the design of the study. Entomologic data was collected by AD, JC, RO, SK. AD, SK and RO carried laboratory analysis. AD, FC, OB and VC have participated in the analysis and interpretation of data. Maps were provided by SBB and NM. The manuscript has been drafted by AD. AD, SBB, NM, M-CH, OB, JC, RO, SK, VC, MA and FC have been involved in manuscript revising. All authors read and approved the final manuscript.

## Competing interests

The authors declare that they have no competing interests.

Received: 14 July 2010 Accepted: 6 September 2010
Published: 6 September 2010

## References

1. WHO: World Malaria Report 2009. World Health Organization, Geneva 2009.

2. Zaim M, Aitio A, Nakashima N: Safety of pyrethroid-treated mosquito nets. *Med Vet Entomol* 2000, **14**:1-5.

3. Santolamazza F, Calzetta M, Etang J, Barrese E, Dia I, Caccone A, Donnelly MJ, Petrarca V, Simard F, Pinto J, della Torre A: Distribution of knock-down resistance mutations in *Anopheles gambiae* molecular forms in west and west-central Africa. *Malar J* 2008, **7**:74.

4. Henry MC, Assi SB, Rogier C, Dossou-Yovo J, Chandre F, Guillet P, Carnevale P: Protective efficacy of lambda-cyhalothrin treated nets in *Anopheles gambiae* pyrethroid resistance areas of Cote d'Ivoire. *Am J Trop Med Hyg* 2005, **73**:859-864.

5. N'Guessan R, Corbel V, Akogbéto M, Rowland M: Reduced efficacy of insecticide-treated nets and indoor residual spraying for malaria control in pyrethroid resistance area, Benin. *Emerg Infect Dis* 2007, **13**:199-206.

6. Djènontin A, Chabi J, Baldet T, Irish S, Pennetier C, Hougard JM, Corbel V, Akogbéto M, Chandre F: Managing insecticide resistance in malaria vectors by combining carbamate-treated plastic wall sheeting and pyrethroid-treated bed nets. *Malar J* 2009, **8**:233.

7. Djènontin A, Chandre F, Dabiré KR, Chabi J, N'Guessan R, Baldet T, Akogbéto M, Corbel V: The Indoor Use of Plastic Sheeting Impregnated with Carbamate in Combination with Long Lasting Insecticidal Mosquito Nets for the Control of Pyrethroid-resistant Malaria. *Am J Trop Med Hyg* 2010, **83**:266-270.

8. Huttel J: Note sur la répartition des moustiques dans le Bas-Dahomey. *Bull Sot Path Exot* 1950, **43**:563-566.

9. Hamon J: Contribution à l'étude des Culicidés de la région de Porto-Novo (Bas-Dahomey). *Ann parasitologie* 1954, **29**:588-594.

10. Edwards F: Mosquitoes of the Ethiopian Region III. Culicine adults and pupae. *British Museum (Nat Hist), London* 1941.

11. Gillies MT, De Meillon BL: The *Anophelinae* of Africa south of the Sahara. Publication of the South African Institute for Medical Research, Johannesburg 1968, 54.
12. Gillies MT, Coetzee M: *A Supplement to the Anophelinae of Africa South of the Sahara (Afrotropical Region)* Publication of the South African Institute for Medical Research, Johannesburg 1987, 55.

13. Wirtz RA, Zavala F, Charoenvit Y, Campbell GH, Burkot T, Schneider I, Esser KM, Beaudoin RL, Andre RG: *Comparative testing of monoclonal antibodies against Plasmodium falciparum sporozoites for ELISA development*. *Bull World Health Organ* 1987, **65**:39-45.

14. Scott JA, Brogdon WG, Collins FH: *Identification of single specimens of the Anopheles gambiae complex by the polymerase chain reaction*. *Am J Trop Med Hyg* 1993, **49**:520-529.

15. Koekemoer LL, Kamau L, Hunt RH, Coetzee M: *A cocktail polymerase chain reaction assay to identify members of the Anopheles funestus* (Diptera: Culicidae) group. *Am J Trop Med Hyg* 2002, **66**:804-811.

16. Favia G, Lanfrancotti A, Spanos L, Siden Kiamos I, Louis C: *Molecular characterization of ribosomal DNA polymorphisms discriminating among chromosomal forms of Anopheles gambiae s.s.* *Insect Mol Biol* 2001, **10**:19-23.

17. Martinez-Torres D, Chandre F, Williamson MS, Darriet F, Berge JB, Devonshire AL, Guillet P, Pasteur N, Pauron D: *Molecular characterization of pyrethroid knockdown resistance (kdr) in the major malaria vector Anopheles gambiae s.s.* *Insect Mol Biol* 1998, **7**:179-184.

18. Weill M, Malcolm C, Chandre F, Mogensen K, Berthomieu A, Marquine M, Raymond M: *The unique mutation in ace-1 giving high insecticide resistance is easily detectable in mosquito vectors*. *Insect Mol Biol* 2004, **13**:1-7.

19. Raymond M, Rousset F: *GENEPOP* (version 1.2): population genetics software for exact tests and ecumenicism. *J Heredity* 1995, **86**:248-249.

20. Akogbéto M: Entomological study on the malaria transmission in coastal and lagoon areas: the case of a village built on a brackish lake. *Ann Soc Belg Med Trop* 1995, **75**:219-227.

21. Fontenille D, Simard F: Unravelling complexities in human malaria transmission dynamics in Africa through a comprehensive knowledge of vector populations. *Comp Immunol Microbiol Infect Dis* 2004, **27**:357-375.

22. Louise A, Kelly-Hope L, Ellis McKenzie F: The multiplicity of malaria transmission: a review of entomological inoculation rate measurements and methods across sub-Saharan Africa. *Malar J* 2009, **8**:19.

23. WHO: Report of the twelfth WHOPES Working group meeting. *WHO/HTM/NTD/WHOPES* 2009, **1**.

24. N'Guessan R, Darriet F, Doannio JM, Chandre F, Carnevale P: Olyset Net efficacy against pyrethroid-resistant *Anopheles gambiae* and *Culex quinquefasciatus* after 3 years' field use in Côte d'Ivoire. *Med Vet Entomol* 2001, **15**:97-104.

25. Djogbénou L, Dabire R, Diabate A, Kengne P, Akogbéto M, Hougard JM, Chandre F: Identification and geographic distribution of the ACE-1R mutation in the malaria vector *Anopheles gambiae* in south-western Burkina Faso, West Africa. *Am J Trop Med Hyg* 2008, **78**:298-302.

doi:10.1186/1756-3305-3-83

**Cite this article as:** Djènontin et al. *Culicidae* diversity, malaria transmission and insecticide resistance alleles in malaria vectors in Ouidah-Kpomasse-Tori district from Benin (West Africa): A pre-intervention study. *Parasites & Vectors* 2010 **3**:83.

Submit your next manuscript to BioMed Central and take full advantage of:

• Convenient online submission

• Thorough peer review

• No space constraints or color figure charges

• Immediate publication on acceptance

• Inclusion in PubMed, CAS, Scopus and Google Scholar

• Research which is freely available for redistribution

Submit your manuscript at
www.biomedcentral.com/submit

BioMed Central