Background
Malaria is one the deadliest vector-borne disease in the world with 1.5 to 3 million deaths a year [
1]. More than 90% of the deaths recorded occur in Africa affecting mainly low immune response individuals, such as children under five years of age and pregnant women [
2,
3]. In 1992, the WHO set up sustainable strategies against malaria, focused on the proper treatment of malaria cases and the use of preventive measures against malaria vectors. Indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) remain the two preventive measures presently used against malaria vectors. Both methods have been very effective in controlling
Anopheles mosquitoes [
4‐
10]. However the emergence of
Anopheles gambiae populations carrying the
kdr gene has become a serious threat to the future effectiveness of these control measures [
3]. N'Guessan
et al [
11] recently established a clear relationship between pyrethroid resistance caused by
kdr and the failure of LLINs and IRS in experimental huts in south Benin.
In the last decade, the emergence of resistance in populations of
Anopheles to common class of insecticides used in public health was reported from many African countries [
7,
8]. Resistance affects the major vectors of malaria,
An. gambiae s.l [
4] and
Anopheles funestus s.l [
5]. The resistance of pyrethroid insecticides in
An. gambiae has been documented in several parts of Africa [
8,
9,
12,
13] and has prompted considerable research activity to investigate mechanisms of resistance and factors contributing to the emergence of insecticide resistance [
11,
14]. There is currently a growing agricultural activity within and around African cities. In Benin, the urban farming has spread in almost all the major cities all over the country. Akogbeto
et al [
14,
15] reported that mosquito species,
An. gambiae in particular, lay their eggs in breeding sites located around agricultural settings. These eggs undergo a selection pressure from agricultural pesticides, which leads to the emergence of resistant strains. There is clear evidence on the implication of agricultural breeding sites in the selection of resistance in the major malaria vectors.
Indeed, a fast development of urban agriculture has recently been recorded in most settings in the Republic of Benin. At all levels of the society, people are devoted themselves to it. The reason which underpins the phenomenon is the impoverishment of the soil far from the town due to its overuse, rural exodus, unemployment, improvement of living standards, and the dietary requirement of urban population to be met.
Being ideal environments for larval growth, it has been reported that vegetable farming uses a large variety of synthetic pesticides for pest control [
16]. Some of these insecticides are registered for pest treatments in vegetable farms, whereas many are not [
14‐
16]. During treatment, insecticide residues are washed away into the mosquito breeding sites thus exerting a selection pressure on larvae population [
14,
15]. This selection leads to the emergence of insecticide resistance in the population of
An. gambiae breeding in these sites. The massive utilization of agricultural pesticides constitutes, therefore, a public health issue in tropical Africa [
14,
15].
This study was designed to assess the impact of the fast growing activities of vegetable farming on the resistance status of malaria vectors in three localities (Cotonou, Porto-Novo and Parakou) in Benin. The study focused on the investigation of agricultural practices in vegetable farming in urban areas, and their impact on the emergence of insecticide resistance in populations of An. gambiae.
Methods
Study sites
The study was conducted in the Republic of Benin, from July 2005 to February 2007 in three vegetable farms: Houeyiho in Cotonou, the economical capital of Benin, Acron in Porto-Novo, the political capital of Benin, and Azèrèkè near Parakou, in the northern part of the country.
The vegetable farm of Houeyiho, Cotonou
This farm is located at 6° 45'N and 2°31'E in the downtown of Cotonou in a highly populated quarter. It is a 14-hectare farm shared between five cooperatives, each led by a chosen cooperative president. Each cooperative approximately consists of 300 individuals making an estimated farmer population of not less than 2,000 persons.
The vegetable farm of Acron, Porto-Novo
Located in south-eastern Benin at 6° 30'N and 2°47'E at the outskirt of Porto-Novo, the vegetable farm of Acron is the oldest one in Benin. This site was established by missionaries in 1945. The farm consists of three hectares, nitially cultivated by 10 farmers. The activities of the farm have now grown and the size has widened from three to 20 hectares. The number of farmers has also increased to about 150 individuals.
The vegetable farm of Azèrèkè, Parakou
This farm is located at 9° 22'N and 2°40'E at the entrance of Parakou town, known as Azèrèkè site. The size of this vegetable plantation is 10 hectares. The farm is crossed by a canalization of rainfall from the main town. The vegetable farm of Azèrèkè is mostly cultivated by men of 35 to 50 years old and their children.
Collection of data on the rapid spread of vegetable farms in Benin
To generate adequate information on the fast spread of vegetable farms in Benin, knowledge-attitude-practice (KAP) studies were organized in the study sites of Houeyiho, Acron and Azèrèkè. A total number of 150 farmers were interviewed at Houeyiho, 80 at Acron and 60 at Azèrèkè. Farmers were subjected to semi-structured questionnaires focused on the history of vegetable farms, the size of farms, the number of workers, their educational levels, the type of vegetable grown, the farming techniques, the pesticides utilization in the farm. Qualitative data were recorded from direct observations, in-depth interview and focus group discussion.
Insecticide susceptibility test
To assess the impact of agricultural pesticides on the selection of resistance in malaria vectors,
Anopheles larvae were collected from vegetable farms and reared to adults in the insectaria. Females mosquitoes aged 2–5 days old were subjected to susceptibility tests using insecticide-impregnated papers, as described by the WHO testing protocol [
17]. Deltamethrin papers, impregnated at the diagnostic concentration of 0.05%, were used in this susceptibility assay. Results with this insecticide were compared with permethrin-impregnated paper (0.75%) and DDT-treated paper (4%). DDT and permethrin were both tested to detect the presence of cross-resistance between pyrethroids and organo-chlorine in
Anopheles populations. Female
Anopheles used in this bio-assay were exposed for one hour to insecticide-treated papers and were monitored at different time intervals (10', 15', 20', 30', 45', 60') to record "knock-down" times.
After 24-hour holding, delayed mortality was recorded. Following the WHO protocol, populations of Anopheles giving mortality rates below 95% after exposure to insecticide-impregnated papers were considered resistant. In this study, these criteria were slightly modified as follow:
Mortality rates between 100-95%: the population was considered fully susceptible
Mortality rates between 94-90%: the population was considered less susceptible
Mortality rates below 90%: the population was considered resistant to the tested insecticides.
Based on the criteria mentioned above, data from insecticide susceptibility tests were used to characterize the susceptibility levels of An. gambiae populations in the three study sites. Dead and survived mosquitoes from this bioassay were separately kept in Carnoy solution at -20°C for further molecular characterization.
Data analysis
Sociological information from focus group discussions, in-depth interviews and questionnaires conducted in studied communities were compiled and tabulated using Excel software and qualitative data were analysed by Text Base-Beta Software. Insecticide susceptibility test on the resistant strains from Houeyiho, Acron and Azèrèkè were compared and analysed using Stat-calc-Epi-info Software to get the status of resistance in the different sites investigated. A Fisher's exact test was performed to determine the differences between the three sites.
Discussion and conclusion
Information collected during the interviews with farmers and the direct observations made confirmed a fast growing activity of vegetable farming and their economical impact in Benin. Urban agriculture contributes to food security and balanced diets. It provides additional incomes to populations throughout the year.
A field study conducted in Benin revealed that vegetable farming yields about 300 million FCFA annually to farmers in Benin with 30 to 40% used for direct consumption in farmers daily diet [
3,
15,
20]. There is a clear evidence that vegetable farming activities do absorb unemployment and eventually reduce hunger [
16,
19‐
21].
Based on its numerous advantages, vegetable farming is becoming a new activity involving the various social groups: men, women, and children, educated, non-educated and even civil servants. More than 3,000 individuals are employed by urban farming and this sector is mainly man-powered by young people between 20 and 40 years of age. The massive use of pesticides in vegetable farms was confirmed during interviews with farmers. Some of the pesticides recorded in vegetable farms were not registered, probably because of the liberalization of the pesticide sector and the elevated cost of registered pesticides (Table
1). Uncontrolled use of pesticides in Benin has resulted in the emergence of insecticide resistance in
An. gambiae larvae breeding in vegetable farms. The emergence of pyrethroid resistance in
An. gambiae has become a serious concern to the success of malaria control in the last decade [
22,
23]. This study showed that pyrethroid insecticides used in vegetable farms are similar to those used in public health against malaria vectors. Pyrethroids remain the only family of insecticides currently registered for the impregnation of bed-nets, the major control strategy against malaria vectors [
6,
24]. Among the pyrethroids, deltamethrin is definitely the most used in both public health and agriculture. Cyfluthrin is one of the pyrethroids used in combination with organophosphates in agriculture [
14,
15]. The
kinikini (in local language), which is a combination of cyfluthrin and malathion, is widely used in vegetable farming and in public health as Solfac [
14,
15]. Studies on insecticide resistance have been currently on most malaria agenda in Africa because of its impact on impregnated bed-nets the major tool against malaria vectors [
6,
24]. The first case of pyrethroids resistance in
An. gambiae has been reported in Africa since the 1993 [
23,
24]. Dieldrin and DDT resistance were reported in Burkina-Faso with populations of
An. gambiae [
23,
24]. Pyrethroids resistance was reported in
An. gambiae in Côte d'Ivoire [
22,
23] and later on many others cases of pyrethroid resistance in Anopheles vectors were detected in West [
25], Central [
24], Eastern [
26] and Southern Africa [
27].
In a recent study, a relatively high frequency of
kdr mutations (
Leu-Phe) was recorded in
An. gambiae collected from cotton farms under massive insecticide treatments compared to farms with no pesticide utilization [
22,
23]. The
kdr mutation is probably responsible for the emergence of resistance of
An. gambiae to DDT and pyrethroids in West Africa. The hypothesis of the implication of the
kdr mutation in the emergence of resistance has been confirmed in this research. In most localities where resistance was detected, the PCR analysis of samples revealed a high frequency of
Kdr genes in localities of Cotonou and Porto-Novo. This study provides further evidence on the contribution of the overuse of insecticide in agriculture to the widespread emergence of insecticide resistance in
Anopheles species. In addition to the
kdr mutation, there are other existing factors, which seem to confer cross-resistance to pyrethroids and DDT, as described by Diabaté
et al in the West African region [
23].
The implication of metabolic mechanisms of resistance was not neglected in this study. Several studies are being currently conducted at CREC to determine the levels of acetylcholinesterase in An. gambiae after exposure to propoxur. In addition, elevated monooxygenase, esterases and glutathion-s-transferases have also been investigated. The increase number of vegetable farming in urban areas of Benin has been confirmed as a result of investigations made during this study from July 2005 to February 2007. This has led to the use of insecticide in improper manner to control vegetable pests, thus exerting a huge selection pressure on mosquito larval population leading to an emergence of mosquito resistance to insecticides.
More investigations need to be carried out in the future in order to better control the use of pesticides in vegetable farming within urban areas, especially in Benin where pyrethroid resistance has been widely reported in An. gambiae. These findings showed an increase emergence of resistance in An. gambiae populations in the vegetable farm breeding sites located not far from human dwellings. It is, therefore, important to set up insecticide management structures to prevent failure from malaria vector control measures using especially those pyrethroid insecticides.
Acknowledgements
We are grateful to the International Development Research Centre (IRDC) who through its AGROPOLIS grant programme. The author would like to thank the Malaria Research Training Center team, and IRD-Benin team, especially Dr Fabrice Chandre, for their help.
Many thanks to Vincent Ishola, Razack Ossé, Mouinath Souradjou and Gil Padonou for their technical assistance. The author is thankful to Jean-Claude Dumais, Paul Viveiros, Mark Redwood and Alison Clegg for the administrative support.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
AWY contributed to design of the study and conceived the protocol, the data analysis and interpretation. MCA contributed in the study design, fully involved financially and in the implementation of this research, guided the study from conception to the manuscript finalization and the write up of the manuscript. JB contributed in the study design and in the implementation of this research. AA, RFD and CDA contributed to the design of the study and substantially helped in drafting the manuscript.