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Abstract Background Pyrethroid insecticides, carbamate and organophosphate are the classes of insecticides commonly used in agriculture for crop protection in Benin. Pyrethroids remain the only class of insecticides recommended by the WHO for impregnation of bed nets. Unfortunately, the high level of pyrethroid resistance in Anopheles gambiae s.l., threatens to undermine the success of pyrethroid treated nets. This study focuses on the investigation of agricultural practices in cotton growing areas, and their direct impact on larval populations of An. gambiae in surrounding breeding sites. Methods The protocol was based on the collection of agro-sociological data where farmers were subjected to semi-structured questionnaires based on the strategies used for crop protection. This was complemented by bioassay tests to assess the susceptibility of malaria vectors to various insecticides. Molecular analysis was performed to characterize the resistance genes and the molecular forms of An. gambiae. Insecticide residues in soil samples from breeding sites were investigated to determine major factors that can inhibit the normal growth of mosquito larvae by exposing susceptible and resistant laboratory strains. Results There is a common use by local farmers of mineral fertilizer NPK at 200 kg/ha and urea at 50 kg/hectare following insecticide treatments in both the Calendar Control Program (CCP) and the Targeted Intermittent Control Program (TICP). By contrast, no chemicals are involved in Biological Program (BP) where farmers use organic and natural fertilizers which include animal excreta. Susceptibility test results confirmed a high resistance to DDT. Mean mortality of An. gambiae collected from the farms practicing CCP, TICP and BP methods were 33%, 42% and 65% respectively. An. gambiae populations from areas using the CCP and TICP programs showed resistance to permethrin with mortality of 50% and 58% respectively. By contrast, bioassay test results of An. gambiae from BP areas gave a high level of susceptibility to permethrin with an average mortality of 94%. Molecular analysis identified An. gambiae s.s, and An. arabiensis with a high predominance of An. gambiae s.s (90%). The two molecular forms, M and S, were also determined with a high frequency of the S form (96%). The Kdr gene seemed the main target- site resistance mechanism detected in CCP, TICP, and BP areas at the rates ranging from 32 to 78%. The frequency of ace-1R gene was very low (< 0.1). The presence of inhibiting factors in soil samples under insecticide treatments were found and affected negatively in delaying the development of An. gambiae larval populations. Conclusions This research shows that Kdr has spread widely in An. gambiae, mainly in CCP and TICP areas where pyrethroids are extensively used. To reduce the negative impact of pesticides use in cotton crop protection, the application of BP-like programs, which do not appear to select for vector resistance would be useful. These results could serve as scientific evidence of the spread of resistance due to a massive agricultural use of insecticides and contribute to the management of pesticides usage on cotton crops hence reducing the selection pressure of insecticides on An. gambiae populations.

Abstract Background Insecticide treated plastic sheeting (ITPS), sometimes known as durable lining, has potential as a long-lasting insecticidal surface for malaria vector control when used as lining for interior walls and ceilings inside the home. Against a backdrop of increasing long lasting net (LN) coverage, we examined the effect of combining permethrin-treated plastic sheeting (ITPS) with LNs in Burkina Faso. Methods A verandah trap experimental hut trial of ITPS with or without Olyset LN was conducted in the Vallée du Kou near Bobo-Dioulasso, where the two molecular forms of Anopheles gambiae s.s., S (frequency 65%) and M (frequency 35%), occur. The S form is mostly pyrethroid resistant (Fkdr = 92%) owing to the kdr mechanism, and the M form is mostly kdr susceptible (Fkdr = 7%). The treatment arms included ITPS, Olyset, ITPS plus Olyset, ITPS plus untreated net (with or without holes), and untreated control. Results ITPS was significantly inferior to Olyset LN in terms of mortality (37% vs 63%), blood feeding inhibition (20% vs 81%) and deterrence (0 vs 42%) effects, and hence altogether inferior as a means of personal protection (16% vs 89%). The addition of ITPS to Olyset did not improve mortality (62%), blood feeding inhibition (75%), deterrence (50%) or personal protection (88%) over that of Olyset used alone. Use of untreated nets - both holed and intact - with ITPS provided greater protection from blood-feeding. The intact net/ITPS combination killed more mosquitoes than ITPS on its own. Conclusions Although ITPS has a potential role for community control of malaria, at low coverage it is unlikely to be as good as Olyset LNs for household protection. The combination of pyrethroid IRS and pyrethroid LN - as practiced in some countries - is unlikely to be additive except, perhaps, at high levels of IRS coverage. A combination of LN and ITPS treated with an alternative insecticide is likely to be more effective, particularly in areas of pyrethroid resistance.

Abstract Background The G119S mutation responsible for insensitive acetylcholinesterase resistance to organophosphate and carbamate insecticides has recently been reported from natural populations of Anopheles gambiae in West Africa. These reports suggest there are costs of resistance associated with this mutation for An. gambiae, especially for homozygous individuals, and these costs could be influential in determining the frequency of carbamate resistance in these populations. Methods Life-history traits of the AcerKis and Kisumu strains of An. gambiae were compared following the manipulation of larval food availability in three separate experiments conducted in an insecticide-free laboratory environment. These two strains share the same genetic background, but differ in being homozygous for the presence or absence of the G119S mutation at the ace-1 locus, respectively. Results Pupae of the resistant strain were significantly more likely to die during pupation than those of the susceptible strain. Ages at pupation were significantly earlier for the resistant strain and their dry starved weights were significantly lighter; this difference in weight remained when the two strains were matched for ages at pupation. Conclusions The main cost of resistance found for An. gambiae mosquitoes homozygous for the G119S mutation was that they were significantly more likely to die during pupation than their susceptible counterparts, and they did so across a range of larval food conditions. Comparing the frequency of G119S in fourth instar larvae and adults emerging from the same populations would provide a way to test whether this cost of resistance is being expressed in natural populations of An. gambiae and influencing the dynamics of this resistance mutation.

Abstract Background Insecticide resistance is a rapid and recent evolutionary phenomenon with serious economic and public health implications. In the mosquito Anopheles gambiae s.s., main vector of malaria, resistance to organophosphates and carbamates is mainly due to a single amino-acid substitution in acetylcholinesterase 1 (AChE1). This mutation entails a large fitness cost. However, a resistant duplicated allele of the gene encoding AChE1 (ace-1), potentially associated to a lower fitness cost, recently appeared in An. gambiae. Methods Using molecular phenotype data collected from natural populations from West Africa, the frequency of this duplicated allele was investigated by statistical inference. This method is based on the departure from Hardy-Weinberg phenotypic frequency equilibrium caused by the presence of this new allele. Results The duplicated allele, Ag-ace-1D, reaches a frequency up to 0.65 in Ivory Coast and Burkina Faso, and is potentially present in Benin. A previous study showed that Ag-ace-1D, present in both M and S molecular forms in different West Africa countries, was generated by a single genetic event. This single origin and its present distribution suggest that this new allele is currently spreading. Conclusion The spread of this less costly resistance allele could represent a major threat to public health, as it may impede An. gambiae control strategies, and thus increases the risk of malaria outbreaks.

Abstract Background Insecticide resistance of the main malaria vector, Anopheles gambiae, has been reported in south-western Burkina Faso, West Africa. Cross-resistance to DDT and pyrethroids was conferred by alterations at site of action in the sodium channel, the Leu-Phe kdr mutation; resistance to organophosphates and carbamates resulted from a single point mutation in the oxyanion hole of the acetylcholinesterase enzyme designed as ace-1R. Methods An entomological survey was carried out during the rainy season of 2005 at Vallée du Kou, a rice growing area in south-western Burkina Faso. At the Vallée du Kou, both insecticide resistance mechanisms have been previously described in the M and S molecular forms of An. gambiae. This survey aimed i) to update the temporal dynamics and the circumsporozoite infection rate of the two molecular forms M and S of An. gambiae ii) to update the frequency of the Leu-Phe kdr mutation within these forms and finally iii) to investigate the occurrence of the ace-1R mutation. Mosquitoes collected by indoor residual collection and by human landing catches were counted and morphologically identified. Species and molecular forms of An. gambiae, ace-1R and Leu-Phe kdr mutations were determined using PCR techniques. The presence of the circumsporozoite protein of Plasmodium falciparum was determined using ELISA. Results Anopheles gambiae populations were dominated by the M form. However the S form occurred in relative important proportion towards the end of the rainy season with a maximum peak in October at 51%. Sporozoite rates were similar in both forms. The frequency of the Leu-Phe kdr mutation in the S form reached a fixation level while it is still spreading in the M form. Furthermore, the ace-1R mutation prevailed predominately in the S form and has just started spreading in the M form. The two mutations occurred concomitantly both in M and S populations. Conclusion These results showed that the Vallée du Kou, a rice growing area formerly occupied mainly by M susceptible populations, is progressively colonized by S resistant populations living in sympatry with the former. As a result, the distribution pattern of insecticide resistance mutations shows the occurrence of both resistance mechanisms concomitantly in the same populations. The impact of multiple resistance mechanisms in M and S populations of An. gambiae on vector control measures against malaria transmission, such as insecticide-treated nets (ITNs) and indoor residual spraying (IRS), in this area is discussed.