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The aim of this study was to evaluate the susceptibility of Anopheles gambiae mosquitoes to bendiocarb, before (2010) and after (2012) the implementation of indoor residual spraying (IRS) interventions and to report the evolution of Ace-1R mutation frequency in Atacora region. Indoor collection was carried out through Morning 7 to 9 a. m in five districts (Kouande, Natitingou, Materi, Tanguieta and Copargo) of the Atacora-Donga region before and after IRS. Anopheles larvae were also reared in each district before and after IRS and emerging adults were exposed to bendiocarb (0.1%) in susceptibility tests. Polymerase chain reaction (PCR) assays were run to determine the members of the An. gambiae complex, as well for insensitive acetylcholinesterase (AChE1) due to Ace-1R mutation. This study showed that the mean Ace-1 mutation frequency had increased significantly from 2010 to 2012 after two years of an IRS. Mortality data indicated that mosquitoes were susceptible in 2010 to bendiocarb 0.1%. By 2012, there was a drastic decline in the An. gambiae susceptibility to bendiocarb in treated districts. An. gambiae s.s. and Anopheles coluzzi were the two members of An. gambiae complex that were found in sympatry in the study area. An. gambiae was predominant in tested samples (92.50%). The Ace-1R mutation was found in both An. gambiae s.s. and An. coluzzi with frequency of 7.33 and 7.35%, respectively. The high proportion of homozygous susceptible specimens that survived from the WHO bioassays suggests the implication of biochemical resistance mechanisms. These results are of prime importance in the effort to document multiple impacts of operational control programmes on mosquito vectors, and to conceive sustainable control strategies for future malaria control programmes. Key words: Anopheles gambiae Ace-1R, increasing, indoor residual spraying, Benin.

The spread of Ace-1R mutation that confers carbamate and organophosphate resistance in Anopheles gambiae s.l. is a critical issue for malaria vector control. The aim of this study was to investigate the seasonal variation of the Ace-1R mutation in A. gambiae s. l populations. The mosquitoes analyzed were collected by human landing catches (HLC) in five districts in the department of Atacora from May 2011 to June 2013. The collection was carried out twice a month by adult volunteers. They caught any mosquitoes that landed on their legs with an aspirator from 7.00 p.m to 6.00 a.m. Anopheles mosquitoes were morphologically identified and polymerase chain reaction (PCR) assays were run to determine the members of the A. gambiae complex, as well as phenotypes for insensitive acetylcholinesterase (AChE1) due to the Ace-1R mutation. This study shows that A. gambiae s. s. and Anopheles coluzzii occurred in sympatry in the Atacora region. However, A. gambiae s. s. was predominant during the rainy season, while A. coluzzii was predominant during the dry season. Moreover, the Ace-1R mutation was detected either in A. gambiae s. s. or in A. coluzzii. Results showed a dramatical increase in Ace-1R allelic frequency during the dry season. The present study provides useful information on the seasonal variation of Ace-1R allelic frequency in A. gambiae populations from Atacora. It showed that Ace-1R allelic frequency has increased in rainy season which can be explained by the various selective insecticide pressures on A. gambiae during this season that consist of agricultural practices and indoor residual spraying using bendiocarb. Key words: Anopheles gambiae Ace-1R, resistance, seasonal, Atacora, Benin.

BACKGROUND Using the same insecticide for multiple successive indoor residual spraying (IRS) cycles is not recommended; instead, the National Malaria Control Program (NMCP) has decided to select another insecticide (insecticide B) in addition to bendiocarb for indoor residual spraying. METHODS An experimental hut trial comparing the effectiveness of three classes of insecticides (one carbamate [bendiocarb], two organophosphates [fenitrothion and pirimiphos methyl] and one pyrethroid [lambdacyalothrin]) was conducted in Malanville, northern Benin, against wild free entered resistant Anopheles gambiae s.l. population to pyrethroids. RESULTS Fenitrothion and pirimiphos methyl yielded the highest rate of deterrence. Their mean rates were respectively 46.6% and 35.4%. Regarding blood feeding inhibition, only fenitrothion has induced a significant inhibition rate (25.4% as mean rate). As regards the exophily rates, only lambdacyhalothrin has induced the highest rate (39.7%). Pirimiphos methyl showed the highest mortality rate and also induced a mortality rate of at least 80% in blood fed An. gambiae population after 24 h observation time. Furthermore, the huts treated with pirimiphos methyl showed the highest residual effect, followed by lambdacyhalothrin. CONCLUSION Benin NMCP has selected pirimiphos methyl as insecticide B to alternate or combine to bendiocarb (carbamate) because of the adverse effects of fenitrothion on the sleepers and its short residual effect on walls.

Background With the rapid spread of pyrethroid resistance in the main malaria vectors from Benin and the various resistance mechanisms involved (metabolic resistance and knock-down resistance (kdr), it is important to foresee effective resistance management strategies. Thus, the knowledge of the insensitive acetylcholinesterase (ace-1R) effects on phenotypes of An. gambiae will help us to strengthen basic and operational research on thedevelopment of strategies that will use organophosphates or carbamates as alternatives against pyrethroids-resistant malaria vectors in the field. Methods Larvae and pupae of Anopheles gambiae s.l. mosquitoes were collected from the breeding sites in Ouemé, Atacora, and Alibori departments. CDC susceptibility tests were conducted on unfed female mosquitoes aged 2–5 days old. CDC bioassays were performed with stock solutions of fenitrothion (50 μg per bottle) and bendiocarb (12.5 μg per bottle). PCR techniques were used to detect species and Ace-1 mutations. Results Anopheles gambiae Seme and Kandi populations were susceptible to fenitrothion whereas Anopheles gambiae Tanguieta and Malanville populations were resistant. An. gambiae populations from Seme, Kandi and Malanville were fully susceptible to bendiocarb whereas those from Tanguieta have developed a strong resistance to the same insecticide. A slight decrease in mortality rate was observed with 97.91% in populations of mosquitoes from Malanville. PCR revealed that all specimens tested were Anopheles gambiae s.s.. The presence of Ace-1R at very low frequency (0.01) was observed in Anopheles gambiae Malanville populations. Conclusion This study demonstrated the need to monitor organophosphate (OPs) and Carbamates resistance among populations of the An. gambiae s.l. in Benin, to determine its spread and anticipate vector control failure where these insecticides are used. However, further studies are needed to understand the current distribution of the Ace-1R mutation in other localities in the south-north transect Benin.

Background The detection of insecticide resistance in natural populations of Anopheles vectors is absolutely necessary for malaria control. In the African region, the WHO insecticide susceptibility test is the most common method for assessing resistance status. In order to search for a simple, rapid and more reliable technique in the assessment of insecticide resistance in malaria vectors, we compared the WHO tests with the CDC bottle bioassay in the Ouemé province of southern Benin where insecticide resistance has been widely reported. Methods Larvae and pupae of Anopheles gambiae s.l. mosquitoes were collected from the breeding sites in Ouemé. WHO and CDC susceptibility tests were conducted simultaneously on unfed female mosquitoes aged 2–5 days old. WHO bioassays were performed with impregnated papers of deltamethrin (0.05%) and bendiocarb (0.1%), whereas CDC bioassays were performed with stock solutions of deltamethrin (12.5 μg per bottle) and bendiocarb (12.5 μg per bottle). PCR techniques were used to detect species, Kdr and Ace-1 mutations. CDC biochemical assays using synergists were also conducted to assess the metabolic resistance. Results A slight decrease in mortality rates was observed with 97.95% and 98.33% obtained from CDC and WHO bioassays respectively in populations of mosquitoes from Adjara and Dangbo. PCR revealed that all specimens tested were Anopheles gambiae s.s. The Kdr mutation was found at high frequency in all populations and both the Kdr mutation and mono-oxygenase enzymes were implicated as mechanisms of pyrethroid resistance in An. gambiae from Misserete. Conclusion This study emphasizes that both WHO and CDC bioassays give similar results with regards to the susceptibility of mosquitoes to insecticides in southern Benin. There were complementarities between both methods, however, some specificity was noted for each of the two methods used. Both Kdr and metabolic mechanisms were implicated in the resistance.

Background Indoor residual spraying (IRS) was implemented in the department of Ouémé-Plateau, southern Benin, in 2008 and withdrawn in 2011, when long lasting insecticidal nets (LLINs) were distributed to the communities that were previously targeted by IRS. Did the LLIN strategy provide a better level of protection against malaria transmission than IRS? Methods Entomological surveillance was carried out to assess indicators of transmission risk during the last year of IRS and the first year after the LLIN intervention was put in place (2010–2011). Mosquito biting density was sampled by human landing collection (HLC). Females of Anopheles gambiae s.l. were dissected to estimate the parity rates and the blood meal index. A subsample of the An. gambiae s.l. collection was tested for presence of Plasmodium falciparum sporozoites. In addition, window exit traps and pyrethrum spray catches were performed to assess exophagic behavior of Anopheles vectors. Results There were significant increases in all the indicators following withdrawal of IRS. Vector biting density (p

Background Since 2008, the National Malaria Control Program (NMCP) has been engaged in the implementation of indoor residual spraying (IRS) in Benin. The first and second round was a success with a drastic decrease of malaria transmission in areas under IRS. We present here the results of the third round. The purpose of this study was to compare the results of the third round of IRS to those achieved during the first two rounds. A second success of IRS will enable the Government of Benin to extend the strategy to other areas. Methods Mosquito collections were carried out in the department of Ouémé where the homes of four districts were treated with bendiocarb. In these districts, more than 350 000 inhabitants were protected by IRS. A fifth untreated district served as control. In the five districts, mosquito collections were organized to follow the dynamics of malaria transmission and possible changes in the behavior of mosquitoes. Results A significant reduction in human biting rate was recorded after the third round of IRS, specifically in Adjohoun (89.78%), Dangbo (56.8%) and Missérété (93.22%) where an inhabitant received less than 2 bites of An. gambiae per night. During this same time, the entomological inoculation rate (EIR) declined dramatically in all areas under intervention (74.26% reduction). We also noted a significant reduction in longevity, the blood feeding rate of the vectors and an increase in exophily induced by bendiocarb on An. gambiae and Mansonia spp. Conclusion The present study showed, once again, the effectiveness of bendiocarb on anopheles populations resistant to pyrethroids. This product can be recommended in combination with other insecticides for the management of vector resistance to insecticides.

Background In many parts of Africa as in Benin, the main strategies of vector control are based on the scaling-up of Long Lasting Insecticide Treated Nets (LLITNs) and indoor residual spraying (IRS). The need to understand the biological implications of IRS in large scale and full coverage of LLITNs is paramount. It is in this context that the present study was conducted. It aims to evaluate the effect of a large scale IRS using a non-pyrethroid insecticide and full coverage of deltamethrin treated nets on the behavior of An. gambiae s.l. in the intervention areas compared to untreated areas used as controls. Methods Mosquitoes were collected using human landing catches, pyrethrum spray catches and window exit traps to assess reduction of entry rate, endophily rate, endophagy rate and overall mortality rate in natural populations of An. gambiae s.l. before IRS and LLITNs intervention (2007) and after in 2008 and 2010. Results In the IRS arm, endophily rate was 67.13% before intervention and 4.5% after intervention, whereas in the control arm it was stable at 51.67% (P > 0 .05). In the LLITN arm endophily rates also decreased after intervention. After the IRS, no gravid mosquitoes were collected from all treated localities, but LLITN performance was not that spectacular. The proportion of mosquitoes biting indoors in the IRS arm decreased from 67.09% before intervention to 42.85% after intervention, compared to a low but significant decrease (71.31% to 57. 46%) in the LLITN arm. The use of vector control tools and behavior of the host would be the main factors that modify the behavior of taking a human blood meal observed on An. gambiae s.l. inside human dwellings. Conclusion The impact on the behavior of An. gambiae s.l. observed with the bendiocarb used in IRS was highly effective compared with the free distribution of LLITNs in terms of mortality and the decrease of proportions of indoor feeding. Despite this efficacy, there is a need for complementary tools and research of alternative strategy oriented on effective health education, and the use of powerful tools such as IRS, LLITNs, larviciding and repellents.

Background In Benin, Indoor Residual Spraying (IRS) and long-lasting insecticidal nets (LLINs) are the cornerstones of malaria prevention. In the context of high resistance of Anopheles gambiae to pyrethroids, The National Malaria Control Program (NMCP) has undertaken a full coverage of IRS in a no-flood zone in the Oueme region, coupled with the distribution of LLINs in a flood zone. We assessed the impact of this campaign on phenotypic resistance, kdr (knock-down resistance) and ace-1R (insensitive acetylcholinesterase) mutations. Methods Insecticides used for malaria vector control interventions were bendiocarb WP (0.4 g/m2) and deltamethrin (55 mg/m2), respectively for IRS and LLINs. Susceptibility status of An. gambiae was assessed using World Health Organization bioassay tests to DDT, permethrin, deltamethrin and bendiocarb in the Oueme region before intervention (2007) and after interventions in 2008 and 2010. An. gambiae specimens were screened for identification of species, molecular M and S forms and for the detection of the West African kdr (L1014F) as well as ace-1R mutations using PCR techniques. Results The univariate logistic regression performed showed that kdr frequency has increased significantly during the three years in the intervention area and in the control area. Several factors (LLINs, IRS, mosquito coils, aerosols, use of pesticides for crop protection) could explain the selection of individual resistant An. gambiae. The Kdr resistance gene could not be the only mechanism of resistance observed in the Oueme region. The high susceptibility to bendiocarb is in agreement with a previous study conducted in Benin. However, the occurrence of ace-1R heterozygous individuals even on sites far from IRS areas, suggests other factors may contribute to the selection of resistance other than those exerted by the vector control program. Conclusion The results of this study have confirmed that An.gambiae have maintained and developed the resistance to pyrethroids, but are still susceptible to bendiocarb. Our data clearly shows that selection of resistant individuals was caused by other insecticides than those used by the IRS and LLINs.

Background Insecticide resistance monitoring is essential to help national programmers to implement more effective and sustainable malaria control strategies in endemic countries. The current study aimed at an exploring the involvement of detoxifying enzymes in the insecticide phenotype resistance in Anopheles gambiae s.l.from Benin, in order to guide future malaria vector control interventions. Methods Larvae and pupae of Anopheles gambiae s.l. mosquitoes were collected from the breeding sites in Oueme, Atacora and Alibori provinces. CDC susceptibility tests were conducted on unfed female mosquitoes aged 2–5 days old. CDC bioassays were performed with stock solutions of permethrin (21.5 μg per bottle), deltamethrin (12.5 μg per bottle) and bendiocarb (12.5 μg per bottle). CDC biochemical assays using synergists were also conducted to assess the metabolic resistance. Results The susceptibility of Anopheles gambiae Agbalilame and Kandi populations to permethrin and deltamethrin respectively, increased significantly when synergized by PBO, suggesting an implication of mono-oxygenases in resistance of Anopheles gambiae s.l. to pyrethroid. Esterases may play a role in bendiocarb resistance in Anopheles gambiae Tanguieta. Conclusion Synergists partially restored susceptibility to pyrethroid and carbamate insecticides and might help mitigate the impact of vector resistance in Anopheles gambiae Agbalilame, Kandi and Tanguieta populations. However, additional vector control tools are needed to further impact on malaria transmission in such settings.This will improve the implementation and management of future control programs against this important malaria vector in Benin and in Africa in general.

Background: Owing to pyrethroid resistance in An. gambiae, the carbamate and organophosphate insecticides are currently regarded as alternatives or supplements to pyrethroids for use on mosquito net treatments. Resistance monitoring is therefore essential to investigate the susceptibility of An. gambiae s.l to these alternative products. Methods: Two to three day old adult female Anopheles mosquitoes were reared from larvae collected in the five districts (Kouande, Natitingou, Materi, Pehunco, Tanguieta) of the Atacora department. Mosquitoes were then exposed to WHO impregnated papers. The four treatments consisted of: carbamates (0.1% bendiocarb, 0.1% propoxur) and organophosphates (0.25% pirimiphosmethyl, 1% fenitrothion). PCR assays were run to determine the members of the An. gambiae complex, the molecular forms (M) and (S), as well as phenotypes for insensitive acetylcholinesterase (AChE1) due to ace-1 R mutation. Results: Bioassays showed bendiocarb resistance in all populations of An. gambiae s.s. tested. Propoxur resistance was observed in Materi, Pehunco and Tanguieta, while it was suspected in Kouande and Natitingou. As for the organophosphates, susceptibility to pirimiphos-methyl was assessed in all populations. Fenitrothion resistance was detected in Kouande, Pehunco and Tanguieta, while it was suspected in Materi and Natitingou. The S-form was predominant in tested samples (94.44%). M and S molecular forms were sympatric but no M/S hybrids were detected. The ace-1 R mutation was found in both S and M molecular forms with frequency from 3.6 to 12%. Although the homozygous resistant genotype was the most prevalent genotype among survivors, the genotypes could not entirely explain the bioassay results. Conclusion: Evidence of bendiocarb resistance in An. gambiae populations is a clear indication that calls for the implementation of insecticide resistance management strategies. The ace-1 R mutation could not entirely explain the resistance to bendiocarb observed and is highly suggestive of involvement of other resistance mechanisms such as metabolic detoxification.

Background: The National Malaria Control Program (NMCP) has been using pirimiphos methyl for the first time for indoor residual spraying (IRS) in Benin. The first round was a success with a significant decrease of entomological indicators of malaria transmission in the treated districts. We present the results of the entomological impact on malaria transmission. Entomologic parameters in the control area were compared with those in intervention sites. Methods: Mosquito collections were carried out in three districts in the Atacora-Dongo region of which two were treated with pirimiphos methyl (Actellic 50EC) (Tanguieta and Kouande) and the untreated (Copargo) served as control. Anopheles gambiae s.l. populations were sampled monthly by human landing catch. In addition, window exit traps and pyrethrum spray catches were performed to assess exophagic behavior of Anopheles vectors. In the three districts, mosquito collections were organized to follow the impact of pirimiphos methyl IRS on malaria transmission and possible changes in the behavior of mosquitoes. The residual activity of pirimiphos methyl in the treated walls was also assessed using WHO bioassay test. Results: A significant reduction (94.25%) in human biting rate was recorded in treated districts where an inhabitant received less than 1 bite of An. gambiae per night. During this same time, the entomological inoculation rate (EIR) dramatically declined in the treated area (99.24% reduction). We also noted a significant reduction in longevity of the vectors and an increase in exophily induced by pirimiphos methyl on An. gambiae. However, no significant impact was found on the blood feeding rate. Otherwise, the low residual activity of Actellic 50 EC, which is three months, is a disadvantage. Conclusion: Pirimiphos methyl was found to be effective for IRS in Benin. However, because of the low persistence of Actellic 50EC used in this study on the treated walls, the recourse to another more residual formulation of pirimiphos methyl is required.