Comparative susceptibility to permethrin of two Anopheles gambiae s.l. populations from Southern Benin, regarding mosquito sex, physiological status, and mosquito age.

OBJECTIVE: To investigate what kind of mosquito sample is necessary for the determination of insecticide susceptibility in malaria vectors. METHODS: Larvae and pupae of Anopheles gambiae s.l. (An. gambiae) mosquitoes were collected from the breeding sites in Littoral and Oueme departments. The Centers for Disease Control and Prevention (CDC) susceptibility tests were conducted on unfed male and female mosquitoes aged 2-5 days old. CDC susceptibility tests were also conducted on unfed, blood fed and gravid female mosquitoes aged 2-5 days old. These susceptibility tests were also conducted on unfed and blood fed female mosquitoes aged 2-5 days old and 20 days old. CDC biochemical assay using synergist was also carried out to detect any increase in the activity of enzyme typically involved in insecticide metabolism. RESULTS: Female An. gambiae Ladji and Sekandji populations were more susceptible than the males when they were unfed and aged 2-5 days old. The mortality rates of blood fed female An. gambiae Ladji and Sekandji populations aged 2-5 days old were lower than those obtained when females were unfed. In addition, the mortality rates of gravid female An. gambiae Ladji and Sekandji populations aged 2-5 days old were lower than those obtained when they were unfed. The mortality rate obtained when female An. gambiae Sekandji populations were unfed and aged 20 days old was higher than the one obtained when these populations were unfed and aged 2-5 days old. The results obtained after effects of synergist penicillin in beeswax on F1 progeny of An. gambiae Ladji populations resistant to permethrin showed that mono-oxygenases were involved in permethrin resistant F1 progeny from Ladji. CONCLUSIONS: The resistance is a hereditary and dynamic phenomenon which can be due to metabolic mechanisms like overproduction of detoxifying enzymes activity. Many factors influence vector susceptibility to insecticide. Among these factors, there are mosquito sex, mosquito age, its physiological status. Therefore, it is useful to respect the World Health Organization criteria in the assessment of insecticide susceptibility tests in malaria vectors. Otherwise, susceptibility testing is conducted using unfed female mosquitoes aged 3-5 days old. Tests should also be carried out at (25±2) °C and (80±10)% relative humidity.

Study of the efficacy of a Wheaton coated bottle with permethrin and deltamethrin in laboratory conditions and a WHO impregnated paper with bendiocarb in field conditions.

OBJECTIVE: To determine the efficacy of WHO impregnated paper and CDC coated bottle based on number of storage days and number of times of consecutive use, in the assessment of insecticide vector susceptibility tests in laboratory and field conditions. METHODS: Larvae and pupae of Anopheles gambiae s.l. mosquitoes were collected from the breeding sites in Seme-Kpodji and Cotonou districts in Southern Benin in April 2013 during the first rainy season. Anopheles gambiae s.l. mosquitoes were also collected from the breeding sites in Parakou district in Northern Benin in May 2013 at the beginning of the rainy season. Susceptibility tests were done using impregnated paper with bendiocarb (0.1%) following WHO protocol and stock solutions of permethrin (21.5 µg per bottle) and deltamethrin (12.5 µg per bottle) following CDC protocol on unfed female mosquitoes aged 2-5 days old. These bioassays were repeated a certain number of times. The temperature and relative humidity were monitored and recorded during the susceptibility tests. RESULTS: This study showed that a WHO impregnated paper with bendiocarb could be used four times during four consecutive days in field conditions. Regarding a Wheaton coated bottle with permethrin or deltamethrin, they could be used at least three times during four consecutive days in laboratory conditions. CONCLUSIONS: The day storage and the number of times that a WHO impregnated paper and a CDC coated bottle maintained their efficacy are useful in the assessment of insecticide vectors susceptibility tests.

A new long-lasting indoor residual formulation of the organophosphate insecticide pirimiphos methyl for prolonged control of pyrethroid-resistant mosquitoes: an experimental hut trial in Benin.

BACKGROUND: Indoor residual spraying (IRS) is widely used for malaria transmission control in sub-Saharan Africa. Resistance to pyrethroids in the mosquito Anopheles gambiae is a growing problem. There is an urgent need to develop long-lasting alternative insecticides to reduce selection pressure for pyrethroid resistance and to provide control with a single IRS application in countries with long transmission seasons. METHODS: Two capsule suspension formulations (CS) of the organophosphate pirimiphos methyl were evaluated as IRS treatments in experimental huts in an area of Benin where the mosquitoes Anopheles gambiae and Culex quinquefasciatus are resistant to pyrethroids but susceptible to organophosphates. The CS formulations were tested alongside an emulsifiable concentrate (EC) formulation of pirimiphos methyl and a CS formulation of the pyrethroid lambdacyhalothrin. RESULTS: The two CS formulations of pirimiphos methyl gave prolonged control of An. gambiae and Cx. quinquefasciatus. In cement huts application rates of 0.5 g/m(2) induced high mortality of An. gambiae for almost a year: overall mortality rates 87% (95% CI 82-91%) and 92% (95% CI 88-94%). In mud huts application rates of 1 g/m(2) induced high mortality of An. gambiae for 10 months: overall mortality rates 75% (95% CI 69-81%) and 76% (95% CI 68-83%). The EC formulation of pirimiphos methyl failed to control An. gambiae two months after spraying. The pyrethroid lambdacyhalothrin demonstrated prolonged residual activity in bioassay tests but failed to control pyrethroid resistant An. gambiae that entered the huts. Pirimiphos methyl CS was highly active against Culex quinquefasciatus and gave control for 10 months in cement huts and 6 months in mud huts. CONCLUSION: Pirimiphos methyl CS (Actellic 300 CS) applied at 1 g/m(2) shows great promise for providing prolonged control of pyrethroid-resistant An gambiae and for delaying pyrethroid resistance. An alternative to DDT, giving year-round transmission control in sub-Saharan Africa is now a realistic prospect.

Loss of household protection from use of insecticide-treated nets against pyrethroid-resistant mosquitoes, benin.

Pyrethroid resistance is becoming widespread in Anopheles gambiae mosquitoes, coinciding with expanded use of insecticide-treated nets (ITNs) throughout Africa. To investigate whether nets in use are still protective, we conducted household trials in northern and southern Benin, where An. gambiae mosquitoes are susceptible and resistant, respectively, to pyrethroids. Rooms were fitted with window traps and monitored for mosquito biting and survival rates before and after the nets were treated with pyrethroid. Sleeping under an ITN in the location with resistant mosquitoes was no more protective than sleeping under an untreated net, regardless of its physical condition. By contrast, sleeping under an ITN in the location with susceptible mosquitoes decreased the odds of biting by 66%. ITNs provide little or no protection once the mosquitoes become resistant and the netting acquires holes. Resistance seriously threatens malaria control strategies based on ITN.

Combining indoor residual spraying with chlorfenapyr and long-lasting insecticidal bed nets for improved control of pyrethroid-resistant Anopheles gambiae: an experimental hut trial in Benin.

BACKGROUND: Neither indoor residual spraying (IRS) nor long-lasting insecticidal nets (LLINs) are able to fully interrupt transmission in holoendemic Africa as single interventions. The combining of IRS and LLINs presents an opportunity for improved control and management of pyrethroid resistance through the simultaneous presentation of unrelated insecticides. METHOD: Chlorfenapyr IRS and a pyrethroid-impregnated polyester LLIN (WHO approved) were tested separately and together in experimental huts in southern Benin against pyrethroid resistant Anopheles gambiae and Culex quinquefasciatus. The bed nets were deliberately holed with either six or 80 holes to examine the effect of increasing wear and tear on protectiveness. Anopheles gambiae were genotyped for the kdr gene to assess the combination's potential to prevent the selection of pyrethroid resistance. RESULTS: The frequency of kdr was 84%. The overall mortality rates of An. gambiae were 37% and 49% with the six-hole and 80-hole LLINs, respectively, and reached 57% with chlorfenapyr IRS. Overall mortality rates were significantly higher with the combination treatments (82-83%) than with the LLIN or IRS individual treatments. Blood feeding (mosquito biting) rates were lowest with the 6-hole LLIN (12%), intermediate with the 80-hole LLIN (32%) and highest with untreated nets (56% with the 6-hole and 54% with the 80-hole nets). Blood feeding (biting) rates and repellency of mosquitoes with the combination of LLIN and chlorfenapyr IRS showed significant improvement compared to the IRS treatment but did not differ from the LLIN treatments indicating that the LLINs were the primary agents of personal protection. The combination killed significantly higher proportions of Cx. quinquefasciatus (51%, 41%) than the LLIN (15%, 13%) or IRS (32%) treatments. CONCLUSION: The chlorfenapyr IRS component was largely responsible for controlling pyrethroid-resistant mosquitoes and the LLIN component was largely responsible for blood feeding inhibition and personal protection. Together, the combination shows potential to provide additional levels of transmission control and personal protection against pyrethroid-resistant mosquitoes, thereby justifying the additional resources required. Chlorfenapyr has potential to manage pyrethroid resistance in the context of an expanding LLIN/IRS strategy.

Cotton pest management practices and the selection of pyrethroid resistance in Anopheles gambiae population in northern Benin.

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.

First report of the infection of insecticide-resistant malaria vector mosquitoes with an entomopathogenic fungus under field conditions.

BACKGROUND: Insecticide-resistant mosquitoes are compromising the ability of current mosquito control tools to control malaria vectors. A proposed new approach for mosquito control is to use entomopathogenic fungi. These fungi have been shown to be lethal to both insecticide-susceptible and insecticide-resistant mosquitoes under laboratory conditions. The goal of this study was to see whether entomopathogenic fungi could be used to infect insecticide-resistant malaria vectors under field conditions, and to see whether the virulence and viability of the fungal conidia decreased after exposure to ambient African field conditions. METHODS: This study used the fungus Beauveria bassiana to infect the insecticide-resistant malaria vector Anopheles gambiae s.s (Diptera: Culicidae) VKPER laboratory colony strain. Fungal conidia were applied to polyester netting and kept under West African field conditions for varying periods of time. The virulence of the fungal-treated netting was tested 1, 3 and 5 days after net application by exposing An. gambiae s.s. VKPER mosquitoes in WHO cone bioassays carried out under field conditions. In addition, the viability of B. bassiana conidia was measured after up to 20 days exposure to field conditions. RESULTS: The results show that B. bassiana infection caused significantly increased mortality with the daily risk of dying being increased by 2.5 × for the fungus-exposed mosquitoes compared to the control mosquitoes. However, the virulence of the B. bassiana conidia decreased with increasing time spent exposed to the field conditions, the older the treatment on the net, the lower the fungus-induced mortality rate. This is likely to be due to the climate because laboratory trials found no such decline within the same trial time period. Conidial viability also decreased with increasing exposure to the net and natural abiotic environmental conditions. After 20 days field exposure the conidial viability was 30%, but the viability of control conidia not exposed to the net or field conditions was 79%. CONCLUSIONS: This work shows promise for the use of B. bassiana fungal conidia against insecticide-resistant mosquitoes in the field, but further work is required to examine the role of environmental conditions on fungal virulence and viability with a view to eventually making the fungal conidia delivery system more able to withstand the ambient African climate.

The impact of the expansion of urban vegetable farming on malaria transmission in major cities of Benin.

BACKGROUND: Urban agricultural practices are expanding in several cities of the Republic of Benin. This study aims to assess the impact of such practices on transmission of the malaria parasite in major cities of Benin. METHOD: A cross sectional entomological study was carried out from January to December 2009 in two vegetable farming sites in southern Benin (Houeyiho and Acron) and one in the northern area (Azèrèkè). The study was based on sampling of mosquitoes by Human Landing Catches (HLC) in households close to the vegetable farms and in others located far from the farms. RESULTS: During the year of study, 71,678 female mosquitoes were caught by HLC of which 25% (17,920/71,678) were Anopheles species. In the areas surveyed, the main malaria parasite, Plasmodium falciparum was transmitted in the south by Anopheles gambiae s.s. Transmission was high during the two rainy seasons (April to July and October to November) but declined in the two dry seasons (December to March and August to September). In the north, transmission occurred from June to October during the rainy season and was vehicled by two members of the An. gambiae complex: Anopheles gambiae s.s. (98%) and Anopheles arabiensis (2%).At Houeyiho, Acron and Azèrèkè, the Entomological Inoculation Rates (EIRs) and the Human Biting Rates (HBRs) were significantly higher during the dry season in Households Close to Vegetable Farms (HCVF) than in those located far from the vegetable areas (HFVF) (p < 0.05.). However, there were no significant differences in HBRs or EIRs between HCFV and HFVF during the rainy seasons at these sites (p > 0.05).The knock-down resistance (kdr) mutation was the main resistance mechanism detected at high frequency (0.86 to 0.91) in An. gambiae s.l. at all sites. The ace-1R mutation was also found but at a very low frequency (< 0.1). CONCLUSION: These findings showed that communities living close to vegetable farms are permanently exposed to malaria throughout the year, whereas the risk in those living far from such agricultural practices is limited and only critical during the rainy seasons. Measures must be taken by African governments to create awareness among farmers and ultimately decentralize farming activities from urban to rural areas where human-vector contact is limited.

An experimental hut evaluation of PermaNet(®) 3.0, a deltamethrin-piperonyl butoxide combination net, against pyrethroid-resistant Anopheles gambiae and Culex quinquefasciatus mosquitoes in southern Benin.

PermaNet 3.0 is a long-lasting combination net with deltamethrin present on the sides and a mixture of deltamethrin and piperonyl butoxide (PBO), an oxidase synergist, on the top panel. An experimental hut trial comparing unwashed and 20 times washed PermaNet 3.0 and PermaNet 2.0, Olyset Net and a conventional deltamethrin-treated net washed three times was conducted in southern Benin. Anopheles gambiae and Culex quinquefasciatus from this area are highly resistant to pyrethroids through kdr and cytochrome P450 mechanisms. The unwashed PermaNet 3.0 killed slightly more A. gambiae (52%) than the unwashed PermaNet 2.0 (44%) (P=0.036), indicating only partial synergism of resistance. After washing there was significant loss of activity to a similar level, with PermaNet 3.0 killing 31%, PermaNet 2.0 killing 29% and the conventional net killing 26%. Blood-feeding rates were partially inhibited for unwashed PermaNet 3.0 and Olyset Net (27% inhibition). Personal protection against A. gambiae derived from PermaNet 3.0 was similar to that from PermaNet 2.0 before washing (50% vs. 47%), and after 20 washes it decreased to 30%. Against C. quinquefasciatus, no treatment killed >24% entering the huts. The synergism from unwashed PermaNet 3.0 was lower than expected, probably due to an unidentified resistance mechanism unaffected by PBO.

The entomopathogenic fungus Beauveria bassiana reduces instantaneous blood feeding in wild multi-insecticide-resistant Culex quinquefasciatus mosquitoes in Benin, West Africa.

BACKGROUND: Mosquito-borne diseases are still a major health risk in many developing countries, and the emergence of multi-insecticide-resistant mosquitoes is threatening the future of vector control. Therefore, new tools that can manage resistant mosquitoes are required. Laboratory studies show that entomopathogenic fungi can kill insecticide-resistant malaria vectors but this needs to be verified in the field. METHODS: The present study investigated whether these fungi will be effective at infecting, killing and/or modifying the behaviour of wild multi-insecticide-resistant West African mosquitoes. The entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana were separately applied to white polyester window netting and used in combination with either a permethrin-treated or untreated bednet in an experimental hut trial. Untreated nets were used because we wanted to test the effect of fungus alone and in combination with an insecticide to examine any potential additive or synergistic effects. RESULTS: In total, 1125 female mosquitoes were collected during the hut trial, mainly Culex quinquefasciatus Say. Unfortunately, not enough wild Anopheles gambiae Giles were collected to allow the effect the fungi may have on this malaria vector to be analysed. None of the treatment combinations caused significantly increased mortality of Cx. quinquefasciatus when compared to the control hut. The only significant behaviour modification found was a reduction in blood feeding by Cx. quinquefasciatus, caused by the permethrin and B. bassiana treatments, although no additive effect was seen in the B. bassiana and permethrin combination treatment. Beauveria bassiana did not repel blood foraging mosquitoes either in the laboratory or field. CONCLUSIONS: This is the first time that an entomopathogenic fungus has been shown to reduce blood feeding of wild mosquitoes. This behaviour modification indicates that B. bassiana could potentially be a new mosquito control tool effective at reducing disease transmission, although further field work in areas with filariasis transmission should be carried out to verify this. In addition, work targeting malaria vector mosquitoes should be carried out to see if these mosquitoes manifest the same behaviour modification after infection with B. bassiana conidia.

Insecticide resistance status in Anopheles gambiae in southern Benin.

BACKGROUND: The emergence of pyrethroid resistance in Anopheles gambiae has become a serious concern to the future success of malaria control. In Benin, the National Malaria Control Programme has recently planned to scaling up long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) for malaria prevention. It is, therefore, crucial to monitor the level and type of insecticide resistance in An. gambiae, particularly in southern Benin where reduced efficacy of insecticide-treated nets (ITNs) and IRS has previously been reported. METHODS: The protocol was based on mosquito collection during both dry and rainy seasons across forty districts selected in southern Benin. Bioassay were performed on adults collected from the field to assess the susceptibility of malaria vectors to insecticide-impregnated papers (permethrin 0.75%, delthamethrin 0.05%, DDT 4%, and bendiocarb 0.1%) following WHOPES guidelines. The species within An. gambiae complex, molecular form and presence of kdr and ace-1 mutations were determined by PCR. RESULTS: Strong resistance to permethrin and DDT was found in An. gambiae populations from southern Benin, except in Aglangandan where mosquitoes were fully susceptible (mortality 100%) to all insecticides tested. PCR showed the presence of two sub-species of An. gambiae, namely An. gambiae s.s, and Anopheles melas, with a predominance for An. gambiae s.s (98%). The molecular M form of An. gambiae was predominant in southern Benin (97%). The kdr mutation was detected in all districts at various frequency (1% to 95%) whereas the Ace-1 mutation was found at a very low frequency (

Development of vegetable farming: a cause of the emergence of insecticide resistance in populations of Anopheles gambiae in urban areas of Benin.

BACKGROUND: A fast development of urban agriculture has recently taken place in many areas in the Republic of Benin. This study aims to assess the rapid expansion of urban agriculture especially, its contribution to the emergence of insecticide resistance in populations of Anopheles gambiae. METHODS: The protocol was based on the collection of sociological data by interviewing vegetable farmers regarding various agricultural practices and the types of pesticides used. Bioassay tests were performed to assess the susceptibility of malaria vectors to various agricultural insecticides and biochemical analysis were done to characterize molecular status of population of An. gambiae. RESULTS: This research showed that:(1) The rapid development of urban agriculture is related to unemployment observed in cities, rural exodus and the search for a balanced diet by urban populations;(2) Urban agriculture increases the farmers' household income and their living standard;(3) At a molecular level, PCR revealed the presence of three sub-species of An. gambiae (An. gambiae s.s., Anopheles melas and Anopheles arabiensis) and two molecular forms (M and S). The kdr west mutation recorded in samples from the three sites and more specifically on the M forms seems to be one of the major resistance mechanisms found in An. gambiae from agricultural areas. Insecticide susceptibility tests conducted during this research revealed a clear pattern of resistance to permethrin (76% mortality rate at Parakou; 23.5% at Porto-Novo and 17% at Cotonou). CONCLUSION: This study confirmed an increase activity of the vegetable farming in urban areas of Benin. This has led to the use of insecticide in an improper manner to control vegetable pests, thus exerting a huge selection pressure on mosquito larval population, which resulted to the emergence of insecticide resistance in malaria vectors.

Evaluation of indoxacarb, an oxadiazine insecticide for the control of pyrethroid-resistant Anopheles gambiae (Diptera: Culicidae).

Owing to the spread of pyrethroid resistance in Anopheles gambiae s.s. (Diptera: Culicidae) and other vector mosquitoes, there is an urgent need to develop alternative insecticides to supplement the pyrethroids for malaria control. Indoxacarb is an oxadiazine insecticide initially commercialized by DuPont for control of agricultural pests. Performance against An. gambiae bearing kdr (pyrethroid and DDT resistance) or Ace-1R insensitive acetylcholinesterase (organophosphate and carbamate resistance) mechanisms was studied using larval and adult bioassays and a simulated experimental hut system (tunnel tests) that allows fuller expression of the behavioral responses to insecticide. Larval and adult bioassays (topical application and cone tests on treated netting) showed a standard probit dosage-mortality response and no evidence of cross-resistance to the kdr and Ace-1R resistance mechanisms. Toxic activity was slow compared with standard insecticides and additional mortality was observed. Indoxacarb induced no excitorepellency in adults. In tunnel tests, indoxacarb induced no inhibition of mosquito penetration or blood feeding through the holed netting, but it induced delayed mortality over 24-96 h. There was > 90% mortality of the kdr strain on netting treated with the 500 mg/m2 dosage, whereas permethin at 500 mg/nm2 only killed 30% of this strain. A mixture of indoxacarb and pyrethroid showed neither synergism nor antagonism. The absence of cross-resistance to current insecticides indicates that indoxacarb has malaria vector control potential as larvicide or adulticide where mosquitoes are pyrethroid resistant.

Experimental hut evaluation of bednets treated with an organophosphate (chlorpyrifos-methyl) or a pyrethroid (lambdacyhalothrin) alone and in combination against insecticide-resistant Anopheles gambiae and Culex quinquefasciatus mosquitoes.

BACKGROUND: Pyrethroid resistant mosquitoes are becoming increasingly common in parts of Africa. It is important to identify alternative insecticides which, if necessary, could be used to replace or supplement the pyrethroids for use on treated nets. Certain compounds of an earlier generation of insecticides, the organophosphates may have potential as net treatments. METHODS: Comparative studies of chlorpyrifos-methyl (CM), an organophosphate with low mammalian toxicity, and lambdacyhalothrin (L), a pyrethroid, were conducted in experimental huts in Côte d'Ivoire, West Africa. Anopheles gambiae and Culex quinquefasciatus mosquitoes from the area are resistant to pyrethroids and organophosphates (kdr and insensitive acetylcholinesterase Ace.1R). Several treatments and application rates on intact or holed nets were evaluated, including single treatments, mixtures, and differential wall/ceiling treatments. RESULTS AND CONCLUSION: All of the treatments were effective in reducing blood feeding from sleepers under the nets and in killing both species of mosquito, despite the presence of the kdr and Ace.1R genes at high frequency. In most cases, the effects of the various treatments did not differ significantly. Five washes of the nets in soap solution did not reduce the impact of the insecticides on A. gambiae mortality, but did lead to an increase in blood feeding. The three combinations performed no differently from the single insecticide treatments, but the low dose mixture performed encouragingly well indicating that such combinations might be used for controlling insecticide resistant mosquitoes. Mortality of mosquitoes that carried both Ace.1R and Ace.1S genes did not differ significantly from mosquitoes that carried only Ace.1S genes on any of the treated nets, indicating that the Ace.1R allele does not confer effective resistance to chlorpyrifos-methyl under the realistic conditions of an experimental hut.