Evidence of increasing L1014F kdr mutation frequency in Anopheles gambiae s.l. pyrethroid resistant following a nationwide distribution of LLINs by the Beninese National Malaria Control Programme

ABSTRACTObjectiveTo determine the susceptibility status to pyrethroid in Anopheles gambiae s.l. (An. gambiae), the distribution of kdr “Leu-Phe” mutation in malaria vectors in Benin and to compare the current frequency of kdr “Leu-Phe” mutation to the previous frequency after long-lasting insecticide treated nets implementation.MethodsLarvae and pupae of An. gambiae s.l. mosquitoes were collected from the breeding sites in Littoral, Zou, Borgou and Alibori provinces. CDC susceptibility tests were conducted on unfed females mosquitoes aged 2-5 d old. An. gambiae mosquitoes were identified to species using PCR techniques. Molecular assays were also carried out to identify kdr mutations in individual mosquitoes.ResultsThe results showed that An. gambiae Malanville and Suru-lere populations were resistant to deltamethrin. Regarding An. gambiae Parakou and Bohicon populations, they were resistant to permethrin. PCR revealed 100% of mosquitoes tested were An. gambiae s.s. The L1014F kdr mutation was found in An. gambiae s.s. Malanville and Parakou at various allelic frequencies. The increase of kdr allelic frequency was positively correlated with CDC bioassays data.ConclusionsPyrethroid resistance is widespread in malaria vector in Benin and kdr mutation is the main resistance mechanism involved. More attention may be paid for the future success of malaria control programmes based on LLINs with pyrethroids in the country

Bendiocarb, a potential alternative against pyrethroid resistant Anopheles gambiae in Benin, West Africa

<p>Abstract</p> <p>Background</p> <p><it>Anopheles gambiae</it>, the main malaria vector in Benin has developed high level of resistance to pyrethroid insecticides, which is a serious concern to the future use of long-lasting insecticidal nets (LLIN) and indoor residual spraying (IRS). In this context, one of the pathways available for malaria vector control would be to investigate alternative classes of insecticides with different mode of action than that of pyrethroids. The goal of this study was to evaluate under field conditions the efficacy of a carbamate (bendiocarb) and an organophosphate (fenitrothion) against pyrethroid-resistant <it>An. gambiae s.s</it>.</p> <p>Methods</p> <p>Wild populations and females from laboratory colonies of five days old <it>An. gambiae </it>were bio-assayed during this study. Two pyrethroids (deltamethrin and alphacypermethrin), an organophosphate (fenitrothion), a carbamate (bendiocarb) and a mixture of an organophosphate (chlorpyriphos + a pyrethroid deltamethrin) were compared in experimental huts as IRS treatments. Insecticides were applied in the huts using a hand-operated compression sprayer. The deterrency, exophily, blood feeding rate and mortality induced by these insecticides against <it>An. gambiae </it>were compared to the untreated control huts.</p> <p>Results</p> <p>Deltamethrin, alphacypermethrin and bendiocarb treatment significantly reduced mosquito entry into the huts (p < 0.05) compared to untreated huts. Blood feeding rates in huts treated with fenitrothion and the mixture chlorpyriphos/deltamethrin were reduced from 10.95% respectively to 3.7% and 4.47% three months after treatment and from 10.20% to 4.4% and 2.04% four months after treatment. Exophily rates in huts with deltamethrin, alphacypermethrin and the mixture chlorpyriphos/deltamethrin were significantly higher than in the huts with fenitrothion. Deltamethrin and alphacypermethrin had the lowest mortality rate while fenitrothion killed 100% of <it>An. gambiae </it>(in the first month) and 77.8% (in the fourth month). Bendiocarb and the mixture chlorpyriphos/deltamethrin mortality rates ranged from 97.9 to 100% the first month and 77.7-88% the third month respectively.</p> <p>Conclusion</p> <p>After four months, fenitrothion, bendiocarb and the mixture chlorpyriphos/deltamethrin performed effectively against pyrethroid-resistant <it>Anopheles</it>. These results showed that bendiocarb could be recommended as an effective insecticide for use in IRS operations in Benin, particularly as the mixture chlorpyriphos/deltamethrin does not have WHOPES authorization and complaints were mentioned by the sleepers about the safety and smell of fenitrothion.</p

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

Estimation of allele-specific Ace-1 duplication in insecticide-resistant Anopheles mosquitoes from West Africa

Background: Identification of variation in Ace-1 copy number and G119S mutation genotype from samples of Anopheles gambiae and Anopheles coluzzii across West Africa are important diagnostics of carbamate and organophosphate resistance at population and individual levels. The most widespread and economical method, PCR–RFLP, suffers from an inability to discriminate true heterozygotes from heterozygotes with duplication. Methods: In addition to PCR–RFLP, in this study three different molecular techniques were applied on the same mosquito specimens: TaqMan qPCR, qRTPCR and ddPCR. To group heterozygous individuals recorded from the PCR–RFLP analysis into different assumptive genotypes K-means clustering was applied on the Z-scores of data obtained from both the TaqMan and ddPCR methods. The qRTPCR analysis was used for absolute quantification of copy number variation. Results: The results indicate that most heterozygotes are duplicated and that G119S mutation must now be regarded as a complex genotype ranging from primarily single-copy susceptible Glycine homozygotes to balanced and imbalanced heterozygotes, and multiply-amplified resistant Serine allele homozygotes. Whilst qRTPCR-based gene copy analysis suffers from some imprecision, it clearly illustrates differences in copy number among genotype groups identified by TaqMan or ddPCR. Based on TaqMan method properties, and by coupling TaqMan and ddPCR methods simultaneously on the same type of mosquito specimens, it demonstrated that the TaqMan genotype assays associated with the K-means clustering algorithm could provide a useful semi-quantitative estimate method to investigate the level of allele-specific duplication in mosquito populations. Conclusions: Ace-1 gene duplication is evidently far more complex in An. gambiae and An. coluzzii than the better studied mosquito Culex quinquefasciatus, which consequently can no longer be considered an appropriate model for prediction of phenotypic consequences. These require urgent further evaluation in Anopheles. To maintain the sustained effectiveness carbamates and organophosphates as alternative products to pyrethroids for malaria vector control, monitoring of duplicated resistant alleles in natural populations is essential to guide the rational use of these insecticides

Etude De L’efficacité Du Cashew Nut Shell Liquid (CNSL) De Anacardium Occidentale L. Extrait À Froid Sur Le Contrôle De Anopheles Gambiae S.L Résistant Aux Pyréthrinoïdes

In order to combat the vectors of malaria, it is necessary to know their behavior with regard to the insecticide products envisaged. The present study is aimed at the CNSL efficiency test extracted from the seed hull of Anacardium occidentale L. for the control of pyrethroid-resistant Anopheles gambiae s.l. Sensitivity tests were carried out on the larvae of stages 2, 3 and 4 of An. gambiae of the wild and Kis-kdr strains carrying the Kdr gene. The "Kisumu" strain devoid of resistance gene served as a control. The mortalities were read after 24 hours of exposure. LD50 and DL90 were determined using the log-probit method of dose determination corresponding to a proportion. The high LD50 determined is 0.0042 mg / ml and corresponds to the LD50 of the larvae of stage 2 of the wild strain. The low LD50 is determined on stage 2 larvae "Kisumu". The high DL90 is determined on stage 4 larvae of the wild strain. The low LD90 determined corresponds to stage 2 larvae "Kisumu". The comparison of the LD between the different strains shows a low sensitivity to CNSL of the larvae of resistant strains compared to the control strain, indicating the influence of the resistance factor kdr on the CNSL efficiency. However, the low-dose CNSL larvicidal activity on the larvae of the kdr-carrying strains provides an alternative advantage in the management of vector resistance to pyrethroid

Seasonal change in species composition and target-site mutations in Anopheles gambiae s.l. in the severe drought area of Kandi, North-eastern Benin

The persistence of malaria transmission in areas with very arid environmental conditions remains enigmatic. The present study investigated seasonal variation of mosquito species composition and Kdr and Ace-1 mutations in Anopheles gambiae s.l. in the very arid district of Kandi in North-eastern Benin.Adult mosquitoes were sampled over 1 year using both human landing catches (HLC) and pyrethrum spray catches in 4 villages belonging to 2 areas of different levels of aridity. The collections were carried out on a bi-monthly basis in the wet season, and once every month in the dry season to better capture the entomological situation in drought period. Females An. gambiae sl specimens were kept aside and analysed by PCR for species identification. Presence of kdr and Ace-1 mutations was also assessed in the An. gambiae s.l. collection.A total of 2,211 host-seeking mosquitoes belonging to 15 species were collected in the study area. An. gambiae s.l. was the most abundant species and represented 67% of the collection. Other Anopheles species were found at very low frequency among which An. funestus, An. pharoensis, An. broheri and An. coustani. Molecular species identification showed in dry season a significantly higher frequency of An. coluzzii over An. gambiae s. s. in both less dry (70% vs 29% with p < 0.001) and driest (70% vs 30% and p = 0.034) areas of the district of Kandi. In the rainy season, there was similar frequency of An. coluzzii and An. gambiae s. s. in the less arid area (53% vs 45%; p = 0.153), while An. coluzzii remained significantly predominant (62% vs 38%; p = 0.012) in the driest zone. The frequency of kdr mutation was significantly higher in dry season than in rainy season (93% vs 84%; p<0.001), while no Ace-1 mutation was detected in the collection.In the current context of climate change marked by increasingly high temperatures and longer droughts, suitable vector control should be designed taking into account characteristics of the vector population maintaining malaria transmission in such arid environmental conditions

Dosage-dependent effects of permethrin-treated nets on the behaviour of Anopheles gambiae and the selection of pyrethroid resistance

BACKGROUND: The evolution and spread of pyrethroid resistance in Anopheles gambiae s.s, the major malaria vector in sub-Saharan Africa, is of great concern owing to the importance of pyrethroid-treated nets in the WHO global strategy for malaria control. The impact of kdr (the main pyrethroid-resistance mechanism) on the behaviour of An. gambiae is not well understood. The objective of this study was to determine whether high or low doses of permethrin differ in their resistance-selection effects. METHODS: The effect of permethrin treatment was assessed under laboratory conditions using the tunnel test technique against susceptible, heterozygous and homozygous genotypes. Experimental huts trials were then carried out in Benin to assess the level of personal protection conferred by nets treated with a variety of permethrin concentrations and their impact on the selection for kdr allele. RESULTS: Tunnel tests showed that nets treated with permethrin at 250 and 500 mg/m(2 )induced higher mortality and blood feeding reduction among susceptible and heterozygous (RS) females as compared to the lower concentration (100 mg/m(2)). The experimental hut trials showed that the best personal protection was achieved with the highest permethrin concentration (1,000 mg/m(2)). Mosquito genotyping revealed a non-linear relationship in the survival of kdr susceptible and resistant genotypes with permethrin dosage. Higher dosages (≥250 mg/m(2)) killed more efficiently the RS genotypes than did lower dosages (50 and 100 mg/m(2)). CONCLUSION: This study showed that nets treated with high permethrin concentrations provided better blood feeding prevention against pyrethroid-resistant An. gambiae than did lower concentrations. Permethrin-treated nets seem unlikely to select for pyrethroid resistance in areas where the kdr mutation is rare and present mainly in heterozygous form

Etude De L’efficacité De L’huile De Thevetia Neriifolia Pour Le Contrôle De Anopheles Gambiae S.L Résistant Aux Pyréthrinoïdes

Effective control of pyrethroid-resistant malaria vectors requires new alternative measures. The purpose of this study is to test the effectiveness of Thevetia oil extracted from seed kernels to control malaria transmission by An. gambiae s.l. Sensitivity tests were carried out on larvae of Stages 3 and 4 of An. gambiae s.s of wild-type and pyrethroid-resistant Kis-kdr. The susceptible reference strain "Kisumu" served as a control. Mortality was read 24 hours and 48 hours after exposure. LD50 and 90 for 24 hours and 48 hours were determined using the log-probit method of determining the dose corresponding to a proportion. The high LD50s determined in 24 hours and 48 hours correspond to doses that kill 50% of Stage 3 larvae in wild populations in 24 hours and 48 hours. Low LD50s refer to stage 4 "Kisumu" larvae. The strong DL90 in 24 hours and 48 hours correspond respectively to stage 3 larvae of the Kis-kdr and wild-type strains. The comparison of the LD between the different strains shows the influence of the resistance on the sensitivity of the larvae of the strains resistant to the oil tested. However, the larvicidal effect of Thevetia oil on the larvae of resistant strains may elicit formulations for alternative measures in vector resistance management to pyrethroids

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

Managing insecticide resistance in malaria vectors by combining carbamate-treated plastic wall sheeting and pyrethroid-treated bed nets

<p>Abstract</p> <p>Background</p> <p>Pyrethroid resistance is now widespread in <it>Anopheles gambiae</it>, the major vector for malaria in sub-Saharan Africa. This resistance may compromise malaria vector control strategies that are currently in use in endemic areas. In this context, a new tool for management of resistant mosquitoes based on the combination of a pyrethroid-treated bed net and carbamate-treated plastic sheeting was developed.</p> <p>Methods</p> <p>In the laboratory, the insecticidal activity and wash resistance of four carbamate-treated materials: a cotton/polyester blend, a polyvinyl chloride tarpaulin, a cotton/polyester blend covered on one side with polyurethane, and a mesh of polypropylene fibres was tested. These materials were treated with bendiocarb at 100 mg/m²and 200 mg/m²with and without a binding resin to find the best combination for field studies. Secondly, experimental hut trials were performed in southern Benin to test the efficacy of the combined use of a pyrethroid-treated bed net and the carbamate-treated material that was the most wash-resistant against wild populations of pyrethroid-resistant <it>An. gambiae </it>and <it>Culex quinquefasciatus</it>.</p> <p>Results</p> <p>Material made of polypropylene mesh (PPW) provided the best wash resistance (up to 10 washes), regardless of the insecticide dose, the type of washing, or the presence or absence of the binding resin. The experimental hut trial showed that the combination of carbamate-treated PPW and a pyrethroid-treated bed net was extremely effective in terms of mortality and inhibition of blood feeding of pyrethroid-resistant <it>An. gambiae</it>. This efficacy was found to be proportional to the total surface of the walls. This combination showed a moderate effect against wild populations of <it>Cx. quinquefasciatus</it>, which were strongly resistant to pyrethroid.</p> <p>Conclusion</p> <p>These preliminary results should be confirmed, including evaluation of entomological, parasitological, and clinical parameters. Selective pressure on resistance mechanisms within the vector population, effects on other pest insects, and the acceptability of this management strategy in the community also need to be evaluated.</p