Small-scale field evaluation of the entomological efficacy and the residual activity of Fludora® Fusion WP-SB indoor residual spraying against Anopheles culicifacies s.l. in Gujarat, India.

OBJECTIVES: To evaluate the entomological efficacy and the residual activity of indoor residual spraying with Fludora® Fusion 562.5 WP-SB, a combination formulation containing clothianidin, a neonicotinoid and deltamethrin, a pyrethroid, against the main rural malaria vector, Anopheles culicifacies s.l., in India in a small-scale trial. METHODS: In three study villages, suitable households were randomly allocated to five treatments: Fludora® Fusion 562.5 WP-SB (target dose 225 mg active ingredient AI/m2 ); clothianidin 70 WG (target dose 200 mg AI/m2 ); K-Othrine 250 WG (deltamethrin, target dose 25 mg AI/m2 ); Ficam VC 80 WP-SB (bendiocarb, target dose 400 mg AI/m2 ) and unsprayed control. Insecticides were sprayed by hand compression sprayers with control flow valves and 8002E nozzles. Post-spray cone bioassays were done on insecticide-treated walls using a colonised, deltamethrin-resistant strain of An. culicifacies. Mosquitoes were collected from treated rooms by different methods. The insecticide content on filter papers collected from the sprayed walls was determined by chemical assay to assess the spray quality. RESULTS: The ratios of applied to target doses of insecticides were within 0.84 to 1.4, showing a good spray quality. The cone bioassays revealed residual action lasting 7 months for all insecticides without significant differences in mortality between different surfaces treated nor between the four treatment arms (P > 0.05). Considering all entomological parameters such as indoor resting density, excito-repellency, blood-feeding inhibition and delayed mortality, the overall efficacy of Fludora® Fusion WG-SB was equal or better compared with other insecticides. CONCLUSIONS: Fludora® Fusion showed overall equal or better efficacy than deltamethrin and bendiocarb alone against a pyrethroid-resistant malaria vector population and can be considered as an alternative product for management of pyrethroid resistance in malaria vectors.

Characterization of resistance profile (intensity and mechanisms) of Anopheles gambiae in three communes of northern Benin, West Africa.

BACKGROUND: The selection and the spread of insecticide resistance in malaria vectors to the main classes of insecticides used in vector control tools are a major and ongoing challenge to malaria vector control programmes. This study aimed to determine the intensity of vector resistance to insecticides in three regions of Benin with different agro-ecological characteristics. METHODS: Larvae of Anopheles gambiae sensu lato (s.l.) were collected from September to November 2017 in different larval sites in three northern Benin communes: Parakou, Kandi and Malanville. Two to five-day-old, non-blood-fed, female mosquitoes were exposed to papers impregnated with deltamethrin, permethrin and bendiocarb at dosages of 1 × the diagnostic dose, 5 × and 10 × to determine the intensity of resistance in these vectors. Molecular frequencies of the kdr L1014F and ace-1R G119S insecticide resistance mutations and levels of detoxification enzymes were determined for mosquitoes sampled at each study site. RESULTS: Resistance to pyrethroids (permethrin and deltamethrin) was recorded in all three communes with mortality rates below 60% using the diagnostic dose (1x). The results obtained after exposure of An. gambiae to permethrin 10 × were 99% in Kandi, 98% in Malanville and 99% in Parakou. With deltamethrin 10x, mortality rates were 100% in Kandi, 96% in Malanville and 73% in Parakou. For the diagnostic dose of bendiocarb, suspected resistance was recorded in the communes of Malanville (97%) and Kandi (94%) while sensitivity was observed in Parakou (98%).Using the 10 × dose, mortality was 98% in Kandi, 100% in Malanville and 99% in Parakou. The frequencies of the kdr L1014F allele varied between 59 and 83% depending on the sites and species of the An. gambiae complex, while the frequency of the ace-1R G119S gene varied between 0 and 5%. Biochemical tests showed high levels of oxidase and esterase activity compared to the susceptible colony strain of An. gambiae sensu stricto (Kisumu strain). CONCLUSION: Anopheles gambiae showed a generalized loss of susceptibility to permethrin and deltamethrin but also showed moderate to high intensity of resistance in different regions of Benin. This high intensity of resistance is a potential threat to the effectiveness of vector control.

Application of ZnS/S/S-RGO three-component nanocomposites in dispersive solid-phase microextraction coupled with ion mobility spectrometry for ultra-trace determination of multiclass pesticides.

A fast, effective, and sensitive dispersive solid-phase microextraction method coupled with ion mobility spectrometry for the simultaneous determination of bendiocarb, butachlor, and diazinon was developed using zinc sulfide/sulfur/sulfur-doped reduced graphene oxide (ZnS/S/S-RGO) nanocomposites. ZnS/S/S-RGO three-component nanocomposites were synthesized through a single-step solvothermal procedure, and their properties were characterized by FT-IR, XRD, X-ray photoelectron spectroscopy (XPS), field emission scanning electron microscopy (FESEM), and energy-dispersive x-ray spectroscopy (EDX). The influence of different parameters was optimized on the efficiency of the extraction including the type and the volume of desorption solvent, pH, type and the volume of buffer, the amount of absorbent, sorption and  desorption times. Under the optimal conditions, linear ranges were achieved 0.8-110, 1.0-110, and 0.5-100 ng mL-1 with detection limits of 0.32±0.01, 0.40±0.02, and 0.27±0.02 ng mL-1 for bendiocarb, butachlor, and diazinon, respectively. The method was employed for the ultra-trace determination of the three pesticides in water, rice, and soil samples with acceptable recovery values within the range 96.6±4.8-104.4±6.4%.

Indoor spraying with chlorfenapyr (a pyrrole insecticide) provides residual control of pyrethroid-resistant malaria vectors in southern Benin.

BACKGROUND: New classes of insecticides with novel modes of action, which can provide effective and prolonged control of insecticide-resistant malaria vector populations, are urgently needed for indoor residual spraying. Such insecticides can be included in a rotation plan to manage and prevent further development of resistance in mosquito vectors of malaria. Chlorfenapyr, a novel pyrrole insecticide with a unique mode of action, is being developed as a long-lasting IRS formulation. METHODS: The efficacy of several formulations of chlorfenapyr alone and as mixtures with alpha-cypermethrin were evaluated in an experimental hut trial against wild pyrethroid-resistant Anopheles gambiae sensu lato in Cové, Benin, in an attempt to identify the most effective and long-lasting formulations for IRS. The trial lasted 12 months. A comparison was made with alpha-cypermethrin and bendiocarb formulations. CDC bottle bioassays were performed to investigate cross-resistance to chlorfenapyr in the local vector population. RESULTS: Mortality rates in World Health Organization (WHO) cylinder bioassays were < 5% with pyrethroids due to high levels of pyrethroid resistance, but > 95% with bendiocarb thus confirming susceptibility to carbamates in the vector population. CDC bottle bioassays showed no cross-resistance between pyrethroids and chlorfenapyr. Overall mortality of free-flying mosquitoes entering the experimental huts over the 12-month trial was 4% with alpha-cypermethrin and 12% with bendiocarb. The chlorfenapyr solo-formulations induced significantly higher levels of mortality (38-46%) compared to the bendiocarb (12% P < 0.001) and to the mixture formulations (18-22%, P < 0.05). The original Sylando 240SC formulation of chlorfenapyr was more efficacious than all other novel chlorfenapyr formulations tested. Bendiocarb induced > 80% mortality in the first month, but this declined sharply to < 20% by the third month while the mortality rates achieved with the chlorfenapyr formulations (38-46%) were persistent lasting 7-10 months. The mixtures induced significantly lower percentage mortality than chlorfenapyr-solo formulations. Wall cone bioassays only showed mortality rates that were consistent with chlorfenapyr IRS treated huts when the exposure time was increased to 2 h. CONCLUSION: Indoor residual spraying with chlorfenapyr (Sylando® 240SC) provides moderate but prolonged control of pyrethroid-resistant malaria vectors compared to pyrethroid and bendiocarb IRS. Wall cone bioassays on chlorfenapyr-treated walls required longer exposure times of 2 h than the customary 30 min indicating that WHO guidelines on residual cone bioassays need to be more insecticide-specific.

Evaluation of the residual efficacy of indoor residual spraying with bendiocarb (FICAM WP 80) in six health districts in Senegal.

BACKGROUND: From 2011 to 2014, an indoor residual spray (IRS) programme for malaria vectors control was implemented in six health districts in Senegal. The main objective of the present study was to evaluate the efficacy of bendiocarb (FICAM WP 80) sprayed on different wall surfaces and its impact on malaria vectors. The entomological monitoring activities were carried out monthly in five treated sentinel villages and one control untreated village in each district. METHODS: The residual efficacy of bendiocarb applied at a dosage of 0.4 g/sq m was monitored for a period up to 9 months post-IRS using WHO cone bioassay method. This assay consisted to expose 2-5 days old unfed susceptible Anopheles coluzzii females to sprayed walls for a period of 30 min. The mortality rates after 24 h post-exposure were estimated and compared between the different types of walls sprayed in each sentinel village. RESULTS: The results showed that the residual efficacy varied between the different sprayed walls, from one sentinel village to another and between the different campaigns. The FICAM had a residual efficacy of 3-6 months post-IRS on mud and cement wall surfaces. In some cases, the observed mortality rates were much higher than those reported elsewhere particularly during the first campaign in all the six districts. CONCLUSIONS: The FICAM was found to be effective with a residual efficacy varying from 3 to 6 months. If the quality of the IRS application is excluded as a possible explanation of the short efficacy duration, the results suggest at least two rounds of treatments in order to cover the rainy season that lasts 5 to 6 months in the area. Such treatments could be carried out before the intensification of the rains in July and August in order to better cover the transmission period that occurs between late August and October in the area.

Menthol Increases Bendiocarb Efficacy Through Activation of Octopamine Receptors and Protein Kinase A.

Great effort is put into seeking a new and effective strategies to control insect pests. One of them is to combine natural products with chemical insecticides to increase their effectiveness. In the study presented, menthol which is an essential oil component was evaluated on its ability to increase the efficiency of bendiocarb, carbamate insecticide. A multi-approach study was conducted using biochemical method (to measure acetylcholinesterase enzyme activity), electrophysiological technique (microelectrode recordings in DUM neurons in situ), and confocal microscopy (for calcium imaging). In the electrophysiological experiments, menthol caused hyperpolarization, which was blocked by an octopamine receptor antagonist (phentolamine) and an inhibitor of protein kinase A (H-89). It also raised the intracellular calcium level. The effect of bendiocarb was potentiated by menthol and this phenomenon was abolished by phentolamine and H-89 but not by protein kinase C inhibitor (bisindolylmaleimide IX). The results indicate that menthol increases carbamate insecticide efficiency by acting on octopamine receptors and triggering protein kinase A phosphorylation pathway.

Genome-wide transcriptional analyses in Anopheles mosquitoes reveal an unexpected association between salivary gland gene expression and insecticide resistance.

BACKGROUND: To combat malaria transmission, the Ugandan government has embarked upon an ambitious programme of indoor residual spraying (IRS) with a carbamate class insecticide, bendiocarb. In preparation for this campaign, we characterized bendiocarb resistance and associated transcriptional variation among Anopheles gambiae s.s. mosquitoes from two sites in Uganda. RESULTS: Gene expression in two mosquito populations displaying some resistance to bendiocarb (95% and 79% An. gambiae s.l. WHO tube bioassay mortality in Nagongera and Kihihi, respectively) was investigated using whole-genome microarrays. Significant overexpression of several genes encoding salivary gland proteins, including D7r2 and D7r4, was detected in mosquitoes from Nagongera. In Kihihi, D7r4, two detoxification-associated genes (Cyp6m2 and Gstd3) and an epithelial serine protease were among the genes most highly overexpressed in resistant mosquitoes. Following the first round of IRS in Nagongera, bendiocarb-resistant mosquitoes were collected, and real-time quantitative PCR analyses detected significant overexpression of D7r2 and D7r4 in resistant mosquitoes. A single nucleotide polymorphism located in a non-coding transcript downstream of the D7 genes was found at a significantly higher frequency in resistant individuals. In silico modelling of the interaction between D7r4 and bendiocarb demonstrated similarity between the insecticide and serotonin, a known ligand of D7 proteins. A meta-analysis of published microarray studies revealed a recurring association between D7 expression and insecticide resistance across Anopheles species and locations. CONCLUSIONS: A whole-genome microarray approach identified an association between novel insecticide resistance candidates and bendiocarb resistance in Uganda. In addition, a single nucleotide polymorphism associated with this resistance mechanism was discovered. The use of such impartial screening methods allows for discovery of resistance candidates that have no previously-ascribed function in insecticide binding or detoxification. Characterizing these novel candidates will broaden our understanding of resistance mechanisms and yield new strategies for combatting widespread insecticide resistance among malaria vectors.

Chitosan-magnetite nanocomposite as a sensing platform to bendiocarb determination.

A novel platform for carbamate-based pesticide quantification using a chitosan/magnetic iron oxide (Chit-Fe3O4) nanocomposite as a glassy carbon electrode (GCE) modifier is shown for an analytical methodology for determination of bendiocarb (BND). The BND oxidation signal using GCE/Chit-Fe3O4 compared with bare GCE was catalyzed, showing a 37.5% of current increase with the peak potential towards less positive values, showing method's increased sensitivity and selectivity. Using square-wave voltammetry (SWV), calibration curves for BND determination were obtained (n = 3), and calculated detection and quantification limits values were 2.09 × 10-6 mol L-1 (466.99 ppb) and 6.97 × 10-6 mol L-1 (1555.91 ppb), respectively. The proposed electroanalytical methodology was successfully applied for BND quantification in natural raw waters without any sample pretreatment, proving that the GCE/Chit-Fe3O4 modified electrode showed great potential for BND determination in complex samples. ᅟ Graphical abstract.

Ultrasound-assisted dispersive liquid-liquid microextraction followed by ion mobility spectrometry for the simultaneous determination of bendiocarb and azinphos-ethyl in water, soil, food and beverage samples.

A sensitive and fast ultrasound-assisted dispersive liquid-liquid microextraction procedure combined with ion mobility spectrometry has been developed for the simultaneous extraction and determination of bendiocarb and azinphos-ethyl. Experimental parameters affecting the analytical performance of the method were optimized: type and volume of extraction solvent (chloroform, 150 µL), pH (9.0), type and volume of buffer (ammonium buffer pH = 9.0, 4.5 mL) and extraction time (3.0 min). Under optimum conditions, the linearity was found to be in the range of 2-40 and 6-100 ng/mL and the limits of detection (LOD) were 1.04 and 1.31 ng/mL for bendiocarb and azinphos-ethyl, respectively. The method was successfully validated for the analysis of bendiocarb and azinphos-ethyl in different samples such as waters, soil, food and beverage samples.

Small-scale (Phase II) evaluation of the efficacy and residual activity of SumiShield® 50 WG (clothianidin 50%, w/w) for indoor residual spraying in comparison to deltamethrin, bendiocarb and pirimiphos-methyl for malaria vector control in Karnataka state, India.

BACKGROUND & OBJECTIVES: There is an urgent need of introducing new insecticide molecules with novel modes of action to counter the ever growing insecticide-resistance in mosquito vectors. In the present study, a new insecticide molecule, SumiShield 50 WG (clothianidin 50%, w/w) was investigated for its efficacy as an indoor residual spray along with its residual action in comparison to deltamethrin, pirimiphos-methyl and bendiocarb. METHODS: The study area included three villages in Almatti Dam catchment area in Bagalkot district, Karnataka, India. Spraying was done using Hudson sprayers with the following dosages-Clothianidin, 300 mg AI/m2; deltamethrin, 25 mg AI/m2; bendiocarb, 400 mg AI/m2; and pirimiphos-methyl, 1 g AI/m2. Cone bioassays were conducted on cement and mud plastered surfaces at fortnightly intervals to assess the bioefficacy and residual activity. Mosquito densities in the sprayed houses were recorded at regular intervals for assessment of the insecticidal efficacy. Filter paper samples collected from the sprayed houses were analyzed for insecticide content sprayed on different wall surfaces at the Walloon Agricultural Research Institute, Gembloux, Belgium. RESULTS: Chemical content analysis of filter paper samples revealed that the applied to target ratios were in the acceptable range (1 + 0.5) for all the treatment types. Duration of persistence of effectiveness of bendiocarb (≥80% mortality in cone bioassays) was 19 to 21 wk on cement plastered surfaces and 15 to 19 wk on mud plastered surfaces. Duration of persistence of effectiveness of deltamethrin was 17 to 21 wk on both mud and cement plastered surfaces and that of pirimiphos-methyl was 15 to 19 wk. For SumiShield, it was 17 to 25 wk on both types of surfaces, indicating slow action of SumiShield. The densities of Anopheles culicifacies were lower in bendiocarb sprayed houses throughout the observation period, followed by pirimiphos methyl, deltamethrin and clothianidin sprayed houses. In case of other mosquitoes also, similar trend was observed. INTERPRETATION & CONCLUSION: Considering the persistence of effectiveness of SumiShield on sprayed surfaces, effectiveness in reducing the density of mosquitoes, operational feasibility, safety and community acceptance, the formulation of clothianidin is a better option for IRS for the control of insecticide-resistant mosquito vectors.

Determination of the residual efficacy of carbamate and organophosphate insecticides used for indoor residual spraying for malaria control in Ethiopia.

BACKGROUND: Indoor residual spraying is one of the key vector control interventions for malaria control in Ethiopia. As malaria transmission is seasonal in most parts of Ethiopia, a single round of spraying can usually provide effective protection against malaria, provided the insecticide remains effective over the entire malaria transmission season. This experiment was designed to evaluate the residual efficacy of bendiocarb, pirimiphos-methyl, and two doses of propoxur on four different wall surfaces (rough mud, smooth mud, dung, and paint). Filter papers affixed to wall surfaces prior to spraying were analyzed to determine the actual concentration applied. Cone bioassays using a susceptible Anopheles arabiensis strain were done monthly to determine the time for which insecticides were effective in killing mosquitoes. RESULTS: The mean insecticide dosage of bendiocarb applied to walls was 486 mg/m2 (target 400/mg). This treatment lasted 1 month or less on rough mud, smooth mud, and dung, but 4 months on painted surfaces. Pirimiphos-methyl was applied at 1854 mg/m2 (target 1000 mg/m2), and lasted between 4 and 6 months on all wall surfaces. Propoxur with a target dose of 1000 mg/m2 was applied at 320 mg/m2, and lasted 2 months or less on all surfaces, except painted surfaces (4 months). Propoxur with a target dose of 2000 mg/m2, was applied at 638 mg/m2, and lasted 3 months on rough mud, but considerably longer (5-7 months) on the other substrates. CONCLUSIONS: It would appear that the higher dose of propoxur and pirimiphos-methyl correspond best to the Ethiopian transmission season, although interactions between insecticide and the substrate should be taken into account as well. However, the insecticide quantification revealed that the dosages actually applied differed considerably from the target dosages, even though care was taken in the mixing of insecticide formulations and spraying of the walls. It is unclear whether this variability is due to initial concentrations of insecticides, poor application, or other factors. Further work is needed to ensure that target doses are correctly applied, both operationally and in insecticide evaluations.

Insecticide resistance patterns in Uganda and the effect of indoor residual spraying with bendiocarb on kdr L1014S frequencies in Anopheles gambiae s.s.

BACKGROUND: Resistance of malaria vectors to pyrethroid insecticides has been attributed to selection pressure from long-lasting insecticidal nets (LLINs), indoor residual spraying (IRS), and the use of chemicals in agriculture. The use of different classes of insecticides in combination or by rotation has been recommended for resistance management. The aim of this study was to understand the role of IRS with a carbamate insecticide in management of pyrethroid resistance. METHODS: Anopheles mosquitoes were collected from multiple sites in nine districts of Uganda (up to five sites per district). Three districts had been sprayed with bendiocarb. Phenotypic resistance was determined using standard susceptibility tests. Molecular assays were used to determine the frequency of resistance mutations. The kdr L1014S homozygote frequency in Anopheles gambiae s.s. was used as the outcome measure to test the effects of various factors using a logistic regression model. Bendiocarb coverage, annual rainfall, altitude, mosquito collection method, LLIN use, LLINs distributed in the previous 5 years, household use of agricultural pesticides, and malaria prevalence in children 2-9 years old were entered as explanatory variables. RESULTS: Tests with pyrethroid insecticides showed resistance and suspected resistance levels in all districts except Apac (a sprayed district). Bendiocarb resistance was not detected in sprayed sites, but was confirmed in one unsprayed site (Soroti). Anopheles gambiae s.s. collected from areas sprayed with bendiocarb had significantly less kdr homozygosity than those collected from unsprayed areas. Mosquitoes collected indoors as adults had significantly higher frequency of kdr homozygotes than mosquitoes collected as larvae, possibly indicating selective sampling of resistant adults, presumably due to exposure to insecticides inside houses that would disproportionately affect susceptible mosquitoes. The effect of LLIN use on kdr homozygosity was significantly modified by annual rainfall. In areas receiving high rainfall, LLIN use was associated with increased kdr homozygosity and this association weakened as rainfall decreased, indicating more frequency of exposure to pyrethroids in relatively wet areas with high vector density. CONCLUSION: This study suggests that using a carbamate insecticide for IRS in areas with high levels of pyrethroid resistance may reduce kdr frequencies in An. gambiae s.s.

Susceptibility of Anopheles gambiae to insecticides used for malaria vector control in Rwanda.

BACKGROUND: The widespread emergence of resistance to pyrethroids is a major threat to the gains made in malaria control. To monitor the presence and possible emergence of resistance against a variety of insecticides used for malaria control in Rwanda, nationwide insecticide resistance surveys were conducted in 2011 and 2013. METHODS: Larvae of Anopheles gambiae sensu lato mosquitoes were collected in 12 sentinel sites throughout Rwanda. These were reared to adults and analysed for knock-down and mortality using WHO insecticide test papers with standard diagnostic doses of the recommended insecticides. A sub-sample of tested specimens was analysed for the presence of knockdown resistance (kdr) mutations. RESULTS: A total of 14,311 mosquitoes were tested and from a sample of 1406 specimens, 1165 (82.9%) were identified as Anopheles arabiensis and 241 (17.1%) as Anopheles gambiae sensu stricto. Mortality results indicated a significant increase in resistance to lambda-cyhalothrin from 2011 to 2013 in 83% of the sites, permethrin in 25% of the sites, deltamethrin in 25% of the sites and DDT in 50% of the sites. Mosquitoes from 83% of the sites showed full susceptibility to bendiocarb and 17% of sites were suspected to harbour resistance that requires further confirmation. No resistance was observed to fenitrothion in all study sites during the entire survey. The kdr genotype results in An. gambiae s.s. showed that 67 (50%) possessed susceptibility (SS) alleles, while 35 (26.1%) and 32 (23.9%) mosquitoes had heterozygous (RS) and homozygous (RR) alleles, respectively. Of the 591 An. arabiensis genotyped, 425 (71.9%) possessed homozygous (SS) alleles while 158 (26.7%) and 8 (1.4%) had heterozygous (RS) and homozygous (RR) alleles, respectively. Metabolic resistance involving oxidase enzymes was also detected using the synergist PBO. CONCLUSION: This is the first nationwide study of insecticide resistance in malaria vectors in Rwanda. It shows the gradual increase of insecticide resistance to pyrethroids (lambda-cyhalothrin, deltamethrin, permethrin) and organochlorines (DDT) and the large presence of target site insensitivity. The results demonstrate the need for Rwanda to expand monitoring for insecticide resistance including further metabolic resistance testing and implement an insecticide resistance management strategy to sustain the gains made in malaria control.

Investigation of mechanisms of bendiocarb resistance in Anopheles gambiae populations from the city of Yaoundé, Cameroon.

BACKGROUND: Resistance to the carbamate insecticide bendiocarb is emerging in Anopheles gambiae populations from the city of Yaoundé in Cameroon. However, the molecular basis of this resistance remains uncharacterized. The present study objective is to investigate mechanisms promoting resistance to bendiocarb in An. gambiae populations from Yaoundé. METHODS: The level of susceptibility of An. gambiae s.l. to bendiocarb 0.1 % was assessed from 2010 to 2013 using bioassays. Mosquitoes resistant to bendiocarb, unexposed and susceptible mosquitoes were screened for the presence of the Ace-1(R) mutation using TaqMan assays. Microarray analyses were performed to assess the pattern of genes differentially expressed between resistant, unexposed and susceptible. RESULTS: Bendiocarb resistance was more prevalent in mosquitoes originating from cultivated sites compared to those from polluted and unpolluted sites. Both An. gambiae and Anopheles coluzzii were found to display resistance to bendiocarb. No G119S mutation was detected suggesting that resistance was mainly metabolic. Microarray analysis revealed the over-expression of several cytochrome P450 s genes including cyp6z3, cyp6z1, cyp12f2, cyp6m3 and cyp6p4. Gene ontology (GO) enrichment analysis supported the detoxification role of cytochrome P450 s with several GO terms associated with P450 activity significantly enriched in resistant samples. Other detoxification genes included UDP-glucosyl transferases, glutathione-S transferases and ABC transporters. CONCLUSION: The study highlights the probable implication of metabolic mechanisms in bendiocarb resistance in An. gambiae populations from Yaoundé and stresses the need for further studies leading to functional validation of detoxification genes involved in this resistance.

First evaluation of bendiocarb in experimental huts using different substrates in Madagascar.

BACKGROUND: Indoor residual spraying with insecticide is recommended for malaria control in high-transmission settings. Determination of residual activity of insecticides is essential for the selection of appropriate indoor spraying policy. The present study was undertaken to evaluate the residual effect of bendiocarb, a carbamate insecticide used in Madagascar, on different indoor surfaces in order to elaborate future vector control interventions. METHODS: The residual activity of bendiocarb was evaluated in both experimental huts and houses. Tests in experimental huts on different substrates represented a small scale-field trials. The houses IRS performed in parallel of experimental huts IRS, was done to compare semi-field results and field results. Bioassays according to the World Health Organization (WHO) standard protocol were carried out on different substrates impregnated with bendiocarb using susceptible strains of Anopheles arabiensis and Aedes albopictus. RESULTS: Bendiocarb induced significantly high mortality in treated huts against exposed mosquito (p < 0.005) compared to untreated huts. The mortality is up to the WHO threshold of 80 % during 5 months post-treatment. Using a multivariate analysis, Ae. albopictus mortality decreased significantly from the 3rd month post-treatment. However, An. arabiensis mortality decreased significantly from the 4th month after treatment. Comparing mosquito mortality results from the mud experimental huts and the mud houses showed no significant difference regarding the persistence of bendiocarb on wall. CONCLUSIONS: Current data suggest variable persistence of bendiocarb according to the type of wall surfaces, highlighting the importance of testing insecticide for IRS in local context before using them in large scale. Data from this study validate also the importance of using experimental huts as representative tool to evaluate the effectiveness of an insecticide.

Trends in the selection of insecticide resistance in Anopheles gambiae s.l. mosquitoes in northwest Tanzania during a community randomized trial of longlasting insecticidal nets and indoor residual spraying.

Anopheles gambiae s.l. (Diptera: Culicidae) in Muleba, Tanzania has developed high levels of resistance to most insecticides currently advocated for malaria control. The kdr mutation has almost reached fixation in An. gambiae s.s. in Muleba. This change has the potential to jeopardize malaria control interventions carried out in the region. Trends in insecticide resistance were monitored in two intervention villages using World Health Organization (WHO) susceptibility test kits. Additional mechanisms contributing to observed phenotypic resistance were investigated using Centers for Disease Control (CDC) bottle bioassays with piperonylbutoxide (PBO) and S,S,S-tributyl phosphorotrithioate (DEF) synergists. Resistance genotyping for kdr and Ace-1 alleles was conducted using quantitative polymerase chain reaction (qPCR). In both study villages, high phenotypic resistance to several pyrethroids and DDT was observed, with mortality in the range of 12-23%. There was a sharp decrease in mortality in An. gambiae s.l. exposed to bendiocarb (carbamate) from 84% in November 2011 to 31% in December 2012 after two rounds of bendiocarb-based indoor residual spraying (IRS). Anopheles gambiae s.l. remained susceptible to pirimiphos-methyl (organophosphate). Bendiocarb-based IRS did not lead to the reversion of pyrethroid resistance. There was no evidence for selection for Ace-1 resistance alleles. The need to investigate the operational impact of the observed resistance selection on the effectiveness of longlasting insecticidal nets and IRS for malaria control is urgent.

Morphometric alterations, steatosis, fibrosis and active caspase-3 detection in carbamate bendiocarb treated rabbit liver.

Increasing use of pesticides all over the world makes it necessary to reveal the toxic risk in populations of nontargeted organisms. Bendiocarb is one of the 12 insecticides recommended by the World Health Organization for use in malaria control in Africa, and is used against a variety of insects. The liver has an important role in its process of detoxication and excretion. In our experiment 56 adult rabbits of breed HY+, 28 males and 28 females were used. Animals were divided into groups (control, days 10, 20, 30 of bendiocarb administration). The presence of many binucleated hepatocytes, the highest number of liver cells and their decreased size at 10 day after bendiocarb administration was observed as an evidence of the hepatic regeneration. After the long-term treatment pronounced changes were presented such as vacuolization and dilatation of hepatocytes, dilatation of sinusoids between hepatocytes, and focal infiltration of inflammatory cells. Numerous cells with caspase-3 activity were present throughout the organ, most commonly around the portal tract and close to the central vein. Short and long-term bendiocarb treatment showed the central vein thickened rim with increased deposition of collagen, spreading of collagen fibers into the perisinusoidal, and pericellular space surrounding the central veins, and septal fibrosis extended from the portal tract. Subsequently, presence of the lipid vacuoles both in the liver parenchyma and inner of the hepatocytes were observed. These results suggest that bendiocarb treatment leads to increased cell death, liver perisinusoidal fibrosis, and steatosis, especially during the long-term administration.

Ultrastructural changes in the rabbit liver induced by carbamate insecticide bendiocarb.

Carbamates (CB) are used as insecticides and some of them have been registered as human drugs. The mechanism of CB poisoning involves reversible inhibition of acetylcholine esterase. In the present study, we investigated changes in liver ultrastructure in rabbits (Oryctolagus cuniculus) which were administered bendiocarb for 3, 10, 20, and 30 days. Rabbits in all experimental groups received capsules of bendiocarb (96% Bendiocarb, Bayer, Germany) per os daily at a dose of 5 mg/kg of body weight, and after day 11 received the same dose every 48 h. The observed changes were only moderate, focal, and the effect on the liver was not uniform. On the third day of the experiment, injured hepatocytes had dilated bile capillaries with reduced microvilli. There were no visible alterations in the intercellular contacts. Nuclei of these cells were irregular in shape. Many hepatocytes showed considerable increase in the number of peroxisomes. On day 10 of the experiment, the number of peroxisomes was reduced. Other changes, such as dilated rough endoplasmic reticulum and proliferation of smooth endoplasmic reticulum were observed on day 20. The number of lipid droplets in hepatocytes gradually increased. Usually they were present in low numbers, but on day 30 of the experiment their number increased significantly. They coalesced and formed a single lipid droplet which changed the shape of the nuclei. The results presented in this study indicate that both short and long-term administration of bendiocarb affects the liver ultrastructure. At the same time we also observed rapid onset of regeneration of the damaged tissue through activation of hepatocytes and oval cells.

Ace-1 Target Site Status and Metabolic Detoxification Associated with Bendiocarb Resistance in the Field Populations of Main Malaria Vector, Anopheles stephensi in Iran.

Background: Anopheles stephensi is the main vector of malaria in Iran. This study aimed to determine the susceptibility of An. stephensi from the south of Iran to bendiocarb and to investigate biochemical and molecular resistance mechanisms in this species. Methods: Wild An. stephensi were collected from Hormozgan Province and reared to the adult stage. The susceptibility test was conducted according to the WHO protocols using bendiocarb impregnated papers supplied by WHO. Also, field An. Stephensi specimens were collected from south of Kerman and Sistan and Baluchistan Provinces. To determine the G119S mutation in the acetylcholinesterase (Ace1) gene, PCR-RFLP using AluI restriction enzyme and PCR direct-sequencing were performed for the three field populations and compared with the available GenBank data. Also, biochemical assays were performed to measure alpha and beta esterases, insensitive acetylcholinesterase, and oxidases in the strains. Results: The bioassay tests showed that the An. stephensi field strain was resistant to bendiocarb (mortality rate 89%). Ace1 gene analysis revealed no G119S in the three field populations. Blast search of sequences revealed 98-99% identity with the Ace1 gene from Pakistan and India respectively. Also, the results of biochemical tests revealed the high activity of non-sensitive acetylcholinesterase, alpha and beta-esterase in the resistant strain compared to the susceptible strain. No G119S was detected in this study additionally the enhanced enzyme activity of esterases and acetylcholinesterase suggesting that resistance was metabolic. Conclusion: The use of alternative malaria control methods and the implementation of resistance management strategies are suggested in the study area.

Sublethal biochemical, behavioral, and physiological toxicity of extremely low dose of bendiocarb insecticide in Periplaneta americana (Blattodea: Blattidae).

Insecticides are dedicated to impair the insect organisms, but also have an impact on other, non-target organisms, including humans. In this way, they became important risk factor for disturbance of physiological homeostasis and can be involved in the development of diseases or in deterioration of existing conditions. The influence of sublethal doses of various insecticides on vertebrates' and invertebrates' organisms has been previously observed. In this paper, we have evaluated the impact of exposure to extremely low dose of neurotoxin, bendiocarb (0.1 nM), a commonly used carbamate insecticide on a model organism in neurobiology-Periplaneta americana. The assessment was performed on all levels of animal organism from molecular (oxidative stress parameters: phosphorylation level of proteins, cAMP level, protein kinase A and C levels, and octopamine) to physiological (heart beat and gas exchange tests) and behavioral (motor skills assay, grooming test). Exposure to such a low level of bendiocarb did not cause direct paralysis of insects, but changed their grooming behavior, decreased heart rate, and increased gas exchange. We also observed the increased parameters of oxidative stress as well as stressogenic response to 0.1 nM bendiocarb exposure. Exposure to a trace amount of bendiocarb also increased sensitivity to effective doses of the same insecticide, thus acts as preconditioning. These results force us to reconsider the possible risk from frequent/continuous exposure to traces of pesticide residues in the environment to human health.