The evolution of insecticide resistance in mosquitoes is threatening the effectiveness and sustainability of malaria control programs in various parts of the world. Through their unique mode of action, entomopathogenic fungi provide promising alternatives to chemical control. However, potential interactions between fungal infection and insecticide resistance, such as cross-resistance, have not been investigated. We show that insecticide-resistant Anopheles mosquitoes remain susceptible to infection with the fungus Beauveria bassiana. Four different mosquito strains with high resistance levels against pyrethroids, organochlorines, or carbamates were equally susceptible to B. bassiana infection as their baseline counterparts, showing significantly reduced mosquito survival. Moreover, fungal infection reduced the expression of resistance to the key public health insecticides permethrin and dichlorodiphenyltrichloroethane. Mosquitoes preinfected with B. bassiana or Metarhizium anisopliae showed a significant increase in mortality after insecticide exposure compared with uninfected control mosquitoes. Our results show a high potential utility of fungal biopesticides for complementing existing vector control measures and provide products for use in resistance management strategies.biopesticide ͉ DDT ͉ pyrethoids ͉ resistance management ͉ vector control

Insecticide resistance poses a significant and increasing threat to the control of malaria and other mosquito-borne diseases. We present a novel method of insecticide application based on netting treated with an electrostatic coating that binds insecticidal particles through polarity. Electrostatic netting can hold small amounts of insecticides effectively and results in enhanced bioavailability upon contact by the insect. Six pyrethroid-resistant Anopheles mosquito strains from across Africa were exposed to similar concentrations of deltamethrin on electrostatic netting or a standard longlasting deltamethrin-coated bednet (PermaNet 2.0). Standard WHO exposure bioassays showed that electrostatic netting induced significantly higher mortality rates than the PermaNet, thereby effectively breaking mosquito resistance. Electrostatic netting also induced high mortality in resistant mosquito strains when a 15-fold lower dose of deltamethrin was applied and when the exposure time was reduced to only 5 s. Because different types of particles adhere to electrostatic netting, it is also possible to apply nonpyrethroid insecticides. Three insecticide classes were effective against strains of Aedes and Culex mosquitoes, demonstrating that electrostatic netting can be used to deploy a wide range of active insecticides against all major groups of disease-transmitting mosquitoes. Promising applications include the use of electrostatic coating on walls or eave curtains and in trapping/ contamination devices. We conclude that application of electrostatically adhered particles boosts the efficacy of WHO-recommended insecticides even against resistant mosquitoes. This innovative technique has potential to support the use of unconventional insecticide classes or combinations thereof, potentially offering a significant step forward in managing insecticide resistance in vector-control operations.electrostatic coating | insecticide | resistance management | mosquito | malaria

DNA or RNA amplification methods for detection of Leishmania parasites have advantages regarding sensitivity and potential quantitative characteristics in comparison with conventional diagnostic methods but are often still not routinely applied. However, the use and application of molecular assays are increasing, but comparative studies on the performance of these different assays are lacking. The aim of this study was to compare three molecular assays for detection and quantification of Leishmania parasites in serial dilutions of parasites and in skin biopsies collected from cutaneous leishmaniasis (CL) patients in Manaus, Brazil. A serial dilution of promastigotes spiked in blood was tested in triplicate in three different runs by quantitative nucleic acid sequence-based amplification (QT-NASBA), quantitative real-time reverse transcriptase PCR (qRT-PCR), and quantitative real-time PCR (qPCR). In addition, the costs, durations, and numbers of handling steps were compared, and 84 skin biopsies from patients with suspected CL were tested. Both QT-NASBA and qRT-PCR had a detection limit of 100 parasites/ml of blood, while qPCR detected 1,000 parasites/ml. QT-NASBA had the lowest range of intra-assay variation (coefficients of variation [CV], 0.5% to 3.3%), while qPCR had the lowest range of interassay variation (CV, 0.4% to 5.3%). Furthermore, qRT-PCR had higher r 2 values and amplification efficiencies than qPCR, and qPCR and qRT-PCR had faster procedures than QT-NASBA. All assays performed equally well with patient samples, with significant correlations between parasite counts. Overall, qRT-PCR is preferred over QT-NASBA and qPCR as the most optimal diagnostic assay for quantification of Leishmania parasites, since it was highly sensitive and reproducible and the procedure was relatively fast.

BackgroundPresented here are a series of preliminary experiments evaluating “eave tubes”—a technology that combines house screening with a novel method of delivering insecticides for control of malaria mosquitoes.MethodsEave tubes were first evaluated with overnight release and recapture of mosquitoes in a screened compartment containing a hut and human sleeper. Recapture numbers were used as a proxy for overnight survival. These trials tested physical characteristics of the eave tubes (height, diameter, angle), and different active ingredients (bendiocarb, LLIN material, fungus). Eave tubes in a hut with closed eaves were also compared to an LLIN protecting a sleeper in a hut with open eaves. Eave tubes were then evaluated in a larger compartment containing a self-replicating mosquito population, vegetation, and multiple houses and cattle sheds. In this “model village”, LLINs were introduced first, followed by eave tubes and associated house modifications.ResultsInitial testing suggested that tubes placed horizontally and at eave height had the biggest impact on mosquito recapture relative to respective controls. Comparison of active ingredients suggested roughly equivalent effects from bendiocarb, LLIN material, and fungal spores (although speed of kill was slower for fungus). The impact of treated netting on recapture rates ranged from 50 to 70 % reduction relative to controls. In subsequent experiments comparing bendiocarb-treated netting in eave tubes against a standard LLIN, the effect size was smaller but the eave tubes with closed eaves performed at least as well as the LLIN with open eaves. In the model village, introducing LLINs led to an approximate 60 % reduction in larval densities and 85 % reduction in indoor catches of host-seeking mosquitoes relative to pre-intervention values. Installing eave tubes and screening further reduced larval density (93 % relative to pre intervention values) and virtually eliminated indoor host-seeking mosquitoes. When the eave tubes and screening were removed, larval and adult catches recovered to pre-eave tube levels.ConclusionsThese trials suggest that the “eave tube” package can impact overnight survival of host-seeking mosquitoes and can suppress mosquito populations, even in a complex environment. Further testing is now required to evaluate the robustness of these findings and demonstrate impact under field conditions.

BackgroundEntomopathogenic fungi are being investigated as a new mosquito control tool because insecticide resistance is preventing successful mosquito control in many countries, and new methods are required that can target insecticide-resistant malaria vectors. Although laboratory studies have previously examined the effects of entomopathogenic fungi against adult mosquitoes, most application methods used cannot be readily deployed in the field. Because the fungi are biological organisms it is important to test potential field application methods that will not adversely affect them. The two objectives of this study were to investigate any differences in fungal susceptibility between an insecticide-resistant and insecticide-susceptible strain of Anopheles gambiae sensu stricto, and to test a potential field application method with respect to the viability and virulence of two fungal speciesMethodsPieces of white polyester netting were dipped in Metarhizium anisopliae ICIPE-30 or Beauveria bassiana IMI391510 mineral oil suspensions. These were kept at 27 ± 1°C, 80 ± 10% RH and the viability of the fungal conidia was recorded at different time points. Tube bioassays were used to infect insecticide-resistant (VKPER) and insecticide-susceptible (SKK) strains of An. gambiae s.s., and survival analysis was used to determine effects of mosquito strain, fungus species or time since fungal treatment of the net.ResultsThe resistant VKPER strain was significantly more susceptible to fungal infection than the insecticide-susceptible SKK strain. Furthermore, B. bassiana was significantly more virulent than M. anisopliae for both mosquito strains, although this may be linked to the different viabilities of these fungal species. The viability of both fungal species decreased significantly one day after application onto polyester netting when compared to the viability of conidia remaining in suspension.ConclusionsThe insecticide-resistant mosquito strain was susceptible to both species of fungus indicating that entomopathogenic fungi can be used in resistance management and integrated vector management programmes to target insecticide-resistant mosquitoes. Although fungal viability significantly decreased when applied to the netting, the effectiveness of the fungal treatment at killing mosquitoes did not significantly deteriorate. Field trials over a longer trial period need to be carried out to verify whether polyester netting is a good candidate for operational use, and to see if wild insecticide-resistant mosquitoes are as susceptible to fungal infection as the VKPER strain.

BackgroundThe increasing global threat of Dengue demands new and easily applicable vector control methods. Ovitraps provide a low-tech and inexpensive means to combat Dengue vectors. Here we describe the development and optimization process of a novel contamination device that targets multiple life-stages of the Aedes aegypti mosquito. Special focus is directed to the diverse array of control agents deployed in this trap, covering adulticidal, larvicidal and autodissemination impacts.MethodsDifferent trap prototypes and their parts are described, including a floater to contaminate alighting gravid mosquitoes. The attractiveness of the trap, different odor lures and floater design were studied using fluorescent powder adhering to mosquito legs and via choice tests. We demonstrate the mosquitocidal impacts of the control agents: a combination of the larvicide pyriproxyfen and the adulticidal fungus Beauveria bassiana. The impact of pyriproxyfen was determined in free-flight dissemination experiments. The effect on larval development inside the trap and in surrounding breeding sites was measured, as well as survival impacts on recaptured adults.ResultsThe developmental process resulted in a design that consists of a black 3 Liter water-filled container with a ring-shaped floater supporting vertically placed gauze dusted with the control agents. On average, 90% of the mosquitoes in the fluorescence experiments made contact with the gauze on the floater. Studies on attractants indicated that a yeast-containing tablet was the most attractive odor lure. Furthermore, the fungus Beauveria bassiana was able to significantly increase mortality of the free-flying adults compared to controls. Dissemination of pyriproxyfen led to >90% larval mortality in alternative breeding sites and 100% larval mortality in the trap itself, against a control mortality of around 5%.ConclusionThis ovitrap is a promising new tool in the battle against Dengue. It has proven to be attractive to Aedes aegypti mosquitoes and effective in contaminating these with Beauveria bassiana. Furthermore, we show that the larvicide pyriproxyfen is successfully disseminated to breeding sites close to the trap. Its low production and operating costs enable large scale deployment in Dengue-affected locations.

We studied the use of African water storage pots for point source application of Metarhizium anisopliae against the malaria vectors Anopheles gambiae s.s. and An. funestus. Clay pots were shown to be attractive resting sites for male and female An. gambiae s.s. and were not repellent after impregnation with fungus. M. anisopliae was highly infective and virulent after spray application inside pots. At a dosage of 4 x 10(10) conidia/m(2), an average of 95 +/- 1.2% of An. gambiae s.s. obtained a fungal infection. A lower dosage of 1 x 10(10) conidia/m(2) infected an average of 91.5 +/- 0.6% of An. gambiae s.s. and 91.8 +/- 1.2% of An. funestus mosquitoes. Fungal infection significantly reduced mosquito longevity, as shown by differences between survival curves and LT(50) values. These pots are suitable for application of entomopathogenic fungi against malaria vectors and their potential for sustainable field implementation is discussed.

In spite of massive progress in the control of African malaria since the turn of the century, there is a clear and recognized need for additional tools beyond long-lasting insecticide-treated bed nets (LLINs) and indoor residual spraying (IRS) of insecticides, to progress towards elimination. Moreover, widespread and intensifying insecticide resistance requires alternative control agents and delivery systems to enable development of effective insecticide resistance management strategies. This series of articles presents a novel concept for malaria vector control, the ‘eave tube’, which may fulfil these important criteria. From its conceptualization to laboratory and semi-field testing, to demonstration of potential for implementation, the stepwise development of this new vector control approach is described. These studies suggest eave tubes (which comprise a novel way of delivering insecticides plus screening to make the house more ‘mosquito proof’) could be a viable, cost-effective, and acceptable control tool for endophilic and endophagic anophelines, and possibly other (nuisance) mosquitoes. The approach could be applicable in a wide variety of housing in sub-Saharan Africa, and possibly beyond, for vectors that use the eave as their primary house entry point. The results presented in these articles were generated during an EU-FP7 funded project, the mosquito contamination device (MCD) project, which ran between 2012 and 2015. This was a collaborative project undertaken by vector biologists, product developers, modellers, materials scientists, and entrepreneurs from five different countries.