Fabrice Chandre

Dynamics of insecticide resistance in malaria vectors in Benin: first evidence of the presence of L1014S kdr mutation in Anopheles gambiae from West Africa

BackgroundInsecticide resistance monitoring is essential to help national programmers to implement more effective and sustainable malaria control strategies in endemic countries. This study reported the spatial and seasonal variations of insecticide resistance in malaria vectors in Benin, West Africa.MethodsAnopheles gambiae s.l populations were collected from October 2008 to June 2010 in four sites selected on the basis of different use of insecticides and environment. WHO susceptibility tests were carried out to detect resistance to DDT, fenitrothion, bendiocarb, permethrin and deltamethrin. The synergist piperonyl butoxide was used to assess the role of non-target site mechanisms in pyrethroid resistance. Anopheles gambiae mosquitoes were identified to species and to molecular M and S forms using PCR techniques. Molecular and biochemical assays were carried out to determine kdr and Ace.1R allelic frequencies and activity of the detoxification enzymes.ResultsThroughout the surveys very high levels of mortality to bendiocarb and fenitrothion were observed in An. gambiae s.l. populations. However, high frequencies of resistance to DDT and pyrethroids were seen in both M and S form of An. gambiae s.s. and Anopheles arabiensis. PBO increased the toxicity of permethrin and restored almost full susceptibility to deltamethrin. Anopheles gambiae s.l. mosquitoes from Cotonou and Malanville showed higher oxidase activity compared to the Kisumu susceptible strain in 2009, whereas the esterase activity was higher in the mosquitoes from Bohicon in both 2008 and 2009. A high frequency of 1014F kdr allele was initially showed in An. gambiae from Cotonou and Tori-Bossito whereas it increased in mosquitoes from Bohicon and Malanville during the second year. For the first time the L1014S kdr mutation was found in An. arabiensis in Benin. The ace.1R mutation was almost absent in An. gambiae s.l.ConclusionPyrethroid and DDT resistance is widespread in malaria vector in Benin and both metabolic and target site resistance are implicated. Resistance was not correlated with a change of malaria species and/or molecular forms. The 1014S kdr allele was first identified in wild population of An. arabiensis hence confirming the expansion of pyrethroid resistance alleles in Africa.

Repellent, Irritant and Toxic Effects of 20 Plant Extracts on Adults of the Malaria Vector Anopheles gambiae Mosquito

Pyrethroid insecticides induce an excito-repellent effect that reduces contact between humans and mosquitoes. Insecticide use is expected to lower the risk of pathogen transmission, particularly when impregnated on long-lasting treated bednets. When applied at low doses, pyrethroids have a toxic effect, however the development of pyrethroid resistance in several mosquito species may jeopardize these beneficial effects. The need to find additional compounds, either to kill disease-carrying mosquitoes or to prevent mosquito contact with humans, therefore arises. In laboratory conditions, the effects (i.e., repellent, irritant and toxic) of 20 plant extracts, mainly essential oils, were assessed on adults of Anopheles gambiae, a primary vector of malaria. Their effects were compared to those of DEET and permethrin, used as positive controls. Most plant extracts had irritant, repellent and/or toxic effects on An. gambiae adults. The most promising extracts, i.e. those combining the three types of effects, were from Cymbopogon winterianus, Cinnamomum zeylanicum and Thymus vulgaris. The irritant, repellent and toxic effects occurred apparently independently of each other, and the behavioural response of adult An. gambiae was significantly influenced by the concentration of the plant extracts. Mechanisms underlying repellency might, therefore, differ from those underlying irritancy and toxicity. The utility of the efficient plant extracts for vector control as an alternative to pyrethroids may thus be envisaged.

Insecticide resistance in Bemisia tabaci Gennadius (Homoptera: Aleyrodidae) and Anopheles gambiae Giles (Diptera: Culicidae) could compromise the sustainability of malaria vector control strategies in West Africa.

Insecticides from the organophosphate (OP) and pyrethroid (PY) chemical families, have respectively, been in use for 50 and 30 years in West Africa, mainly against agricultural pests, but also against vectors of human disease. The selection pressure, with practically the same molecules year after year (mainly on cotton), has caused insecticide resistance in pest populations such as Bemisia tabaci, vector of harmful phytoviruses on vegetables. The evolution toward insecticide resistance in malaria vectors such as Anopheles gambiae sensus lato (s.l.) is probably related to the current use of these insecticides in agriculture. Thus, successful pest and vector control in West Africa requires an investigation of insect susceptibility, in relation to the identification of species and sub species, such as molecular forms or biotypes. Identification of knock down resistance (kdr) and acetylcholinesterase gene (Ace1) mutations modifying insecticide targets in individual insects and measure of enzymes activity typically involved in insecticide metabolism (oxidase, esterase and glutathion-S-transferase) are indispensable in understanding the mechanisms of resistance. Insecticide resistance is a good example in which genotype-phenotype links have been made successfully. Insecticides used in agriculture continue to select new resistant populations of B. tabaci that could be from different biotype vectors of plant viruses. As well, the evolution of insecticide resistance in An. gambiae threatens the management of malaria vectors in West Africa. It raises the question of priority in the use of insecticides in health and/or agriculture, and more generally, the question of sustainability of crop protection and vector control strategies in the region. Here, we review the susceptibility tests, biochemical and molecular assays data for B. tabaci, a major pest in cotton and vegetable crops, and An. gambiae, main vector of malaria. The data reviewed was collected in Benin and Burkina Faso between 2008 and 2010 under the Corus 6015 research program. This review aims to show: (i) the insecticide resistance in B. tabaci as well as in An. gambiae; and (ii) due to this, the impact of selection of resistant populations on malaria vector control strategies. Some measures that could be beneficial for crop protection and vector control strategies in West Africa are proposed.

Electrophysiological and behavioral characterization of bioactive compounds of the Thymus vulgaris, Cymbopogon winterianus, Cuminum cyminum and Cinnamomum zeylanicum essential oils against Anopheles gambiae and prospects for their use as bednet treatments

BackgroundLaboratory and field studies showed that repellent, irritant and toxic actions of common public health insecticides reduce human-vector contact and thereby interrupt disease transmission. One of the more effective strategies to reduce disease risk involves the use of long-lasting treated bednets. However, development of insecticide resistance in mosquito populations makes it imperative to find alternatives to these insecticides. Our previous study identified four essential oils as alternatives to pyrethroids: Thymus vulgaris, Cymbopogon winterianus, Cuminum cyminum, Cinnamomum zeylanicum. The objectives of this study were to identify active compounds of these essential oils, to characterize their biological activity, and to examine their potential as a treatment for bednets.MethodsWe evaluated the electrophysiological, behavioural (repellency, irritancy) and toxic effects of the major compounds of these oils against Anopheles gambiae strain ‘Kisumu’.ResultsAldehydes elicited the strongest responses and monoterpenes the weakest responses in electroantennogram (EAG) trials. However, EAG responses did not correlate consistently with results of behavioral assays. In behavioral and toxicity studies, several of the single compounds did exhibit repellency, irritancy or toxicity in An. gambiae; however, the activity of essential oils did not always correlate with activity expected from the major components. On the contrary, the biological activity of essential oils appeared complex, suggesting interactions between individual compounds and the insect under study. Data also indicated that the three effects appeared independent, suggesting that repellency mechanism(s) may differ from mechanisms of irritancy and toxicity.ConclusionsBased on the bioassays reported here, some of the compounds merit consideration as alternative bednet treatments.

Insecticide resistance in disease vectors from Mayotte: an opportunity for integrated vector management

BackgroundMayotte, a small island in the Indian Ocean, has been affected for many years by vector-borne diseases. Malaria, Bancroftian filariasis, dengue, chikungunya and Rift Valley fever have circulated or still circulate on the island. They are all transmitted by Culicidae mosquitoes. To limit the impact of these diseases on human health, vector control has been implemented for more than 60xa0years on Mayotte. In this study, we assessed the resistance levels of four major vector species (Anopheles gambiae, Culex pipiens quinquefasciatus, Aedes aegypti and Aedes albopictus) to two types of insecticides: i) the locally currently-used insecticides (organophosphates, pyrethroids) and ii) alternative molecules that are promising for vector control and come from different insecticide families (bacterial toxins or insect growth regulators). When some resistance was found to one of these insecticides, we characterized the mechanisms involved.MethodsLarval and adult bioassays were used to evaluate the level of resistance. When resistance was found, we tested for the presence of metabolic resistance through detoxifying enzyme activity assays, or for target-site mutations through molecular identification of known resistance alleles.ResultsResistance to currently-used insecticides varied greatly between the four vector species. While no resistance to any insecticides was found in the two Aedes species, bioassays confirmed multiple resistance in Cx. p. quinquefasciatus (temephos: ~ 20 fold and deltamethrin: only 10% mortality after 24xa0hours). In An. gambiae, resistance was scarce: only a moderate resistance to temephos was found (~5 fold). This resistance appears to be due only to carboxyl-esterase overexpression and not to target modification. Finally, and comfortingly, none of the four species showed resistance to any of the new insecticides.ConclusionsThe low resistance observed in Mayotte’s main disease vectors is particularly interesting, because it leaves a range of tools useable by vector control services. Together with the relative isolation of the island (thus limited immigration of mosquitoes), it provides us with a unique place to implement an integrated vector management plan, including all the good practices learned from previous experiences.

Naturally occurring bioactive compounds from four repellent essential oils against Bemisia tabaci whiteflies

BACKGROUNDnIn tropical countries, netting is an effective sustainable tool for protecting horticultural crops against Lepidoptera, although not against small pests such as Bemisia tabaci, while smaller mesh netting can be used in temperate regions. A solution is to combine a net with a repellent. Previously we identified repellent essential oils: lemongrass (Cymbopogon citratus), cinnamon (Cinnamomum zeylanicum), cumin (Cuminum cyminum) and citronella (Cymbopogon winternarius). The present study was designed to identify the active compounds of these essential oils, characterise their biological activity and examine their potential for coating nets. We investigated the efficiency and toxicity of nets dipped in different solutions. We then studied the repellent effect with an olfactometer and the irritant effect by videotracking.nnnRESULTSnGeraniol and citronellol were the most promising net coatings owing to their repellent effect. The repellency, irritancy or toxicity varied with the product and concentration, and these features were independent, indicating that the repellent and the irritant/toxic mechanisms were not the same. The combined effects of these different compounds account for the bioactivity of the mixture, suggesting interactions between the compounds.nnnCONCLUSIONnThis new sustainable strategy for protecting vegetable crops against whiteflies is discussed, in addition to the use of companion plants that could produce such bioactive compounds.

Field Efficacy of Vectobac GR as a Mosquito Larvicide for the Control of Anopheline and Culicine Mosquitoes in Natural Habitats in Benin, West Africa

Introduction The efficacy of Vectobac GR (potency 200 ITU/mg), a new formulation of bacterial larvicide Bacillus thuringiensis var. israelensis Strain AM65-52, was evaluated against Anopheles gambiae and Culex quinquefasciatus in simulated field and natural habitats in Benin. Methods In simulated field conditions, Vectobac GR formulation was tested at 3 dosages (0.6, 0.9, 1.2 g granules/m2 against An. gambiae and 1, 1.5, 2 g granules/m2 against Cx. quinquefasciatus) according to manufacturer’s product label recommendations. The dosage giving optimum efficacy under simulated field conditions were evaluated in the field. The efficacy of Vectobac GR in terms of emergence inhibition in simulated field conditions and of reduction of larval and pupal densities in rice fields and urban cesspits was measured following WHO guidelines for testing and evaluation of mosquito larvicides. Results Vectobac GR caused emergence inhibition of ≥80% until 21 [20]–[22] days for An. gambiae at 1.2 g/m2 dose and 28 [27–29] days for Cx. quinquefasciatus at 2 g/m2 in simulated field habitats. The efficacy of Vectobac GR in natural habitats was for 2 to 3 days against larvae and up to 10 days against pupae. Conclusions Treatment with Vectobac GR caused complete control of immature mosquito within 2–3 days but did not show prolonged residual action. Larviciding can be an option for malaria and filariasis vector control particularly in managing pyrethroid-resistance in African malaria vectors. Since use of larvicides among several African countries is being emphasized through Economic Community of West Africa States, their epidemiological impact should be carefully investigated.

Prospects for repellent in pest control: current developments and future challenges

The overall interest in environmentally safe pest control methods and the rise of insecticide resistance in pest populations have prompted medical and agricultural entomology research on insect repellents in recent years. However, conducting research on repellent is challenging for several reasons: (1) the different repellent phenomena are not well defined; (2) it is difficult to test for and quantify repellent; (3) the physiological mechanisms are poorly known; (4) the field efficacy appears to be highly variable. Here, we identified five different types of repellent: expellency, irritancy, deterrency, odor masking and visual masking, and described behavioral bioassays to differentiate between them. Although these categories are currently defined by their behavioral response to different stimuli, we suggest new definitions based on their mechanism of action. We put forward three main hypotheses on the physiological mechanism: (1) a dose effect that modifies the behavior, (2) a repellent mechanism with specific receptors, or (3) inhibition of the transduction of neural information.

A user-friendly software to easily count Anopheles egg batches

BackgroundStudies on malaria vector ecology and development/evaluation of vector control strategies often require measures of mosquito life history traits. Assessing the fecundity of malaria vectors can be carried out by counting eggs laid by Anopheles females. However, manually counting the eggs is time consuming, tedious, and error prone.MethodsIn this paper we present a newly developed software for high precision automatic egg counting. The software written in the Java programming language proposes a user-friendly interface and a complete online manual. It allows the inspection of results by the operator and includes proper tools for manual corrections. The user can in fact correct any details on the acquired results by a mouse click. Time saving is significant and errors due to loss of concentration are avoided.ResultsThe software was tested over 16 randomly chosen images from 2 different experiments. The results show that the proposed automatic method produces results that are close to the ground truth.ConclusionsThe proposed approaches demonstrated a very high level of robustness. The adoption of the proposed software package will save many hours of labor to the bench scientist. The software needs no particular configuration and is freely available for download on: http://w3.ualg.pt/∼hshah/eggcounter/.

Insights into the epigenomic landscape of the human malaria vector Anopheles gambiae

The epigenome of the human malaria vector Anopheles gambiae was characterized in midgut cells by mapping the distribution and levels of two post-translational histone modifications, H3K27ac and H3K27me3. These histone profiles were then correlated with levels of gene expression obtained by RNA-seq. Analysis of the transcriptome of A. gambiae midguts and salivary glands led to the discovery of 13,898 new transcripts not present in the most recent genome assembly. A subset of these transcripts is differentially expressed between midgut and salivary glands. The enrichment profiles of H3K27ac and H3K27me3 are mutually exclusive and associate with high and low levels of transcription, respectively. This distribution agrees with previous findings in Drosophila showing association of these two histone modifications with either active or inactive transcriptional states, including Polycomb-associated domains in silenced genes. This study provides a mosquito epigenomics platform for future comparative studies in other mosquito species, opening future investigations into the role of epigenetic processes in vector-borne systems of medical and economic importance.