Rise of multiple insecticide resistance in Anopheles funestus in Malawi: a major concern for malaria vector control

Riveron et al. Malar J (2015) 14:344 DOI 10.1186/s12936-015-0877-y Open Access RESEARCH Rise of multiple insecticide resistance in Anopheles funestus in Malawi: a major concern for malaria vector control Jacob M. Riveron1*, Martin Chiumia2, Benjamin D. Menze1,3, Kayla G. Barnes1, Helen Irving1, Sulaiman S. Ibrahim1, Gareth D. Weedall1, Themba Mzilahowa2 and Charles S. Wondji1 Abstract Background: Deciphering the dynamics and evolution of insecticide resistance in malaria vectors is crucial for successful vector control. This study reports an increase of resistance intensity and a rise of multiple insecticide resistance in Anopheles funestus in Malawi leading to reduced bed net efficacy. Methods: Anopheles funestus group mosquitoes were collected in southern Malawi and the species composition, Plasmodium infection rate, susceptibility to insecticides and molecular bases of the resistance were analysed. Results: Mosquito collection revealed a predominance of An. funestus group mosquitoes with a high hybrid rate (12.2 %) suggesting extensive species hybridization. An. funestus sensu stricto was the main Plasmodium vector (4.8 % infection). Consistently high levels of resistance to pyrethroid and carbamate insecticides were recorded and had increased between 2009 and 2014. Furthermore, the 2014 collection exhibited multiple insecticide resistance, notably to DDT, contrary to 2009. Increased pyrethroid resistance correlates with reduced efficacy of bed nets (<5 % mortality by Olyset® net), which can compromise control efforts. This change in resistance dynamics is mirrored by prevalent resistance mechanisms, firstly with increased over-expression of key pyrethroid resistance genes (CYP6Pa/b and CYP6M7) in 2014 and secondly, detection of the A296S-RDL dieldrin resistance mutation for the first time. However, the L119F-GSTe2 and kdr mutations were absent. Conclusions: Such increased resistance levels and rise of multiple resistance highlight the need to rapidly implement resistance management strategies to preserve the effectiveness of existing insecticide-based control interventions. Keywords: Malaria, Insecticide resistance, Vector control, Anopheles funestus, Malawi Background Malaria remains a major public health burden in Africa [1], notably in Malawi, where it is highly endemic with an estimated six million annual cases [2, 3]. Current malaria control efforts in Malawi rely heavily on insecticide-based interventions such as long-lasting insecticidetreated nets (LLINs) and indoor residual spraying (IRS) [4]. However, reports of increasing resistance against the main insecticides used in public health are of concern *Correspondence: 1 Vector Biology Department, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK Full list of author information is available at the end of the article for the continued effectiveness of these control tools. In Malawi, the concern is greater for the increasing cases of resistance against pyrethroids (the only insecticide class used in bed nets) reported in the major malaria vector Anopheles funestus [5–8]. Insecticide resistance is a dynamic process and resistance pattern can change rapidly with time, as reported in other vector species such as Anopheles gambiae [9, 10]. However, changes due to ongoing control programmes in the profiles, intensity and underlying resistance mechanisms in Malawi remain largely uncharacterized. Understanding the dynamics and evolution of the resistance pattern in such a major malaria vector is crucial for the design and implementation of successful resistance management strategies. © 2015 Riveron et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Riveron et al. Malar J (2015) 14:344 Design of effective control strategies relies also on a good knowledge of the vector population in term of species composition, vectorial capacity and behaviour. Such information remains patchy in Malawi, notably in the southern region where resistance has previously been reported [6]. An. funestus belongs to a group of ten to 11 species morphologically indistinguishable as adults [11]. However, the local species composition of this group, their role in malaria transmission, the hybridization between these species, and its impact on the introgression of genes of interests, such as resistance genes, remains largely uncharacterized. To fill this knowledge gap and to facilitate the design and implementation of suitable vector control strategies, this study reports an extensive investigation of the dynamic changes in resistance profile and resistance mechanisms associated with ongoing insecticidebased control interventions in Malawi between 2009 and 2014. This study reveals an increase of resistance intensity and a rise of multiple insecticide resistance in An. funestus in Malawi causing a reduction in bed net efficacy. Methods Study area and mosquito collection Adult Anopheles and Culex mosquitoes were collected in Chikwawa district (16°1′S; 34°47′E), southern Malawi, in January 2014. Geographical details of this location have been described previously [7]. Indoor-resting, blood-fed or gravid mosquitoes were collected between 06.00 and 12.00 h inside households using electric insect aspirators, after obtaining the consent of village chiefs and house owners. Collected mosquitoes were kept until fully gravid and induced to lay eggs in individual 1.5-ml microcentrifuge tubes, as described previously [12]. All F0 females that laid eggs were morphologically identified as belonging to either the An. funestus group or the An. gambiae complex according to a morphological key [13]. Dead adult mosquitoes and egg batches were transported to the Liverpool School of Tropical Medicine under a DEFRA license (PATH/125/2012). Species identification To identify the different species within the An. funestus group, a cocktail PCR was performed as previously described [14] after genomic DNA extraction from whole mosquitoes using the DNeasy Blood and Tissue kit (Qiagen. Hilden, Germany). In addition, 50 females belonging to the An. gambiae complex were identified as previously described [15] after gDNA extraction [16]. Eggs were hatched in small paper cups and larvae transferred to plastic larvae trays, according to species, for rearing as previously described [12, 17]. Page 2 of 9 Plasmodium sporozoite infection rate The Plasmodium infection rate of An. funestus group mosquitoes (167 An. funestus, s.s., (...truncated)