Malaria vectors in the Democratic Republic of the Congo: the mechanisms that confer insecticide resistance in Anopheles gambiae and Anopheles funestus.

BACKGROUND: The Democratic Republic of the Congo (DRC) is characterized as a holoendemic malaria area with the main vectors being Anopheles funestus and members of the Anopheles gambiae complex. Due to political instability and socio-economic challenges in the region, knowledge of insecticide resistance status and resistance mechanisms in these vectors is limited. Mosquitoes were collected from a mining site in the north-eastern part of the country and, following identification, were subjected to extensive testing for the target-site and biochemical basis of resistance. Quantitative real-time PCR was used to assess a suite of 10 genes frequently involved in pyrethroid and dichlorodiphenyltrichloroethane (DDT) resistance in An. gambiae females and males. In An. funestus, gene expression microarray analysis was carried out on female mosquitoes. RESULTS: In both species, deltamethrin resistance was recorded along with high resistance and suspected resistance to DDT in An. gambiae and An. funestus, respectively. A total of 85% of An. gambiae carried the kdr mutations as either homozygous resistant (RR) (L1014S, L1014F or both) or heterozygous (RS), however only 3% carried the rdl mutant allele (RS) and no ace-1 mutations were recorded. Synergist assays indicated a strong role for P450s in deltamethrin resistance in both species. In An. gambiae, analysis of transcription levels showed that the glutathione-S-transferase, GSTS1-2, produced the highest fold change in expression (7.6-fold in females and 31-fold in males) followed by GSTE2, thioredoxin peroxidase (TPX2), and cytochrome oxidases (CYP6M2 and CYP6P1). All other genes tested produced fold change values below 2. Microarray analysis revealed significant over-transcription of cuticular proteins as well as CYP6M7, CYP6P9a and CYP6P9b in insecticide resistant An. funestus. CONCLUSIONS: These data show that high levels of deltamethrin resistance in the main malaria vector species, conferred by enzymatic detoxification, are present in the DRC.

An online tool for mapping insecticide resistance in major Anopheles vectors of human malaria parasites and review of resistance status for the Afrotropical region.

BACKGROUND: Malaria control programmes across Africa and beyond are facing increasing insecticide resistance in the major anopheline vectors. In order to preserve or prolong the effectiveness of the main malaria vector interventions, up-to-date and easily accessible insecticide resistance data that are interpretable at operationally-relevant scales are critical. Herein we introduce and demonstrate the usefulness of an online mapping tool, IR Mapper. METHODS: A systematic search of published, peer-reviewed literature was performed and Anopheles insecticide susceptibility and resistance mechanisms data were extracted and added to a database after a two-level verification process. IR Mapper ( http://www.irmapper.com) was developed using the ArcGIS for JavaScript Application Programming Interface and ArcGIS Online platform for exploration and projection of these data. RESULTS: Literature searches yielded a total of 4,084 susceptibility data points for 1,505 populations, and 2,097 resistance mechanisms data points for 1,000 populations of Anopheles spp. tested via recommended WHO methods from 54 countries between 1954 and 2012. For the Afrotropical region, data were most abundant for populations of An. gambiae, and pyrethroids and DDT were more often used in susceptibility assays (51.1 and 26.8% of all reports, respectively) than carbamates and organophosphates. Between 2001 and 2012, there was a clear increase in prevalence and distribution of confirmed resistance of An. gambiae s.l. to pyrethroids (from 41 to 87% of the mosquito populations tested) and DDT (from 64 to 91%) throughout the Afrotropical region. Metabolic resistance mechanisms were detected in western and eastern African populations and the two kdr mutations (L1014S and L1014F) were widespread. For An. funestus s.l., relatively few populations were tested, although in 2010-2012 resistance was reported in 50% of 10 populations tested. Maps are provided to illustrate the use of IR Mapper and the distribution of insecticide resistance in malaria vectors in Africa. CONCLUSIONS: The increasing pyrethroid and DDT resistance in Anopheles in the Afrotropical region is alarming. Urgent attention should be afforded to testing An. funestus populations especially for metabolic resistance mechanisms. IR Mapper is a useful tool for investigating temporal and spatial trends in Anopheles resistance to support the pragmatic use of insecticidal interventions.

DDT and pyrethroid resistance in Anopheles arabiensis from South Africa.

BACKGROUND: Pyrethroid resistance has been well documented in Anopheles arabiensis, one of the major African malaria vectors, and the predominant malaria vector in South Africa. METHODS: In this study, the genetic basis of pyrethroid resistance in a selected laboratory strain of An. arabiensis from South Africa was investigated using a custom-made microarray, known as the An. gambiae detoxification chip. RESULTS: A large number of P450 genes were over-transcribed, as well as a suite of redox genes and glutathione S-transferases. The five genes that showed the highest level of gene transcription when compared with an insecticide susceptible strain were: CYP6AG2, CYPZ1, TPX2, CYPZ2 and CYP6P1. CONCLUSIONS: Permethrin resistance in South African An. arabiensis is associated with increased transcription of multiple genes, and a large proportion of these genes were also previously recorded as over-transcribed in another An. arabiensis strain selected for resistance to DDT with cross-resistance to deltamethrin. The deltamethrin resistance developed de novo in the DDT-selected strain and is most likely due to increased transcription of those genes associated with DDT resistance. However, of particular interest was the fact that the strain selected for resistance to pyrethroids did not develop de novo resistance to DDT. These differences are compared and discussed.

Detoxification enzymes associated with insecticide resistance in laboratory strains of Anopheles arabiensis of different geographic origin.

BACKGROUND: The use of insecticides to control malaria vectors is essential to reduce the prevalence of malaria and as a result, the development of insecticide resistance in vector populations is of major concern. Anopheles arabiensis is one of the main African malaria vectors and insecticide resistance in this species has been reported in a number of countries. The aim of this study was to investigate the detoxification enzymes that are involved in An. arabiensis resistance to DDT and pyrethroids. METHODS: The detoxification enzyme profiles were compared between two DDT selected, insecticide resistant strains of An. arabiensis, one from South Africa and one from Sudan, using the An. gambiae detoxification chip, a boutique microarray based on the major classes of enzymes associated with metabolism and detoxification of insecticides. Synergist assays were performed in order to clarify the roles of over-transcribed detoxification genes in the observed resistance phenotypes. In addition, the presence of kdr mutations in the colonies under investigation was determined. RESULTS: The microarray data identifies several genes over-transcribed in the insecticide selected South African strain, while in the Sudanese population, only one gene, CYP9L1, was found to be over-transcribed. The outcome of the synergist experiments indicate that the over-transcription of detoxification enzymes is linked to deltamethrin resistance, while DDT and permethrin resistance are mainly associated with the presence of the L1014F kdr mutation. CONCLUSIONS: These data emphasise the complexity associated with resistance phenotypes and suggest that specific insecticide resistance mechanisms cannot be extrapolated to different vector populations of the same species.

Multiple insecticide resistance in Anopheles gambiae (Diptera: Culicidae) from Pointe Noire, Republic of the Congo.

Successful implementation of an integrated vector control program will rely on availability of accurate vector information in the specific location. However, such information can be limited in some countries. The aim of this study was to obtain baseline vector information from Pointe Noire on the Congo coast (Republic of the Congo). Field sampling was conducted during April 2009 in the village of Boutoto and its surrounds, close to the city of Pointe Noire. Anopheles gambiae sensu lato mosquitoes were collected resting indoors. Samples were analyzed for insecticide susceptibility, species identification, and Plasmodium sporozoite infection. Molecular and biochemical assays were conducted to characterize insecticide resistance mechanisms. The malaria vector A. gambiae S-form was the only mosquito species identified, and it had a high Plasmodium falciparum infection rate (9.6%). Multiple insecticide resistance was detected in this population with full susceptibility to only one insecticide class, the organophosphates. Dieldrin and DDT resistance was mainly attributed to target-site resistance (the Rdl and L1014F/L1014S kdr mutations respectively), whereas pyrethroid resistance was mainly attributed to P450 metabolic enzyme-mediated detoxification in addition to kdr. The role of various insecticide resistance mechanisms revealed a complex association between metabolic detoxification and reduced target-site sensitivity.