Microarray analysis of a pyrethroid resistant African malaria vector, Anopheles funestus, from southern Africa

Abstract

Anopheles funestus is one of the major malaria vectors in southern Africa and several populations in this region are resistant to pyrethroids. The current study uses a microarray based approach to identify genes up-regulated in the pyrethroid resistant population, FUMOZ, from Mozambique. As the full set of detoxification genes in An. funestus are unknown, this study investigated the utility of the An. gambiae 'detox chip' to screen for differentially expressed detoxification genes in An. funestus. Differential expression of detoxification genes in three day old adult females and males from the FUMOZ resistant strain and the FANG susceptible strain was identified using the An. gambiae 'detox chip'.After optimization of the hybridization conditions, over 90% of the probes showed a positive signal. Only three genes were significantly (p<0.001) differentially expressed in the females, CYP6P9 (5.4-fold), COI (2.7-fold) and CYP6M7 (1.8-fold). The same genes were also significantly differentially expressed in the adult males, CYP6P9 (6.0-fold), COI (2.9-fold) and CYP6M3 (3.6-fold) together with an additional 21 transcripts.Quantitative PCR (qPCR) analysis was conducted to validate the microarray results. This study demonstrated that heterologous hybridization is a helpful tool in identifying detoxification genes differentially expressed in An. funestus strains. Keywords:Microarrays, An. funestus, 'detox chip', pyrethroid resistance, cytochrome P450. 3 IntroductionIn 2008 In this study we investigated the use of the An. gambiae 'detox chip' in determining potential genes associated with insecticide resistance in a southern African An. funestus resistant strain (FUMOZ-R) and a susceptible strain (FANG). Selected significantly overexpressed genes from the microarray results were validated using qPCR. 5 Materials and methods Mosquito strainsTwo An. funestus strains were used in this study. The FUMOZ-R strain originated from Mozambique and has been maintained under selection pressure with permethrin. FANG ori ginates f rom Angol a and is susceptible to all known insecti cides. Details on the insecticide resistance status of these colonies can be found in the study by Hunt et al.[20]. Both strains are maintained in standard insectary conditions of 25°C with 80% relative humidity and 12 h day/night, 45 min dusk/dawn lighting cycle. Sample preparation and microarray hybridizationsFemale and male An. funestus adults were separated on day of emergence and fed on 10%sugar solution until they were three days old (without prior exposure to pyrethroids). The transcription levels in the three day old resistant FUMOZ-R adult females were compared to the susceptible females of the same age. The same was done for the males. Each comparison in all experiments consisted of three independent biological replicates and two technical repeats which included dye swaps to control for dye bias. There were four within-array replicate spots per microarray slide. Total RNA was extracted from three batches of fifteen three day old adult fema...

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

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DDT and pyrethroid resistance in Anopheles arabiensis from South Africa

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