Abstract
BackgroundPyrethroid resistance has been slower to emerge in Anopheles arabiensis than in An. gambiae s.s and An. funestus and, consequently, studies are only just beginning to unravel the genes involved. Permethrin resistance in An. arabiensis in Lower Moshi, Tanzania has been linked to elevated levels of both P450 monooxygenases and β-esterases. We have conducted a gene expression study to identify specific genes linked with metabolic resistance in the Lower Moshi An. arabiensis population.MethodsMicroarray experiments employing an An. gambiae whole genome expression chip were performed on An. arabiensis, using interwoven loop designs. Permethrin-exposed survivors were compared to three separate unexposed mosquitoes from the same or a nearby population. A subsection of detoxification genes were chosen for subsequent quantitative real-time PCR (qRT-PCR).ResultsMicroarray analysis revealed significant over expression of 87 probes and under expression of 85 probes (in pairwise comparisons between permethrin survivors and unexposed sympatric and allopatric samples from Dar es Salaam (controls). For qRT-PCR we targeted over expressed ABC transporter genes (ABC ‘2060’), a glutathione-S-transferase, P450s and esterases. Design of efficient, specific primers was successful for ABC ‘2060’and two P450s (CYP6P3, CYP6M2). For the CYP4G16 gene, we used the primers that were previously used in a microarray study of An. arabiensis from Zanzibar islands. Over expression of CYP4G16 and ABC ‘2060’ was detected though with contrasting patterns in pairwise comparisons between survivors and controls. CYP4G16 was only up regulated in survivors, whereas ABC ‘2060’ was similar in survivors and controls but over expressed in Lower Moshi samples compared to the Dar es Salaam samples. Increased transcription of CYP4G16 and ABC ‘2060’ are linked directly and indirectly respectively, with permethrin resistance in Lower Moshi An. arabiensis.ConclusionsIncreased transcription of a P450 (CYP4G16) and an ABC transporter (ABC 2060) are linked directly and indirectly respectively, with permethrin resistance in Lower Moshi An. arabiensis. Our study provides replication of CYP4G16 as a candidate gene for pyrethroid resistance in An. arabiensis, although its role may not be in detoxification, and requires further investigation.
Highlights
Pyrethroid resistance has been slower to emerge in Anopheles arabiensis than in An. gambiae s.s and An. funestus and, studies are only just beginning to unravel the genes involved
The genes that were down regulated include multiple detoxification genes, including several that have come up as top upregulated hits in previous An. gambiae s.s. arrays, including Cytochrome P450 monooxygenase enzymes (P450) repeatedly resistance-associated in previous work on An. gambiae s.s. (CYP6M2 and CYP6P3)
CYP6P3 is one of the leading candidate genes for metabolic resistance in West African An. gambiae and its down regulation suggests that quite different mechanisms may operate in the different species
Summary
Pyrethroid resistance has been slower to emerge in Anopheles arabiensis than in An. gambiae s.s and An. funestus and, studies are only just beginning to unravel the genes involved. We have conducted a gene expression study to identify specific genes linked with metabolic resistance in the Lower Moshi An. arabiensis population. Continuous usage of insecticides under such malaria control programmes and/or agricultural application of insecticides has resulted in the development of resistance in major malaria vector species [3,4,5,6,7]. Metabolic resistance and target-site resistance are major mechanisms assumed to be responsible for insecticide resistance [11] and are known to contribute to pyrethroid resistance in malaria vectors. The most common target site resistance mechanism constitutes two point mutations at amino acid position 1014 of the voltage-gated sodium channel gene, resulting in either a leucine-phenylalanine (L1014F) [13], or a leucine-serine (L1014S) substitution [14]
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