Over expression of a Cytochrome P450 (CYP6P9) in a Major African Malaria Vector, <i>Anopheles Funestus,</i> Resistant to Pyrethroids

Amenya, Dolphine A.; Naguran, R.; Lo, Tsun‐Ho; Ranson, Hilary; Spillings, Belinda L; Wood, Oliver R; Brooke, Basil D.; Coetzee, Maureen; Koekemoer, Lizette L.

doi:10.1111/j.1365-2583.2008.00776.x

Cited by 120 publications

(104 citation statements)

References 20 publications

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“…In 2008, Amenya et al [31] found the CYP6P9 gene to be over-expressed in the egg and adult stages of a pyrethroid resistant laboratory strain (FUMOZ-R) originating from southern Mozambique. Wondji et al [32] identified a quantitative trait locus (QTL) associated with pyrethroid resistance in the same An.…”

Section: Discussionmentioning

confidence: 99%

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

Christian

Strode

Ranson

et al. 2011

Pesticide Biochemistry and Physiology

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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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Section: Discussionmentioning

confidence: 99%

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

Christian

Strode

Ranson

et al. 2011

Pesticide Biochemistry and Physiology

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“…funestus (Amenya et al, 2008). A partial sequence of CYP6P9 was used in Northern, dot blot and real-time PCR expression studies.…”

Section: Discussionmentioning

confidence: 99%

“…funestus, higher levels of CYP6P9 mRNA are found in pyrethroid-resistant strains than in pyrethroid-susceptible strains (Amenya et al, 2008;Wondji et al, 2009). Consequently, P450 may play a protective role against insecticides.…”

Section: Introductionmentioning

confidence: 99%

Sequence characterization of cytochrome P450 CYP6P9 in pyrethroid resistant and susceptible Anopheles funestus (Diptera: Culicidae)

Matambo¹,

Paine²,

Coetzee³

et al. 2010

Genet. Mol. Res.

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ABSTRACT. Anopheles funestus, one of the main African malaria vectors, caused a major malaria outbreak in South Africa during 1999/2000, even though South Africa had an effective vector control program in place. The outbreak was due to pyrethroid resistant An. funestus invading KwaZulu/Natal. Increased activity of cytochrome P450 (monooxygenase) was responsible for the pyrethroid resistance in this species. A monooxygenase gene, CYP6P9, was highly overexpressed in the pyrethroid-resistant strain compared with a susceptible strain. Characterization of this gene as well as the redox partners involved in the catalytic cycle of P450s was investigated. The full length of the CYP6P9 sequence was isolated, sequenced and compared between the pyrethroid-resistant and -susceptible strains. Sequence identity between the two strains was 99.3%; the sequence differences were mainly outside of the conserved regions. The functional significance is still unknown, but it is feasible that these variations are associated with differences in insecticide metabolism. A second CYP6 gene (CYP6P13) was also isolated; it shared close similarities with CYP6P9. The putative redox partners, cytochrome b 5 (cyt b 5 ) and NADPH-cytochrome P450 reductase (CPR), were isolated from An. funestus (resistant strain) and showed high levels of sequence identity to other insect cyt b 5 and CPRs. Isolation of the coding sequences CYP6P9 and its cognate redox partners enables expression of functional recombinant protein for biochemical and structural analysis.

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“…Pyrethroid resistance in A. funestus from South Africa and southern Mozambique was subsequently shown to be mediated by a monooxygenase (P450) detoxification mechanism and no target site insensitivity was identified (Brooke et al, 2001;Wondji et al, 2007;Okoye, 2008). Recently, the molecular characterization of monooxygenase-based pyrethroid resistance in A. funestus was initiated (Amenya et al, 2005(Amenya et al, , 2008Wondji et al, 2007Wondji et al, , 2009Matambo et al, 2010). Amenya et al (2005) isolated the first 31 partial monooxygenase P450 sequences and subsequently reported that one of these, CYP6P9, was over-expressed in the egg and adult stages of a pyrethroid-resistant laboratory strain (FUMOZ-R) originating from southern Mozambique (Amenya et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

“…Recently, the molecular characterization of monooxygenase-based pyrethroid resistance in A. funestus was initiated (Amenya et al, 2005(Amenya et al, , 2008Wondji et al, 2007Wondji et al, , 2009Matambo et al, 2010). Amenya et al (2005) isolated the first 31 partial monooxygenase P450 sequences and subsequently reported that one of these, CYP6P9, was over-expressed in the egg and adult stages of a pyrethroid-resistant laboratory strain (FUMOZ-R) originating from southern Mozambique (Amenya et al, 2008). Wondji et al (2007) identified a quantitative trait locus (QTL) associated with pyrethroid resistance in the same A. funestus strain and confirmed the increase in CYP6P9 transcription.…”

Section: Introductionmentioning

confidence: 99%

Age-related pyrethroid resistance is not a function of P450 gene expression in the major African malaria vector, Anopheles funestus (Diptera: Culicidae)

Christian¹,

Matambo²,

Spillings³

et al. 2011

Genet. Mol. Res.

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ABSTRACT. Anopheles funestus is a major vector of malaria in most of the African region. Resistance to pyrethroid and carbamate insecticides has been recorded in populations of this species in South Africa and Mozambique. The P450 gene, CYP6P9, has been shown to be highly transcribed in a permethrin (pyrethroid)-resistant laboratory strain, FUMOZ-R, originating from southern Mozambique. We examined the relationship between pyrethroid resistance and gene transcription levels of two closely related genes, CYP6P9 and CYP6P13, in FUMOZ-R. Levels of resistance to 0.75% permethrin were determined based on standard WHO insecticide susceptibility assays using females and males of different ages, ranging from 3 to 30 days old. The transcription Pyrethroid resistance and P450 gene expression in A. funestus levels of the two genes were quantified using qPCR for each age cohort. In the WHO insecticide susceptibility assays, survival of both males and females significantly decreased as age increased. Quantitative analysis of the two genes CYP6P9 and CYP6P13 showed the highest levels of expression at 10 days of age. There was no correlation between expression of these two genes and pyrethroid survival by age. We conclude that the resistance of this mosquito strain to permethrin is not directly related to age-mediated differences in CYP6P9 and CYP6P13 expression.

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Over expression of a Cytochrome P450 (CYP6P9) in a Major African Malaria Vector, Anopheles Funestus, Resistant to Pyrethroids

Cited by 120 publications

References 20 publications

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

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

Sequence characterization of cytochrome P450 CYP6P9 in pyrethroid resistant and susceptible Anopheles funestus (Diptera: Culicidae)

Age-related pyrethroid resistance is not a function of P450 gene expression in the major African malaria vector, Anopheles funestus (Diptera: Culicidae)

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