Student Number : 0318930A -
PhD thesis -
School of Animal, Plant and Environmental Studies -
Faculty of Science / The widespread use of pyrethroid insecticides has led to the emergence of significant
insecticide resistance in various parts of the world. An unprecedented increase in the
number of annual malaria cases reported in Kwazulu Natal, South Africa in 1999 to
2000 was attributed to the re-emergence of pyrethroid-resistant Anopheles funestus
Giles. Resistance was metabolic-based with increased monooxygenase (P450)
metabolising the pyrethroid insecticides. This emphased the need to understand the
molecular mechanisms conferring pyrethroid resistance in An. funestus. The present
study aimed to firstly isolate P450 genes in An. funestus and secondly, to identify
P450 gene over-expressed in a resistant (FUMOZ-R) strain compared to a
susceptible (FANG) strain. A third aim was to construct an An. funestus cDNA
library to lay the foundation for future studies on P450 monooxygenses.
Degenerate primers based on conserved regions of three An. gambiae P450 families
were used to amplify cDNAs from An. funestus. Eleven CYP4, four CYP6 and five
CYP9 partial genes were isolated and sequenced. BLAST results revealed that An.
funestus P450s have a high sequence similarity to An. gambiae with above 75%
identity at the amino acid level. The exception was CYP9J14. The An. gambiae
P450 with the closest similarity to CYP9J14 exhibited only 55% identity suggesting
a recent duplication event in CYP9J14. Molecular phylogenetic analysis also
supported this hypothesis. Intron positions were highly conserved between the two
species.
Expression studies using blot analysis implicated CYP6P9, an ortholog of CYP6P3
in An. gambiae, as the over-expressed P450. Dot blot analysis revealed a 500-fold
expression higher in FUMOZ-R strain compared with FANG strain. Semiquantitative
PCR revealed that CYP6P9 was developmentally regulated. Expression
was not detected in eggs and was higher in larvae compared to pupae. Quantitative
real time PCR showed that CYP6P9 expression was 4.5-fold higher in 3-day old
FUMOZ-R males than females and 3.5-fold higher in the 14-day old males than 14-
day old females. Statistically, this difference was not significant suggesting that
CYP6P9 expression is not sex specific.
The An. funestus cDNA library construction in λTriplEx2 vector was successful with
a titre of 4.9 x108 pfu/ml and a transformation efficiency of 98%.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/2115 |
| Date | 26 February 2007 |
| Creators | Amenya, Dolphine Achieng' |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 1063968 bytes, application/pdf, application/pdf |
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