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Title: In vivo functional genetic analysis of cytochromes P450 involved in insecticide resistance in the malaria vector Anopheles gambiae
Author: Adolfi, A.
ISNI:       0000 0004 6495 9739
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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Insecticide-based vector control strategies have greatly contributed to malaria control, yet their success is hindered by the spread of resistance. It is thus essential to discover the molecular basis of resistance to use the current insecticides effectively and to search for new ones informatively. In Anopheles gambiae, overexpression of single detoxifying enzymes of the P450 (CYP) family has been strongly associated with resistance, yet in vivo analysis has only been performed in the fruit fly. Furthermore, patterns of P450 overexpression that lead to resistance, which are crucial to identify detoxification pathways, remain unclear. This study aims to investigate the role of two genes, Cyp6m2 and Cyp6p3, in conferring resistance in vivo in An. gambiae when overexpressed tissue-specifically in the midgut, oenocytes, or Malpighian tubules or in multiple tissues. To obtain spatially-controlled gene overexpression the GAL4/UAS system was employed. Responder lines for the expression of Cyp6s were created by PhiC31-RMCE and crossed with two tissue-specific drivers specific for the midgut or the oenocytes. Resistance phenotype assessment, conducted according to WHO guidelines, revealed no difference in mortality between mosquitoes overexpressing Cyp6 genes and controls after exposure to four different insecticides, suggesting that overexpression confined to these tissues is not sufficient to drive resistance. We therefore aimed to examine overexpression in the Malpighian tubules and in multiple tissues. Since promoters for these locations had not been characterised, putative regulatory regions were isolated from genes showing desired patterns of expression. Two main candidates, PUBc and GPI, were chosen after assessing their activity by luciferase assay in mosquito cells, and were used to create driver lines using the piggyBac transposon. Random insertion was chosen to identify single genomic sites able to sustain widespread expression and at the same time create new PhiC31 docking lines at these permissive sites. Patterns of expression induced by PUBc drivers carrying single insertions were investigated by crossing with a responder UAS:mCherry line. Two drivers were isolated that induced multi-tissue expression in all life stages and both sexes at relatively higher (A10) and lower (A8) levels. In similar experiments, the ubiquitous GPI and the Malpighian-specific VATG candidate promoters did not show visible mCherry expression. The PUBc driver lines were finally used to assess resistance resulting from multi-tissue overexpression. Ubiquitous expression of Cyp6m2 under the control of the A10 driver correlated with acquisition of WHO levels of resistance to pyrethroids, while that of Cyp6p3 with resistance to pyrethroids and bendiocarb; DDT resistance was not affected. Lower levels of overexpression driven by A8 did not significantly alter the resistance phenotype except for permethrin resistance when Cyp6m2 is overexpressed. Furthermore, Cyp6 overexpression driven by A10 or A8 caused an increase in susceptibility to the pro-insecticide malathion, supporting the role of specific CYP6s in providing negative cross-resistance between insecticide classes. This study provides the first in vivo evidence of the role of Cyp6s in causing WHO-defined resistance in An. gambiae, suggesting that location and level of overexpression are critical. Overall, this work contributed to the optimisation of a system for in vivo functional analysis in transgenic An. gambiae through the characterisation of a novel ubiquitous promoter and the ability to modulate level of expression using alternative drivers. Such a system is physiologically more relevant than the current Drosophila-based system for in vivo validation of mosquito resistance gene. In addition, metabolically resistant lines created here can be included alongside field-caught populations for screening new insecticides.
Supervisor: Lycett, G. J. ; Ranson, H. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral