title: NMR METABOLIC PROFILING OF MOSQUITO SPECIES TO UNDERSTAND INSECTICIDE RESISTANCE
Creators: name: Grosman, Rudi
Publisher: name: University of Liverpool Repository
Year: 2020

description: The work presented in this thesis explores insecticide resistance using NMR metabolomics in pupa, early pupa and early adult mosquitoes of both sexes. Firstly, sex differences were investigated in early pupa and early adult strains of Anopheles gambiae and Aedes aegypti. Secondly, Cyp4g16 and Cyp4g17 knock-down strains of An. gambiae were studied to further understand the cuticular hydrocarbon (CHC) biosynthesis mechanism. Lastly, wild type susceptible and resistant strains of An. gambiae and Ae. aegypti were studied to understand metabolic differences in resistance. A common theme in this work is the utilisation of NMR metabolomics, focusing on polar metabolic profiles and its application in insecticide resistant mosquitoes. Improvements on the analyses of the NMR data to make the identification of metabolites from the NMR spectra more robust was also undertaken in the course of this project. The vast majority of mosquito studies focus on females since only females bite. This means that there is often a great amount of information on male mosquitoes that is not utilised. In order to investigate sex differences in wild type An. gambiae (and knock-down strains) and Ae. aegypti, a metabolomics protocol was established. This study found that early female pupae and adults have higher levels of lactate and glucose compared to males, whereas, their pyruvate and propionate levels were found to be lower. Cuticular resistance is a relatively newly discovered mechanism, with much about it that is still unknown, Electron microscopy studies have shown that insecticide resistant mosquitoes of An. gambiae possess a thicker cuticular layer. Furthermore, the decarbonylases, Cyp4g16 and Cyp4g17, have been found to be critical in CHC biosynthesis and are highly expressed in insecticide resistant strains. Although not fully characterised, these enzymes are thought to catalyse a decarbonylation step in CHC biosynthesis. The study here showed evidence that both Cyp4g16 and Cyp4g17 take part in the biosynthesis of methyl branched-alkanes. Additionally, these enzymes have temporal activity where Cyp4g16 activity can be observed in both pupa and adult stages, whereas Cyp4g17 activity is only observable in the adult stage. Using resistant and susceptible strains of both An. gambiae and Ae. aegypti, metabolic differences were observed. Trehalose was found consistently higher in all resistant strains IIand so it has been proposed as a biomarker candidate. Furthermore, systems using trehalose were suggested as potential targets in insecticide development. In conclusion, the work in this thesis shows that it is possible to use NMR metabolomics to study the metabolic responses found in insect pupae and adults, to distinguish sex differences and the obtained development stage- dependent temporal information on the activity of specific enzymes. Furthermore, through the identification of elevation in trehalose in resistant mosquitoes, this study has proposed a potential biomarker for identification of resistant mosquito species.