Adedeji, Eunice Oluwatobiloba and Covenant University, Theses (2022) IN SILICO PREDICTION AND BIOCHEMICAL VALIDATION OF PROTEIN TARGETS IN Anopheles gambiae. ["eprint_fieldopt_thesis_type_phd" not defined] thesis, Covenant University Ota.
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Abstract
Malaria, an endemic disease in sub-Saharan Africa, is transmitted by female Anopheles mosquitoes. The major malaria vector control strategy has remained the use of insecticides. However, resistance in Anopheles to all classes of existing insecticides motivates the identification of novel targets for malaria vector control. Anopheles metabolic proteins represent a repertoire of possible targets, however, finding potential targets using experimental methods alone is a tall order. This study aimed to identify and modulate genes genes central for An. gambiae’s survival or Plasmodium berghei clearance in the mosquito using in silico and biochemical techniques. A multi-compartment central metabolic model of An. gambiae was constructed using Reconstruction, Analysis and Visualization of Metabolic Networks (RAVEN) toolbox, manual curation was performed, and essential genes were predicted using chokepoint and percentage of unreached products larger than zero criteria. Experimental validation of selected genes was carried out using RNA interference (RNAi) and chemical inhibition methods. Double-stranded RNA (138 nL of 5 μg/μL) for selected genes and LacZ was injected into respective groups of between 2 to 4 days old female An. gambiae mosquitoes. Seventy (70) female mosquitoes per treatment were injected for survival experiments, and survival was monitored from day 2 post-injection until the death of all mosquitoes. Two hundred (200) female mosquitoes per treatment group (LacZ and Arginase) were injected for experiments involving infection with P. berghei. Parasite infection was performed 48 h after injection, and oocyte count was determined. Validamycin was administered at varying concentrations to third-stage (L3) larvae and first-stage (L1) larvae, then biochemical parameters and effect on gene-expression were investigated. Appropriate statistical analyses were carried out on the results. The central metabolic model consisted of 570 reactions, 471 metabolites, and 833 genes distributed across cytoplasm, mitochondria, and extracellular compartments. Three genes out of the 106 genes predicted from the network were selected, and three other non-metabolic genes for experimental validation in An. gambiae G3 mosquitoes based on established literature alluding to their essentiality. The six genes validated were Trehalase, Arginase, 3-hydroxykynurenine transaminase (3HKT), Heat shock 70kDa protein (HSP), elongation factor 2 (2elf), and elongation factor 1-alpha. Knockdown of HSP and 2elf resulted in a significant reduction (p<0.05) in the percentage survival of mosquitoes compared to control groups. Similarly, knockdown of arginase led to a marked reduction (p<0.05) in the number of oocytes count per midgut compared to the control group. In addition, larval treatment with validamycin A, an inhibitor of trehalase resulted in marked larval death at 48 h in a dose-dependent manner and retarded development of mosquitoes. Lethal concentration 50 (LC50) was 167.1 ppm in L3 larvae and 30.71 ppm in L1 larvae. Treatment with validamycin increased trehalose concentration at all concentrations considered and expression of insulin-like peptide2 at 500 ppm, 24 h after validamycin A exposure. This study suggests trehalase as a possible larvicide target, revealed the importance of HSP and 2elf for mosquito survival, and arginase for parasite development. These may serve as potential targets for vector control. Further studies to identify suitable inhibitors for these targets is recommended.
| Item Type: | Thesis (["eprint_fieldopt_thesis_type_phd" not defined]) |
|---|---|
| Uncontrolled Keywords: | Anopheles, Elongation factor, Heat shock, Plasmodium, Trehalase, Validamycin, Vector control |
| Subjects: | Q Science > QH Natural history |
| Divisions: | Faculty of Engineering, Science and Mathematics > School of Chemistry Faculty of Medicine, Health and Life Sciences > School of Biological Sciences |
| Depositing User: | nwokealisi |
| Date Deposited: | 01 Feb 2023 15:19 |
| Last Modified: | 01 Feb 2023 15:19 |
| URI: | http://eprints.covenantuniversity.edu.ng/id/eprint/16576 |
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