Induction of Molecular and Physiologic Changes in Larvae and Adult Anopheles Gambiae s.s. Mosquito by Murraya Koenigii Leaf Phytochemicals

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Maikuri, Mang’era Clarence
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Kenyatta University
Plant-based constituents have been proposed as eco-friendly alternatives to synthetic insecticides for control of mosquito vectors of malaria. These synthetic products are increasingly becoming problematic mainly due mosquito resistance, and hence the need to search and identify alternative control agents. In this study, we first screened the effects of methanolic leaf extracts of curry tree (Murraya koenigii) growing in tropical (Mombasa, Malindi) and semi-arid (Kibwezi, and Makindu) ecological zones of Kenya on late third and early fourth instar (L3/L4) An. gambiae s.s. larvae. Extracts of the plant from the semi-arid region, and particularly from Kibwezi, led to high mortality of the larvae. Bioassay-guided fractionation of the methanolic extract of the leaves of the plants from Kibwezi was then undertaken and the most active fraction (20 fold more potent than the crude extract) was then analysed by Liquid chromatography quadruple time of flight coupled with mass spectrometry (LC-QtoF-MS) and a number of constituents were identified, including a major alkaloid constituent, Neplanocin A (5). Exposure of the third instar larvae to a sub-lethal dose (below 4.43 ppm) of this fraction, induced gross morphogenetic abnormalities in the larvae, with reduced locomotion, a 3-fold reduction in pupation rates and protracted larval-pupa moulting by up to 15 days post-exposure relative to controls. In order to explain these hormetic responses and gain the molecular insights on the observed phenotypic effects conferred by sub-lethal doses of M. koenigii bioactive fractions, differential gene expression profiles were assessed by analysing RNA-Seq datasets from An. gambiae mosquito immature stages and validated through RT-qPCR. These deformities were accompanied by significant induction (up to 780-fold increase) of transcripts predominantly associated with hard cuticular proteins, juvenile hormone esterases, immunity and detoxification in the larvae samples exposed to the extract relative to the non-exposed control samples coupled with alteration of pathways involved in putrescine metabolism and structural constituents of the cuticle. Additionally, essential oils (EO) from M. koenigii leaves showed significant fumigant toxicity against adult An. gambiae (100% mortality in 3.30 ± 0.071 hrs, p = .00). These results demonstrate subtle growth-disrupting effects of the phytochemical blend from M. koenigii leaves on aquatic stages of An. gambiae mosquito and provides significant molecular evidence for differentially expressed genes linked to developmental abnormalities observed in An. gambiae mosquito larvae exposed to a bioactive fraction of M. koenigii leaf extract. Also, fumigant toxicity assays demonstrate that M. koenigii has a unique EO complex blend with an insecticidal potential. The study lays down some useful groundwork for the downstream development of phytochemical blends for eco-friendly control of An. gambiae vector population that can be swiftly deployed concurrently with existing Integrated Vector Management (IVM) technologies. Functional studies into the potential roles of the candidate genes as molecular targets that can be evaluated to shed further light on the molecular mechanisms underlying the effects of the phytochemicals.
A Thesis Submitted in Fulfilment of the Requirement for the Award of the Degree of Doctor of Philosophy (Biochemistry) in the School of Pure and Applied Sciences of Kenyatta University, May, 2021
Induction of Molecular, Physiologic Changes, Larvae, Adult Anopheles Gambiae s.s. Mosquito, Murraya Koenigii Leaf Phytochemicals