Maina, Sharon Mwagira2026-05-082026-05-082025-10https://ir-library.ku.ac.ke/handle/123456789/33044A Thesis Submitted in Partial Fulfillment of the Requirements for the Award of the Degree of Master of Science (Biotechnology) in the School of Pure and Applied Sciences of Kenyatta University, October 2025. Supervisors 1. Prof. Steven Runo 2. Dr. Luna KamauMolecular diagnostic tools have been incorporated in insecticide resistance monitoring programs to identify underlying genetic basis of resistance and develop early warning systems of vector control failure. Identifying genetic markers of insecticide resistance is crucial in enhancing the ability to mitigate potential effects of resistance. The knockdown resistance (kdr) mutation associated with resistance to DDT and pyrethroids, the acetylcholinesterase-1 (ace-1R) mutation associated with resistance to organophosphates and carbamates and 2La chromosomal inversion associated with indoor resting behavior, were investigated in the present study. Anopheles mosquitoes were collected from four study sites representative of four out of five malaria epidemiological zones in Kenya. The collection was within the context of malaria vector surveillance in the country. Mosquitoes were collected indoors using light traps, pyrethrum spray and hand catches between August 2016 and November 2017. Mosquitoes were initially identified using morphological keys and Anopheles gambiae s.l. mosquitoes further identified into sibling species by the Polymerase Chain Reaction method following DNA extraction by alcohol precipitation. Anopheles gambiae s.s. and Anopheles arabiensis were analyzed for the presence of the kdr and ace-1R mutations while 2La inversion was only screened for in An. gambiae s.s. where it is polymorphic. Chi-square statistics were used to determine correlation between the 2La inversion karyotype and kdr mutation. The kdr-east mutation occurred at frequencies ranging between 0.5%-65.6%. The kdr-west mutation was only found in Migori at a total frequency of 5.3% (n=124). No kdr mutants were detected in Tana River. The ace-1R mutation was absent in all populations which implies that there hasn’t been enough buildup of insecticidal pressure from the agricultural and domestic sector to select for this mutation. The observed phenotypic resistance to organophosphates and carbamates in the country could therefore be as a result of other mechanisms of resistance. The 2La chromosomal inversion screened in An. gambiae s.s. occurred at frequencies of 87% (n=30), 80% (n=10) and 52% (n=50) in Baringo, Tana River and Migori, respectively. A significant association between the 2La chromosomal inversion and the kdr mutation was found (Fisher’s exact test statistic value, F=36.967, P =0.000; Likelihood Ratio=33.068, P=0.000). The significant association between the 2La inversion karyotype and kdr mutation suggests that pyrethroid resistant An. gambiae s.s. continue to rest indoors regardless of the presence of treated bed nets and residual sprays, a persistence further substantiated by studies documenting continued mosquito abundance indoors. Behavioral resistance by which Anopheles vectors prefer to exit the indoor environment may therefore not be a factor of concern in our study populations. This therefore means that we can continue deploying interventions indoors. Absence of ace-1R implies that organophosphates and carbamates can be used as alternative insecticides to manage pyrethroid resistance.enThesis