Bionomics, Insecticide Susceptibility and Characterization of Mid-Gut Micro biota In Anopheles Mosquitoes in Ethiopia

Abstract

Background: This PhD dissertation explores the complex ecology of malaria vectors in Ethiopia, where malaria transmission patterns remain spatially heterogeneous and shaped by the interplay of diverse Anopheles mosquito species with distinct ecological, behavioural, and physiological characteristics, and human interventions.Vector control efforts have been challenged by gaps in knowledge regarding species composition, seasonal dynamics, insecticide resistance, infection rates, and microbiota profiles. Objective: This dissertation aimed to characterize the bionomics, insecticide susceptibility, infection rates, blood meal sources, and midgut microbiota diversity of key Anopheles species vectors across multiple eco-epidemiological settings in Ethiopia. Methods: Data for the overall PhD research were collected between June 2018 and February 2023 across three sites: Lare in Nuwer Zone (Gambella), Asendabo in Jimma Zone (Oromia), and Batu in East Shewa Zone (Oromia). Adult Anopheles mosquitoes were sampled using CDC light traps, human landing catches, and pyrethrum spray catches, while larvae were sampled by dipping from breeding sites. Species identification was performed via morphological keys and species-specific PCR. Plasmodium infection rates were assessed using sporozoite infection rates (SRs), circumsporozoite proteins (CSP-ELISA) and TaqMan qPCR. Insecticide susceptibility was determined using WHO standard bioassays and molecular screening for resistance markers (kdr L1014F/S, ace-1 N485I, CYP6P9a). Midgut microbiota were isolated from aseptically dissected mosquitoes and characterized using culture-based techniques and biochemical assays. Results: A total of over 14,800 Anopheles mosquitoes, representing four species: An. arabiensis, An. pharoensis, An. coustani, and An. funestus were collected. Anopheles coustani was the most abundant species, accounting for more than 40% of the total collection and 42% in Lare. Anopheles pharoensis was the next most common, while An. arabiensis was dominant species in Asendabo and Batu. Seasonal abundance peaked between June and November, except for An. funestus, which was more prevalent during post malaria peak months in the dry season. All An.gambiae s.l. were confirmed as An. arabiensis via PCR while more than 90% An. funestus s.l. from Lare were all confirmed as An. funestus s.s. Despite the absence of insecticide resistance mutations (CYP6P9a, kdr L1014F/S, ace-1 N485I) in An. funestus, phenotypic resistance to pyrethroids and organochlorines was widespread in An. arabiensis, An. pharoensis, and An. coustani. Plasmodium infected mosquitoes were identified across multiple species including, An. funestus, An. arabiensis, An. coustani, and An. pharoensis, with An. funestus showing the highest entomological inoculation IV rate (10.52 bites/person/month), positive for both Plasmodium falciparum and P. vivax. Blood meal analysis confirmed zoophilic tendencies and mixed feeding behaviour with spatio-temporal and species-specific variation. Midgut microbiota analyses revealed 659 bacterial and five fungal isolates from 129 mosquitoes across four species. Eleven bacterial genera were identified with Staphylococcus, Klebsiella, Proteus, and Bacillus dominating. Microbial composition and colonization intensity varied by species, site, life stage, and insecticide exposure status. Remarkably, An. arabiensis survivors of DDT and permethrin exposure (from Batu) harbored higher microbial loads compared to susceptible counterparts, possibly suggesting potential microbiota-mediated resistance mechanisms. Conclusions: This work highlights complex spatiotemporal patterns in Anopheles species composition, insecticide resistance, infection rates, host choices, and midgut microbiota diversity across regions in Ethiopia. The dry-season persistence and infection potential of An. funestus, along with the widespread distribution and insecticide resistance observed in secondary and suspected vectors like An. pharoensis and An. coustani in addition to the primary vector underscore the urgent need for expanded and targeted vector surveillance. Moreover, the association between microbiota diversity and insecticide resistance may offer new insights into vector competence and suggest potential for microbiome-targeted interventions to complement existing malaria control strategies. Sustained integrative entomological and microbiological monitoring is crucial for adaptive and effective vector control in Ethiopia.