Assessment of Physico-Chemical Water Parameters, Habitat Characteristics, and Bacterial Influence on Anopheles Funestus Mosquito Larvae in Fihoni, Kwale County

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Date
2024-08
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Kenyatta University
Abstract
Anopheles funestus is a primary malaria vector yet remains understudied in East Africa. Resistance to available control measures has led to increased prevalence of the vector. Studies have revealed that chemical cues mediating oviposition attraction may be associated with bacterial communities. However, the bacteria responsible have not been profiled and characterized. This study, therefore, provides missing information on bacterial species associated with oviposition attraction in Anopheles funestus. This study aimed to isolate and identify bacteria isolates from the larval and non-larval sites of A. funestus, characterize the larval habitats using physiochemical parameters, and then conduct a bioassay-guided determination of oviposition attraction by A. funestus. Larval habitats were characterized using an estimation method on habitat parameters like size, distance from homestead, and water depth, among others. Physiochemical parameters of the larval water were determined using multi-parameter equipment and other standard laboratory methods. The experiment used two larval water samples (obtained from mixing samples from the four different transects in Fihoni) categorized as either preferred or non-preferred. Bacteria were isolated from the preferred and non-preferred larval water and grown on TSA agar. Isolates were then characterized using morphological, biochemical, and molecular techniques. 16S rRNA gene was used in amplification and Sanger sequencing. Bacterial primer pairs (27F and 1492R) were used in sequencing. Data on the number of A. funestus and non-A. funestus larvae were compared using the Mann-Whitney U test in Scientific Analytical System (SAS) version 9.4, and results were presented in table format. Data on counts of oviposition due to inoculation of substrate (Media) by different bacteria isolates was analyzed by Kruskal Wallis test in SAS version 9.4. Data on the association between habitat parameters and the number of larvae was analyzed using chi-square (α = 0.05) and Pearson correlation coefficient in SAS version 9.4. Significance medians were compared using Steel-Dwass-Critchlow-Fligner in SAS version 9.4. Phylogenetic analysis was conducted on MEGA X version 11.0, and alignment was done using the MUSCLE program in MEGA X version 11.0. Conductivity, total dissolved solids, pH, sulfate, conductivity, and Biological Oxygen Demand had an effect (p<0.0001) on the presence of Anopheles and non-Anopheles funestus larvae at the oviposition sites. Phylogenetic analysis shows that the bacterial isolates had a sequence identity between 82.05% and 96.56%. They included Staphylococcus saprophyticus, Enterobacter sp strain EDB2, Uncultured actinobacterium clone SGR 192, Enterobacter sp MLB, and Citrobacter werkmanii which attracted A. funestus to their oviposition cups. The vector's attraction rate to the preferred oviposition cup was stronger (median=52.00) with crude bacterial isolates than with pure isolates (median=38.00). Bacteria (Enterobacter sp strain EDB2 and Enterobacter sp MLB) synergistically increased oviposition attraction. The rate of attraction was substantially increased from (median= 23.00 for CFI3 [Pluralibacter sp] and median=35.00 for CPIp [Bacillus sp]) when isolates were pure to (median =123.00 for mixture M) when isolates were mixed. No oviposition (median=0.00) was seen in cups amended with Pluralibacter gergoviae, Citrobacter sp, and Enterobacter sp MLB07, among others, when used individually. There are no sequence differences between CPI7 [Enterobacter sp. strain EDB2], CNPI3 [Pluralibacter gergoviae], and CPI5 [Pluralibacter gergoviae]. CFI4 [Uncultured actinobacterium] was most attractive (median=58.00), followed by CPI11 [Enterobacter sp] (median=57.00). The present study shows the ability of some bacterial isolates from A. funestus larval sites to influence oviposition site preference. Information on bacteria isolates with positive oviposition response adds knowledge crucial for researchers and policymakers dealing with malaria control. For example, government agencies concerned with public health could use the oviposition attraction of mosquitoes by bacteria to reduce human mosquito contact and the spread of malaria.
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Supervisor: 1.James Nonoh 2.Regina Ntabo A Thesis Submitted to the Department of Biochemistry, Microbiology and Biotechnology in the School of Pure and Applied Science in Partial Fulfillment of the Requirements for the Award of the Degree of Master of Science (Microbiology) of Kenyatta University August, 2024
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