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.
Description
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