AUTHOR=Reyes Jerica Isabel L. , Suzuki Takahiro , Suzuki Yasutsugu , Watanabe Kozo 

TITLE=Detection and quantification of natural Wolbachia in Aedes aegypti in Metropolitan Manila, Philippines using locally designed primers

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=14

YEAR=2024

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2024.1360438

DOI=10.3389/fcimb.2024.1360438

ISSN=2235-2988

ABSTRACT=<sec><title>Background</title><p>The Philippines bears health and economic burden caused by high dengue cases annually. Presently, the Philippines still lack an effective and sustainable vector management. The use of <italic>Wolbachia</italic>, a maternally transmitted bacterium, that mitigate arbovirus transmission has been recommended. Cytoplasmic incompatibility and viral blocking, two characteristics that make <italic>Wolbachia</italic> suitable for vector control, depend on infection prevalence and density. There are no current <italic>Wolbachia</italic> release programs in the Philippines, and studies regarding the safety of this intervention. Here, we screened for <italic>Wolbachia</italic> in <italic>Aedes aegypti</italic> collected from Metropolitan Manila, Philippines. We designed location-specific primers for qPCR to test whether this improved <italic>Wolbachia</italic> detection in <italic>Ae. aegypti.</italic> We explored if host sex and <italic>Wolbachia</italic> strain could be potential factors affecting <italic>Wolbachia</italic> density.</p></sec><sec><title>Methods</title><p><italic>Ae. aegypti</italic> mosquitoes (n=429) were screened for natural <italic>Wolbachia</italic> by taqman qPCR using location-specific <italic>Wolbachia</italic> surface protein primers (<italic>wsp</italic>AAML) and known <italic>16S rRNA</italic> primers. Samples positive for <italic>wsp</italic>AAML (n=267) were processed for Sanger sequencing. We constructed a phylogenetic tree using IQ-TREE 2 to further characterize <italic>Wolbachia</italic> present in the Philippine <italic>Ae. aegypti</italic>. We then compared <italic>Wolbachia</italic> densities between <italic>Wolbachia</italic> groups and host sex. Statistical analyses were done using GraphPad Prism 9.0.</p></sec><sec><title>Results</title><p><italic>Wolbachia</italic> prevalence for 16S rRNA (40%) and <italic>wsp</italic>AAML (62%) markers were high. <italic>Wolbachia</italic> relative densities for 16S rRNA ranged from −3.84 to 2.71 and <italic>wsp</italic>AAML from −4.02 to 1.81. Densities were higher in male than female mosquitoes. <italic>Wolbachia</italic> strains detected in <italic>Ae. aegypti</italic> clustered into supergroup B. Some 54% (123/226) of these sequences clustered under a group referred to here as “wAegML,” that belongs to the supergroup B, which had a significantly lower density than wAegB/<italic>w</italic>AlbB, and <italic>w</italic>AlbA strains.</p></sec><sec><title>Conclusion</title><p>Location-specific primers improved detection of natural <italic>Wolbachia</italic> in <italic>Ae. aegypti</italic> and allowed for relative quantification. <italic>Wolbachia</italic> density is relatively low, and differed between host sexes and <italic>Wolbachia</italic> strains. An economical way of confirming sporadic or transient <italic>Wolbachia</italic> in <italic>Ae. aegypti</italic> is necessary while considering host sex and bacterial strain.</p></sec>