Discovery of prevalent natural Wolbachia in Aedes aegypti in Metropolitan Manila, Philippines using locally designed primers: bacterial density is influenced by strain and host sex

Wolbachia are maternally transmitted bacteria that are utilized for arboviral disease prevention. Cytoplasmic incompatibility (CI) and viral blocking, two characteristics that make Wolbachia suitable for vector control, both depend on bacterial infection prevalence and density. Although numerous mosquito species naturally harbor Wolbachia, the prevalence of Wolbachia in Aedes aegypti varies from minimal to absent. The absence of natural Wolbachia in Ae. aegypti is considered advantageous for novel Wolbachia transinfection because interference caused by incompatible Wolbachia strains will not occur. In this study, we screened for Wolbachia density in 429 individual Ae. aegypti collected from Metropolitan Manila, Philippines using locally designed primers to determine whether primer compatibility improves Wolbachia detection in naturally infected Ae. aegypti. We also investigated the effects of host sex and Wolbachia strain on Wolbachia density. We found a high Wolbachia prevalence for 16S rRNA (40%) and wsp (62%) markers. Relative Wolbachia densities ranged from -3.840 to 2.710 for 16S rRNA and from -4.020 to 1.810 for wsp, and these relative densities were higher among male mosquitoes than female mosquitoes. Phylogenetic analysis revealed that most of the Ae. aegypti were clustered into supergroup B, with 63% (142/226) of these sequences representing a unique strain referred to here as "wAegML," which exhibited a significantly lower density in Ae. aegypti than the other two Wolbachia strains detected (wAegB/wAlbB and wAlbA) as well as a higher density in male Ae. aegypti than in females. Overall, locally designed primers improved Wolbachia detection in naturally infected Ae. aegypti. The unique strain wAegML occurs at a low density and is related to the use of wAlbB in mass-release programs; therefore, it is unlikely to interfere with such programs. Further studies on natural Wolbachia infection in Ae. aegypti combining methodological and biological factors are warranted to help resolve current conflicts in the field.