Molecular Identification and Characterization of Spiroplasma in Anopheles Arabiensis from Kenya

Abstract

Vector-borne diseases (VBDs) are a devastating global health problem. Mosquitoes are amongst the most important vectors of human VBDs. Several measures have been put in place to manage and eliminate these vectors, however they have all faced a variety of setbacks and raising the need to develop other methods of control. The use of bacterial endosymbionts is a highly promising new method to explore for this purpose. This study aimed to identify and characterize Spiroplasma, a maternally transmitted endosymbiont in Anopheles arabiensis mosquitoes as a candidate to block vector transmission in Africa. The study involved the development and validation of a PCR-based pan-Spiroplasma detection procedure that can be used for the screening of Spiroplasma in other mosquitoes as well as other insects/vectors. The Spiroplasma detection strategy was utilized for the examination of Spiroplasma prevalence in natural Anopheles arabiensis mosquito. Miseq illumina sequencing was used for validation of the developed PCR-based method. Moreover, this study also investigated the diversity and prevalence of microsporidian protozoan parasites in natural Anopheles arabiensis populations. Microsporidia are amongst the most important mosquito parasites that can be transmitted both vertically and horizontally and studying the infection of microsporidia and Spiroplasma has the potential to give insights into the protection of Spiroplasma to the mosquito. Two strains of Spiroplasma were found in one sampling location (Mwea), that is Spiroplasma insolitumtype and Spiroplasma melliferum-type while mosquitoes collected from the other sampling site (Mbita) having no Spiroplasma infection. In Mwea, the Spiroplasma insolitum-type was abundant in females with an overall population prevalence of 2% while the Spiroplasma melliferum-type was found only in males with a prevalence of approximately 7%.In addition, Miseq illumina sequencing results showed the prevalence of Spiroplasma insolitum to be 0.02%. Analysis on the mosquito ND5 mitochondrial DNA (mtDNA) gene showed that the two types of Spiroplasma were evenly distributed with the mtDNA haplotypes. The microsporidia infection rate varied between sites (a range of 9% to 35%). Notably, no samples had a coinfection of Spiroplasma and microsporidia. These results showed two strains of Spiroplasma circulating in the Mwea population with the possibility of being transmitted both horizontally and vertically, lack of coinfection with microsporidia suggested that the Spiroplasma found in mosquitoes confers protection to the mosquito against microsporidia.