Novel Wolbachia strains in Anopheles malaria vectors from Sub-Saharan Africa

Abstract

<b>Background: </b> <i>Wolbachia</i>, a common insect endosymbiotic bacterium that can influence pathogen transmission and manipulate host reproduction, has historically been considered absent from the <i>Anopheles (An.) </i>genera, but has recently been found in <i>An. gambiae </i>s.l. populations in West Africa. As there are numerous <i>Anopheles </i>species that have the capacity to transmit malaria, we analysed a range of species across five malaria endemic countries to determine <i>Wolbachia </i>prevalence rates, characterise novel <i>Wolbachia </i>strains and determine any correlation between the presence of <i>Plasmodium</i>, <i>Wolbachia</i> and the competing bacterium <i>Asaia</i>. <b>Methods: </b> <i>Anopheles</i> adult mosquitoes were collected from five malaria-endemic countries: Guinea, Democratic Republic of the Congo (DRC), Ghana, Uganda and Madagascar, between 2013 and 2017. Molecular analysis was undertaken using quantitative PCR, Sanger sequencing, <i>Wolbachia </i>multilocus sequence typing (MLST) and high-throughput amplicon sequencing of the bacterial <i>16S rRNA</i> gene. <b>Results</b>: Novel <i>Wolbachia </i>strains were discovered in five species: <i>An. coluzzii</i>, <i>An. gambiae </i>s.s., <i>An. arabiensis</i>, <i>An. moucheti </i>and <i>An.</i> species A, increasing the number of <i>Anopheles </i>species known to be naturally infected. Variable prevalence rates in different locations were observed and novel strains were phylogenetically diverse, clustering with <i>Wolbachia </i>supergroup B strains. We also provide evidence for resident strain variants within <i>An</i>. species A. <i>Wolbachia</i> is the dominant member of the microbiome in <i>An. moucheti </i>and <i>An.</i> species A but present at lower densities in <i>An. coluzzii</i>. Interestingly, no evidence of <i>Wolbachia/Asaia </i>co-infections was seen and <i>Asaia</i> infection densities were shown to be variable and location dependent. <b>Conclusions: </b>The important discovery of novel <i>Wolbachia </i>strains in <i>Anopheles </i>provides greater insight into the prevalence of resident <i>Wolbachia </i>strains in diverse malaria vectors. Novel <i>Wolbachia </i>strains (particularly high-density strains) are ideal candidate strains for transinfection to create stable infections in other <i>Anopheles </i>mosquito species, which could be used for population replacement or suppression control strategies.