Assessing the Use of Oxford Nanopore Technologies Sequencing for Detection of Stable Wolbachia Bacterial Strain in Symbiosis in Wild Anopheles Mosquitoes From the WHO African Region

ABSTRACT Malaria remains a significant global health challenge with 263 million cases in 2023. Anopheles insecticide resistance threatens current vector control methods, and novel strategies are crucial to combat malaria transmission. Wolbachia , an endosymbiotic bacteria that can invade mosquito populations and inhibit pathogen replication, has only recently been shown as a high‐density symbiont in two wild Anopheles species. Detection of Wolbachia in symbiosis with Anopheles mosquitoes has relied on PCR amplification of Wolbachia genes and/or Sanger and Illumina sequencing. This study assesses Oxford Nanopore Technology (ONT) to detect Wolbachia , which could facilitate screening for symbiosis with wild Anopheles mosquitoes in endemic settings. We used 46 samples; 10 were pooled for a single MinION flow cell run and the rest processed individually using Flongle flow cells, comparing three mosquito species with three kit combinations. Using the ONT Field sequencing kit, there were 13X more Wolbachia reads in An. demeilloni (genuine symbiosis) than An. gambiae (no evidence of Wolbachia ). The ONT 16S barcoding kit and Rapid barcoding kit were then assessed using more economical Flongle flow cells. Wolbachia was successfully identified in An . demeilloni and An. moucheti but not in An. gambiae . This study demonstrates the feasibility of ONT for field‐based detection of Wolbachia .