Optimizing bacterial enrichment procedures to improve mosquito midgut microbiome research

Abstract Mosquitoes are major vectors of infectious diseases, with their midgut microbiota playing a crucial role in host development, reproduction, and pathogen interactions. However, characterizing midgut microbial communities remains challenging because host DNA often dominates metagenomic libraries, reducing the resolution of bacterial community profiling and hindering the recovery of microbial genomes. To evaluate strategies for bacterial DNA enrichment in this context, we compared three DNA extraction protocols applied to pooled Aedes aegypti midguts: (i) the QIAGEN DNeasy Blood and Tissue kit without filtration (DNeasy NF), (ii) the same kit preceded by a 5 μm filtration step to reduce eukaryotic material (DNeasy F), and (iii) the QIAGEN QIAamp Microbiome kit, which combines differential host cell lysis and enzymatic DNA depletion. Each method was assessed using qPCR-based quantification of bacterial and host DNA, SSU rRNA-based taxonomic profiling of metagenomic reads, and Metagenome-Assembled Genomes (MAGs) reconstruction using advanced binning approaches. Our results show that while the Microbiome kit provided the highest bacterial enrichment and captured a large fraction (> 85%) of the bacterial taxa detected with the other kits, it yielded a higher relative abundance of Gammaproteobacteria . This occurred without a loss of overall diversity, suggesting improved recovery of microbial taxa rather than a compositional bias. The filtration step associated with the DNeasy kit significantly reduced host DNA but also lowered bacterial material, allowing MAG reconstruction, though less efficiently than the Microbiome kit. Despite this, the DNeasy methods allowed recovering a broader range of bacterial phyla with a more even distribution across taxa, while each extraction method also detected a unique set of NTUs. Overall, a combination of DNA extraction methods may be necessary to optimize microbiome analysis in mosquito midguts, facilitating a more comprehensive understanding of microbial diversity and its potential applications in vector control strategies.