1. DNA barcoding of the mitochondrial cytochrome c oxidase I (COI) gene is widely used to characterise insect diversity and distributions; however, its potential to reveal information on species interactions, including host-symbiont' associations, remains largely unexplored. Here, we assess whether COI amplicon data can be used to identify Wolbachia - one of the most widely distributed bacterial symbionts known to profoundly affect their hosts biology. 2. We demonstrate that several commonly used invertebrate COI primer sets perfectly match many reference Wolbachia genomes, leading to frequent co-amplification. 3. By screening 7,901 individual-insect COI amplicon libraries obtained with the popular BF3-BR2 primer set, we detected Wolbachia sequences in over 35% of samples, revealing that co-amplification is indeed widespread. After removing low-abundance reads, Wolbachia detection based on COI amplicons showed over 90% agreement with simultaneously generated 16S-V4 rRNA amplicon data from the same specimens. The degree of agreement, however, varied depending on the thresholds used, among datasets and insect clades. 4. Further, we show that Wolbachia abundance inferred from COI amplicons correlated with their abundance in metagenomic datasets for 152 specimens, supporting the quantitative relevance of the signal. 5. Finally, we find that Wolbachia COI sequences provide greater phylogenetic resolution than 16S-V4 rRNA data (mean pairwise genetic distance of COI sequences - 9.6%, 16S-V4 rRNA - 2.8%), and the reconstructed Wolbachia COI-based genotype network largely agrees with genome-based phylogenies. Collectively, our results demonstrate that off-target Wolbachia sequences recovered from standard insect COI barcoding data may reliably detect symbiont presence, provide phylogenetic insight, and guide sample selection for metagenomics. Given the rapid expansion of global insect barcoding initiatives, these findings highlight an opportunity for cost-effective monitoring of their most prevalent bacterial symbionts, offering new perspectives on how host-microbe interactions may shape insect communities.
Nowak, K. H., Buczek, M., Marszałek, M., Prus-Frankowska, M., Valdivia, C., Deng, J., Shropshire, J. D., Łukasik, P.
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