Counting rare Wolbachia endosymbionts using digital droplet PCR

Wolbachia is the most widespread animal-associated intracellular microbe, living within the cells of over half of insect species. Since they can suppress pathogen replication and spread rapidly through insect populations, Wolbachia is at the vanguard of public health initiatives to control mosquito-borne diseases. Wolbachia's abilities to block pathogens and spread quickly are closely linked to their abundance in host tissues. The most common method for counting Wolbachia is quantitative polymerase chain reaction (qPCR), yet qPCR can be insufficient to count rare Wolbachia, necessitating tissue pooling and consequently compromising individual-level resolution of Wolbachia dynamics. Digital droplet PCR (ddPCR) offers superior sensitivity, enabling the detection of rare targets and eliminating the need for sample pooling. Here, we report three ddPCR assays to measure total Wolbachia abundance, Wolbachia abundance adjusted for DNA extraction efficiency, and Wolbachia density relative to host genome copies. Using Drosophila melanogaster with wMel Wolbachia as a model, we show these ddPCR assays can reliably detect as few as 7 to 12 Wolbachia gene copies in a 20 µL reaction. The designed oligos are homologous to sequences from at least 106 Wolbachia strains across supergroup A and 53 host species from the Drosophila, Scaptomyza, and Zaprionus genera, suggesting broad utility. These highly sensitive ddPCR assays are expected to significantly advance Wolbachia-host interactions research by enabling the collection of molecular data from individual insect tissues. Their ability to detect rare Wolbachia will be especially valuable in applied and natural field settings where pooling samples could obscure important variation.IMPORTANCEWolbachia bacteria live inside the cells of many animals, especially insects. In many insect species, almost every individual carries Wolbachia. How common Wolbachia becomes within a population often depends on how much of it is present in the insect's body. Therefore, accurately measuring Wolbachia levels is crucial for understanding how these bacteria interact with their hosts and spread. However, traditional molecular assays can lack the sensitivity needed for accurate, individual-level quantification of rare Wolbachia. Here, we present three highly sensitive digital droplet PCR assays for Wolbachia detection, offering superior sensitivity compared to existing methods. These assays will be useful for studies that measure Wolbachia abundance and related phenotypes in individual insects, providing enhanced resolution and improving efforts to characterize the mechanisms that govern phenotypic variation.