Counting rare <i>Wolbachia</i> endosymbionts using digital droplet PCR

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