Interactions among symbiotic organisms and their hosts are major drivers of ecological and evolutionary processes. Monitoring the infection patterns among natural populations and identifying factors affecting these interactions is critical for understanding symbiont-host relationships. However, many of these interactions remain understudied since the knowledge about the symbiont species is lacking and hinders the development of appropriate tools. In this study, we developed a digital droplet PCR (ddPCR) assay based on apicomplexan COX1 gene to detect an undescribed agamococcidian symbiont. We show that the method gives precise and reproducible results and enables detecting cryptic symbionts in low target concentration. We further exemplify the assay’s use to survey seasonally sampled natural host (Pygospio elegans) populations for symbiont infection dynamics. We found that symbiont prevalence differs spatially but does not show seasonal changes. Infection load differed between populations and was low in spring and significantly increased towards fall in all populations. We also found that the symbiont prevalence is affected by host length and population density. Larger hosts were more likely to be infected and high host densities were found to have lower probability of infection. The observed variations could be due to characteristics of both symbiont and host biology, especially the seasonal variation in encounter rates. Our findings show that the developed ddPCR assay is a robust tool for detecting undescribed symbionts that are otherwise difficult to quantify, enabling further insight into the impact cryptic symbionts have on their hosts.

Molecular detection of agamococcidian COX1 gene was done using Bio-Rad's QX200 droplet digital PCR system with QX Eva Green chemitsry from whole host DNA samples. Infection load is calculated in number of copies/ ng of host DNA, infection status is defined infected (= 1), if more than 0 copies/ ng of host DNA is detected, and non-infected (= 0) otherwise.

Environmental data (temperature, salinity, and sediment characteristics), host length and density is from original paper: Thonig A., Knott K.E., Kesäniemi J.E., Winding Hansen B. & Banta G.T. 2016. Population and reproductive dynamics of the polychaete Pygospio elegans in a boreal estuary complex. Invertebrate Biology, 135(4): 370–384.

Missing values are marked as NA.

Environmental data and host density is missing from October and host length from 2 individuals.