Quantifying larval trematode infections in hosts: A comparison of method validity and implications for infection...

Accurately estimating parasite transmission success and subsequent infection load has important ramifications for a wide range of disease-related questions and research disciplines. Recent interest in the role of parasites in amphibian population declines and deformities, for instance, has prompted increased interest in approaches to quantify infection and pathology. Here, we introduce a novel method of fluorescently labeling trematode cercariae and optimize its application to interactions between the pathogenic trematode Ribeiroia ondatrae and amphibian hosts (Lithobates sphenocephalus). We then compare the efficacy of this method with two other approaches commonly used to assess infection in second intermediate hosts – necropsy and tissue clearing – with a focus on accuracy, precision, and bias. Dye (Invitrogen, BODIPY® FL C12) concentrations of <200 nM and DMSO solvent concentrations <0.2% offered a highly visible, long-lasting marker with no detectable effects on cercariae survival or metacercariae establishment. Among methods, the necropsy approach yielded the highest proportion of detected parasites and the lowest standard error around the mean. However, the fluorescent labeling method offered highly similar results (r = 0.99), with an estimated 75% of administered parasites establishing successfully. At low to moderate parasite exposure dosages, tissue clearing was comparable to the other two methods, but tended to underpredict infection at the higher exposures (i.e., 'proportional bias'), likely because individual parasites became more difficult to distinguish. Conceptually, these findings illustrate that initial infection within amphibian hosts is a consistent, linear function of exposure dosage, suggesting that parasite density dependence does not regulate initial establishment. From an applied standpoint, our results offer a methodological foundation for subsequent research using fluorescently labeled parasites, which offer distinct advantages such as allowing in vivo parasite tracking within living hosts.