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

Abstract

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.

Host and parasite thermal acclimation responses depend on the stage of infection

“…; LaFonte & Johnson ; LaFonte et al . ). Within 5 h of emergence, groups of 30–40 cercariae were exposed to a 100 nM dye solution (made by adding 10 μL of a dye‐dimethyl sulfoxide mixture to 10 mL of water) for 45 min, taking care to maintain cercariae at the appropriate temperature.…”

“…Because tadpoles can clear R. ondatrae metacercariae post‐encystment (LaFonte & Johnson ; LaFonte et al . ), R. ondatrae cercariae were tagged with a fluorescent dye to allow for tracking the clearance of trematode metacercariae through time (described below). To provide time for this additional data collection, this experiment was conducted in six temporal blocks, with one replicate per treatment combination in each block.…”

“…To allow in vivo tracking of R. ondatrae metacercariae, cercariae were labelled with a green fluorescent fatty acid analogue (BOD-IPY FL C12; Invitrogen, Carlsbad, CA, USA) prior to exposing cercariae to tadpoles (Keeney et al 2008;LaFonte & Johnson 2013;LaFonte et al 2015). Within 5 h of emergence, groups of 30-40 cercariae were exposed to a 100 nM dye solution (made by Fig.…”

Experimental investigation of alternative transmission functions: Quantitative evidence for the importance of nonlinear transmission dynamics in host–parasite systems

“…We did not account for parasites that penetrated the host but failed to successfully infect (“irreversible contact,” Civitello et al., ), but previous research found that (1) the proportion of R. ondatrae cercariae successfully forming metacercariae was consistent across exposure levels, and (2) the loss or clearance of established metacercariae within P. regilla hosts is minimal over the time span of the current study (LaFonte & Johnson, ). The necropsy methods used here have high accuracy and precision in detecting infections, such that it is further unlikely that encysted parasites were not counted (LaFonte, Raffel, Monk, & Johnson, ).…”

In vivo fluorescent cercariae reveal the entry portals of Cardiocephaloides longicollis (Rudolphi, 1819) Dubois, 1982 (Strigeidae) into the gilthead seabream Sparus aurata L.

“…In vivo fluorescent dyes can be an adequate tool to study cercarial penetration patterns into fish. This method has been used with platyhelminths for several applications, including post-infection identification of trematode genetic clones [4], lipid utilization in S. mansoni [18], individual cestode tracking within intermediate host [19] and quantification of trematode larval infection success [20]. The dyes suggested in this study have been previously employed to reveal, in particular, attachment, distribution and development of monogenean larvae on fish (CFSE, [21, 22]), to localize protein kinase C in cercariae (CFSE, [23]) and to discriminate live and dead S. mansoni eggs (NB, [24]) and S. mansoni cercariae exposed to human serum (NB, [25]).…”