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

Global climate change is expected to alter patterns of temperature variability, which could influence species interactions including parasitism. Species interactions can be difficult to predict in variable-temperature environments because of thermal acclimation responses, i.e. physiological changes that allow organisms to adjust to a new temperature following a temperature shift. The goal of this study was to determine how thermal acclimation influences host resistance to infection and to test for parasite acclimation responses, which might differ from host responses in important ways. We tested predictions of three, non-mutually exclusive hypotheses regarding thermal acclimation effects on infection of green frog tadpoles (Lithobates clamitans) by the trematode parasite Ribeiroia ondatrae with fully replicated controlled-temperature experiments. Trematodes or tadpoles were independently acclimated to a range of 'acclimation temperatures' prior to shifting them to new 'performance temperatures' for experimental infections. Trematodes that were acclimated to intermediate temperatures (19-22 °C) had greater encystment success across temperatures than either cold- or warm-acclimated trematodes. However, host acclimation responses varied depending on the stage of infection (encystment vs. clearance): warm- (22-28 °C) and cold-acclimated (13-19 °C) tadpoles had fewer parasites encyst at warm and cold performance temperatures, respectively, whereas intermediate-acclimated tadpoles (19-25 °C) cleared the greatest proportion of parasites in the week following exposure. These results suggest that tadpoles use different immune mechanisms to resist different stages of trematode infection, and that each set of mechanisms has unique responses to temperature variability. Our results highlight the importance of considering thermal responses of both parasites and hosts when predicting disease patterns in variable-temperature environments.

Role of Antimicrobial Peptides in Amphibian Defense Against Trematode Infection.

Antimicrobial peptides (AMPs) contribute to the immune defenses of many vertebrates, including amphibians. As larvae, amphibians are often exposed to the infectious stages of trematode parasites, many of which must penetrate the host's skin, potentially interacting with host AMPs. We tested the effects of the natural AMPs repertoires on both the survival of trematode infectious stages as well as their ability to infect larval amphibians. All five trematode species exhibited decreased survival of cercariae in response to higher concentrations of adult bullfrog AMPs, but no effect when exposed to AMPs from larval bullfrogs. Similarly, the use of norepinephrine to remove AMPs from larval bullfrogs, Pacific chorus frogs, and gray treefrogs had only weak (gray treefrogs) or non-significant (other tested species) effects on infection success by Ribeiroia ondatrae. We nonetheless observed strong differences in parasite infection as a function of both host stage (first- versus second-year bullfrogs) and host species (Pacific chorus frogs versus gray treefrogs) that were apparently unrelated to AMPs. Taken together, our results suggest that AMPs do not play a significant role in defending larval amphibians against trematode cercariae, but that they could be one mechanism helping to prevent infection of post-metamorphic amphibians, particularly for highly aquatic species.

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

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 diversity jointly control disease risk in complex communities.

Host-parasite interactions are embedded within complex communities composed of multiple host species and a cryptic assemblage of other parasites. To date, however, surprisingly few studies have explored the joint effects of host and parasite richness on disease risk, despite growing interest in the diversity-disease relationship. Here, we combined field surveys and mechanistic experiments to test how transmission of the virulent trematode Ribeiroia ondatrae was affected by the diversity of both amphibian hosts and coinfecting parasites. Within natural wetlands, host and parasite species richness correlated positively, consistent with theoretical predictions. Among sites that supported Ribeiroia, however, host and parasite richness interacted to negatively affect Ribeiroia transmission between its snail and amphibian hosts, particularly in species-poor assemblages. In laboratory and outdoor experiments designed to decouple the relative contributions of host and parasite diversity, increases in host richness decreased Ribeiroia infection by 11-65%. Host richness also tended to decrease total infections by other parasite species (four of six instances), such that more diverse host assemblages exhibited ∼40% fewer infections overall. Importantly, parasite richness further reduced both per capita and total Ribeiroia infection by 15-20%, possibly owing to intrahost competition among coinfecting species. These findings provide evidence that parasitic and free-living diversity jointly regulate disease risk, help to resolve apparent contradictions in the diversity-disease relationship, and emphasize the challenges of integrating research on coinfection and host heterogeneity to develop a community ecology-based approach to infectious diseases.

Experimental infection dynamics: using immunosuppression and in vivo parasite tracking to understand host resistance in an amphibian-trematode system.

Although naturally occurring hosts often exhibit pronounced differences in infection and pathology, the relative importance of factors associated with host life history and immunity in explaining such patterns often remains speculative. Research in eco-immunology highlights the trade-offs between host physiology and immunity, for which natural variations in disease susceptibility offer a valuable platform to test predictions within this framework. Here, we combined use of a novel, in vivo assay for tracking parasite fate and an experimental manipulation of host immune function (via chronic corticosterone exposure) to assess the role of host immunity in regulating susceptibility of amphibian hosts to three larval trematodes: Ribeiroia ondatrae, Echinostoma trivolvis and Alaria sp. 2. Results from the in vivo parasite-tracking assay revealed marked differences in initial parasite penetration and subsequent host clearance. Relative to infections in a highly susceptible species (Pseudacris regilla), the virulent trematode R. ondatrae was -25% less successful at penetrating larvae of three hylid frog species and was cleared > 45(×) faster, such that all parasites were rapidly cleared from hylid hosts over 72 h following a Weibull distribution. Immune suppression of Hyla versicolor sharply reduced this resistance and increased infection of all three trematodes by 67 to 190%, with particularly strong increases for R. ondatrae. Diminished resistance correlated with a 62% decrease in circulating eosinophils. Correspondingly, 10 days after corticosterone exposures ended, infections declined dramatically while eosinophil levels returned to normal. In light of ongoing declines and deformities in amphibian populations, these findings have application potential for mitigating disease-driven effects.