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ABSTRACT Helminths infect humans, livestock, and wildlife, yet remain understudied despite their significant impact on public health and agriculture. Because many of the most prevalent helminth‐borne diseases are zoonotic, understanding helminth transmission among wildlife could improve predictions and management of infection risks across species. A key challenge to understanding helminth transmission dynamics in wildlife is accurately and quantitatively tracking parasite load across hosts and environments. Traditional methods, such as visual parasite identification from environmental samples or infected hosts, are time‐consuming, while standard molecular techniques (e.g., PCR and qPCR) often lack the sensitivity to reliably detect lower parasite burdens. These limitations can underestimate the prevalence and severity of infection, hindering efforts to manage infectious diseases. Here, we developed a multiplexed droplet digital PCR (ddPCR) assay to quantify helminth loads in aquatic habitats using 18S rRNA target genes. UsingSchistocephalus solidusand their copepod hosts as a case study, we demonstrate ddPCR's sensitivity and precision. The assay is highly reproducible, reliably detecting target genes at concentrations as low as 1 pg of DNA in lab standards and field samples (multi‐species and eDNA). Thus, we provide a toolkit for quantifying parasite load in intermediate hosts and monitoring infection dynamics across spatio‐temporal scales in multiple helminth systems of concern for public health, agriculture, and conservation biology. 

Abstract Host populations often vary in the magnitude of coinfection they experience across environmental gradients. Furthermore, coinfection often occurs sequentially, with a second parasite infecting the host after the first has established a primary infection. Because the local environment and interactions between coinfecting parasites can both drive patterns of coinfection, it is important to disentangle the relative contributions of environmental factors and within‐host interactions to patterns of coinfection.Here, we develop a conceptual framework and present an empirical case study to disentangle these facets of coinfection. Across multiple lakes, we surveyed populations of five damselfly (host) species and quantified primary parasitism by aquatic, ectoparasitic water mites and secondary parasitism by terrestrial, endoparasitic gregarines. We first asked if coinfection is predicted by abiotic and biotic factors within the local environment, finding that the probability of coinfection decreased for all host species as pH increased. We then asked if primary infection by aquatic water mites mediated the relationship between pH and secondary infection by terrestrial gregarines.Contrary to our expectations, we found no evidence for a water mite‐mediated relationship between pH and gregarines. Instead, the intensity of gregarine infection correlated solely with the local environment, with the magnitude and direction of these relationships varying among environmental predictors.Our findings emphasize the role of the local environment in shaping infection dynamics that set the stage for coinfection. Although we did not detect within‐host interactions, the approach herein can be applied to other systems to elucidate the nature of interactions between hosts and coinfecting parasites within complex ecological communities. 

Abstract Eco‐evolutionary experiments are typically conducted in semi‐unnatural controlled settings, such as mesocosms; yet inferences about how evolution and ecology interact in the real world would surely benefit from experiments in natural uncontrolled settings. Opportunities for such experiments are rare but do arise in the context of restoration ecology—where different “types” of a given species can be introduced into different “replicate” locations. Designing such experiments requires wrestling with consequential questions. (Q1) Which specific “types” of a focal species should be introduced to the restoration location? (Q2) How many sources of each type should be used—and should they be mixed together? (Q3) Whichspecificsource populations should be used? (Q4) Which type(s) or population(s) should be introduced into which restoration sites? We recently grappled with these questions when designing an eco‐evolutionary experiment with threespine stickleback (Gasterosteus aculeatus) introduced into nine small lakes and ponds on the Kenai Peninsula in Alaska that required restoration. After considering the options at length, we decided to use benthic versus limnetic ecotypes (Q1) to create a mixed group of colonists from four source populations of each ecotype (Q2), where ecotypes were identified based on trophic morphology (Q3), and were then introduced into nine restoration lakes scaled by lake size (Q4). We hope that outlining the alternatives and resulting choices will make the rationales clear for future studies leveraging our experiment, while also proving useful for investigators considering similar experiments in the future.