Abstract
Identifying factors influencing infection patterns among hosts is critical for our understanding of the evolution and impact of parasitism in natural populations. However, the correct estimation of infection parameters depends on the performance of detection and quantification methods. In this study, we designed a quantitative PCR (qPCR) assay targeting the 18 S rRNA gene to estimate prevalence and intensity of Hepatozoon infection and compared its performance with microscopy and PCR. Using qPCR, we also compared various protocols that differ in the biological source and the extraction methods. Our results show that the qPCR approach on DNA extracted from blood samples, regardless of the extraction protocol, provided the most sensitive estimates of Hepatozoon infection parameters; while allowed us to differentiate between mixed infections of Adeleorinid (Hepatozoon) and Eimeriorinid (Schellackia and Lankesterella), based on the analysis of melting curves. We also show that tissue and saline methods can be used as low-cost alternatives in parasitological studies. The next step was to test our qPCR assay in a biological context, and for this purpose we investigated infection patterns between two sympatric lacertid species, which are naturally infected with apicomplexan hemoparasites, such as the genera Schellackia (Eimeriorina) and Hepatozoon (Adeleorina). From a biological standpoint, we found a positive correlation between Hepatozoon intensity of infection and host body size within each host species, being significantly higher in males, and higher in the smaller sized host species. These variations can be associated with a number of host intrinsic factors, like hormonal and immunological traits, that require further investigation. Our findings are relevant as they pinpoint the importance of accounting for methodological issues to better estimate infection in parasitological studies, and illustrate how between-host factors can influence parasite distributions in sympatric natural populations.
Highlights
Parasites can be major drivers of host ecology and evolution and play key roles in ecosystem functioning and structure
Results quantitative PCR (qPCR) assay validation Intensity of Hepatozoon infection estimated by microscopy and qPCR were significantly correlated (r = 0.893, P,0.001), with qPCR being much more sensitive and detecting as few as 5 copies of parasite DNA
Sequences retrieved from qPCR amplifications were BLASTed on GenBank
Summary
Parasites can be major drivers of host ecology and evolution and play key roles in ecosystem functioning and structure. The estimation of biological relevant and realistic infection patterns is constrained by differences in the accuracy, specificity and sensitivity of the detection and quantification protocols used [14,15]. The use of less accurate protocols may lead to erroneous ecological and epidemiological inferences, which is critical in host-parasite systems with low intensity levels or with mixed parasite infections. Quantitative PCR (qPCR) has been used in parasitological studies as it allows the simultaneous detection and quantification of parasite DNA from various biological sources, such as host and vector blood and tissues [16,17,18,19,20]. Compared to more traditional approaches, such as microscopy or conventional PCR (PCR), qPCR has increased accuracy and sensitivity of detection [21,22]
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