DNA Barcoding of Trichobilharzia (Trematoda: Schistosomatidae) Species and Their Detection in eDNA Water Samples

Abstract

We designed and tested species-specific PCR primers to detect Trichobilharzia species via environmental DNA (eDNA) barcoding in selected Austrian water bodies. Tests were performed with eDNA samples from the field as well as with artificial samples from the lab, where snails releasing cercariae were kept in aquariums. From two localities, Trichobilharzia was documented based on the release of cercariae from snails, enabling morphological species identification. In both cases, the corresponding species were detected via eDNA: Trichobilharzia szidati and Trichobilharzia physellae. Nonetheless, the stochasticity was high in the replicates. PCR tests with aquarium water into which the cercariae had been released allowed eDNA detection even after 44 days. As in the PCRs with eDNA samples from the field, positive results of these experiments were not obtained for all samples and replicates. PCR sensitivity tests with dilution series of T. szidati genomic DNA as well as of PCR amplification products yielded successful amplification down to concentrations of 0.83 pg/µL and 0.008 pg/µL, respectively. Our results indicate that the presumed species specificity of PCR primers may not be guaranteed, even if primers were designed for specific species. This entails misidentification risks, particularly in areas with incomplete species inventories.

Molecular identification of Trichobilharzia species in recreational waters in North-Eastern Poland

“…Most avian schistosomes reported in European water bodies represented either of the two genera, Trichobilharzia or Bilharziella ( Helmer et al, 2023 ; Lashaki et al, 2020 ). Generally, the prevalence of infections caused by Trichobilharzia species in intermediate hosts is not high and does not exceed a few percent.…”

“…In Europe, Trichobilharzia spp. have, among others, been reported in Lymnaea stagnalis (Linnaeus, 1758), Stagnicola palustris (Müller, 1774), Radix auricularia (Linnaeus, 1758), R. lagotis (Schrank, 1803), R. ampla (Hartmann, 1821), R. peregra (Müller, 1774) (synonymous with R. ovata (Draparnaud, 1805), R. balthica (Linnaeus, 1758) and R. labiata (Rossmässler, 1835)) as well as Physella acuta (Draparnaud, 1805) ( Horák, Kolářová & Adema, 2002 ; Helmer et al, 2023 ; Bargues et al, 2001 ). Definitive hosts, in turn, are some waterfowl species.…”

“…First, the snails should be collected and screened for cercariae, which may be responsible for skin lesions. Alternatively, the cercariae or their e-DNA can be collected and concentrated directly from water samples ( Helmer et al, 2023 ; Rudko et al, 2019 ). Other detection methods include the analysis of bird droppings for parasite eggs and autopsies of birds ( Horák et al, 2015 ).…”

Underreported and taxonomically problematic: characterization of sanguinicolid larvae from freshwater limpets (Burnupiidae), with comments on the phylogeny and intermediate hosts of sanguinicolids

“…Soft tissues of the snails (that harboured parasites) were preserved in 96% ethanol for genetic identification. For morphometric analyses, sporocysts and 20 cercariae of each morphotype were processed following the procedures of Helmer et al (2023). Representative morphotypes were studied using a Zeiss Axioplan 2 epifluorescence microscope and measurements were obtained using AxioVision 4.3 imaging software (Göttingen, Germany).…”

DNA Barcodes for Evolution and Biodiversity

“…The monitoring of pathogens and parasites to identify high-risk-infection areas, enabling disease control, is also facilitated by eDNA methods [33,34]. A study included in this Special Issue [35] provides reference DNA barcodes for the Austrian avian schistosomes of the genus Trichobilharzia. Based on these data, an eDNA-based PCR assay was developed to identify Trichobilharzia in water samples.…”