A multi-source approach for the detection and diversity assessment of avian schistosomes (Trematoda, Schistosomatidae) in freshwater ecosystems

Avian schistosomes (AS) are trematodes of significant medical importance as the causative agents of cercarial dermatitis, a re-emerging waterborne human disease. Their monitoring has traditionally relied on examination of snail hosts; however, this approach is time-consuming, requires taxonomic expertise and often underestimates their diversity due to low prevalence of infection. Although environmental DNA (eDNA)-based methods have shown promise in detecting AS, most studies have employed species-specific assays that restrict the discovery of novel genetic lineages. This study integrated three complementary methodologies to comprehensively assess AS diversity in Central Europe: (i) metabarcoding of environmental water samples from various localities; (ii) metabarcoding of naturally infected snail hosts to detect pre-patent stages and (iii) standard parasitological examination and collection of cercariae released from snail hosts. In total, 34 species/genetic lineages were detected at 19 localities. Of these, three species were identified by all three techniques, two lineages by both water sample and snail tissue metabarcoding; 17 exclusively through water sample metabarcoding, 10 only in snail tissues and two solely by the standard method. Notably, metabarcoding of environmental water samples was the most powerful tool for capturing most of the AS diversity, while snail tissue metabarcoding uncovered early pre-patent infections and additional unique lineages not detected in water. These findings underscore the critical value of a multi-method approach to minimise detection bias associated with any single technique.