Elucidating the life cycle of opossum parasites: DNA sequences reveal the involvement of planorbid snails as intermediate hosts of Rhopalias spp. (Trematoda: Echinostomatidae) in Brazil.

Echinostomatid digeneans belonging to the genus Rhopalias are intestinal trematodes found mainly in opossums in the New World. The genus comprises seven species, but their life cycles and intermediate hosts have been unknown until now. During our long-term study carried out in freshwater habitats within the state of Minas Gerais, Southeast Brazil, echinostomatid cercariae lacking collar spines were found in planorbid snails Biomphalaria glabrata, Biomphalaria straminea, Drepanotrema lucidum and Gundlachia ticaga in six different batches of snail samples collected between 2010 and 2019. Morphologically, the larvae reported herein are morphologically consistent with each other and characterized by the presence of 2-3 large ovoid or spherical corpuscles in each main duct of the excretory system, resembling to Cercaria macrogranulosa previously described from the same region of Brazil. Partial sequences of the ITS (ITS1-5.8S-ITS2) region and 28S gene of the nuclear ribosomal RNA operon, and partial sequences of mitochondrial nad1 and cox1 genes were obtained and compared with the data available for members of the family Echinostomatidae. Nuclear markers indicate that all samples of cercariae evaluated in the present study can be assigned to Rhopalias, but distinct from North American isolates of Rhopalias macracanthus, Rhopalias coronatus and Rhopalias oochi (divergence 0.2-1.2% in 28S and 0.8-4.7% in ITS). The lack of differences verified in both 28S and ITS in 5 out 6 studied samples suggested that they belong to the same species. However, nad1 sequences revealed that our cercariae correspond to three distinct species of Rhopalias (interspecific divergence: 7.7-9.9%), named here as Rhopalias sp. 1, found in B. straminea and G. ticaga, Rhopalias sp. 2 found in B. glabrata and D. lucidum, and Rhopalias sp. 3 also found in D. lucidum. They also differ by 10.8-17.2% from a North American isolate of R. macracanthus sequenced in this study. The cox1 sequences obtained for Rhopalias sp. 1 and Rhopalias sp. 2 (but not Rhopalias sp. 3) reveal that they are distinct from North American isolates of R. macracanthus (genetic divergence 16.3-16.5% and 15.6-15.7%, respectively), R. coronatus (9.2-9.3% and 9.3-9.5%) and Rhopalias oochi (9.0% and 9.5-10.1%). Encysted metacercariae with general morphology similar to that of the body of cercariae were found in tadpoles of Rhinella sp. from the same stream where snails harbored Rhopalias sp. 2, suggesting that the amphibians could act as second intermediate hosts of species of Rhopalias. Data obtained provide the first insights into the life cycle of this unusual echinostomatid genus.