Authors

Discussion

Knowledge of the life cycle of parasites is crucial for numerous reasons such as integrating them into food webs or for studying parasite transmission pathways. The common cockle (Cerastoderma edule) is parasitized by a diverse community of digenean trematodes of which the life cycles are elucidated, except for one. Indeed, the trematode Curtuteria arguinae can be found as metacercariae infecting cockles as their second intermediate host in southern Europe. Cockles and trematodes in the National Nature Reserve of Banc d’Arguin (Atlantic coast of France) were monitored for more than 20 years, revealing the constant presence of C. arguinae in cockles, sometimes reaching very high abundances (up to 850 metacercariae per cockle). However, despite previous efforts to identify its first intermediate host, C. arguinae’s life cycle remained unelucidated. Indeed, the typically low prevalence at this stage makes sampling several species of potential intermediate hosts and the recovery of trematode juvenile stages from these samples a massive undertaking with traditional methods (i.e. cercarial emission, dissection). Similarly, recovering adult stages in birds (potential final hosts) can be ethically problematic, especially when it concerns protected species.
Therefore, we used a non-invasive eDNA-type approach to detect C. arguinae in different samples collected in Banc d’Arguin. On the one hand, the investigation of the first intermediate host consisted in massively collecting the five dominant marine gastropod species in an infestation hotspot. Individuals were kept separately per species in a container with seawater for cercarial emission and DNA was extracted from the remaining fraction of the filtered seawater. On the other hand, we collected feces of individual seabird taxa to investigate the presence of eggs as a proxy of the birds’ infestation as final hosts by adult worms. Using newly developed species-specific cox1 primers, we conducted quantitative PCR assays to test the presence of C. arguinae DNA in the samples. Finally, in parallel, we sampled the benthic macrofauna along the Banc d’Arguin to study its spatial distribution.
Out of 25 water samples containing five potential host species, all five replicates of one gastropod species tested positive for C. arguinae, while the other 20 samples remained negative. Then, the putative first intermediate host was tested by cercarial emission technique, which resulted in an average C. arguinae prevalence of 1,6 %. The molecular sequences obtained from cercariae were identical to those isolated from C. arguinae metacercariae in cockles. Concerning birds, out of 167 analyzed feces, 30% tested positive for C. arguinae, all positive samples (except one) belonging to a unique seabird species. Our results provided new essential information that allowed us to identify the first intermediate host and the final host of C. arguinae, hereby elucidating its complete life cycle for the first time. Our results demonstrate the usefulness of molecular techniques to identify potential host species in a non-destructive way before confirming with an integrative molecular and morphological approach. In addition, the benthic macrofauna sampling revealed that the first intermediate host of C. arguinae was most present and abundant in the same area which constituted the infestation hotspot in cockles. This shows that the spatial proximity between the first and the second intermediate hosts is of the utmost importance for parasite transmission, even for free-living larval stages, and supports the hypothesis that the spatial distribution of an upstream host strongly influences the spatial distribution of the parasite in a downstream host.