Finding a needle in a haystack: larval stages of Didymozoidae (Trematoda: Digenea) parasitizing marine zooplankton.

Larval didymozoids (Trematoda: Digenea) were discovered parasitizing the hemocoel of the heteropod Firoloida desmarestia (redia mean intensity = 13) and the chaetognaths Flaccisagitta enflata and Flaccisagitta hexaptera (metacercaria mean intensity = 1) during a 2014-2016 systematic study of parasites of zooplankton collected in the central and southern regions of the Gulf of California, Mexico. Didymozoid infection route during the early life cycle was inferred combining morphological (light microscopy) and molecular (mitochondrial cytochrome c oxidase subunit I gene, cox1) evidence. Didymozoid rediae parasitizing F. desmarestia were observed, just after field collection of the host, containing hundredths of completely developed cystophorous cercariae, releasing them though the birth pore at approximately one cercaria every 12 s. Cercariae lost their tails developing into a 'young metacercaria' in 1 d at 22 °C without need of an intermediate host. Molecular analysis of cox1 showed that rediae found in F. desmarestia belong to two distinct didymozoid species (Didymozoidae sp. 1 and sp. 2). Metacercariae parasitizing chaetognaths were morphologically identified as Didymozoidae type Monilicaecum and cox1 sequences showed that metacercariae of chaetognaths matched with these two Didymozoidae sp. 1, and sp. 2 species found parasitizing F. desmarestia, plus a third distinct Didymozoidae sp. 3. These are the first DNA sequences of cox1 gene from didymozoid larvae for any zooplankton taxonomic group in the world. We concluded that F. desmarestia is the first intermediate host of rediae and cercariae, and the chaetognaths are the second intermediate hosts where non-encysted metacercariae were found. The definitive host is still unknown because cox1 sequences of present study did not genetically match with any available cox1 sequence of adult didymozoid. Our results demonstrate a potential overlap in the distribution of two carnivorous zooplankton taxonomic groups that are intermediate hosts of didymozoids in the pelagic habitat. The didymozoid specimens were not identified to species level because any of the cox1 sequences generated here matched with the sequences of adult didymozoids currently available in GenBank and Bold System databases. This study provides baseline information for the future morphological and molecular understanding of the Didymozoidae larvae that has been previously based on the recognition of the 12 known morphotypes.