Assessing the Efficiency of Molecular Markers for the Species Identification of Gregarines Isolated from the Mealworm and Super Worm Midgut

Protozoa, of the taxon Gregarinasina, are a heterogeneous group of Apicomplexa that includes ~1600 species. They are parasites of a large variety of both marine and terrestrial invertebrates, mainly annelids, arthropods and mollusks. Unlike coccidians and heamosporidians, gregarines have not proven to have a negative effect on human welfare; thus, they have been poorly investigated. This study focuses on the molecular identification and phylogeny of the gregarine species found in the midgut of two insect species that are considered as an alternative source of animal proteins for the human diet: the mealworm Tenebriomolitor, and the super-worm Zophobasatratus (Coleoptera: Tenebrionidae). Gregarine specimens were isolated from the gut of both larval and adult stages of T.molitor specimens, as well as from Z.atratus larvae. The morphological analyses were restricted to the identification of the different parasite morpho-types, likely corresponding either to different life-cycle stages or to alternative gregarine species. The samples were also used for the DNA extraction necessary for their genetic characterization. Finally, the efficiency of different molecular markers (i.e., 18S rDNA gene alone or combined with the Internal Transcribed Spacer 1) was assessed when applied either to gregarine species identification and to phylogenetic inference.