Cytochrome c oxydase I phylogenetic analysis of Haemogregarina parasites (Apicomplexa, Coccidia, Eucoccidiorida, Haemogregarinidae) confirms the presence of three distinct species within the freshwater turtles of Tunisia.

Species of Haemogregarina are apicomplexan blood parasites that use vertebrates as intermediate hosts. Due to limited interspecific morphological characters within the genus during the last decade, 18S rRNA gene sequences were widely used for species identification. As coinfection patterns were recently reported from nuclear molecular data for two sympatric freshwater turtles Mauremys leprosa and Emys orbicularis from Tunisia, our objectives were to design COI specific primers to confirm the presence of three distinct species in both host species. Blood samples were collected from 22 turtles, from which DNAs were extracted and used as templates for amplification. Following different rounds of PCR and nested PCR, we designed specific Haemogregarina COI primers that allowed the sequencing of nine distinct haplotypes. Phylogenetic Bayesian analysis revealed the occurrence of three well-differentiated sublineages that clustered together into a single clade. Based on pairwise genetic distances (p-distance), we confirmed the occurrence of three distinct but phylogenetically closely related species coinfecting M. leprosa and E. orbicularis in the same aquatic environments. Our results demonstrate that the use of fast evolving genes within Haemogregarina will help to investigate the parasite diversity within both intermediate vertebrate and definitive invertebrate hosts, and to assess the evolution, historical biogeography and specificity of haemogregarines.

The genetic diversity of blood parasites within the freshwater turtles Mauremys leprosa and Emys orbicularis in Tunisia reveals coinfection with Haemogregarina spp.

Haemogregarina species are apicomplexan blood parasites infecting vertebrates such as fish, lizards, and turtles. Due to the high morphological similarity of the erythrocytic stages infecting host species, it has always been a challenge to identify the true diversity of these parasites. Therefore, taxonomic studies are presently based on the combination of morphological and molecular data. In Tunisia, two species of Haemogregarina have been reported within the freshwater turtle Mauremys leprosa (Geoemydidae) for more than 40 years. Since M. leprosa occurs in the same aquatic environments as Emys orbicularis (Emydidae) in Tunisia, our objectives were to assess parasite diversity and specificity on the basis of both morphological and molecular approaches. The turtles were surveyed and sampled across six aquatic areas of Tunisia. Among the 39 specimens of M. leprosa and seven of E. orbicularis that were trapped and investigated, the presence of haemogregarines was detected in the blood of turtles only at sites where leeches were observed. Three 18S variants were identified, which corresponded to three distinct Haemogregarina species, among which one was identified as Haemogregarina stepanowi. The two other species that were detected are likely new to science. Because we show the occurrence of more than one blood parasite species within a single host specimen, our study provides the first report of coinfection with molecularly distinct Haemogregarina spp.