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

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

“…Two freshwater turtle species, namely M. leprosa, the Mediterranean pond turtle, and E. orbicularis, the European pond turtle, were trapped using hoop nets baited with fish from three localities in Northern Tunisia (see Table 1 for GPS coordinates and also ref [32]).…”

“…A drop from each blood sample was also smeared on a glass slide, air-dried, fixed in absolute methanol for 5 min and stained with 10% Giemsa for 20 min in the laboratory for the presence of hemoparasites. Results from microscopic observations were reported in [32].…”

“…Six DNA samples, four from M. leprosa (Ml36AM, Ml34AM, Ml44HS and Ml10HB) and two from E. orbicularis (Eo18HS and Eo19HS) were selected to test the PCR efficiency of the newly designed degenerated primers used two by two. These DNA samples were also studied earlier in [32] to investigate the genetic diversity of Haemogregarina based on 18S rRNA gene sequences. All PCRs were performed in a final volume of 25 µL under the following conditions: 5 min at 95°C for initial denaturation; 35 cycles of 30 sec each at 94°C for denaturation, 30 sec at 48°C for annealing, 1 min at 72°C for elongation; 10 min at 72°C for final extension.…”

“…Due to limited interspecific morphological differences, traditional approaches were subsequently combined with molecular methods using 18S rRNA gene sequences for species identification [31]. Genetic diversity of blood parasites within the sympatric freshwater turtles Mauremys leprosa and Emys orbicularis of Tunisia, based on 18S sequences, revealed the occurrence of three distinct Haemogregarina variants including 6 H. stepanowi [32]. As coinfection patterns were clearly reported from molecular data for both turtle species, morphological identification of these variants was challenging, if not impossible [32].…”

“…Genetic diversity of blood parasites within the sympatric freshwater turtles Mauremys leprosa and Emys orbicularis of Tunisia, based on 18S sequences, revealed the occurrence of three distinct Haemogregarina variants including 6 H. stepanowi [32]. As coinfection patterns were clearly reported from molecular data for both turtle species, morphological identification of these variants was challenging, if not impossible [32]. Thus, our objectives in the present study were to design COI specific primers to investigate the COI Haemogregarina variations from blood samples in both turtle species and to then validate the presence of three distinct species of Haemogregarina.…”