Haemoproteus minutus and Haemoproteus belopolskyi (Haemoproteidae): complete sporogony in the biting midge Culicoides impunctatus (Ceratopogonidae), with implications on epidemiology of haemoproteosis.

Species of Haemoproteus (Haemoproteidae) are cosmopolitan haemosporidian parasites, some of which cause severe diseases in birds. Numerous recent studies address molecular characterization, distribution and genetic diversity of haemoproteids. However, the information about their vectors is scarce. We investigated sporogonic development of two widespread species of Haemoproteus (Haemoproteus minutus and Haemoproteus belopolskyi) in the experimentally infected biting midge Culicoides impunctatus. Wild-caught flies were allowed to take blood meals on naturally infected common blackbirds Turdus merula and icterine warblers Hippolais icterina harboring mature gametocytes of H. minutus (lineage hTURDUS2) and H. belopolskyi (hHIICT1), respectively. The engorged flies were collected, transported to the laboratory, held at 15-18°C, and dissected daily in order to obtain ookinetes, oocysts and sporozoites. Mature ookinetes of H. minutus developed blisteringly rapidly; they were numerous in the midgut content between 1 and 4 h post exposure. Ookinetes of H. belopolskyi developed slower and were reported 1 day post exposure (dpe). Oocysts of both parasites were seen in the midgut wall 3-4 dpe. Sporozoites of H. minutus and H. belopolskyi were first observed in the salivary glands preparations 7 dpe. The percentage of experimentally infected flies with sporozoites of H. minutus was 82.1% and 91.7% with H. belopolskyi. In accordance with microscopy data, polymerase chain reaction amplification and sequencing confirmed presence of the corresponding parasite lineages in experimentally infected biting midges. Sporogonic stages of these parasites were described and illustrated. This study indicates that C. impunctatus is involved in the transmission of deadly H. minutus, which kills captive parrots in Europe. This biting midge is an important vector of avian haemoproteids and worth more attention in epidemiology research of avian haemoproteosis.