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Background   Haemoproteus  parasites are widespread, and several species cause diseases both in birds and blood-sucking insects. These pathogens are transmitted by dipterans belonging to the Ceratopogonidae and Hippoboscidae, however certain vector species remain unknown for the majority of  Haemoproteus  spp. Owls are often infected by  Haemoproteus  parasites, but experimental studies on vectors of these infections are lacking. The aim of this study was to investigate sporogonic development of two widespread  Haemoproteus  parasites of owls,  H. noctuae  and  H. syrnii  in experimentally infected biting midges  Culicoides impunctatus  and  Culicoides nubeculosus.  We also followed  in vitro  sporogonic development of these infections and determined their phylogenetic relationships with  Haemoproteus  spp., for which vectors have been identified.    Methods  Wild-caught  C. impunctatus  and laboratory reared  C. nubeculosus  were infected experimentally by allowing them to take blood meals on one individual long-eared owl ( Asio otus ) and one tawny owl ( Strix aluco ) harbouring mature gametocytes of  H. noctuae  (lineage hCIRCUM01) and  H. syrnii  (hCULCIB01), respectively. The engorged insects were maintained in the laboratory at 16–18 °C, and dissected at intervals in order to follow the development of ookinetes, oocysts and sporozoites. We also observed  in vitro  development of sexual stages of both parasites by exposure of infected blood to air. The parasite lineages were determined by polymerase chain reaction-based methods. Bayesian phylogeny was constructed in order to determine the relationships of owl parasites with other avian  Haemoproteus  spp., for which vectors have been identified.    Results  Both  H. noctuae  and  H. syrnii  completed sporogony in  C. nubeculosus,  and  H. noctuae  completed sporogony in  C. impunctatus . Ookinetes, oocysts and sporozoites of these parasites were reported and described. Gametes and ookinetes of both species readily developed  in vitro . In accordance with sporogony data, the phylogenetic analysis placed both parasite lineages in a clade of  Culicoides  spp.-transmitted avian  Haemoproteus (Parahaemoproteus)  spp.    Conclusions   Culicoides nubeculosus  and  C. impunctatus  are vectors of  H. noctuae  and  H. syrnii . Phylogenies based on cytochrome  b  gene indicate parasite-vector relationships, and we recommend using them in predicting possible parasite-vector relationships and planning research on avian  Haemoproteus  spp. vectors in wildlife.

Biting midges (Culicoides, Diptera) transmit Haemoproteus parasites of owls: evidence from sporogony and molecular phylogeny