title: Biting midges (Culicoides, Diptera) transmit Haemoproteus parasites of owls: evidence from sporogony and molecular phylogeny.
abstract: 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.
authors.affiliation: Institute of Ecology, Nature Research Centre, Akademijos 2, Vilnius 21, LT-09412, Lithuania. dovilebu7@gmail.com.
authors.forename: Dovilė
authors.initials: D
authors.lastname: Bukauskaitė
authors.affiliation: Institute of Ecology, Nature Research Centre, Akademijos 2, Vilnius 21, LT-09412, Lithuania. rita.kazlauskien@gmail.com.
authors.forename: Rita
authors.initials: R
authors.lastname: Žiegytė
authors.affiliation: Institute of Ecology, Nature Research Centre, Akademijos 2, Vilnius 21, LT-09412, Lithuania. palinauskas@gmail.com.
authors.forename: Vaidas
authors.initials: V
authors.lastname: Palinauskas
authors.affiliation: Institute of Ecology, Nature Research Centre, Akademijos 2, Vilnius 21, LT-09412, Lithuania. tatjana@ekoi.lt.
authors.forename: Tatjana A
authors.initials: TA
authors.lastname: Iezhova
authors.affiliation: Institute of Ecology, Nature Research Centre, Akademijos 2, Vilnius 21, LT-09412, Lithuania. ddmitko@gmail.com.|Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 2 Gagarin Street, Sofia, 1113, Bulgaria. ddmitko@gmail.com.
authors.forename: Dimitar
authors.initials: D
authors.lastname: Dimitrov
authors.affiliation: Institute of Ecology, Nature Research Centre, Akademijos 2, Vilnius 21, LT-09412, Lithuania. ilgunasmikas@gmail.com.
authors.forename: Mikas
authors.initials: M
authors.lastname: Ilgūnas
authors.affiliation: Institute of Ecology, Nature Research Centre, Akademijos 2, Vilnius 21, LT-09412, Lithuania. bern.rasa@gmail.com.
authors.forename: Rasa
authors.initials: R
authors.lastname: Bernotienė
authors.affiliation: Biological Station Rybachy of the Zoological Institute, Russian Academy of Sciences, Rybachy, 238535, , Kaliningrad Region, Russia. mikhail.markovets@gmail.com.
authors.forename: Mikhail Yu
authors.initials: MY
authors.lastname: Markovets
authors.affiliation: Institute of Ecology, Nature Research Centre, Akademijos 2, Vilnius 21, LT-09412, Lithuania. gedvalk@ekoi.lt.
authors.forename: Gediminas
authors.initials: G
authors.lastname: Valkiūnas
mesh_terms: D000818:Animals; D001483:Base Sequence; D001715:Bird Diseases / Q000469:parasitology / Q000635:transmission*; D002519:Ceratopogonidae / Q000469:parasitology*; D045303:Cytochromes b / Q000235:genetics; D005260:Female; D016787:Haemosporida / Q000145:classification / Q000235:genetics / Q000502:physiology*; D007303:Insect Vectors / Q000469:parasitology*; D008969:Molecular Sequence Data; D033661:Oocysts; D010802:Phylogeny; D011529:Protozoan Infections, Animal / Q000635:transmission*; D017422:Sequence Analysis, DNA / Q000662:veterinary; D020305:Strigiformes / Q000469:parasitology*
chemical_list: D045303:Cytochromes b