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Background  Haemosporidian parasites of the genus  Haemoproteus  (Haemoproteidae) are widespread and cause haemoproteosis in birds and therefore, their diversity, ecology and evolutionary biology have become subjects of intensive research. However, the vectors and transmission patterns of haemoproteids as well as the epidemiology of haemoproteosis remain insufficiently investigated. Several species of  Culicoides  (Ceratopogonidae) support complete sporogony of haemoproteids belonging to the subgenus  Parahaemoproteus.  However, experimental research with these fragile insects is difficult to design in the field, particularly because their abundance markedly depends on seasonality. This is an obstacle for continuous sampling of sufficient numbers of naturally infected or experimentally exposed midges from wildlife. We developed simple methodology for accessing sporogonic development of haemoproteids in laboratory-reared  Culicoides nubeculosus . This study aimed to describe the mosaic of methods constituting this methodology, which was applied for investigation of the sporogonic development of  Haemoproteus  ( Parahaemoproteus )  pastoris , a widespread parasite of the common starling  Sturnus vulgaris .    Methods  The methodology consists of the following main stages: (i) laboratory rearing of  C. nubeculosus  from the egg stage to adult insects; (ii) selection of naturally infected birds, the donors of mature gametocytes to expose biting midges; (iii) experimental exposure of insects and their laboratory maintenance; and (iv) dissection of exposed insects. Biting midges were exposed to  H. pastoris  ( cytochrome b  lineage hLAMPUR01) detected in one naturally infected common starling. Engorged insects were dissected at intervals in order to follow sporogony. Microscopic examination and PCR-based methods were used to identify the sporogonic stages and to confirm the presence of the parasite lineage in infected insects, respectively.    Results   Culicoides nubeculosus  females were successfully reared and exposed to  H. pastoris , which completed sporogonic development 7–9 days post-infection when sporozoites were observed in the salivary glands.    Conclusions  The new methodology is easy to use and non-harmful for birds, providing opportunities to access the sporogonic stages of  Parahaemoproteus  parasites, which might be used in a broad range of parasitology and genetic studies.  Culicoides nubeculosus  is an excellent experimental vector of subgenus  Parahaemoproteus  and is recommended for various experimental studies aiming investigation of sporogony of these pathogens.

parasites and vectors

A new methodology for sporogony research of avian haemoproteids in laboratory-reared Culicoides spp., with a description of the complete sporogonic development of Haemoproteus pastoris