In spite of their potential as model organisms and their medical importance, parasite speciation processes have been insufficiently discussed in the general literature. Scarcity of experimental data regarding reproductive isolation of parasites is a serious obstacle. Toward this end we developed a method for the investigation of hybridization of hemosporidian parasites in vitro. Five species of Haemoproteus (Haemosporida, Haemoproteidae) were isolated from naturally infected passerine birds. They were identified to species based on morphology of their gametocytes and PCR amplification of a segment of the parasite's mitochondrial cytochrome b gene. Hybridization of Haemoproteus spp. was initiated in vitro by mixing blood containing mature gametocytes of different species with a 3.7% solution of sodium citrate and exposure of the mixture to air. The following hybridization experiments were performed: (1) Haemoproteus minutus × Haemoproteus pallidus, (2) Haemoproteus balmorali × Haemoproteus tartakovskyi, and (3) Haemoproteus fringillae × H. tartakovskyi. The development of ookinetes of both species was blocked in the first experiment. Ookinetes of all species developed in other experiments, but presumed hybrids were distinguished only in the third experiment. Illustrations of ookinetes of all species are given. The present communication indicates that controlled experiments for direct hybridization of hemosporidians can be carried out in vitro. Such experimental research can be used to reconcile molecular and morphological data and to define biological species for this group of parasites.
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