Tissue stages of Haemoproteus parasites (Haemosporida, Apicomplexa) in Ficedula flycatchers with evidence for phenotypic variation in Haemoproteus balmorali and implications for haemosporidian taxonomy.

Abstract

During exo-erythrocytic merogony, avian Haemoproteus parasites (Apicomplexa, Haemosporida) develop distinct tissue stages: small meronts and large megalomeronts. Recent molecular studies suggest that Haemoproteus species develop either of the two morphotypes in naturally infected birds but earlier research indicates that certain species form both depending on the replicating cycle. Because data on tissue stages are limited to a few described species, patterns and mechanisms of (megalo-)merogony are unclear but required to better understand pathologies during haemoproteosis. In this study, we aimed to characterize exo-erythrocytic stages of Haemoproteus species in naturally infected flycatchers (Muscicapidae). Tissue samples of eight Ficedula hypoleuca and two Ficedula parva positive by PCR and blood smear microscopy were subjected to histology and in situ hybridization applying Haemoproteus lineage-specific probes. Additionally, laser capture microdissection was performed to enable molecular identification of individual tissue stages. A phylogenetic analysis based on the parasites' cytochrome b gene (886 bp) was performed to explore the relationship of meront- and megalomeront-forming species within the genus Haemoproteus. In F. hypoleuca, we found meronts and megalomeronts, and molecular data indicate that both belong to Haemoproteus balmorali, suggesting phenotypic variation of this parasite. Phylogenetically, H. balmorali was closely related to Haemoproteus attenuatus, which develops similar meronts but no megalomeronts, suggesting varying phenotypic variation among closely related species. In F. parva, we detected megalomeronts and gametocytes of Haemoproteus majoris hPHSIB1, presenting the first record in that host. By contrast, in F. hypoleuca showing high H. pallidus gametocytaemia, no exo-erythrocytic stages were found, implying that merogony was completed and not coinciding with gametocytaemia. These results demonstrate that exo-erythrocytic development varies considerably between species and patterns of tissue merogony may not be generalized at genus level. Furthermore, we report significant nuclear hypertrophy of the host cell in Haemoproteus megalomeronts, a feature formerly considered unique to Leucocytozoon, challenging it's use for genus identification.