New experimental insights into the biology of avian malaria parasites

Despite much research on avian malaria, the details of development of Plasmodium species in birds remain insufficiently investigated. This study aimed to gain new experimental information on avian malaria parasites regarding their host specificity, molecular characterization, persistence, and association with Matryoshka RNA viruses (MaRNAV), which are linked to haemosporidian infections. Three species of widespread avian Plasmodium—P. circumflexum pTURDUS1, P. vaughani pSYAT05, and P. relictum pCOLL7—were found in naturally infected wild birds of the Muscicapidae, Turdidae and Phylloscopidae, respectively. The parasites were transferred to non-infected Eurasian siskins Spinus spinus (Fringillidae) by inoculation of infected blood, and infections were monitored up to 185 days. Microscopy, histology, chromogenic in situ hybridization (CISH), PCR, RNA sequencing and bioinformatic methods were applied in this study. All recipient birds were susceptible to these pathogens. Extensive histology and CISH testing did not detect any tissue stages in all birds, suggesting that long-lasting persistence is mainly due to erythrocytic merogony. However, in the case of pCOLL7, parasitaemia consisted almost exclusively of gametocytes, presenting an unusual mode of parasitaemia in avian malaria. Based on morphological and phylogenetic data, pCOLL7 was linked to P. relictum. Morphological characterization showed that taxonomic characters of blood stages of all three parasite species are relatively stable across host species, supporting the value of the morphological characters of blood stages in Plasmodium species identification in birds belonging to different families. Furthermore, this study detected and associated a Matryoshka RNA virus (MaRNAV-9) with avian Plasmodium parasites for the first time. This virus was found in association with the bird malaria parasite P. (Giovannolaia) circumflexum (pTURDUS1) widespread in Eurasia, which can be used as a model host-parasite system to better understand the biology of MaRNAVs and their relationship with haemosporidians. This study provides new information to better understand diagnostics and persistence of avian malaria parasites in avian hosts. It also contributes to new knowledge about the diversity of MaRNAVs associated with Plasmodium pathogens, opening opportunities for detecting these viruses in association with malaria parasites.