Description of Haemoproteus multivacuolatus n. sp.

Haemoproteus multivacuolatus n. sp. (lineage hBUBT1, Figs. 3a–3p, Table1).

urn:lsid:zoobank.org:act: 22936DEA-BBFD-4FEA-9CB1- B64138A17035

Type-host: Common buzzard Buteo buteo ( Accipitriformes).

Type locality: Osnabrück, Lower Saxony, Germany .

Type specimens: Hapantotype (intensity of parasitemia is 8.3%, Buteo buteo, approximately 4-week-old nestling, Osnabrück, Lower Saxony, Germany, N52.12535 ° E8.37812 °, coll. N. Chakarov, July 2, 2019, Leucocytozoon toddi lMILANS04 is present) was deposited in the Muséum National d’ Histoire Naturelle, Paris (accession no. MNHN-IR-2023-03). Parahapantotypes (no. 49491NS, a duplicate of the hapantotype and no. 49492NS, intensity of parasitemia 12.2%, B. buteo, Austria, Leucocytozoon toddi lMILANS04 is present) were deposited in the Nature Research Centre, Vilnius.

Additional material: Voucher blood films were deposited in the Muséum National d’ Histoire Naturelle, Paris (accession no. MNHN-IR-2023-04).

Representative DNA sequences: Mitochondrial cytb lineage hBUBT1 (GenBank accession OR078932 and OR078930).

Distribution: Gametocytes were found in the type host B. buteo in Austria, Germany, and France. Transmission probably occurs throughout the breeding range of B. buteo . The lineage hBUBT1 was also found in five A. nisus individuals from France. However, the presence of gametocytes was not documented, so it remains unclear if the parasite completes the life cycle in this bird species.

Etymology: The species name reflects the markedly vacuolated appearance of the cytoplasm in both macrogametocytes and microgametocytes, resulting in minor differences between these types of gametocytes regarding the staining intensity (compare Figs. 3e–3h and Figs. 3i–3p).

Young gametocytes (Figs. 3a–3d) were seen anywhere in infected erythrocytes but more often occupied polar or subpolar positions in the host cells (Figs. 3a, 3b). As the gametocytes grow, they adhere to the nuclei of the erythrocytes and extend along the nuclei; they often assume asymmetrical positions regarding the nuclei, and this often leads to the appearance of comma-like gametocyte forms (Figs. 3c, 3d). Growing gametocytes often do not adhere to the erythrocyte envelope (Fig. 3c).

Macrogametocytes (Figs. 3e–3h; Table 1) are markedly vacuolated, lack volutin granules, and the cytoplasm stains relatively pale in comparison to microgametocytes (compare Figs. 3e–3h and Figs. 3i–3p), which is a characteristic feature of this parasite. Gametocytes extend along nuclei of erythrocytes (Fig. 3e); they markedly enclose the erythrocyte nuclei with their ends (Figs. 3f, 3g) and finally completely encircle the nuclei (Fig. 3h). Advanced and fully grown gametocytes are closely appressed to the nucleus and envelope of the erythrocytes; however, they usually do not fill poles of erythrocytes completely, resulting in the presence of more or less visible unfilled spaces on the poles (Figs. 3f–3h). The outline of gametocytes is predominantly wavy, sometimes slightly ameboid or even. Parasite nucleus median or submedian in position, markedly variable in form and outline; its boundaries were poorly visible (Figs. 3e–3h), which is a rare character in macrogametocytes of avian haemoproteids. Pigment granules are predominantly oval, sometimes roundish, of small (<0.5 µm) and average (0.5–1 µm) size, randomly scattered throughout the cytoplasm. Nuclei of infected erythrocytes only slightly displaced laterally (Figs. 3e–3h); however, advanced growing gametocytes (Figs. 3e– 3g) and fully grown gametocytes (Fig. 3h) cause marked enlargement of infected erythrocytes in length (p <0.01) and particularly in area (p <0.001) in comparison to uninfected erythrocytes (Table 1), a characteristic feature of this species, which was readily recognisable in blood films even without statistical analysis (see Figs. 3e, 3g, 3k, 3m for comparison of the size of the infected and uninfected erythrocytes).

Microgametocytes (Figs. 3i–3p; Table 1) are similar to macrogametocytes in general configuration and other features. Gametocyte nuclei are markedly diffuse, of irregular form. The outline of nuclei is poorly recognisable, so the nuclei were difficult to measure.

Taxonomic remarks: Four distinct morphospecies of Haemoproteus parasites were described in birds of the order Accipitriformes . These are H. elani Mello, 1935, H. buteonis Wingstrand, 1947, H. janovyi Greiner, Mundy, 1979, and H. nisi Peirce, Marquiss, 1983 . Haemoproteus multivacuolatus n. sp. can be readily distinguished from all these parasites, primarily due to remarkable hypertrophy of infected erythrocytes in area (Table 1; compare the appearance of infected and uninfected erythrocytes in Fig. 3). Slight enlargement of infected erythrocytes by fully grown gametocytes might occur in other haemoproteids, but this feature is not as distinct in other species parasitising accipitriform birds (Table 1). It is important to note that even growing gametocytes of H. multivacuolatus n. sp. induce marked enlargement of infected erythrocytes (Figs. 3d, 3e); that is not the case in any other haemoproteids parasitising accipitriform birds. Additionally, fully grown gametocytes of H. buteonis and H. elani are markedly halteridial [58], which is not the case in H. multivacuolatus n. sp.

It is important to note that the identification of H. multivacuolatus n. sp. might be difficult if only growing gametocytes are present in a sample. Such blood samples from B. buteo were common in this study. If parasitaemia is represented only by growing gametocytes (Figs. 3e, 3i–3k) but fully grown (circumnuclear) gametocytes (Figs. 3h, 3o, 3p) are absent, H. multivacuolatus n. sp. identification might be difficult due to similarities with fully grown gametocytes of H. buteonis and H. elani . Molecular characterisation would be particularly helpful in this case.

Circumnuclear fully grown gametocytes predominate in both lineages of H. nisi (hACCNIS06 and hCIAE08) (Figs. 1, 2) and H. janovyi [56]. Haemoproteus multivacuolatus n. sp. can be readily distinguished from both H. nisi lineages due to the lack of volutin and the marked vacuolisation of gametocyte cytoplasm in the former. Fully grown gametocytes of H. janovyi predominantly occupy all available space in infected erythrocytes and are also markedly pleomorphic in morphology [58]; both these characters are not features of H. multivacuolatus n. sp.