Description of parasite
Plasmodium (Novyella) collidatum n. sp.
DNA-sequence: Mitochondrial cyt b lineage pFANTAIL01 (478 bp, GenBank accession no. MW175901)
Type host: The Common rosefinch Carpodacus erythrinus (Passeriformes, Fringillidae).
Additional hosts: According to literature data the lineage pFANTAIL01 (synonym codes AP63, GenBank accession no. AY714196; C028, DQ212193 and ASI-2012, JX418225) have been recorded in18 species of 15 families in 7 orders of naturally infected birds (see Table 1). Eurasian siskin was susceptible to experimental infection and could be a competent host.
Type locality: The Curonian Spit of the Baltic Sea (55º05'N, 20º44'E).
Prevalence: 71 Common rosefinches were collected and examined for haemosporidian infection on Curonian spit in 2010-2019 years. About 7% of all rosefinches were infected with different species of blood parasites. P. collidatum was reported in one bird individual in 2019. According to MalAvi database, this lineage is rare in Europe.
Site of infection: Erythrocytic meronts and gametocytes developed in mature red blood cells (Fig. 1); no other data.
Vectors: Natural vectors are unknown. Mosquitoes of C. pipiens pipiens form molestus and C. quinquefasciatus were not susceptible to P. collidatum.
Distribution: According to the molecular data, Plasmodium collidatum pFANTAIL01 has been reported in South, SE Asia, Australia, and neighboring islands as well as Europe (Table 1). In South, SE and Australia, P. collidatum has been found in numerous species of resident and migratory birds. Among infected European birds in the temperate zone, this lineage was confirmed only in adult birds of those species which are wintering in South Asia (Carpodacus erythrinus, Pastor roseus).
Type specimens: Hapantotype (accession nos. 49242-49244 NS, intensity of parasitemia is approximately 0.3%, Carpodacus erythrinus, the Curonian spit, Kaliningrad district, Russia, 36° 44′ N, 119° 29′ W, collected 6 June 2019 by E. Platonova) is deposited in the Nature Research Centre, Vilnius, Lithuania. Parahapantotypes (accession nos. G466222, G466223 [1149b/19C, 1220b/19C]), the intensity of parasitemia is approximately 10% and 3,3% respectively, of experimentally infected C. spinus, collected 26-30 July 2019 by E. Platonova) are deposited in the Queensland Museum, Queensland, Australia.
Additional material: Blood films from experimentally infected C. spinus (accession nos. 1149/19C, 1219/19c, 1220/19C, 1367/19C) and blood samples fixed in SET-buffer (accession nos. 1149/19C, 1219/19c, 1220/19C, 1367/19C) are deposited in the Nature Research Centre, Vilnius, Lithuania.
Etymology: The specific name reflects the morphological feature – markedly indented (lobular-like) appearance of the pellicle, which is typical for advanced gametocytes.
Exoerythrocytic meronts: Primary exoerythrocytic merogony (cryptozoites and metacryptozoites) was not investigated and remain unknown. Numerous secondary exoerythrocytic meronts (phanerozoites) were found in the lungs (Fig. 2a-c) of the infected individuals and appeared elongated (Fig. 2a), oval (Fig. 2b) or irregularly shaped (Fig. 2c). Phanerozoites in the lungs contained over 30 roundish merozoites. In smaller numbers, phanerozoites were also seen in the liver (Fig. 2d-f), spleen (Fig. 2g-i) and kidneys (Fig. 2j-l) of the infected individuals. In these organs phanerozoites appeared roundish or slightly oval and contained less than 30 merozoites.
Trophozoites (Fig. 1a, b) are most often seen in mature erythrocytes, however, rarely can be observed in polychromatophilic red blood cells. Round or oval trophozoites are located at the poles of infected erythrocytes. As trophozoites mature, they attach to the nucleus of the infected erythrocyte and this contact is maintained.
Erythrocytic meronts (Fig. 1c-j; Table 2) found only in mature erythrocytes. Growing meronts contain little, but readily visible, blue in color cytoplasm and small pigment granules (Fig. 1c); in mature meronts cytoplasm is rarely seen. Both, in growing and mature meronts a small bluish roundish and non-refractive globule is often seen (Fig. 1f, h). Meront with developing merozoites touches the erythrocyte nucleus, located laterally or close to the pole of the infected cell and this contact is maintained during the whole time of maturation (Fig. 1c-i). Grown meronts are oval or irregularly shaped and contain 4-8 merozoites (Fig. 1e-i). Pigment granules in mature meronts are small and clumped, however, scattered granules can also be seen in some cells (Fig. 1j). Effect of the gametocyte on the infected erythrocyte is not expressed. Mature merozoites contain prominent irregular-shaped nuclei, and each contains a small portion of readily visible cytoplasm (Fig. 1g, j).
Macrogametocytes (Fig. 1k-y; Table 2) found only in mature erythrocytes. The cytoplasm is markedly heterogeneous and is unevenly stained: more dense stained portions of the cytoplasm are intermediated with pale stained portions, which look like large vacuole-like pale-stained spaces (Fig. 1q-s, w, y), a characteristic feature of this species. Young forms usually found in the poles of infected erythrocytes. In the earliest growing gametocytes, long outgrowths often appear (Fig. 1l). Gametocytes vary in outline from amoeboid in growing to wavy in mature parasites. Gametocytes are located laterally to the nucleus of the infected erythrocyte; maturing and mature gametocytes are strictly nucleophilic, however, growing forms not touching the nucleus can also be seen occasionally (Fig. 1o). A central part of the pellicle of some growing gametocytes does not extend to erythrocyte envelope causing a ‘dip’, which gives dumbbell-shaped form (Fig. 1q, r). Typically, the growing gametocytes are asymmetric in appearance, with one end being broader than the other one (Fig. 1p, s, t, x). Fully-grown gametocytes often do not adhere to the envelope of the erythrocyte and do not fill the poles of erythrocytes (Fig. 1x, y). Markedly wavy lobular-like appearance of the pellicle (on the opposite side to erythrocytes nucleus) of mature gametocytes is an important distinctive feature of this species (Fig. 1u, w). The cytoplasm stains more densely in lobules than in indented areas. The nucleus of the gametocyte is diffused and of unclear outline; it consists of several chromatin clumps, which visually look to be non-connected with each other in Giemsa-stained preparation and can be seen closer to center or anywhere in the gametocyte, a characteristic feature of this species (Fig. 1s-y). Due to the pale cytoplasm staining and diffuse nucleus, macrogametocytes are difficult to distinguish from microgametocytes. Pigment granules are roundish, of small (˂0.5µm) size, most often grouped in one relatively large distinct spot, but occasionally also were seen scattered in the cytoplasm (Fig. 1s-x). The area of the pigment granule groups is relatively large, and this feature attracts attention during microscopic examination (Fig. 1y). Individual pigment granules do not change size and shape during the development of gametocytes, a rare feature in avian malaria parasites. Effect of the gametocyte on the infected erythrocyte is not expressed.
Microgametocyte (Fig. 1z-dd; Table 2) are difficult to distinguish from the macrogametocytes based on the intensity of the cytoplasm staining and appearance of parasite nuclei. The cytoplasm is relatively paler in the microgametocytes, all other characters are as in macrogametocytes. In the blood of type host, microgametocytes were seen extremely rare, and this prevented the complete picture of the morphometry of parasite.
Taxonomic summary
Morphology of P. collidatum – small meronts, a small amount of cytoplasm in meronts and elongated shape of grown gametocytes allows linking this species with the subgenus Novyella. Nucleophilic stages developing in the blood of the infected host allows to discriminate this species from other non-nucleophilic species of Novyella.This parasite can be readily distinguished from all nucleophilic Novyella species due to the following morphological characteristic of its mature gametocytes: i) the cytoplasm consists of intermediated readily visible large pale-stained and dense-stained areas, providing markedly heterogeneous appearance, ii) the majority of advanced gametocytes possesses the markedly indented (lobular-like) appearance of pellicle and iii) macro- and microgametocytes are difficult to distinguish based on their size and morphology of their nuclei. Additionally, the presence of a relatively large loose group on small pigment granules is also helpful during this parasite identification (see our description and [12, 13, 55, 56, 57]). The described parasite morphologically is the most similar to P. delichoni however, some features, like small roundish granules clearly distinguish this parasite from P. delichoni. Other mentioned nucleophilic species also have additional features, which are not found in the described parasite. Growing meronts of P. nucleophilum often displace the nuclei of the infected erythrocytes and its macrogametocytes have a compact nucleus; P. paranucleophilum possesses gametocytes, which push the nucleus in infected erythrocyte laterally; gametocytes of P. homonucleophilum are not strictly nucleophilic. None of the above-mentioned features is characteristics of the newly described species. The main morphological features of described parasite blood stages, which were observed in type host (C. erythrinus) also maintained in Eurasian siskins after our experimental infection. Very occasionally meronts with 9 and 10 merozoites appeared in experimentally infected siskins, but not in type host.
Phylogenetic analysis
According to the phylogenetic analysis, the lineage pFANTAIL01 clusters together with Plasmodium nucleophilum lineage pDENPET03 (Fig. 3). The genetic distance between these two lineages is 4.31%. Both lineages cluster within a bigger clade with other two Novyella parasites e.g. P. ashfordi (pGRW2) and P. delichoni (pCOLL6). However, the genetic difference between pFANTAIL01 and the latter two cyt b lineages is 8.43% and 10.45%, respectively. P. collidatum (pFANTAIL01) morphologically is the most similar to pCOLL6, but distribution and host range of these lineages differ.
Development and caused virulence in experimentally infected birds
All experimentally exposed siskins were susceptible to P. collidatum (pFANTAIL01) and showed the complete development of all blood stages of the parasite.
Prepatent period of infection, when the first infected erythrocytes were detected in the blood smears varied between 12-20 days post infection (dpi), (Fig. 4). Dynamics of parasitemia were highly variable between individuals (Fig. 4); in some reaching up to 70-80%, while in others only < 1%. In half of infected birds after sharp increase of parasitemia, there was typical bell-shaped form of primary parasitemia with clearly decreasing slope after the sharp initial increase. High parasitemia maintained in most of the experimental birds up to 36 dpi, reaching up to 11-75% (Fig. 4-5). Two birds died with parasitemias of 15% and 18% on 32 dpi. All control birds survived until the end of the experiment.
The average body mass of the infected birds did not differ significantly from the control group throughout the experiment (W = 4415, p-value = 0.6502, Fig. 6a).Hematocrit value was decreasing gradually during the experiment in all infected birds. However, significant differences between infected and control siskins were not detected until 28 dpi (W = 4427, p-value = 0.4631; Fig. 6b). On day 32 the difference between two groups of birds was the biggest (W = 53, p-value = 0.08689)
Development in experimentally infected mosquitoes
After screening C. p. p. f. molestus and C. quinquefasciatus midguts and salivary glands preparations it was determined that ookinetes, oocysts and sporozoites in all exposed to P. collidatum (pFANTAIL01) mosquitoes within 22 days after blood feeding were absent. Authors also did not detect any zygotes within 48 h in preparations of mosquito midgut contents.