Leucocytozoon (Leucocytozoon) grallariae, Lotta & Valkiūnas & Pacheco & Escalante & Hernández & Matta, 2019

Lotta, Ingrid A., Valkiūnas, Gediminas, Pacheco, M. Andreína, Escalante, Ananías A., Hernández, Sandra Rocío & Matta, Nubia E., 2019, Disentangling Leucocytozoon parasite diversity in the neotropics: Descriptions of two new species and shortcomings of molecular diagnostics for leucocytozoids, International Journal for Parasitology: Parasites and Wildlife 9, pp. 159-173 : 164-165

3.2.1. Leucocytozoon (Leucocytozoon) grallariae sp. nov

Young gametocytes ( Fig. 1 ) markedly influence the shape of host cells from earliest stages of their development. Growing parasites were of oval or ellipsoid shapes; they closely adhered to the host cell nuclei, which were markedly enlarged, deformed and assumed crescent shapes ( Fig. 1A–C ). The host cell cytoplasm was present around growing gametocytes, and it was very evident. Advanced young gametocytes often possessed invaginations on their sides, which were opposite to the host cell nuclei, and that gave the growing gametocytes the shapes of giant beans with approximately equally rounded ends ( Fig. 1 B ). Host cells assumed ellipsoid shapes from early stages of gametocyte development ( Fig. 1 A ).

Macrogametocytes developed in fusiform host cells ( Fig. 1 ). In Leucocytozoon species, as the gametocytes develop, they cause considerable distortion of the host cells, producing two distinct host cellparasite complex forms: roundish and fusiform. In this Leucocytozoon species, the gametocytes only in fusiform host cells were observed. Gametocytes induced marked hypertrophy and deformation of the host cells and displacement of their nuclei, which lay on the periphery of gametocytes. The host cell nuclei acquired slender waning moon shapes; usually extending up to ½ of the circumference of gametocytes, and they could reach the fusiform processers, but they never extended into the processes ( Fig. 1C–E ). In both types of host cells, the nuclei looked homogenous.

The host-cell cytoplasm forms two short cytoplasmic processes located close to ends of gametocytes. Fusiform processes were variable in form ( Fig.1 D, F–I ) nevertheless, those processes, which length was similar to their largest width were common ( Table 2), and that was a characteristic feature of the development of this species. It is worth mentioning that the cytoplasmic processes were occasionally observed as being unequally long at both sides of the same parasite ( Fig. 1H and I ), probably as a consequence of the host cell deformation during blood film preparation. Some remnants of host cell cytoplasm were seen covering around half of the perimeter of the gametocyte-host cell complex as a thin rim ( Fig. 1G–I ), which is uncommon in avian leucocytozoids. Additionally, host cells with fully grown gametocytes could possess irregular shapes and blunt processes, while they were often pointed in host cells with immature growing gametocytes ( Fig. 1G–I ).

The gametocyte cytoplasm was granular in appearance; it often possessed small vacuoles and tiny volutin granules ( Fig. 1E and F ). Vacuoles were of different sizes, but not greater than 3.0 μm in their maximum diameter; they were observed in 97 gametocytes. Vacuoles were seen in growing (immature) gametocytes ( Fig. 1 B ). Parasite nuclei were compact; they were seen mainly in the central position (67% of reported cases) in gametocytes and were of roundish ( Fig. 1D–F ) or various oval ( Fig. 1 E ) shapes. Nucleoli were visible in 50% of the gametocytes.

The general configuration of the Microgametocytes and other features ( Fig. 1G–I ) were as for macrogametocytes with the usual haemosporidian sexual dimorphic characters that were the pale stained

Feature Leucocytozoon g rallariae sp. nov

Macrogametocyte n = 15 Microgametocyte n = 10 Parasite

Length 14.7–23.6 (20.2 ± 2.5) 15.8–21.3 (18.5 ± 2.1) Width 4,5-5,5 (5,4 ± 0,5) 4.1–6.1 (5.1 ± 0.6)

Area 79.7–109.0 (97.4 ± 9.6) 71.3–93.4 (81.7 ± 6.0) Perimeter 39.9–54.4 (47.1 ± 4.3) 39.6–49.3 (43.5 ± 3.4) Parasite nucleus

Length 2.7–4.9 (3.6 ± 0.6) 7.2–12.9 (10.0 ± 1.9) Width 3.1–5.4 (4.4 ± 0.6) 3.0–4.5 (3,7 ± 0.4)

Area 9.9–18.0 (13.1 ± 2.2) 23.4–49.4 (32.8 ± 9.0) Host-cell parasite complex

Length 24.8–32.1 (29.6 ± 2.5) 24.7–33.9 (29.4 ± 2.7) Width 7.3–10.3 (8.5 ± 1.0) 7.0–10.3 (8.4 ± 0.9)

Area 162.6–196.4 152.2–188.1

(185.1 ± 12.8) (168.8 ± 11.0)

Host-cell nucleus

Length 15.8–22.9 (21.1 ± 1.8) 17.5–21.6 (18.9 ± 1.2) Width 1.5–3.6 (2.2 ± 0.5) 2.0–2.6 (2.3 ± 0.2)

Area 27.0–49.1 (32.6 ± 5.5) 25.7–34.8 (31.3 ± 2.3) Perimeter of parasite 15.3–22.1 (21.1 ± 1.8) 17.2–20.5 (18.2 ± 1.1)

covered

Cytoplasmic processes a

Length 3.3–7.6 (5.0 ± 1.0) 3.8–7.8 (5.5 ± 1.3)

Width 3.7–8.0 (5.7 ± 1.0) 4.4–8.0 (5.8 ± 1.1)

Area 15.4–35.3 (24.1 ± 3.7) 20.0–35.3 (26.4 ± 4.5)

a Measurements are given in μm or μm 2 (for area). Minimum and maximum values as well as mean ± SD are provided.

a Only one of 2 cytoplasmic processes was measured for each parasite.

cytoplasm and large diffuse nuclei ( Table 2). The proportion of microgametocytes and macrogametocytes in the type material was approximately 3:5. A prominent azurophilic granule was seen only in the cytoplasm of 90% of microgametocytes ( Fig. 1H and I ) located close to the parasite nuclei.

3.2.2. Taxonomic summary

Type host: Undulated Antpitta Grallaria squamigera ( Grallaridae , Passeriformes ).

Additional hosts: unknown.

Type locality: Palacio Forest at the transition zone of Chingaza NNP (4̊ 41′ N, 73̊ 50′ W, 2950 masl), Cundinamarca, Colombia .

Distribution: This parasite has been recorded only at the type locality.

Type specimens: Hapantotype (accession No. UNAL: GERPH: PA340 -XI, intensity of parasitemia 0.25%, collected by Nubia E. Matta, 18 January 2015) and deposited in the biological collection GERPH (Grupo de Estudio Relación Parásito Hospedero) at the Universidad Nacional de Colombia, Bogotá, Colombia. Parahapantotypes (accession Nos., UNAL: GERPH: PA340 – I to UNAL: GERPH: PA340 –X and UNAL: GERPH: PA340 –XII to UNAL: GERPH: PA340 -XIX) are deposited in the same collection. Digital images of blood stages of the parasite in the type preparations are available on request from GERPH .

DNA sequences: The partial mitochondrial DNA genome (5891 bp) that includes cox1, cox3 and cytb genes (GenBank accession number MK103895 ) was obtained from the type host Grallaria squamigera .

Site of infection: Blood cells, of which the origin is unclear due to marked deformation caused by gametocytes.

Prevalence: Only one individual of the host species was collected and found to be infected, so the sample size does not allow to estimate of prevalence. The parasite was morphologically detected in 1 of out of 684 examined birds (0.12%). This or similar parasites were not found in 840 sampled birds of different species at Palacio Forest and Los Nevados NNP ( Table 1).

Etymology: The species name refers to the host genus name Grallaria ,

which is the type host of the parasite belongs.

3.2.3. Remarks

Seventeen species of Leucocytozoon , which gametocytes develop in fusiform host cells are reported to date (Supplentary Table S2), but only four of them, Leucocytozoon maccluri ( Greiner, 1976) , Leucocytozoon balmorali ( Peirce, 1984) , Leucocytozoon hamiltoni ( Valkiūnas et al., 2002) , and Leucocytozoon pterotenuis ( Lotta et al., 2015) are described in birds of the.

Passeriformes . The new species described here, Leucocytozoon grallariae , found in a passerine bird, can be readily distinguished from the four Leucocytozoon species mentioned above due to two distinctive morphological characters. First, the fusiform processes are short ( Table 2) in the host cells with fully grown gametocytes of L. grallariae ( Fig. 1D–I ). The length of these processes often does not exceed their largest width ( Table 2, Fig. 1 G, H ). This is not the case in L. maccluri , L. balmorali , L. hamiltoni and L. pterotenuis ; their gametocytes develop fusiform host cells, which fusiform processes are greater in length than in largest width. Second, the host cell nuclei assume the slender waning moon shapes, and the nuclei could reach the cytoplasmic processes, but never extended into them. None of these two characters are features of L. maccluri , L. balmorali , L. hamiltoni , L. pterotenuis (see Valkiūnas, 2005; Lotta et al., 2015).

Gametocytes in many Leucocytozoon species described in non-passerine birds develop in fusiform host cells ( Table S2). During L. grallariae infection, the host cell nuclei extend up to ½ of the circumference of the gametocytes, and the host cell nuclei might reach the beginning of cytoplasmic processes. This feature is not characteristic of other leucocytozoids where gametocytes developed fusiform host cells, and this feature can be used during identification of L. grallariae .

During microscopic examination of the type material, a co-infection with Leucocytozoon species with gametocytes developing in roundish host cells were detected ( Fig. 3 , Table S3). Gametocytes developing in fusiform host cells were ten times more often observed (parasitemia intensity is 0.25%) than gametocytes developing in roundish host cells (0.01%). In addition to the lineage associated with the GenBank No. MK103895 for the partial mtDNA genome reported above, one cytb lineage of 476 bp (L_GRSQU 02 GenBank No. MH909275) was amplified from the same blood sample of this type host. Genetic distance between both lineages was 0.23 ( Table 5), which suggest that they belong to a different species. However, from these two lineages obtained in the same sample, we propose that lineage MK103895 obtained by cloning corresponds to the parasite, which gametocytes develop in fusiform host cells in the sample (see discussion below).

Figures

Fig. 1. Leucocytozoon grallariae sp. nov. from Undulated Antpitta (Grallaria squamigera) captured at Palacio forest in the Chingaza National Natural Park (NNP), Colombia. Immature gametocytes (A–C), macrogametocytes (D–F) and microgametocytes (G–I) in fusiform host cells. Nucleus of host cells (black arrows) possessing mature gametocytes assumes a slender waning moon shape (D–I). Parasite nuclei are indicated by white arrows () and parasite nucleolus – by double white arrow tips (). Host cell cytoplasm is distorted by developing parasites forming a thin rim that develops into the cytoplasmic processes (asterisk *). In mature gametocytes, vacuoles are indicated by white arrow tips (). Volutin granules are indicated by double black arrow tips () and azurophilic granule by black arrow tips (). Giemsa-stained thin blood films. Scale bar = 10 μm.

Fig. 3. Gametocytes developing roundish host cells observed in type material of Leucocytozoon grallariae (A–C) and Leucocytozoon neotropicalis (D–F). Host cell nucleus (black arrows) deformed as a cap re- sembles the parasites of the Leucocytozoon fringilli- narum group. The white arrow indicates the parasite nuclei (), double white arrow tips shows the nu- cleolus (). Vacuoles (white arrow tips) are pre- sent, while volutin granules (double black arrow tips ) are few or absent in some gametocytes. Giemsa- stained thin blood films. Although gametocytes in roundish host cells were observed in both samples, those co-existing with Leucocytozoon grallariae (A–C), are significative smaller than Leucocytozoon sp. ga- metocytes found in type host of L. neotropicalis (D–F) (see Table S3). Scale bar = 10 μm.