Haemoproteus (Parahaemoproteus) minchini, Chavatte & Okumura & Landau, 2017

Chavatte, Jean-Marc, Okumura, Chiharu & Landau, Irène, 2017, Haematozoa of the Great Blue Turacos, Corythaeola cristata (Vieillot, 1816) (Aves: Musophagiformes: Musophagidae) imported to Singapore Jurong Bird Park with description and molecular characterisation of Haemoproteus (Parahaemoproteus) minchini new species (Apicomplexa: Haemosporidia: Haemoproteidae), Raffles Bulletin of Zoology 65, pp. 325-340 : 327-334

Genus Haemoproteus Kruse, 1890

Haemoproteus (Parahaemoproteus) minchini new species ( Fig. 1 )

Material examined. Hapanthotype: one thin blood film numbered JBP7-1, made from the blood of Corythaeola cristata number H2910, location Jurong Bird Park, Singapore, coll. Dr. C. Okumura, 22 October 2013; deposited into the Parasitology collection of the Muséum National d’Histoire Naturelle, Paris under the accession number 617YY-PXX50, parasitemia is approximately: 0.5%.

Parahapanthotype: a second thin blood film numbered JBP7- 2, made from the blood of Corythaeola cristata number H2910, other information as for the hapantotype. Paratypes: thin blood films numbered JBP11, JBP12, JBP16 from Corythaeola cristata H2914, H2915, H2909, other data as for the hapanthotye are deposited into the Parasitology collection of the Muséum National d’Histoire Naturelle, Paris under the accession numbers: 222BF-PXX108, 221BF-PXX109 and 218BF-PXX106 respectively.

Type host. Corythaeola cristata ( Vieillot, 1816) – Great Blue Turaco, Musophagiformes , Musophagidae .

Site of infection. Mature erythrocytes; tissue stages unknown.

Locality. Tanzania.

Distribution and additional hosts. Tanzania and Uganda where a similar parasite has been recorded by Minchin (1910) from the blood of the type host. No other record neither DNA sequences were found identical.

DNA sequences. Fragment of the cytb (1,258bp) and cox1 (1,336bp) genes from the mitochondrial genome and fragment of the clpC (647bp) and tufA (814bp) genes from the apicoplast genome; all isolated from the blood of the type specimen C. cristata H2910; respective GenBank accession numbers are [ KU 160476 ] to [ KU 160479 ]; sequences will also be deposited into MalAvi database .

Vector. Unknown.

Etymology. The species name is given in honour of Edward Alfred Minchin, Professor of Protozoology in the University of London who first reported this parasite from the blood of C. cristata .

Description. Rings and young gametocytes ( Fig. 1A–C ): Develop in mature red blood cell (RBC), are round or oval in shape, with a peripheral dark nucleus and a large white vacuole. They are usually found in sub-apical or lateral position in the RBC and are rarely in contact with the nucleus or the membrane of the RBC. Growing they elongate in lateral position parallel to the nucleus of the RBC and extend up to a similar size. Their nucleus stains a bright fuchsia colour, adopts a subapical and transversal position, the vacuole becomes wider and the first thin granules of volutin start to appear peripherally.

Macrogametocytes ( Fig. 1E–K ): When young they continue to elongate, present an even thickness, and are almost straight or only very slightly curved. Their cytoplasm is loose and stains a pale blue, their nucleus becomes submedian and transversal; the first thin grains of dark brown pigment appear; the vacuole recesses while more granules of volutin accumulate at the apexes and around the vacuole. Thereafter the macrogametocytes continue to elongate, their maximum expand is from one pole of the RBC to the other but the majority remains shorter, lying onto the membrane of the RBC and separated or just loosely touching the nucleus of the RBC; they take a slightly more curved shape but never encircled the nucleus of the RBC. Their cytoplasm becomes denser, thinly granular and stains a deeper blue, with few small, clear and round vacuoles; the dark brown pigment grains coarse and more abundant with yellow reflects can be found anywhere but tend to scatter near the nucleus and the ends; the nucleus no longer transversal recesses in a dense, deeper fuchsia mass of variable size often attached to the membrane of the parasite; the amount of volutin continues to increase creating clumps of dark violet aggregates filing the polar regions. All along the growth the margin of the parasite remains even and the extremity rounded, sometime a subapical throttling is observed, accentuating the accumulation of volutin granules and the darkness of the extremities of the parasites. Only the largest fully grown macrogametocytes touch the nucleus of the RBC and induced a slight lateral displacement of it.

Microgametocytes ( Fig. 1D, L–P ): General development and configuration are similar to the macrogametocytes with the usual sexual variations. However, they display a slightly bigger size at all stages; a larger nucleus made of a very loose and diffused chromatin, only differentiated from the cytoplasm by its deeper pink colour; less condensed volutin granules accumulating onto the membrane of the polar regions of the parasite and usually not filling them entirely. Due to their larger size, they tend to hypertrophy more the RBC, to touch more often the nucleus of the RBC and to displace it more laterally. Parasites that curved above one of the nucleus pole are seldom seen.

Remarks. Minchin (1910) gave the following brief description of a Haemopoteus sp., he observed into the blood of a C. cristata from Uganda: “The Halteridia are abundant and of a distinct type, of even thickness, very slightly curved, with coarse pigment-grains, and with red-staining grains at the two ends of the body in addition to a more diffuse red patch which apparently represents the nucleus and is usually situated near the middle of the body”. Along with this short description Minchin (1910) published six coloured drawings that illustrated the above mentioned characters of his parasite and also clearly depicted the yellowish reflect of the pigmentgrains, the even margin of the extremities, the condensed nucleus of the macrogametocyte, the presence of a space between the macrogametocyte and the nucleus of the RBC and its absence for the microgametocyte. When compared with our parasite, we noticed that all the morphological features described or illustrated by Minchin are encompassed into our description. Therefore, it seems very likely that we are looking at the same parasite that Minchin a century ago and we have therefore decided to dedicate this parasite to him.

Differential diagnosis. One other haemoproteid species has been described from the Musophagiformes : Haemoproteus montezi Travassos Santos Dias, 1953 from the blood of the Purple-crested Turaco, Tauraco porphyreolophus ( Vigors, 1831) in Mozambique. This parasite has been reported from several species of Turacos ( Bennett & Herman, 1976; Bennett et al., 1982; Bennett & Pierce, 1990; Bennett et al., 1992; Valkiūnas, 2005; Vrána et al., 2005), and also from some Nectariniidae ( Bennett & Herman, 1976) but never from the Great Blue Turaco. Travassos Santos Dias (1953), in the original description based on blood samples recovered from two sacrificed birds noted the presence of round forms that displace the nucleus of the RBC to the periphery and the presence of halteridian forms that did not displace it. He might have dealt with a mixed infection or with parasites that have undergone typical post-mortem changes as suggested by Bennett & Pierce (1990). Focusing onto the halteridian forms described by Travassos Santos Dias (1953) they are readily distinctive of H. minchini new species by several characters: the more or less narrowed extensions at the extremities in both sex; the margin sometime tortuous; the presence in the macrogametocytes of one or two large refractile vacuoles located into the polar region; the dark chatain almost black, mid-size grains of pigment scattered throughout the cytoplasm but that tend to accumulate at the extremities; and their lack of volutin granules.

Bennett & Pierce (1990) redescribed H. montezi based on material they deposited into the IRCAH and designated as: i) neohapantotype: blood film No. 39042a obtained from Tauraco hartlaubi ( Fischer & Reichenow, 1884) collected by C.M. Herman, Kabete, Kenya in March 1939 and ii) several paraneohapantotypes: blood film No. 39042b (same as above); blood film No. 36767 obtained from Tauraco livingstoni ( Gray, 1864) collected by M. Lips, near Lubumbashi, Zaire in February 1956. On their material, these authors noted an amoeboid margin of the young stages; noticed as Travassos Santos Dias (1953) the slightly amoeboid polar margin of the gametocytes; highlighted the parasite tendency to displace the nucleus of the RBC longitudinally toward one extremity of the RBC and to loop around the other pole of the nucleus giving it an asymmetrical aspect; mentioned that the yellow-brown pigment was small, difficult to see and scattered throughout the cytoplasm while the volutin granules were rarely seen but when present located in the poles. All these morphological features made their parasite distinct from H. minchini new species.

Valkiūnas (2005) gave also a description of H. montezi from the IRCAH material. He highlighted the same morphological features that Bennett & Pierce (1990); presented their morphometric measurements; insisted on the asymmetrical aspect of the parasite, the narrowed extremities and the “cleft” left between the young and growing stages and the nucleus of the RBC; but to their contrary mentioned that the outline of parasites is even and the volutin granules usually present and gathered at the ends of the parasite.

Minchin (1910) also observed a Haemoproteus sp. from the blood of the Ross’s Turaco Musophaga rossae Gould, 1852 from Uganda. The description and illustrations he provided led him to readily differentiate this parasite from the one he observed in C. cristata and actually seem to be more corresponding to the description of H. montezi .

Morphometric measurements. Bennett & Pierce (1990) provided measurements of H. montezi that are recalled for reference along with those of H. minchini new species in Table 2. As the only results and not the measurement data of H. montezi are known, it was not possible to perform a statistical comparisons between the morphometric measurement of H. minchini new species and H. montezi , however some difference can be noticed with caution: i) the macrogametocytes and the microgametocytes of H. minchini new species appear respectively slightly larger in both length and width than those of H. montezi ( Table 2); ii) the infected RBC seem more enlarged in both length and width by the gametocytes of H. minchini new species than by the gametocytes of H. montezi ( Table 2); iii) the gametocytes of H. minchini new species tend to displace more laterally the nucleus of the infected RBC (NDR reduced) than the gametocytes of H. montezi ( Table 2); iv) the gametocytes of H. minchini new species harbour less pigment granules than the gametocytes of H. montezi ( Table 2). All these comparisons apparently confirm again the differences between these two parasites.

Morphometric measurements of the gametocytes of H. minchini new species, were assessed by Shapiro-Wilk test ( Shapiro & Wilk, 1965) and did not follow a normal distribution. Therefore, they were analysed by the Mann- Whitney U test ( Mann & Whitney, 1947) (α=0.05) and highlight a sexual dimorphism. Mature microgametocytes are significantly larger in both length (p =0.00126) and width (p =0.01072) than the macrogametocytes. The sexual dimorphism is also noted on the impact onto the infected RBC. Gametocytes of H. minchini new species significantly enlarge the infected RBCs in both length and width (p <0.00001 and p =0.00006 respectively) for RBCs infected by macrogametocytes and (p <0.00001 and p <0.00001 respectively) for RBCs infected microgametocytes. The enlargement induced by the microgametocytes is significantly bigger in both directions than the one induced by the macrogametocytes (p <0.00001 and p <0.00001 respectively). The sexual dimorphism is not observed on the impact onto nucleus of the infected RBC. Gametocytes of H. minchini new species do not significantly modify the length and width of the nucleus of the infected RBCs (p =0.48803 and p =0.08226 respectively) for RBCs infected by macrogametocytes and (p =0.17879 and p =0.06178 respectively) for RBCs infected microgametocytes. No significant difference between the microgametocytes and the macrogametocytes are observed on their impact onto the nucleus of the infected RBCs neither in its length and width (p =0.17619 and p =0.08226 respectively) nor in its displacement (p =0.05821).

Molecular data and phylogenetic analysis. The PCRs for haemosporidian parasite detection are congruent with the microscopy and confirm the 3 negative birds, the 2 mixed Plasmodium species infected birds and the single pure Haemoproteus infection. Haemoproteus minchini new species is further characterised at the genetic level by four sequences of conserved genes namely cytb (GenBank: KU160476 ), cox1 (GenBank: KU160477 ), clpC (GenBank: KU160478 ) and tufA (GenBank: KU160479 ) obtained from the type specimen, no cloning and sequencing were attempted from the mixed infected samples. Alignment and BLAST comparison of the sequences for cytb, cox1 and clpC confirm that H. minchini new species belongs to the Haemoproteidae and it is distinct from all other deposited sequences. Regarding tufA, it is the first time that a sequence of this gene is obtained from a Haemoproteus parasite.

Phylogenetic analysis of cytb gene: It was performed with our new sequence [ KU160476 ] and a selection of 79 identified morpho-species sequences recorded into the Avian haemosporidian uniform database: MalAvi (sequence [ KU160476 ] was trimmed according to the standard fragment size of the MalAvi database to a fragment of 479bp) ( Fig. 2 ). It shows that H. minchini new species is a distinct from all other sequences of identified Haemosporidia parasites deposited into the MalAvi database. Analysis of this short fragment shows that H. minchini new species belongs to the Parahaemoproteus subgenus and falls within a small clade forms by Haemoproteus balmorali Pierce, 1984 ; Haemoproteus attenuatus Valkiūnas, 1989 ; Haemoproteus n: sample size, X: mean, SD: standard deviation. L: length, W: width, NL: nucleus length, NW: nucleus width, NDR: nuclear displacement ratio (calculated according to Bennett & Campbell, 1972), NPG: number of pigment granules. * Measurements of Haemoproteus montezi are from Bennett & Pierce, 1990. All sizes are given in µm.

gavrilovi Valkiūnas & Iezhova, 1990; Haemoproteus paranucleophilus Iezhova, Dodge, Sehgal, Smith & Valkiūnas, 2011 ; and Haemoproteus ptilotis Clark, Adlard & Clegg, 2015 ; with divergences ranging from 2.8% to 5.2% ( Fig. 2 ). A similar analysis conducted with all the sequences deposited into Genebank and MalAvi (data not shown) highlights a more closely related sequence: H_AFR59 [ KM056456 ] that diverges by only 1% on this short fragment. Interestingly this sequence derivate from an unidentified Parahaemoproteus species isolated from another Musophagiformes host, the Tauraco corythaix schalowi ( Reichenow, 1891) screened during a blood parasites survey in Malawi ( Lutz et al., 2015).

Phylogenetic analysis of cox1 gene: It was performed with our new sequence [ KU160477 ] and 28 other sequences of identified avian Haemosporidia downloaded from Genbank (fragment size of 918 bp) ( Fig. 3 ). This analysis confirms that H. minchini new species belongs to the Parahaemoproteus subgenus. It also shows that H. minchini new species is distinct from all the other sequences included into the analysis and forms a sister group to two others clades containing parasites of the Passeriformes : Haemoproteus fringillae ( Labbé, 1894) , Haemoproteus magnus Valkiūnas & Iezhova, 1992 , Haemoproteus lanii Mello, 1936 , Haemoproteus belopolskyi Valkiūnas, 1989 and Haemoproteus parabelopolskyi Valkiūnas, Križanauskienė, Iezhova, Hellgren & Bensch, 2007 , and Haemoproteus tartakovskyi Valkiūnas, 1986 , Haemoproteus passeris Kruse, 1890 and Haemoproteus vireonis Bennett, Caines & Woodworth-Lynas, 1987 respectively with divergences ranging from 5.1% to 9.3% ( Fig. 3 ).

Phylogenetic analysis of clpC gene: It was performed with our new sequence [ KU160478 ] and 31 other sequences of identified avian Haemosporidia downloaded from Genbank (fragment size of 505bp) ( Fig. 4 ). Analysis of this gene from the apicoplast confirms also that H. minchini new species belongs to the Parahaemoproteus subgenus. Similarly to the results obtained from the cox1 gene, it appears related to some parasites of the Passeriformes and is placed at the base of clade containing H. lanii , H. belopolskyi , H. magnus and H. fringillae with divergence ranging from 8.9% to 13.2% ( Fig 4 ).

Phylogenetic analysis of tufA gene: It was performed with our new sequence [ KU160479 ] and 19 other sequences of identified Haemosporidia downloaded from Genbank (fragment size of 814 bp) ( Fig. 5 ). It is the first time that a sequence of tufA gene is obtained from a haemoproteid parasite. Phylogenetic analysis on this gene confirms that H. minchini new species is not related to the parasites of the genus Plasmodium and forms a sister group to all the Plasmodium species from Birds, Rodents and Primates, included in to the analysis, that cluster within a single clade. The genetic divergence is ranging from 12.7% to 15.5% and equal to 14.3% with the Plasmodium species and Leucocytozoon caulleryi Mathis & Leger, 1909 , respectively, clearly separating H. minchini new species from both Plasmodium and Leucocytozoon genera ( Fig. 5 ).

Figures

Fig. 1. Microphotographs of Haemoproteus (Parahaemoproteus) minchini new species from the blood of the Great Blue Turaco (Corythaeola cristata). A, ring; B–C, young gametocytes; E–K, macrogametocytes; D, L, early microgametocyte; M–P, microgametocytes. Giemsa stained thin blood film from hapantotype material. Scale bar = 10 µm.

Fig. 2. Molecular phylogeny based on the mitochondrial cytb gene illustrating the position of Haemoproteus (Parahaemoproteus) minchini new species within the avian haemosporidian parasites. The analysis was performed by Maximum Likelihood (ML) method with GTR+Γ+I model on 79 identified morpho-species sequences (49 Haemoproteus, 21 Plasmodium and 9 Leucocytozoon used as out-group to root the tree) recorded into the MalAvi database (fragment size 479bp) and our sequence of H. minchini new species [KU160476]. Statistical branch support (>60%) is provided by bootstrap values (1,000 replicates) on the branches. MalAvi accession numbers are in square bracket and GenBank accession numbers are in vertical bars.

Fig. 3. Molecular phylogeny based on the mitochondrial cox1 gene illustrating the position of Haemoproteus minchini new species within the avian haemosporidian parasites. The analysis was performed by Maximum Likelihood method (ML) with GTR+Γ+I model on 28 sequences from identified species (16 Haemoproteus, 10 Plasmodium and 2 Leucocytozoon used as out-group to root the tree) downloaded from GenBank (fragment size 918bp) and our sequence of H. minchini new species [KU160477]. Statistical branch support (>60%) is provided by bootstrap values (1,000 replicates) on the branches. GenBank accession numbers are in vertical bars.

Fig. 4. Molecular phylogeny based on the apicoplast clpC gene illustrating the position of Haemoproteus (Parahaemoproteus) minchini new species within the avian haemosporidian parasites. The analysis was performed by Maximum Likelihood (ML) method with GTR+Γ+I model on 31 sequences from identified species (16 Haemoproteus, 13 Plasmodium and 2 Leucocytozoon used as out-group to root the tree) downloaded from GenBank (fragment size 505bp) and our sequence of H. minchini new species [KU160478]. Statistical branch support (>60%) is provided by bootstrap values (1,000 replicates) on the branches. GenBank accession numbers are in vertical bars.

Fig. 5. Molecular phylogeny based on the apicoplast tufA gene illustrating the position of Haemoproteus (Parahaemoproteus) minchini new species within the haemosporidian parasites. The analysis was performed by Maximum Likelihood (ML) method with GTR+Γ+I model on 19 sequences from identified species (18 Plasmodium from Bird, Rodents and Primates and 1 Leucocytozoon used as out-group to root the tree) downloaded from GenBank (fragment size 814 bp) and our sequence of H. minchini new species [KU160479]. Statistical branch support (>50%) is provided by bootstrap values (1,000 replicates) on the branches. GenBank accession numbers are in vertical bars.