The haemosporidian parasites of bats with description of Sprattiella alecto gen. nov., sp. nov.

Four species of Haemoproteidae were found in Pteropus alecto Temminck, 1837 in Queensland, Australia: i) Johnsprentia copemani, Landau et al., 2012; ii) Sprattiella alecto gen. nov., sp. nov., characterised by schizonts in the renal vessels; iii) Hepatocystis levinei, Landau et al., 1985, originally described from Pteropus poliocephalus Temminck, 1825 and, experimentally from Culicoides nubeculosus and found in this new host and for which features of the hepatic schizonts are reported; iv) gametocytes of Hepatocystis sp. which are illustrated but cannot be assigned to a known species. A tentative interpretation of phylogenetic characters of haemosporidians of bats is provided from the morphology of the gametocytes and localisation of the tissue stages with respect to recent data on the phylogeny of bats.


INTRODUCTION
G ametocytes of haemoproteids have frequently been reported in flying foxes (Pteropodidae). In the absence of information on the localization of their schizonts, they have been placed in the genus Hepatocystis. This genus was defined by Garnham (1966) as having "megaloschizonts" in the liver and gametogony stages in the blood. In reality, the haemoproteids of bats are highly variable with respect to such characters and their representatives in Pteropus alecto Temminck, 1837 from Queensland, provide an excellent example of this diversity. They are: i) Hepatocystis levinei described by Landau et al. (1985) from Pteropus poliocephalus Temminck, 1825. The life cycle was completed using Culicoides nubecu-losus under laboratory conditions (Landau et al., 1985). This species was found in four of 11 P. alecto examined from Townsville, Queensland, Australia, and we here provide additional data on the hepatic schizonts; ii) Johnsprentia copemani described by Landau et al. (2012) in P. alecto from Queensland. Co-infection with other haemoproteids was reported in the same paper; iii) Hepatocystis sp. represented by gametocytes in the blood of four of the 11 bats examined in the present study, but no schizonts were found which could be attributed to this species; iv) Sprattiella alecto gen. nov., sp. nov., herein described, gametocytes in the blood of six of 11 bats examined and characteristic schizonts in vessels of the kidney in one of the bats examined. Recent advances of phylogeny studies of the Pteropodidae based on molecular methods prompted us to reevaluate the morphological features of haemoproteids of bats and to compare them with the molecular data currently available for the hosts.

MATERIALS AND METHODS
T he methods used were essentially the same as those used in the description of Johnsprentia copemani by Landau et al. (2012). Original contribution Parasite, 2012, 19, 137-146 Eleven P. alecto, captured at Townsville using a mist net and exhibiting a parasitaemia with Haemoproteidae, were transported to Paris, to the Muséum National d'Histoire Naturelle, shortly after their capture, arriving on the 15.12.1978 and the 07.06.1979, respectively. Blood samples from each animal were collected by pricking the radial vein and the blood was smeared onto a slide, air dried, fixed with absolute methanol and stained using Giemsa stain (8 % in phosphate buffer, pH 7.4). The bats were examined over a period of several months. At autopsy, internal organs were fixed in Carnoy's fluid and serial sections of each organ were stained by the giemsa-colophonium method (Bray & Garnham, 1962;Garnham, 1966) and examined for tissue stages of the parasites. Type material has been deposited in the Muséum National d'Histoire Naturelle (MNHN), Paris. Bat number 850-932PX in which we found renal schizonts was autopsied on the 03-01-1979, after 19 days in captivity. DESCRIPTIONS SPRATTIELLA ALECTO SP. NOV. (Figs 1 -8, 11-24) H olotype material: schizont in section of kidney from P. alecto (850-932PX), MNHN P2 XXXI 10 ( Fig. 7). Paratypes: blood smears and sections of schizonts in kidney, deposited in MNHN.

SYSTEMATICS
• Renal schizonts Found in the lumen of renal veins, inside hypertrophied cells invariably with two nuclei (Fig. 4); nuclei also hypertrophied and modified. Host cell nuclei ellipsoidal, up to 10.5 μm with chromatin concentrated around the periphery and a large nucleolus. Host cell surrounded by fibrous, bright pink substance, up to 2 μm in thickness.
Parasitized cells free in vessels in most sections (Fig. 1), but in some serial sections, apparently attached to the walls of veins (Fig. 2) or wedged by a shrinkage of vessels (Figs 5, 6); round or oval depending on the angle of the section, sometimes deformed when trapped in shrunken vessel (Fig. 6). The smallest schizont observed was in an enlarged, binucleate rounded cell measuring 16.5 μm, with a dense basophilic cytoplasm. The cell was surrounded by an irregular, pink, tufted layer. The schizont, by contrast, was small, 4 μm in diameter, containing a dozen small nuclei with little, pale cytoplasm. The schizont develops as cordons, at first straight with small well-spaced nuclei (Figs 2, 5), then reflected upon themselves (Figs 7, 8). The enlarged host cell (Fig. 3) is pyriform with a uniformly granular, grey cytoplasm, containing distinctive nuclei: oval 2-2.5 μm long with a clear centre and a thin membrane with masses of chromatin attached to its inner surface. These nuclei subsequently fragment and the oldest schizont observed (Fig. 8) measured 83 × 21 μm with elongated masses within the host cell with a basophilic cytoplasm and numerous nuclei.
• Gametocytes  In the serial blood films examined, gametocytes were more numerous in the middle and terminal thirds of the films than in other regions. They lodge in red blood cells (RBC) and from the young trophozoite stage undergo significant transformation. The RBC is generally rounded but may become deformed with young parasites either irregularly ( Fig. 12) or pyriform (Fig. 13). The parasite is either a clear reddish-brown, or more commonly darker, reddish or brick-red. The gametocytes increase in size slightly (7 μm); the intact host cell is still visible surrounding the gametocyte which against the dark background appears clear, measuring between 6 and 7 μm. The microgametocytes (Figs 19-22) are pale pink or pink with several vacuoles and a more or less elongated zone of chromatin forming small, loosely attached masses. The macrogametocyte (Figs 23, 24) is pale blue to almost white against the darkly staining red blood cell background; its nucleus is smaller, and more dense than that of the microgametocyte. Pigment in both micro-and macrogametocytes is fine, scattered and more abundant towards the periphery. The microgametocytes of Sprattiella belong within the vivax group (vide infra).
• Relationship between the gametocytes of Sprattiella and renal schizonts In multiple infections it is often difficult to associate gametocytes with their corresponding schizonts. In the blood of bat 850-932PX, we observed the gametocytes of four species of Haemoproteidae. We already Original contribution know the gametocytes of three species: Johnsprentia copemani, Hepatocystis levinei and Hepatocystis sp. It is therefore logical to associate the renal schizonts with the distinctive gametocytes described here. Consequently, the holotype of Sprattiella alecto has been designated as the renal schizont and the name is therefore attached to this stage.
• Differential diagnosis The gametocytes of Sprattiella differ from those of the other three known species present in P. alecto due to their striking effect on the host cell and the following morphological features: the nucleus of the microgametocyte is not peripheral in contrast to the other species; in addition, the nucleus of Sprattiella is diffuse in contrast to that of Johnsprentia which is in the form of a crescent and that of H. levinei which has a condensed center surrounded by a clear space. Sprattiella alecto is also compared with the other two haemoproteids in which the schizont and its hypertrophied host cell are found in the lumen of medium to large blood vessels.
i) The schizonts of Dionisia bunoi are found in the blood vessels of the liver of the microchiropteran Hipposideros cyclops (Landau et al., 1980b). They exhibit several characters in common with the present parasite: an intravascular localisation, host cell and nucleus hypertrophied and the presence of a hirsute pink substance around the periphery. However, they are exclusively hepatic and not renal. The schizont of Dionisia is anchored by prolongations from the host cell to the hepatic capillaries of the surrounding tissue while that of Sprattiella is free. The schizont of Dionisia forms a solid rounded mass while that of Sprattiella consists of elongated cords. The unique host nucleus of Dionisia becomes very large and highly modified while those of Sprattiella only undergo a moderate increase in volume. Finally and of most significance, the microgametocytes of Dionisia are of the "falciparum" type and the microgametocytes of the "malariae" type; those from Pteropus -host are of the "vivax" type.
ii) The schizonts of Polychromophilus are found in the lumen of vessel, primarily in the lung, but also in other organs (liver, kidney, adrenals) (Landau et al., 1980a); the host cell is only visible in very young schizonts and is reduced in older forms to a slightly hypertrophied nucleus and a peripheral band of homogeneous, bright red 'colloid', different to the tufted band in Sprattiella. The gametocytes of Polychromophilus are of the "malariae" type.

HEPATOCYSTIS LEVINEI
• Additions to the description of the hepatic schizonts Schizonts and gametocytes belonging to H. levinei were found in the blood and in liver sections of several P. alecto, having first been reported from Pteropus poliocephalus by Landau et al. (1985). We had not, at the time, observed all of the hepatic schizogonic stages, in particular the intermediate stage between the young, stellate schizont lacking colloid and the mature schizont. Here, we illustrate ( Fig. 29) a characteristic microgametocyte with the condensed center and a mature schizont in a section through several lobes, each lobe comprising a peripheral zone with numerous small nuclei and a central zone filled with purple colloid. In the section in which the parasite is largest, the parasite and the surrounding zone of inflammation measure 1,700 × 2,700 μm; the parasite itself measures 630 × 370 μm.

HEPATOCYSTIS SP.
The microgametocytes of this species are of the "vivax" type with a diffuse nucleus (Figs 25-28). Macrogametocytes could not be distinguished from those of other species.

GAMETOCYTES IN MAMMALS
T he morphology of haemosporidian gametocytes in mammals is generally considered to be of limited taxonomic interest. By contrast, we consider that because of their stability and their archaic nature (they represent a fundamental primitive stage in the cycle of all coccidiomorphs), they are probably of considerable importance from a phyletic and systematic perspective. Schizogony, be it haematogenous or in tissues, is a superimposed character with numerous morphological variants including convergences between distant species. Landau et al. (1976a), based on the morphology of the gametocytes of mammalian haemosporidians, divided them into three types (Table I) which were designated by the name of the best known species in each group ("falciparum", considered the most archaic, then "malariae" and finally "vivax"). The classification is based on the morphology of the microgametocytes which are the most characteristic and most readily recognizable features of Plasmodium.  Rosin, Landau et al., 1978Garnham & Heisch, 1953Rosin, Landau et al., 1978Garnham & Heisch, 1953 Landau, 1981Garnham, 1966Miltgen, Landau et al., 1977(Rodhain, 1926) Miltgen, Landau et al., 1980Van der Kaay, 1964Landau & Adam, 1971Landau, Chavatte et al., 2012Landau, Chavatte et al., 2012Landau, Humpherey-Smith et al., 1985(Breinl, 1913) Landau, Humpherey-Smith et al., 1985Van Riel, Herniaux-L'Hoëst et al., 1951 Kuntz, 1966Eyles, Dunn et al., 1962Landau, Chabaud et al., 1980bLandau & Chabaud, 1978Mialhe & Landau, 1977Landau, Miltgen et al., 1976bLandau, Baccam et al., 1984Duval, Robert et al., 2007Landau, Baccam et al., 1984  Type of gametocytes: M = "malariae", F = "falciparum" and V = "vivax"; site of schizogony: HEP = hepatocyte and RES = reticulo-endothelial system. (*) indicate the potential location of schizogony which is unknown; (**) redescribed from Eptesicus fuscus by Marinkelle (1995) who change the genus name Polychromophilus by Bioccala. The reference of the authors of each taxa are listed in the references. The principal morphological characters of the three types are summarised in Table I and their distribution in different types of bats is presented in Table II. In addition, a dichotomous key for the identification of the genera of haemoproteids in bats is presented in Table III.

AFFINITIES WITH THE HAEMOSPIRIDIANS
OF SAUROPSIDS Garnham (1966), in his treatise of the Haemosporidia, separated them into two distinct lineages, those which multiplied in the reticulo-endothelial system including the haemosporidians of birds and reptiles, and in mammals Bioccala, designated at the time as a species of Polychromophilus; the others multiplying in hepatocytes, comprising Nycteria and Hepatocystis. Development in the reticulo-endothelial system is close to the primitive model seen in reptiles and birds and the evolution of schizonts in hepatocytes, is in our view a specialisation. The gametocytes of the "malariae" and "falciparum" groups have morphological characteristics in common with parasites of Sauropsidae and are therefore, according to Landau et al. (1976a), more archaic than those of the "vivax" group. Thus, Bioccala, a parasite of vepertilionids and miniopterids is, for us, the genus closest to the primitive species. Its gametocyte is ellipsoidal, more or less elongate, does not fill the erythrocyte, the pigment consists of large, irregularly grouped granules, the nucleus is poorly defined, and an accessory chromatin granule is present; the schizonts are small, invasive and are found in the reticuloendothelial system.
The "malariae" group is represented by two different genera: Polychromophilus in Vespertilionidae and Miniopteridae, and Nycteria in Nycteris and Rhinolophus. The archaic characters of the gametocytes are the fact that the erythrocyte remains partially visible when the gametocyte is mature, the presence of an accessory chromatin granule and the irregularly grouped, large chromatin granules. However, in contrast to Bioccala, the nucleus is well delimited and is rounded. The two genera also differ in their schizonts which are relatively large and non-invasive, in the reticulo-endothelial system for Polychromophilus and in hepatocytes for Nycteris.
The gametocytes of the "vivax" group are homogeneous morphologically and differ from the preceding forms. At maturity, they fill or nearly fill the erythrocyte, lack an accessory chromatin granule, have fine, abundant pigment, dispersed in mature forms, and a distinct boundary between nucleus and cytoplasm. Two species are reticulo-endothelial parasites (Johnsprentia copemani and Sprattiella alecto), but most of the species known from the Pteropodidae have megaloschizonts, which develop in hepatocytes (Hepatocystis) The chromatin in the nucleus of some Hepatocystis species instead of being diffuse is aggregated in the Original contribution  (2005) have proposed, using molecular techniques, a phylogenetic tree and dates for the appearance of the different genera of bats.
The hosts of haemosporidians of bats belong to five families: i) The Nycteridae harbour parasites of the "malariae" group. They separated from other Emballonuroidea 58-47 mya.
ii) The Miniopteridae and Vespertilionidae harbour a pair of cosmopolitan parasite genera, Bioccala and Polychromophilus, often in the same individual, which belong respectively to the "falciparum" and "malariae" groups. These two families are also ancient and separated according to Miller-Butterworth et al. (2007) 48-39 mya.
iii) The Rhinolophidae, where Rhinolophus harbours species of the "malariae" group and Hipposideros harbours species of three groups, "vivax", "malariae" and "falciparum". They diversified more recently, 39 mya. iv) The Pteropodidae, which harbour only species of the "vivax" group are even more recent and diverged from the preceding families 24 mya. The oblique doted line separates species with schizonts in the reticuloendothelial system (RES) on the right and the species with schizonts in hepatocytes (HEP) on the left. The green line (low left) encircles parasites with gametocytes of the "malariae" type, the blue line (low right) gametocytes of the "falciparum" type and the red line (top) gametocytes of the "vivax" type.