Syst Parasitol (2009) 74:219–223 DOI 10.1007/s11230-009-9209-3 A new species of Isospora Schneider, 1881 (Apicomplexa: Eimeriidae) in Ruppell’s agama Agama rueppelli (Vaillant) (Sauria: Agamidae) from East Africa, with a review of this genus in agamid lizards Andrei Daniel Mihalca Æ Miloslav Jirků Æ Patrick Kenyatta Malonza Æ David Modrý Received: 15 April 2009 / Accepted: 4 June 2009 Ó Springer Science+Business Media B.V. 2009 Abstract Coprological examinations of eight Ruppell’s agamas Agama rueppelli (Vaillant) revealed the presence of a coccidium of the genus Isospora Schneider, 1881 that represents a previously undescribed species. Oöcysts of Isospora farahi n. sp. are spherical or subspherical, 29.1 (26–31) 9 28.8 (26–31) lm, with a shape-index of 1.01 (1–1.07). An oöcyst residuum, polar granules and micropyle are absent. The oöcyst wall is bilayered, brownish and smooth, c. 1.5–2 lm thick. The sporocysts are oval, 16.6 (15–18) 9 11.4 (11–12) lm, with a shape-index of 1.46 (1.25–1.64) and both Stieda and substieda bodies. A sporocyst residuum is present as medium- A. D. Mihalca (&) Department of Parasitology and Parasitic Disease, University of Agricultural Sciences and Veterinary Medicine, Calea Mănăştur 3-5, 400372 Cluj-Napoca, Romania e-mail: amihalca@usamvcluj.ro M. Jirků
D. Modrý Institute of Parasitology, Academy of Sciences of the Czech Republic, Branišovská 31, 37005 České Budějovice, Czech Republic P. K. Malonza Department of Herpetology, National Museums of Kenya, Museum Hill, Nairobi, Kenya D. Modrý Department of Parasitology, University of Veterinary and Pharmaceutical Sciences, Palackého 1-3, 61242 Brno, Czech Republic sized granules scattered irregularly among the sporozoites. The sporozoites are vermiform, with a large posterior spherical refractile body. Endogenous development is intranuclear in the epithelial cells of the small intestine. Sporulation is unknown, as oöcysts were recovered from the faeces. Introduction Eimeriid coccidian parasites (Apicomplexa: Eimeriorina) of poikilotherm hosts, especially those of reptiles, represent a neglected part of our biodiversity. In contrast to eimeriids from birds and mammals, the assemblage of eimeriid coccidia from reptiles represents a phylogenetically and taxonomically complex group of protists, as reflected by repeated attempts to solve their higher-level taxonomy (Paperna & Landsberg, 1989; Jirků et al., 2002). Comparison of the diversity of lizards of the family Agamidae with the number of coccidian species described from these hosts clearly shows the under-sampling and limits of our knowledge. The Agamidae comprises over 50 genera widely distributed in Africa, Asia and Australia (Zug et al., 2001). Of these, only two genera are found in East Africa, namely Acanthocercus and Agama (see Spawls et al., 2004). Only two species of coccidia have previously been described from African agamids, Eimeria agamae (Laveran & Petit, 1910) Reichenow, 1921 and E. colonorum Prasad, 1960, both from Agama agama Linnaeus. 123 220 In present paper, we describe a new species of Isospora Schneider, 1875 from Agama rueppelli (Vaillant), an abundant agamid from dry, low-altitude savanna and semi-desert areas of Eastern Africa. Materials and methods Eight specimens of Agama rueppelli were collected at various localities in Kenya. Animals were killed using an intra-coelomic overdose of barbiturates (ThiopentalÒSpofa) and dissected. Fresh contents from the terminal part of the large intestine were preserved with 2.5% (w/v) potassium dichromate (K2Cr2O7) and the gastrointestinal tract of each animal was preserved in 10% buffered formalin. Faecal samples were examined microscopically after concentration by flotation with Sheather’s sugar solution (specific gravity 1.25). Oöcysts and endogenous stages were measured and photographed using differential interference contrast (DIC) optics on an Olympus AX70 microscope. Measurements were made using a calibrated ocular micrometer and are reported in micrometres, as the means, followed by the range in parentheses. After coprological examination, fixed tissues of a single infected lizard, were processed for histology using standard methods. Paraffin sections, 5–6 lm thick, were stained with haematoxylin and eosin (H&E) and examined using light microscopy. Syst Parasitol (2009) 74:219–223 Isospora farahi n. sp. Type-host: Agama rueppelli (Vaillant) (Sauria: Agamidae), Ruppell’s agama. Type-locality: Kalkumpe (02°310 5400 N, 36°490 2000 E), Marsabit District, Kenya. Type-material: Photosyntypes are deposited in the protozoological collection of the Institute of Parasitology of the Biology Centre of the Academy of Sciences of Czech Republic, České Budějovice, Czech Republic, under collection number IP ProtColl 6. Symbiotype: An ethanol-preserved host specimen is deposited in the herpetological collection of the National Museum Prague, Czech Republic, under collection number NMP6V 73614. Prevalence: One of eight examined Ruppell’s agamas had oöcysts of I. farahi n. sp. in its intestinal contents. Etymology: The species is named for Dr Idle Farah, the General Director of the National Museums of Kenya, in recognition of his support for collaborative research. Description (Figs. 1–7) Oöcysts Fully-sporulated oöcysts (Figs. 1–2, 7) spherical or subspherical, 29.1 (26–31) 9 28.8 (26–31); shapeindex (SI, length/width ratio) 1.01 (1–1.07), n = 30. Figs. 1–6 Micrographs of the developmental stages of Isospora farahi n. sp. 1–2. DIC micrographs of fully-sporulated oöcysts, both at the same scale. 3. Young trophozoite. 4. Macrogamont with peripherally localised, fine, wall-forming bodies. 5. Microgamont with microgametes. 6. Zygote with well-defined oöcyst wall. 3–6. Histological sections stained with H&E, all at the same scale. Scale bars: 20 lm 123 Syst Parasitol (2009) 74:219–223 221 Sporulation Unknown; oöcysts recovered from faeces after few weeks of storage in potassium dichromate; but it is probably exogenous, as in other members of this genus known from saurians. Discussion Fig. 7 Composite line drawing of sporulated oöcyst of Isospora farahi n. sp. Oöcyst residuum, polar granules and micropyle absent. Oöcyst wall bilayered, c. 1.5–2 thick (inner layer much thinner, c. 0.5), brownish and smooth. Wall striations absent. Sporocysts oval, 16.6 (15–18) 9 11.4 (11–12), SI = 1.46 (1.25–1.64), n = 30. Stieda body discoid, c. 1 high and 2–3 wide (Fig. 1). Substieda body globular, c. 2 high and 3 wide. Sporocyst residuum consists of medium-sized granules scattered irregularly among sporozoites (Fig. 1). Sporozoites vermiform, with large posterior spherical refractile body, c. 5 9 5 (Fig. 2), and centrally located spherical nucleus, c. 3 in diameter. Site of infection and endogenous stages Endogenous developmental stages scattered throughout mucosa of small intestine. All developmental stages intranuclear in enterocytes, localised in distinct parasitophorous vacuole. Trophozoites (Fig. 3) most prevalent. Mature macrogamonts spherical, 19–25 in diameter, containing centrally localised nucleus and fine wall-forming bodies scattered close to surface (Fig. 4). Single mature microgamont found, spherical, 20 in diameter, contains numerous curved, 2–3 long, intensively stained microgametes (Fig. 5). Few zygotes in final stages of development observed, lack wall-forming bodies and surrounded by fine oöcyst wall (Fig. 6). No species of Isospora have previously been reported from African agamids. However, 10 species have been described from agamid lizards in Asia and Australia (Table 1). I. farahi n. sp. differs from all these species in oöcyst morphology. Four species (I. amphiboluri Cannon, 1967; I. cannoni Finkelman & Paperna, 1994; I. caryophila Rogier & Colley, 1976; and I. gonocephali Maupin, Diong & McQuistion, 1998) have much smaller oöcysts than I. farahi (see Cannon, 1967; Rogier & Colley, 1976; Finkelman & Paperna, 1994; Maupin et al., 1998). Moreover, I. caryophila also differs by having a rather ellipsoidal oöcyst shape. Sporocyst shape differentiates I. farahi from other three species (I. phrynocephali Ovezmukhammedov, 1971; I. rayi Mandal, 1966; and I. rustamovi Ovezmukhammedov, 1977) (see Mandal, 1966; Ovezmukhammedov, 1971, 1977). I. choochotei Finkelman & Paperna, 1994 lacks a substieda body (Finkelman & Paperna, 1994), whereas in I. farahi the substieda body is clearly visible. I. lacertae Saum, Diong & McQuistion, 1997 has light perpendicular striations in the outer oöcyst wall (Saum et al., 1997), which are evidently absent in I. farahi. I. deserti Finkelman & Paperna, 1994 is the most similar species in terms of oöcyst morphology, but differs by possessing slightly smaller oöcysts, relatively smaller sporocysts and less prominent Stieda bodies (Finkelman & Paperna, 1994). However, further distinguishing features can be drawn from the localisation and appearance of the stages of endogenous development. Basically, isosporan coccidia from reptilian hosts exhibit two modes of endogenous development: intracytoplasmic or intranuclear. Although it is exceptional among coccidia from homeotherms, intranuclear localisation, which is the case in I. farahi n. sp., is a rather common feature of Isospora spp. from saurian hosts (Finkelman & Paperna, 1994; Paperna & Finkelman, 1998). Also, among species parasitising agamid lizards, intranuclear localisation is known in at least in four other species (Table 1). Among them, I. deserti, the 123 222 Syst Parasitol (2009) 74:219–223 Table 1 Revised checklist with key taxonomic characters of Isospora species from lizards of the family Agamidae Species Host Oöcyst shape and size I. farahi n. sp. Agama rueppelli Subspherical 29.1 (26–31) 9 28.8 (26–31) I. amphiboluri Cannon, 1967 Sporocyst shape and size Endogenous development Geographical origin Ovoid Intranuclear Kenya 16.6 (15–18) 9 11.4 (11–12) Pogona barbata, (Sub)spherical P. vitticeps 24.9 (22.1–26.8) 9 24.2 (22.1–26.8) Oval 14.6 (13.8–15.7) 9 10.3 (9.1–11.0) I. cannoni Finkelman & Diporiphora Paperna, 1994 australis Subspherical Ovoid 22.8 (20–25) 9 24.8 (22.5–27.5) 14.7 (14–15.5) 9 10.2 (10–11.5) I. caryophila Rogier & Colley, 1976 Ellipsoidal–subspherical Ovoid 23.5 (21–30) 9 21.9 (18–29) 13.2 (9–15) 9 8.2 (7–10) I. choochotei Finkelman Calotes & Paperna, 1994 mystaceus (Sub)spherical Ovoid 29.3 (24–32) 9 29.5 (28–32.5) 16.5 (15.5–18) 9 11.2 (11) I. deserti Finkelman & Paperna, 1994 Spherical Ovoid 27.7 (25–28) 9 27.7 (25–28) 16.1 (14–17.5) 9 10.7 (10–11) I. gonocephali Maupin, Gonocephalus Diong & McQuistion, grandis 1998 Subspherical–ovoid Almond-shaped 22.3 (19–25) 9 18.7 (17–23) 13.5 (12–15) 9 9.2 (8.5–10.0) I. lacerate Saum, Diong Calotes & McQuistion, 1997 versicolor Subspherical–ovoid Ovoid 28.1 (23.0–31.0) 9 26.5 (23.0–28.0) 14.6 (13.0–15.0) 9 10.3 (7.0–11.0) Gonocephalus grandis Trapelus pallidus, T. mutabilis I. phrynocephali Ovezmukhammedov, 1971 Phrynocephalus helioscopus Spherical Subspherical 26.2 (24.3–27.0) 9 26.2 (24.3–27.0) 14.7 (13.5–18.9) 9 9.2 (8.1–13.5) I. rayi Mandal, 1966 Ptyctolaemus gularis Spherical Naviculoid 26.3 (25.5–27.4) 9 26.3 (25.5–27.4) 15.4 (14.5–16.3) 9 8.6 (9.5–10.5) Phrynocephalus reticulatus Spherical Pyriform 26.2 (18.9–32.4) 9 26.2 (18.9–32.4) 16.5 (13.5–18.9) 9 11.7 (10.8–13.5) I. rustamovi Ovezmukhammedov, 1977 Intracytoplasmic Australia Intranuclear Australia Intranuclear Malaysia Intranuclear Thailand Intranuclear Israel No data Malaysia No data Singapore No data Turkmenia No data India No data Turkmenia All sizes are in micrometres only species with a similar oöcyst morphology, differs in the appearance of wall-forming bodies which are evidently larger than those of I. farahi (cf. our Fig. 4 with figure 8 of Finkelman & Paperna, 1994). Only limited information exists on the pathogenicity of reptilian species of Isospora. In the case of I. amphiboluri from an Australian agamid in captivity, the destruction of the intestinal epithelium and resulting deaths have been reported (McAllister et al., 1995). However, under natural condition, these coccidia appear to have little effect on parasitised 123 hosts, which is also evident in the absence of histopathological changes in our material. Acknowledgements Research on the diversity of parasites of East African vertebrates was facilitated by Biota East Africa, and we are deeply indebted to Jörn Koehler for generous help. We also thank: Richard Bagine (Kenyan Wildlife Service) for assistance and issuing necessary permits; and Damaris Rotich (National Museums of Kenya) for help with the organisation of the trip and kindly providing necessary laboratory space in Nairobi. 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