Isospora ptyodactyli n. sp. (Apicomplexa: Eimeriidae), a new coccidian parasite of the fan-footed gecko Ptyodactylus puiseuxi Boutan, 1893 (Reptilia: Gekkonidae) from Jordan

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Systematic Parasitology 39: 45–48, 1998. c 1998 Kluwer Academic Publishers. Printed in the Netherlands.

Isospora ptyodactyli n. sp. (Apicomplexa: Eimeriidae), a new coccidian parasite of the fan-footed gecko Ptyodactylus puiseuxi Boutan, 1893 (Reptilia: Gekkonidae) from Jordan David Modr´y1 , Bˇretislav Koudela2, Ratib M. Al-Oran3 , Zuhair S. Amr4 and David Doleˇzel2;5 1

Department of Parasitology, University of Veterinary and Pharmaceutical Sciences, Palack´eho 1-3, CZ - 612 42 Brno, Czech Republic 2 ˇ e Institute of Parasitology, Academy of Sciences of the Czech Republic, Braniˇsovsk´a 31, CZ – 370 05 Cesk´ Budˇejovice, Czech Republic 3 Department of Biology, Mutah University, Mutah, Jordan 4 Department of Biology, Faculty of Science, Jordan University of Science and Technology, Irbid, Jordan 5 ˇ e Budˇejovice, Czech Republic Department of Parasitology, Faculty of Biology, University of South Bohemia, Cesk´ Accepted for publication 25th April, 1997

Abstract Faecal samples from 17 fan-footed geckoes Ptyodactylus puiseuxi Boutan were examined for coccidian parasites. Five geckoes (29%) were found to be passing o¨ocysts of the genus Isospora Schneider. Comparison with other members of the genus Isospora indicates that the coccidian found represents a new species. Sporulated o¨ocysts of I. ptyodactyli n. sp. are spherical or subspherical, 22.1 (19.0–24.0)  21.2 (18.0–23.0) m, with a shape-index (length/width) of 1.04; and a smooth and bilayered o¨ocyst wall, 1.0–1.5 m thick. A micropyle, o¨ocyst residuum and polar granule are absent. The sporocysts are ellipsoidal, 12.2 (11.0–14.0)  8.0 (7.5–9.0) m, with a shape index of 1.5 (1.4–1.9). Stieda and substieda bodies are present, the Stieda body being knob-like and the substieda body spherical to subspherical. A sporocyst residuum is present and composed of numerous granules of different size scattered among the sporozoites. The sporozoites are vermiform, with a slightly granulated surface appearance, and are arranged head to tail within the sporocyst. Most o¨ocysts have still to sporulate when excreted; sporulation was completed within 12 h at 25  C. Endogenous development occurs inside the nuclei of enterocytes in the small intestine. Introduction

Materials and methods

Petricollous geckos of the genus Ptyodactylus Goldfuss inhabit arid and semiarid regions of North Africa and the Near and Middle East. Five species have recently been suggested as valid (R¨osler, 1994). Other than Schellackia ptyodactyli Paperna & Finkelman, 1996 from Ptyodactylus hasselquistii Donndorf (see Paperna & Finkelman, 1996), no other coccidian species has been described from these geckoes to date. Herein, we present a description of a new species of Isospora Schneider from fan-footed geckoes Ptyodactylus puiseuxi Boutan in Jordan.

Seventeen specimens of fan-footed geckoes Ptyodactylus puiseuxi were collected by hand during a parasitological and herpetological survey in Jordan during April, 1996. Five specimens originated from the basalt desert around Safawi (32 120 N; 37 100 E) and another 12 were collected from Mediterranean oak forests around Zoubia (32 250 N; 35 450 E). Lizards were kept separately or in groups of 2–3 specimens in small plastic boxes prior to transportation to the University of Veterinary and Pharmaceutical Sciences Brno, the Czech Republic. In the laboratory, lizards were housed individually in small glass or plastic terraria with 15–25 W heating lamps. The ground temperature of the terraria reached

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Figures 1–4. Isospora ptyodactyli n. sp. 1. Nomarski interference contrast (NIC) photograph of sporulated o¨ocyst; note characteristic Stieda and substieda body (circle). 2–4. Endogenous development and endogenous stages stained with H&E: 2. Atrophy of the villi in the small intestine in infected fan-footed gecko. 3. Villi in the small intestine in uninfected fan-footed gecko. 4. The intranuclear endogenous stages; note early trophozoite (arrow), mature macrogamonts and young o¨ocyst (arrowhead). Scale-bars: 1, 10 m; 2, 3, 100 m; 4, 20 m.

27–29  C during the daytime and 19–21  C at night. Lizards were fed daily on laboratory-reared crickets with vitamin and mineral supplementation. Individual faecal samples were collected repeatedly from the substrata of the terraria, or by carefully manipulating the lizards, and examined microscopically after concentration using flotation in modified Sheather’s sugar solution (specific gravity 1.30). Three infected and two uninfected lizards were killed with an overdose of barbiturate (Thiopental, Spofa, Czech Republic) and necropsied. At necropsy, tissue samples of the stomach, duodenum, small and large intestine, heart, lung, liver, gall-bladder and kidney were fixed in 10% buffered formalin. Fixed tissues were processed for a light microscopy using standard methods. Paraffin sections were stained with hematoxylin and eosin (H&E) and Giemsa stains. In order to determine the sporulation time, o¨ocysts from the intestinal contents were suspended in 2.5% potassium dichromate solution and incubated at 25  C. O¨ ocysts during different stages of sporogony were

observed and photographed using Nomarski interference contrast (NIC) microscopy. Thirty fresh sporulated o¨ocysts were measured using a calibrated ocular micrometer; measurements are reported in micrometres (m), as means followed by the range in parentheses.

Isospora ptyodactyli n. sp. (Figures 1–5) Description O¨ocysts spherical or subspherical, 22.1 (19.0–24.0)  21.2 (18.0–23.0); shape-index (length/width) 1.04 (1.0–1.1). O¨ocyst wall bilayered, 1.0–1.5 thick, smooth and slightly brownish; outer layer 0.75–1.0; inner layer 0.25–0.5. Micropyle, o¨ocyst residuum and polar granule absent. Sporocysts ellipsoidal, 12.2 (11.0–14.0)  8.0 (7.5–9.0), with smooth, thin wall (c. 0.5 thick); shape-index 1.5 (1.4–1.9). Knob-like Stieda body present, c. 0.75 high and 1.5 wide. Substieda

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Figure 5. Composite line drawings of sporulated o¨ocyst of Isospora ptyodactyli n. sp. Scale-bar: 10 m.

body spherical to subspherical, homogenous, 2.0 high (1.5–2.0) and 1.5 wide (1.0–2.0). Sporocyst residuum present, composed of numerous granules of irregular size scattered among sporozoites. Sporozoites vermiform, with slightly granulated surface appearance, arranged head to tail within sporocyst. Both posterior and anterior refractile body present, c. 3.5 in diameter. Type-host: Ptyodactylus puiseuxi Boutan (Sauria: Gekkonidae). Type-locality: Safawi, northern Jordan (32 120 N; 37 100 E). Other locality: Zoubia, northern Jordan (32 250 N; 35 450 E). Prevalence: 2/5 (40%) in Safawi and 3/12 (25%) in Zoubia. Sporulation: Exogenous. Intestinal contents of infected geckos contained unsporulated o¨ocysts, o¨ocysts at the beginning of sporulation and also some almost sporulated o¨ocysts. Fully sporulated o¨ocysts were observed within 12 hours at 25  C. Site of infection: Histological examination revealed all endogenous stages developed within the nuclei of enterocytes in the small intestine. Histological sections

of the small intestine of all infected lizards exhibited pathological lesions consisting of villus atrophy and inflammatory infiltration of the lamina propria (Figure 3). All infected cell nuclei were displaced toward the apical part of enterocytes. During endogenous development, nuclei were gradually consumed and transformed into a thin envelope around the parasite. Endogenous stages: Early trophozoites (Figure 4) spherical to ovoidal (3.5–6.0  3.5–8.0). Developing meronts not found; mature meront contained c. 40 merozoites (3.5–5.0  1.0–1.5). Young microgamonts contained up to 40 nuclei per section plane. Mature microgamonts with centrally located residual body (5.0–8.0  3.0–5.0), contained up to 60 peripherally arranged microgametes. Young macrogamonts (12.0–14.0  8.0–10.0) with large nucleus and distinct nucleolus, but few eosinophilic granules. Zygotes and young o¨ocysts (15.0–18.0  14.0–16.0) contained eosinophilic granules and vacuoles (Figure 4). Type-specimens: Phototypes and histological slides deposited in the Institute of Parasitology, Acadˇ emy of Sciences of the Czech Republic, Cesk´ e Budˇejovice (Coll. No. R 96/96). Symbiotype is deposited in the herpetological collection of Zoologisches Forschungsinstitut und Museum Alexander Koenig, Bonn, Germany (Coll. No. ZFMK 63428).

Discussion Thirteen species of coccidia of the genus Isospora Schneider have previously been described and named from gekkonid hosts (Cannon, 1967; Carini, 1936; El-Toukhy, 1994; Else & Colley, 1975; Matuschka & Bannert, 1986; Ovezmukhammedov, 1972; Ray & Das Gupta, 1936; Upton & Barnard, 1987; Upton & Freed, 1988; Upton & Freed, 1990; Upton et al., 1990; Upton et al., 1991; Upton et al., 1992). The species described as a new herein possesses smaller o¨ocysts than, I. ablephari, I. albogularis, I. canariensis, I. frenatus, I. pachydactyli, I. stenodactyli and I. tarentolae. Conversely, o¨ocysts of I. schlegeli are significantly smaller than those of I. ptyodactyli n. sp. Of the five species most similar in size, I. gekkonis has slightly larger o¨ocysts and those of I. thavari are slightly larger and have a single-layered o¨ocyst wall. I. thavari sporocysts just overlap with the new species in length and the Stieda body is reduced. I. gekkonis sporocysts contain a much larger substieda body and have a significantly different shape than the new species. O¨ocysts of I. hemidactyli are mostly subspherical, containing one

48 polar granule. Although detailed data on morphology of I. knowlesi and I. gymnodactyli are lacking in the original descriptions, conspecifity can be excluded by differences in oocyst and sporocyst dimensions, and also by different host groups and geographical origin. The endogenous development of gekkonid Isospora spp. has been relatively poorly studied. Basic data on endogenous stages are available only for I. ablephari and I. stenodactyli, which have intranuclear endogenous development (Cannon, 1967; El-Toukhy, 1994), and for I. thavari, with a cytoplasmatic localisation of the endogenous stages (Else & Colley, 1975). Basic morphological features and localisation of the stages of merogony and gamogony of I. ptyodactyli n. sp. are very similar to those of I. stenodactyli.

Acknowledgements We are indebted to the Royal Society for the Conservation of the Nature, especially to Adnan Budieri and Sultan A. Al-Mashaqbah, and to all staff of the Badia Research and Development Program, Safawi, for help and gracious hospitality during our stay in Jordan. We also thanks Ahmad M. Disi and staff at the Zoubia Reserve for their kind help and facilitating our stay. The Faculty of Science of Mutah University kindly provided transportation, laboratory facilities and accommodation. We thank Veronika Schacherlov´a for preparing tissue samples for histology and Jaroslaˇ va Ruˇziˇckov´a and Jan Slapeta for the line drawing. This study was supported by Grant No. 508/95/0273 of the Grant Agency of the Czech Republic.

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