Karyophagus ranarum, Duszynski & Bolek & Upton, 2007

Duszynski, Donald W., Bolek, Matthew G. & Upton, Steve J., 2007, Coccidia (Apicomplexa: Eimeriidae) of amphibians of the world, Zootaxa 1667 (1667), pp. 1-77 : 39-44

Host genus Pelophylax Fitzinger 1843

(24 spp.)

Eimeria prevoti ( Laveran and Mesnil 1902a, b) Doflein 1909 ( Fig. 25) Synonyms: Paracoccidium prevoti Laveran and Mesnil 1902a ; Eimeria prevunti ( Laveran and Mesnil 1902a) , Yakimoff and Matikaschwili 1933, lapsus.

Type host: Pelophylax lessonae (Camerano 1882) Pool frog.

Other hosts: None reported to date.

Type locality: EUROPE: France, west of Paris .

Geographic distribution: EUROPE: France.

Description of sporulated oocyst: Oocyst shape: mostly globoidal to ovoidal; number of walls: 1; wall thickness: “thin;” wall characteristics: smooth; L x W: variable; globoidal forms are 16–18, while ovoidal forms are 20–22 x 12–15; L/W ratio: variable; M and PG: absent; OR: present; OR characteristics: spheroidal, ~5–6 wide, composed of a large vacuole surrounded by coarse granules. Distinctive features of oocyst: thin, single-layered wall and an OR with a vacuole surrounded by granules.

Description of sporocyst and sporozoites: Sporocyst shape: initially fusiform, but the sporocyst wall eventually disappears leaving 8 SZ free in oocyst; L x W: unknown; L/W ratio: unknown; SB: present as a small, nipple-like structure at one end of sporocyst; SSB and PSB: absent; SR: present; SR characteristics: small mass composed of coarse granules between SZ; SZ: elongate, ~10–12 long, with 1 obvious RB. Distinctive features of sporocyst: sporocyst wall disappears soon after sporulation is completed, leaving the 8 SZ and the remaining OR and SR free within the oocyst.

Prevalence: Unknown.

Sporulation: Unknown. The initial sporoblast, ~ 7 µm wide, elongates to form the sporocysts and OR. Upon sporocyst formation, the SZ are arranged head to tail therein.

Prepatent and patent periods: Unknown.

Site of infection: Mid-gut epithelial cells.

Endogenous stages: Both merogony and gamogony occur above the N of the intestinal epithelial cell. Young meronts were round, ~4, and limited by a fine membrane. Ripe meronts were ovoidal, ~21.5 x 16.8, and produced up to 40 merozoites (38–44), each ~6.2 x 1.5. Mature microgamonts were 21.4 x 19.8 and give rise to comma-shaped microgametes, 5–6 x 0.7, with 2 flagella which are unequal in length (11 and 8 long). A vacuole is visible within the granular cytoplasm of the mature macrogamonts, which are round, ~10, and have a distinct N with a conspicuous karyosome. Doflein (1909) said that the macrogamont formed the covering only after fertilization and that the sporoblast of the zygote did not go through a pyramid stage, as seen in many coccidia.

Pathology: Unknown.

Materials deposited: None.

Remarks: Laveran and Mesnil (1902a) created the subgenus Paracoccidium in their belief that disintegration of the sporocyst wall soon after sporulation was completed might represent an intermediate phylogenetic type of development; however, work with other amphibian coccidia (e.g. E. canaliculata ; E. fragilis ) shows that this is not an uncommon phenomenon. Doflein’s (1909) line drawing of E. prevoti ( Fig. 25) shows that the oocysts he saw were ellipsoidal. Boulard (1975) redescribed the species and its endogenous development, also from pool frogs, which he caught in Normandy and most of the measurements above are from his paper. Boulard (1975) provided a detailed line drawing of a sporulated oocyst that showed a distinct SB on the sporocysts before they disintegrated, a detail omitted by Laveran and Mesnil (1902a) in their original work. Boulard’s (1975) young oocysts taken from feces measured 16.5 x 12.8 (15–17 x 12–14), with a L/W ratio of 1.3; after sporulation, the oocysts were 17.4 x 12.6 with a L/W ratio of 1.4. The OR was a spherical mass of granules, ~4.5 x 3.

Eimeria ranarum ( Labbé 1894b) Doflein 1909 ( Fig. 26)

Synonyms: Acystis parasitica ( Labbé 1894b) , pro parte; Caryophagus ranarum Labbé 1899 ; Coccidium ranarum ( Labbé 1894b) Laveran and Mesnil 1902a ; Karyophagus ranarum Labbé 1894b .

Type host: Pelophylax esculenta , ( L. 1758), Pool frog .

Other hosts: Rana temporaria , L. 1758, Grass frog.

Type locality: EUROPE: France .

Geographic distribution: EUROPE: France.

Description of sporulated oocyst: Oocyst shape: ellipsoidal to ovoidal; number of walls: 1; wall thickness: “thin;” wall characteristics: smooth; L x W: 18–20 x 12–16; L/W ratio: unknown; M: present (line drawing in Laveran & Mesnil 1902a); OR and PG: both absent. Distinctive features of oocyst: presence of a small M.

Description of sporocyst and sporozoites: Sporocyst shape: spindle-shaped with both ends tapering to a point; L x W: 7 x 4; L/W ratio: 1.8; SB, SSB, PSB: all absent; SR: present; SR characteristics: scattered ovoid globules; SZ: about the same length as the sporocysts (line drawing). Distinctive features of sporocyst: very small and spindle shaped.

Prevalence: Unknown.

Sporulation: Exogenous; but oocysts passed in the feces in an advanced state of sporulation.

Prepatent and patent periods: Unknown.

Site of infection: Development is exclusively within the N of small intestinal epithelial cells.

Endogenous stages: Meronts contain 25–30 small merozoites, ~5–6 long, arranged in irregular rows. Microgametes are also small, ~3 long. Ellipsoidal macrogametes are filled with large granules and are ~17 x 12 when discharged into the lumen of the small intestine; at that time they have a distinct M, through which Laveran and Mesnil (1902a) believed the microgametes entered.

Pathology: Atrophy of the infected host cell N.

Materials deposited: None.

Remarks: The history of the discovery and naming of this coccidium is convoluted and confusing and is best summarized by Dobell (1909) who wrote that Labbé (1894b) mentioned that he found a parasite, like that occurring in newts, in the N of the intestinal epithelium of Rana temporaria . Without giving any further description he bestowed the name Karyophagus ranarum n. sp. upon it. But on the very next page (p. 212) he said that he believed that this parasite is identical with Karyophagus salamandrae Steinhaus , and Cytophagus salamandrae Steinhaus , and he proposed to call them all Acystis parasitica! Later, Labbé (1899) retained the name Caryophagus ranarum Labbé for the intestinal coccidium of the frog, but gave the host as Rana esculenta (= Pelophylax esculenta ), and gave no further description of it. It is obviously useless to attach much importance to these names, and impossible to identify the animal(s). Walton (1941, 1961) erroneously reported 2 Ambystoma spp. as hosts for this coccidium.

Goussia neglecta ( Nöller 1920) Molnár 1995 ( Fig. 27)

Synonym: Eimeria neglecta Nöller 1920 .

Type host: Tadpoles of Pelophylax esculenta ( L. 1758), Pool frog .

Other hosts: Tadpoles of Pelophylax ridibundus (Pallas 1771) , Marsh frog; possibly tadpoles of Pelophylax lessonae (Camerano 1882) , Little water frog; possibly tadpoles of Rana temporaria L. 1758, Grass frog.

Type locality: EUROPE: Germany, near Hamburg .

Geographic distribution: EUROPE: Germany, Hungary.

Description of sporulated oocyst: Oocyst shape: round occasionally in groups of 2 or 3 enclosed in yellow bodies; number of walls: 1; wall thickness: thin, fragile; wall characteristics: smooth (see Remarks); L x W: 10.6 (8.5–12.5); L/W ratio: 1.0; M, OR, PG: all absent. Distinctive features of oocyst: very thin, singlelayered wall that is often carried with the intestinal epithelial cell when these are shed into the lumen.

Description of sporocyst and sporozoites: Sporocyst shape: ovoidal ( Nöller 1920) to ellipsoidal ( Molnár 1995), with 1 side slightly flattened, with distinct longitudinal sutures (see Remarks); L x W: 8.8 x 4.8 (6.5–10 x 4–6); L/W ratio: 1.8; SB, SSB, PSB: all absent; SR: present; SR characteristics: spheroidal to subspheroidal body composed of large granules/globules (line drawings), 5–7.5 x 3–4 in young oocysts and spheroidal, ~2, in older oocysts; SZ: vermiform with one end reflexed, 8.2 x 1.8 (6–10 x 1.5–2) with indistinct, centrally located N. Distinctive features of sporocyst: very thin walls with one side slightly flattened and with distinct longitudinal sutures, characteristic of the genus, recurved SZs.

Prevalence: Unknown for type host in Germany; 19 of 38 (50%) P. ridibundus and 7 of 10 (70%) P. esculenta in Hungary ( Molnár 1995).

Sporulation: Endogenous.

Prepatent and patent periods: Unknown.

Site of infection: Epithelial cells of the intestine.

Endogenous stages: Nöller (1920) found merogony to occur in the epithelial cells of the tadpole, but not in those of the adult. Molnár (1995) indicated that meronts contained 8–16 merozoites arranged in parallel. Meronts, gamonts, and immature oocysts were observed both in apical and basal cytoplasm of the epithelial cells.

Pathology: Unknown.

Materials deposited: None.

Remarks: This was the first coccidium described from tadpoles ( Nöller 1920) and to date is only the second named species of Goussia known from amphibians. The description given above is a composite from Nöller’s (1920) original description (which was grossly inadequate, but did include a crude line drawing) and Molnár’s (1995) re-description. Finally, Walton (1949b), in an abstract, mentioned G. (E.) neglecta as a parasite of R. temporaria from Europe, but provided no other information. Molnár (1995) indicated that the oocysts of this species consisted of 2 halves joined by a suture and because of this observation transferred Eimeria neglecta to the genus Goussia . However, his line drawing of the oocyst does not show the 2 halves, and we suspect that he meant that the sporocysts contained 2 halves joined by a suture which is the characteristic feature of the genus Goussia and this was misstranslated from Hungarian to English. Both Nöller (1920) and Molnár (1995) reported G. neglecta in tadpoles of Pelophylax spp. and possibly tadpoles of Pelophylax lessonae , but not adult frogs. Molnár (1995) noted that the highest prevalence of infection was observed during July in tadpoles that had hindlimbs, but that the prevalence was lower in tadpoles with hindlimbs and emerged forelimbs and lowest in those with no limbs. Sporulated oocysts were found in mucus and feces and in groups of 2 or 3 enclosed in yellow bodies. Molnár (1995) speculated how the oocysts of G. neglecta might survive after leaving the tadpole, given the very fragile nature of their oocyst and sporocyst walls; he suggested the species might overwinter as sporozoites in tubificid oligiochaetes as was shown for G. carpelli in the common carp (Steinhagen & Körting 1990). Molnár (1995) also mentioned seeing developmental stages of another coccidium in the N of the epithelial cells in the frogs he examined, but that these seem to represent another species. Recently Jirků and Modrý (2006b) reported a Goussia sp. similar in morphology to G. neglecta from a forest pond at Zajetchee potok, Brno, Czeck Republic in 23 of 45 (51%) tadpoles of the agile frog, Rana dalmatina, Fitzinger In Bonaparte, 1839 . Because measurements of the sporocysts of their isolate differed slightly, but overlapped in range with G. neglecta from the original description by Nöller (1920) and as reported by Molnár (1995), they took the conservative approach and refer to their isolate as a species of Goussia . Importantly, their examinations of tadpoles indicated that they found developmental stages of Goussia sp. in the intestine, and fully sporulated oocysts in the intestine and the sinuses of the liver. They allowed tadpoles of R. dalmatina to metamorphose to assess the fate of Goussia infections in these hosts. All juvenile and subadult frogs which metamorphosed in the laboratory and were examined 2 wk to 15 mo after metamorphosis contained oocysts of Goussia sp. in the liver, but not in the intestine. Only individual or clusters of oocysts were observed in the liver, and these were not enclosed by yellow bodies or macrophages. This study was the first to demonstrate Goussia sp. infections in an extraintestinal location in an amphibian host. The authors suggested that the extraintestinal oocysts of amphibian Goussia sp. in metamorphosed frogs may serve as a reservoir for colonization of tadpoles at new breeding sites or when ponds dry up. They suggested that oocysts of Goussia sp. in the liver of adult frogs are not likely to leave the host by the feces, and tadpoles may become infected by consuming dead frogs with oocysts in their liver.

Hyaloklossia lieberküehni ( Labbé 1894b) Laveran and Mesnil 1902b ( Fig. 28)

Synonyms: Diplospora lieberkühni ( Labbé 1894b) Grasse 1953 ; Hyaloklossia lieberkühni ( Labbe 1894b) Labbé 1896 ; Klossia lieberkühni Labbé 1894b ; Isospora lieberküehnk ( Labbe 1894b) Laveran and Mesnil 1902b .

Type host: Pelophylax esculenta ( L. 1758), Pool frog .

Other hosts: Lithobates pipiens (Schreber 1782) , Northern leopard frog; Pelophylax ridibunda (Pallas, 1771) , Marsh frog; Rana temporaria L. 1758, Grass frog; Bombina variegata (L. 1758), Yellowbelly toad.

Type locality: EUROPE: France .

Geographic distribution: EUROPE: Bulgaria, Czeck Republic, France, Poland; NORTH AMERICA: Wisconsin.

Description of sporulated oocyst: Oocyst shape in situ: asymmetrically ellipsoidal; number of walls: 1; wall thickness: “very thin;” wall characteristics: transparent, elastic, and fragile; L x W: 35–45 x 20–25; L/W ratio: unknown; M, OR, PG: all absent. Distinctive feature of oocyst: intracellular development and sporulation.

Description of sporocyst and sporozoites: Sporocyst shape: broadly spindle-shaped to ellipsoidal; L x W 25–30 x 14–16; L/W ratio: unknown; SB, SSB, PSB: all absent; SR: present; SR characteristics: spheroidal to subspheroidal cluster of irregular granules; 9.5–17 x 7–12; SZ: elongate, banana-shaped, 17–21 x 3–4 with a small, round RB at their rounded end (line drawing). Distinctive feature of sporocyst: very thin wall.

Prevalence: Usually not stated, but 1 of 137 (<1%) L. pipiens in Wisconsin ( Levine & Nye 1977); in 2 of>2000 (<0.1%) P.esculenta in Poland (Kazubski & Grabda-Kazubski 1973); 4 of 16 (25%) frogs during April and May and 0 of 18 (0%) frogs in July in the Czeck Republick ( Modrý et al. 2001) and Vojtková (1976) not stated; and Laveran and Mesnil (1902b) in France and Nöeller (1923) in Germany both reported a high degree of infection in young P. esculenta .

Sporulation: Endogenously, within the tubular epithelial cells of the kidney.

Prepatent and patent periods: Unknown, but oocysts may rupture intracellularly releasing sporocysts into the renal tubules.

Site of infection: Epithelium of the renal tubules.

Endogenous stages: According to Laveran and Mesnil (1902b), within only 48 h after ingesting oocysts, the parasite produces an intense infection in various organs of the body with both meronts and gamonts. Nöller (1923) found sporozoites in the spleen of 16 day old tadpoles after he had infected them with sporocysts. Merogony occurred in the spring in the glomerular epithelium of the kidneys of young frogs, but did not develop to gamonts until the summer. Merozoites were short and crescent-shaped with a central N. Gamonts also developed in glomerular epithelial cells. Macrogamonts were elongate ovoidal to ellipsoidal bodies with a large N and a granular cytoplasm. Mature microgamonts had peripherally arranged N and each microgamete had 2 flagella. Levine and Nye (1977) found from 1–12 merozoites per host cell in the cytoplasm of kidney tubule epithelial cells where they completely destroyed the cytoplasm, but left the N intact although sometimes shrunken. The merozoites they saw were elongated and slightly curved, ~6–7 long, with a central or subcentral spherical or squarish N, ~1, and similar to those described by Nöller (1923).

Pathology: Produces marked pathological changes in the kidneys where they form large, whitish cyst-like structures filled with granular contents consisting of mostly immature oocysts (Kazubski & Grabda-Kazubski 1973).

Material deposited: SSU rRNA sequences in GenBank ( AF298623 ).

Remarks: Lieberkühn (1854) was the first to find this renal coccidium in P. esculenta in France. Later, Labbé (1894b) described it under the name Klossia lieberkühni and two years later, in 1896, after re-evaluating its status, he erected the genus Hyaloklossia to accommodate this species. Later authors ( Laveran & Mesnil 1902b; Minchin 1903) placed H. leiberküehni into the genus Diplospora Labbé 1893 ; still others ( Nöller 1923; Doflein & Reichenow 1953) placed it into the genus Isospora ( Schneider 1881) . Recognizing that the genus Isospora is now known to be polyphyletic, Modrý et al. (2001) collected “ I. ” lieberküehni from P. esculenta in the Czech Republic, extracted DNA, and amplified and sequenced the SSU rRNA gene. Using various phylogenetic analyses to examine the phylogenetic position of this enigmatic organism to species in both the Eimeriidae ( Isospora , Eimeria, Cyclospora ) and the Sarcocystidae ( Sarcocystis, Toxoplasma, Hammondia, Neospora , and others) they concluded that, the unique combination of morphological, biological and phylogenetic features was sufficient to re-erect and emend the oldest available synonym, Hyaloklossia with H. liberküehni as the type species.

Nöller (1923) believed that the endogenous stages reported in organs other than the kidneys by Laveran and Mesnil (1902b) were actually Lankesterella minima , and that the endogenous stages of H. (= I.) lieberküehni are limited to the kidney. Nöller (1923) also gave the details of the life history noting that oocysts deposited in the water were swallowed by tadpoles in which the SZ make their way to the glomeruli of the kidneys, where merogony takes place in the epithelial cells. Resulting merozoites then invade the tubule epithelium, which was found heavily infected with other meronts in young frogs during late April and early May. Several weeks later, macro- and microgamonts were found in tubule cells where the oocysts formed ( Wenyon 1926). Nöller (1923) said microgametes were biflagellate. Levine and Nye (1977) reported the infection in 1 L. pipiens from Wisconsin, USA, as a new host and geographic record based solely on merozoites seen in the kidney tubule epithelial cells. We feel that Levine and Nye (1977) had no justification in identifying H. lieberküehni based on merozoites seen in the kidney tubules and their report may represent an undescribed species of apicomplexan from the northern leopard frog. Pellérdy (1974, p. 725) indicated that this species, along with I. mesnili , E. ranarum and E. salamandrae , are all intranuclear parasites.