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Gorilloflasca africana n.g., n.sp., (Entodiniomorphida) from wild habituated Virunga mountain gorillas (Gorilla beringei beringei) in Rwanda
Accepted Manuscript Title: Gorilloflasca africana n.g., n.sp., (Entodiniomorphida) from wild habituated Virunga mountain gorillas (Gorilla beringei beringei) in Rwanda Authors: Akira Ito, Winnie Eckardt, Tara S. Stoinski, Thomas R. Gillespie, Toshihiro Tokiwa PII: DOI: Reference:
S0932-4739(17)30032-9 http://dx.doi.org/doi:10.1016/j.ejop.2017.06.002 EJOP 25512
To appear in: Received date: Accepted date:
5-2-2017 6-6-2017
Please cite this article as: Ito, Akira, Eckardt, Winnie, Stoinski, Tara S., Gillespie, Thomas R., Tokiwa, Toshihiro, Gorilloflasca africana n.g., n.sp., (Entodiniomorphida) from wild habituated Virunga mountain gorillas (Gorilla beringei beringei) in Rwanda.European Journal of Protistology http://dx.doi.org/10.1016/j.ejop.2017.06.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 Gorilloflasca
africana
n.g.,
n.sp.,
(Entodiniomorphida)
from
wild
habituated Virunga mountain gorillas (Gorilla beringei beringei) in Rwanda
Akira Itoa, *, Winnie Eckardtb,c, Tara S. Stoinskib, Thomas R. Gillespiec, Toshihiro Tokiwad
aOokusa bDian
Animal Clinic, Ookusa 503, Matsue, Shimane 690-0032, Japan
Fossey Gorilla Fund International, Atlanta, USA
cDepartments
of Environmental Sciences and Environmental Health, Emory
University and Rollins School of Public Health, Atlanta, USA dDivision
of Pathobiological Analysis, Department of Veterinary Pathobiology,
School of Veterinary Medicine, Nippon Veterinary and Life Science University, Tokyo, Japan
*Corresponding
author. Tel.: +81 852237780; fax: +81 852237780.
E-mail address: [email protected] (A. Ito).
Abstract
A new entodiniomorphid ciliate species, Gorilloflasca africana n. g., n. sp. was described from the Virunga mountain gorillas, Gorilla beringei beringei, in Rwanda. It is characterized by a flask-shaped body, a long tubular vestibulum, a round frontal lobe, a large posterior cavity, an ellipsoidal or peanut-shaped
2 macronucleus and a single contractile vacuole. G. africana has the adoral and the vestibular ciliary zones in the buccal area. The adoral ciliary zone is non-retractable, encircling the vestibular opening. The vestibular ciliary zone extends posteriorly in the vestibulum. The somatic ciliary zones are the cavity ciliary zone in the posterior cavity along the ventral side of its opening and two longitudinal ciliary zones on the dorsal body surface. The buccal infraciliary bands of G. africana are a C-shaped adoral polybrachykinety, a stick-shaped vestibular kinety band, and paralabial kineties. The anterior region of the vestibular kinety band is composed of short kineties whereas, kineties in the remaining region are longitudinal. The somatic infraciliary bands are a cavity polybrachykinety and two longitudinal polybrachykineties. Gorilloflasca is a member of the family Blepharocorythidae based on the non-retractable adoral ciliary zone, the frontal lobe, the large posterior cavity and the vestibular longitudinal kineties.
Keywords: Apes; Infraciliature; Gorilloflasca africana n. g., n. sp. Introduction
Study of Entodiniomorphid ciliates inhabiting the intestines of primates, such as western and eastern gorillas (Gorilla gorilla and Gorilla beringei), chimpanzees (Pan troglodytes), bonobos (Pan paniscus), orangutans (Pongo pygmaeus), siamangs (Hylobates syndactylus), pygmy siamangs (Hylobates klossi)
and
guinea
baboons
(Papio
hamadryas),
revealed
four
genera,
3 Gorillophilus, Prototapirella, Troglocorys and Troglodytella (Ashford et al. 2000; Bonhomme et al. 1989; Brumpt and Joyeux 1912; Dollinger and Rüedi 1974; Freeman et al. 2004; Gillespie et al. 2010; Goussard et al. 1983; Hasegawa et al. 1983; Howells et al. 2011; Imai et al. 1991; Irbis et al. 2008; Ito et al. 2016; Landsoud-Soukate et al. 1995; Modrý et al. 2009; Mortelmans et al. 1971; Murray et al. 2000; Myers and Kunts 1972; O’Donoghue et al. 1993; Pomajbíková et al. 2012; Reichenow 1917, 1920; Tokiwa et al. 2010). We examined entodiniomorphid ciliates in the feces of wild habituated Virunga mountain gorillas (Gorilla beringei beringei) monitored by the Dian Fossey Gorilla Fund’s Karisoke Research Center in Rwanda and described two new and one known Prototapirella species (Ito et al. 2016). In the same samples, a new species was found. To accommodate it, a new genus in the family Blepharocorythidae was established. This report will focus on the description of the morphology of Gorilloflasca africana n. g., n. sp.
Material and Methods
From August 2011 to August 2012, 2,601 fecal samples were obtained from 120 wild habituated Virunga mountain gorilla (Gorilla beringei beringei) in the Volcanoes National Park, Rwanda. All gorillas are individually known and monitored daily by staff of the Karisoke Research Center. These fecal contents were fixed in approximately two times the volume of 10% formalin solution and transported to Emory University for microscopic examination of gastrointestinal
4 parasites and commensals after their isolation using flotation and sedimentation techniques (Gillespie 2006). This initial examination revealed an unexpected high diversity of ciliates. A selection of 40 preserved fecal samples collected during four different seasons with individuals of all identified ciliate types were sent to the Laboratory of the Veterinary Pathology in Japan for further examination. The infraciliary bands were stained by the pyridinated silver carbonate impregnation method, following Ito and Imai (1998). The end of the cell where the vestibulum opens was termed the anterior and the opposite end was termed the posterior. The side from which the frontal lobe arises was termed the dorsal side and the opposite one was termed the ventral side, defining the right and left sides. Cell measurements were made from a sample of 20 fixed individuals using a calibrated micrometer. Body length was determined as the distance between the anterior and posterior ends of the cell. The term, polybrachykinety, refers to infraciliary
bands
composed
of
numerous,
short,
parallel
kineties
(Fernández-Galiano et al. 1985; Ito and Imai 1998).
Results
A new entodiniomorphid species was found in fecal samples of the mountain gorillas in Rwanda. It has the flask-shaped body composed of an anterior cylindrical region and a posterior spherical region, which is distinct from other entodiniomorphid ciliates. This new species has a frontal lobe, a tubular vestibulum, and a non-retractable adoral ciliary zone. Silver impregnation
5 revealed a vestibular ciliary zone constructed of longitudinal kinety rows. These morphological features indicate that the new species is a member of the family Blepharocorythidae. This new species also has a cavity with a ciliary zone similar to the cavity, “the left concavity”, of Troglocorys cava Tokiwa, Modrý, Ito, Pomajbíková, Petrželková, and Imai, 2010. However, this new species is not a member of the genus Troglocorys since it has two parallel longitudinal ciliary bands on the dorsal surface and lacks the dorsoadoral polybrachykinety and the dorsal polybrachykinety on the frontal lobe. Therefore, a new genus Gorilloflasca is proposed and Gorilloflasca africana n.g., n. sp. is described. G. africana was detected in five out of 40 fecal samples.
Gorilloflasca n. g.
Diagnosis.
Gorilloflasca
has
characteristics
of
the
family
Blepharocorythidae. The body is flask-shaped and composed of an anterior cylindrical region and a posterior spherical region. The vestibulum is long and tubular and extends posteriorly in the anterior cylindrical region of body. The frontal lobe arises from the anterior end of body. The large cavity opens at the posterior end of the body. The contractile vacuole is single. The macronucleus is ellipsoidal or peanut-shaped. The buccal ciliary zone consists of an adoral zone and a vestibular zone. Somatic zones are a cavity ciliary zone and two parallel longitudinal ciliary zones.
6 Type species. Gorilloflasca africana n. g., n. sp. Etymology. Gorilloflasca is named after the host animal and the body shape (Gorillo-, Gorilla; L. flasca, flask). Gender: female. Remarks. The genus Gorilloflasca is closely related to the genus Troglocorys Tokiwa, Modrý, Ito, Pomajbíková, Petrželková, and Imai, 2010 due to the presence of a large cavity with a ciliary zone, an ellipsoidal or peanut-shaped macronucleus, and a round frontal lobe. Troglocorys has a ciliary zone on the frontal lobe and a ciliary zone anterior to the vestibular longitudinal kineties. However, Gorilloflasca lacks these ciliary zones but has two parallel longitudinal ciliary zones on the dorsal surface of the posterior spherical region of the body.
Gorilloflasca africana n. g., n. sp.
Description (Figs. 1 - 5, 12, 13, Table 1). Cell measurements and morphometric ratios are shown in Table 1. The body is flask-shaped characterized by a slender and cylindrical anterior region and a large and spherical posterior region. The frontal lobe is round, arising from the anterior end of the body. The posterior cavity is large with a thick wall and opens widely posteriorly. A cytoproct is not present. The vestibulum is long and tubular, extending posteriorly in the anterior cylindrical region up to the posterior spherical region of the body. The adoral ciliary zone is non-retractable, extending along the vestibular opening except for the base of the frontal lobe. An adoral lip is present. The vestibular ciliary zone is distributed from the frontal lobe to the
7 vestibular bottom. The cavity ciliary zone lies in the posterior cavity, extending transversely along the ventral edge of the posterior cavity opening. Right and left longitudinal ciliary zones extend parallel on the dorsal surface of the body. The macronucleus is ellipsoidal or peanut-shaped, lying in the posterior spherical region of the body mostly near the posterior end of the vestibulum. The micronucleus is small and ellipsoidal, lying on various sides of the macronucleus. One contractile vacuoles occurs in the posterior spherical region of the body. Habitat, type host, locality. The intestine of the mountain gorillas (Gorilla beringei beringei) in the Volcanoes National Park, Rwanda. Etymology. Gorilloflasca africana is named after Africa. Type material. The slide containing the holotype specimen (accession number) is deposited in Meguro Parasitological Museum, Tokyo, Japan. Infraciliature (Figs. 6 -11, 13 - 20). Gorilloflasca africana has a buccal infraciliature composed of an adoral polybrachykinety (AP), a vestibular kinety band (VK), and paralabial kineties (PK). The AP is C-shaped along the vestibular opening, except for the base of the frontal lobe. The VK is stick-shaped with a bending anterior region, extending longitudinally from the frontal lobe to the vestibular bottom. The anterior bending region of the VK is composed of short kineties, ranging from eight to 11 in number (mode = 10). The remaining region of the VK is composed of long kineties, ranging from five to seven in number (mode = 6). Numerous bifurcate fibers arise from the dorsal side of the VK and stretch to the dorsal side turning to the ventral side. The PK are several rows of larger kinetids along the right ventral side of the AP. The kinetids in the PK are often
8 randomly disposed and do not form kinety rows. Gorilloflasca africana has three somatic infraciliary bands; a cavity polybrachykinety (CP), a left longitudinal polybrachykinety (LP) and a right longitudinal polybrachykinety (RP). The CP is crescent-shaped and incurved, extending transversely along the ventral edge of the posterior cavity opening. The LP and the RP are long wedge-shaped with tapering anterior ends, prolonging longitudinally on the dorsal surface near the cavity opening. Remarks. In this paper, since the side from which the frontal lobe arises is termed the dorsal side, two longitudinal ciliary zones are located on the dorsal surface of the body, while the cavity ciliary zone is at the ventral side. However, cells of G. africana are very deformable and the anterior cylindrical region is flexible relative to the posterior spherical region of the body. Therefore, it is often difficult to precisely apply the terminology of orientation to both the anterior and posterior regions of the body. The macronucleus is ellipsoidal or peanut-shaped found in 40% and 35 % of the examined cells (n = 20). However, 25% of the cells had a spherical macronucleus, which represent a long axis view of an ellipsoidal or peanut-shaped macronucleus. Troglocorys cava has a large cavity called “the left concavity”, which is analogous to the posterior cavity of G. africana. The cavity of T. cava has small projections and a groove and opens to the left side, whereas the cavity of G. africana does not have such structures and opens to the posterior side. The cytoproct was absent in G. africana, but exists in Troglocorys cava,
9 being slit like-shaped and located at the posterior dorsal side (Tokiwa et al. 2010).
Discussion
Gorilloflasca africana has a flask-shaped body which is distinct from other entodiniomorphid
species.
Initial
difficulties
with
the
morphological
interpretation of this species were resolved by applying the silver impregnation which revealed a new species and required the establishment of the new genus Gorilloflasca. The morphological features of the vestibular kinety band (VK) and the adoral ciliary zone (AC) are important when we discuss the taxonomical position of the genus Gorilloflasca. The VK of Gorilloflasca is made up of short kineties in its anterior region and long longitudinal kineties in its posterior region and the AC of Gorilloflasca is non-retractable. Such VK and AC are also found in Raabena and Pararaabena ciliates in the family Blepharocorythidae (Wolska 1967, 1968). In addition to the VK and the AC, the frontal lobe, the tubular vestibulum, and numerous lateral vestibular fibers of G. africana are morphological features characteristic of Blepharocorythidae. Therefore, we could describe the new genus Gorilloflasca as a member of the family Blepharocorythidae. Raabena and Pararaabena ciliates have the VK similar to that of Gorilloflasca although they have extremely different appearances; Raabena and Pararaabena have a laterally compressed ellipsoidal body. Wolska (1967, 1968, 1971) discussed the evolution within the family Blepharocorythidae based on the
10 development of the buccal and dorsal ciliary zones and showed that Raabena is the most primitive blepharocorythid form. The anterior end of the VK in Raabena extends dorsally to continue as the ciliary band on the frontal lobe, whereas the VK in Pararaabena is completely separated from its dorsal region which forms a ciliary zone on the frontal lobe. Wolska regarded Raabena as the primitive blepharocorythid because of a single ciliary band composed of the VK and the dorsal ciliary band. Following Wolska’s hypothesis that blepharocorythids with the VK combined with the dorsal ciliary zone are primitive, Gorilloflasca africana would be the most primitive blepharocorythid form rather than Raabena species because the VK of Gorilloflasca does not extend dorsally and lacks the dorsal ciliary zone on the frontal lobe. The VK composed of only longitudinal kineties are found in other ciliates in the families, Blepharocorythidae, Parentodiniidae and Pseudoentodiniidae (Ito et al 2002; Wolska 1971, 1986). Blepharocorys, Ochoterenaia and Troglocorys ciliates in the Blepharocorythidae have the VK composed of only longitudinal kineties, but they have a small ciliary zone lying anterior to the VK and a dorsal ciliary zone on the frontal lobe. Ciliates in the families Parentodiniidae and Pseudoentodiniidae also have the VK composed of only longitudinal kineties but their AC are retractable and ciliates in Parentodiniidae have a small ciliary zone anterior to the VK. Such ciliary zones and the retractable AC are not present in Gorilloflasca. The large posterior cavity with the ciliary zone of G. africana is a distinct morphological feature of this species. Raabena bella and Pararaabena dentata do
11 not have such cavity in spite of similarities in the buccal infraciliature (Wolska 1967, 1968). The cavity with the ciliary zone was also described in Troglocorys cava in the family Blepharocorythidae, inhabiting the intestine of chimpanzee (Tokiwa et al. 2010). T. cava has numerous round projections and a deep groove in its cavity, whereas G. africana lacks such structures in the posterior cavity but is characterized by many granules inside the posterior cavity. Granules are not structures of the posterior cavity but could derive from debris in the feces of the host gorilla or from excrements of G. africana. This cavity is an interesting structure and should be studied in the future to know the phylogenetic and morphological meaning of the space, ciliary zone, groove and projections in the cavity. T. cava has a slit-like cytoproct near the cavity (Tokiwa et al. 2010). However, we could not find the cytoproct in G. africana. If granules in the posterior cavity are excrements of G. africana, the cytoproct may be found inside the posterior cavity or may be the cavity itself. Goussard et al. (1983) reported “Entodiniomorph Type B” from lowland gorillas (Gorilla gorilla gorilla) in Gabon with two micrographs. The cell in one of their two microphotographs closely resembles Gorilloflasca africana in the flask-shaped body and the large cavity. Entodiniomorph Type B is much smaller than G. africana; the body width of the Entodiniomorph Type B and G. africana are 28.7 (23-34) μm and 51.1 (40.8 -84.2) μm, respectively. In addition, the Entodiniomorph Type B ciliate has only one ciliary zone (a tuft of cirri) in the anterior cytostome and lacks the ciliary zones as found in G. africana; the
12 vestibular ciliary zone and three somatic ciliary zones in the posterior cavity and on the dorsal surface of the body. Therefore, the Entodiniomorph Type B differs from G. africana and represents one or more new entodiniomorphid species which remain undescribed.
Acknowledgements
We would like to thank the Government of Rwanda and the Rwandan Development Board for permission to carry out this study with the mountain gorillas. Our sincere thank also goes to the Dian Fossey Gorilla Fund’s Karisoke Research Center for their immense help in collecting fecal samples and to G. Andrle, D. Ryu, and G. De La Torre for assistance in isolation and initial identification of ciliates. We also thank Mrs. Takako Ito for assistance in the silver impregnation. Funding for this study was provided by the Dian Fossey Gorilla Fund, Emory University, the Sasakawa Scientific Research Grant from The Japan Science Society.
13 References
Ashford, R. W., Reid, G. D. F., Wrangham, R. W., 2000. Intestinal parasites of the chimpanzee Pan troglodytes in Kibale Forest, Uganda. Ann. Trop. Med. Parasitol. 94, 173-179. Bonhomme, A., Grain, J., Collet, J. Y., 1989. Etude ultrastructurale de Troglodytella gorillae, Cilié de l’intestin des Gorilles. Eur. J. Protistol. 24, 225-237. Brumpt, E., Joyeux, C., 1912. Sur un infusoire nouveau parasite du Chimpanzé Troglodytella abrassarti, n. g. n. sp. Bull. Soc. Path. Exot. 5, 499-503. Dollinger, P., Rüedi, D., 1974. Endoparasiten im Affenbestand des Zoologoschen Gartens Basel und Behandlungsversuche mit Mebendazol und Levamisol. Zoologische Garten 44, 329-337. Fernández-Galiano, T., Serrano, S., Fernández-Galiano,D., 1985. General morphology and stomatogenesis of two species of the genus Entodinium (Ciliophora, Entodiniomorphida). Acta Protozool. 24, 181-186. Freeman, A. S., Kinsella, J. M., Cipolletta, C., Deem, S. L., Karesh, W. B., 2004. Endoparasites of western lowland gorillas (Gorilla gorilla gorilla) at Bai Hokou, Central African Republic. J. Wildl. Dis. 40, 775-781. Gillespie, T. R., 2006. Noninvasive assessment of gastrointestinal parasite infections in free-ranging primates. Int. J. Primatol. 27, 1129-1143. Gillespie, T. R., Lonsdorf, E. V., Canfield, E. P., Meyer, D. J., Nadler, Y., Raphael, J., Pusey, A. E., Pond, J., Pauley, J., Mlengeya, T., Travis, D. A., 2010.
14 Demographic and Ecological Effects on Patterns of Parasitism in Eastern Chimpanzees (Pan troglodytes schweinfurthii) in Gombe National Park, Tanzania. American Journal of Physical Anthropology 143, 534-544. Goussard, B., Collet, J. –Y., Garin, Y., Tutin, C. E. G., Fernandez, M., 1983. The intestinal entodiniomorph ciliates of wild lowland gorillas (Gorilla gorilla gorilla) in Gabon, West Africa. J. Med. Primatol. 12, 239-249. Hasegawa, H., Kano, T., Mulavwa, M., 1983. A parasitological survey on the feces of pygmy chimpanzees, Pan paniscus, at Wamba, Zaïre. Primates 24, 419-423. Howells, M. E., Pruetz, J., Gillespie, T. R., 2011. Patterns of gastro-intestinal commensals and parasites as an index of population and ecosystem health: the case of sympatric western chimpanzees (Pan troglodytes verus) and guinea baboons (Papio hamadryas papio) at Fongoli, Senegal. American Journal of Primatology 73, 173-179. Imai, S., Ikeda, S., Collet, J. Y., Bonhomme, A., 1991. Entodiniomorphid ciliates from the wild lowland gorilla with the description of a new genus and three new species. Eur. J. Protistol. 26, 270-278. Irbis, C., Garriga, R., Kabasawa, A., Usida, K., 2008. Phylogenic analysis of Troglodytella abrassarti isolated from Chimpanzees (Pan troglodytes verus) in wild and in captivity. J. Gen. Appl. Microbiol. 54, 409-413. Ito, A., Eckardt, W., Stoinski, T., Gillespie, T., Tokiwa, T., 2016. Prototapirella ciliates from wild habituated Virunga mountain gorillas (Gorilla beringei beringei) in Rwanda with the description of two new species. Eur. J.
15 Protistol. 54, 47-58. Ito, A., Imai, S., 1998. Infraciliary bands in the rumen ophryoscolecid ciliate Ostracodinium gracile (Dogiel, 1925), observed by light microscopy. J. Euk. Microbiol. 45, 628-636. Ito, A., Miyazaki, Y., Imai, S., 2002. Descriptions of new Parentodinium ciliates in the family Parentodiniidae n. fam. from Hippopotamus amphibius in comparison with some entodiniomorphs from horses and cattle. Eur. J. Protistol. 37, 405-426. Landsoud-Soukate, J., Tutin, C. E. G., Fernandez, M., 1995. Intestinal parasites of sympatric gorillas and chimpanzees in the Lope Reserve, Gabon. Ann. Trop. Med. Parasitol. 89, 73-79. Modrý, D. Petrželková, K. J., Pomajbíková, K., Tokiwa, T., Křížek, J., Imai, S., Vallo, P., Profousová, I., Šlapeta, J., 2009. The occurrence and ape-to-ape transmission of the entodiniomorphid ciliate Troglodytella abrassarti in captive gorillas. J. Eukaryot. Microbiol. 56, 83-87. Mortelmans, J., Vercruysse, J. Kaguruka, P., 1971. Three pathogenic intestinal protozoa of anthropoid apes: Entoamoeba histolytica, Balantidium coli and Troglodytella abrassarti. Proc. 3rd int. Congr. Primat., Zurich 1970 2, 187-191. Murray, S., Stem, C., Boudreau, B., Goodall, J., 2000. Intestinal parasites of baboons (Papio cynocephalus anubis) and chimpanzees (Pan troglodytes) in Gombe National Park. J. Zoo. Widl. Med. 31, 176-178. Myers, B. J., Kuntz, R. E., 1972. A checklist of parasites and commensals
16 reported for the Chimpanzee (Pan). Primates 13, 433-471. O’Donoghue, P.J., Gasser, R.B. Tribe, A., 1993. New host record for entodiniomorphid
ciliate,
Troglodytella
abrassarti,
from
siamangs
(Hylobates syndactylus). International Jour. Parasitol. 23, 415-418. Pomajbíková, K., Petrželková, K. J., Petrášová, J., Profousová, I, Kalousová, B., Jirků, M., Modrý, D., 2012. Distribution of the entodiniomorphid ciliate Troglocorys cava Tokiwa, Modrý, Ito, Pomajbíková, Petrzelková, & Imai, 2010, (Entodiniomorphida: Blepharocorythidae) in wild and captive cimpanzees. J. Eukaryot. Microbiol. 59, 97-99. Reichenow, E., 1917. Parasitos de la sangre y del intestino de los monos antropomorfos africanos. Bol. R. Soc. Esp. Hist. Nat. 17, 312-332. Reichenow, E., 1920. Den Wiederkäuer-Infusorien verwandte Formen aus Gorilla und Schimpanse. Arch. Protistenk. 41, 1-33. Tokiwa, T., Modrý, D., Ito, A., Pomajbíková, K., Petrželková, K. J., Imai, S., 2010. A new entodiniomorphid ciliate, Troglocorys cava n. g. n. sp., from the wild eastern chimpanzee (Pan troglodytes schweinfurthii) from Uganda. J. Eukaryot. Microbiol. 57, 115-120. Wolska, M., 1967. Studies on the family Blepharocorythidae Hsiung. III. Raabena bella gen. n., sp. n. from the intestine of the Indian elephant. Acta Protozool. 4, 285-290. Wolska, M., 1968. Studies on the family Blepharocorythidae Hsiung. IV. Pararabena dentata gen. n., sp. n. from the intestine of Indian elephant. Acta Protozool. 5, 219-224.
17 Wolska, M., 1971. Studies on the family Blepharocorythidae Hsiung. VI. Phylogenesis of the family and the description of the new genus Circodinium gen. n. with the species C. minimum (Gassovsky, 1918). Acta Protozool. 9, 171-194 Wolska, M., 1986. Pseudoentodinium gen. nov., sp. n. from the Order Entodiniomorphida. Proposition of the new family Pseudoentodiniidae. Acta Protozool. 25, 139-146.
18 Figs 1-5. Schematic figures of Gorilloflasca africana n. g., n. sp. 1. Cell from left side. 2. Cell from dorsal side. 3-5. Macronucleus and micronucleus. 3. Ellipsoidal macronucleus. 4. Peanut-shaped macronucleus. 5. Spherical macronucleus. AC, anterior cylindrical region; ACZ, adoral ciliary zone; CCZ, cavity ciliary zone; CO, cavity opening; CV, contractile vacuole; FL, frontal lobe; LCZ, left longitudinal ciliary zone; MA, macronucleus; MI, micronucleus; PC, posterior cavity; PS, posterior spherical region; RCZ, right longitudinal ciliary zone; VCZ, vestibular ciliary zone; VS, vestibulum. Bar = 10µm.
19 Figs 6-11. Gorilloflasca africana n. g., n. sp. after silver impregnation. 6. Cell from left side. 7. Vestibular kinety band from left side. 8. Anterior cylindrical region of body from right side. 9. Anterior cylindrical region of body from left side viewed diagonally frontal. 10. Posterior spherical region from dorsal side. 11. Cell from dorsal side viewed diagonally backward. AC, anterior cylindrical region; AL, adoral lip; AP, adoral polybrachykinety; CO, cavity opening; CP, cavity polybrachykinety; CV, contractile vacuole; FL, frontal lobe; LP, left longitudinal polybrachykinety; MA, macronucleus; MI, micronucleus; PC, posterior cavity; PK, paralabial kineties; PS, posterior spherical region; RP, right longitudinal polybrachykinety; VK, vestibular kinety band; VF, vestibular fiber; VS, vestibulum. Bar = 10µm.
20
21 Figs 12-20. Micrographs of Gorilloflasca africana n. g., n. sp. 12. Cell from right side. 13-20. Cells after silver impregnation. 13. Cell from left side. 14. Anterior cylindrical region from left side viewed diagonally forward. 15. Anterior cylindrical region from right side. 16-18. Anterior cylindrical region from left side. 19.
Right longitudinal polybrachykinety. 20. Posterior cavity. AC, anterior
clindrical region; AP, adoral polybrachykinety; CO, cavity opening; CP, cavity polybrachykinety; FL, frontal lobe; LCZ, left longitudinal ciliary zone; MA, macronucleus; MI, micronucleus; PC, posterior cavity; PK, paralabial kineties; PS, posterior spherical region; RCZ, right longitudinal ciliary zone; RP, right longitudinal polybrachykinety; VK, vestibular kinety band; VF, vestibular fiber; VS, vestibulum. Bar = 10µm.
22
23 Table 1. Cell measurements (μm) and morphometric ratios (n = 20) of Gorilloflasca africana. Mean
SD
Min
Max
Body length
80.3
15.6
63.8
137.7
Length of anterior cylindrical region of body
30.2
5.8
25.5
86.7
Length of posterior spherical region of body
50.2
10.3
38.3
86.7
Body width ( width of posterior spherical region)
51.1
9.2
40.8
84.2
Width of anterior cylindrical region of body
17.6
3.2
15.3
28.1
Body length / body width
1.58
0.23
1.33
2.44
Macronuclear length
12.1
2.7
7.7
17.9
S0932-4739(17)30032-9 http://dx.doi.org/doi:10.1016/j.ejop.2017.06.002 EJOP 25512
To appear in: Received date: Accepted date:
5-2-2017 6-6-2017
Please cite this article as: Ito, Akira, Eckardt, Winnie, Stoinski, Tara S., Gillespie, Thomas R., Tokiwa, Toshihiro, Gorilloflasca africana n.g., n.sp., (Entodiniomorphida) from wild habituated Virunga mountain gorillas (Gorilla beringei beringei) in Rwanda.European Journal of Protistology http://dx.doi.org/10.1016/j.ejop.2017.06.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 Gorilloflasca
africana
n.g.,
n.sp.,
(Entodiniomorphida)
from
wild
habituated Virunga mountain gorillas (Gorilla beringei beringei) in Rwanda
Akira Itoa, *, Winnie Eckardtb,c, Tara S. Stoinskib, Thomas R. Gillespiec, Toshihiro Tokiwad
aOokusa bDian
Animal Clinic, Ookusa 503, Matsue, Shimane 690-0032, Japan
Fossey Gorilla Fund International, Atlanta, USA
cDepartments
of Environmental Sciences and Environmental Health, Emory
University and Rollins School of Public Health, Atlanta, USA dDivision
of Pathobiological Analysis, Department of Veterinary Pathobiology,
School of Veterinary Medicine, Nippon Veterinary and Life Science University, Tokyo, Japan
*Corresponding
author. Tel.: +81 852237780; fax: +81 852237780.
E-mail address: [email protected] (A. Ito).
Abstract
A new entodiniomorphid ciliate species, Gorilloflasca africana n. g., n. sp. was described from the Virunga mountain gorillas, Gorilla beringei beringei, in Rwanda. It is characterized by a flask-shaped body, a long tubular vestibulum, a round frontal lobe, a large posterior cavity, an ellipsoidal or peanut-shaped
2 macronucleus and a single contractile vacuole. G. africana has the adoral and the vestibular ciliary zones in the buccal area. The adoral ciliary zone is non-retractable, encircling the vestibular opening. The vestibular ciliary zone extends posteriorly in the vestibulum. The somatic ciliary zones are the cavity ciliary zone in the posterior cavity along the ventral side of its opening and two longitudinal ciliary zones on the dorsal body surface. The buccal infraciliary bands of G. africana are a C-shaped adoral polybrachykinety, a stick-shaped vestibular kinety band, and paralabial kineties. The anterior region of the vestibular kinety band is composed of short kineties whereas, kineties in the remaining region are longitudinal. The somatic infraciliary bands are a cavity polybrachykinety and two longitudinal polybrachykineties. Gorilloflasca is a member of the family Blepharocorythidae based on the non-retractable adoral ciliary zone, the frontal lobe, the large posterior cavity and the vestibular longitudinal kineties.
Keywords: Apes; Infraciliature; Gorilloflasca africana n. g., n. sp. Introduction
Study of Entodiniomorphid ciliates inhabiting the intestines of primates, such as western and eastern gorillas (Gorilla gorilla and Gorilla beringei), chimpanzees (Pan troglodytes), bonobos (Pan paniscus), orangutans (Pongo pygmaeus), siamangs (Hylobates syndactylus), pygmy siamangs (Hylobates klossi)
and
guinea
baboons
(Papio
hamadryas),
revealed
four
genera,
3 Gorillophilus, Prototapirella, Troglocorys and Troglodytella (Ashford et al. 2000; Bonhomme et al. 1989; Brumpt and Joyeux 1912; Dollinger and Rüedi 1974; Freeman et al. 2004; Gillespie et al. 2010; Goussard et al. 1983; Hasegawa et al. 1983; Howells et al. 2011; Imai et al. 1991; Irbis et al. 2008; Ito et al. 2016; Landsoud-Soukate et al. 1995; Modrý et al. 2009; Mortelmans et al. 1971; Murray et al. 2000; Myers and Kunts 1972; O’Donoghue et al. 1993; Pomajbíková et al. 2012; Reichenow 1917, 1920; Tokiwa et al. 2010). We examined entodiniomorphid ciliates in the feces of wild habituated Virunga mountain gorillas (Gorilla beringei beringei) monitored by the Dian Fossey Gorilla Fund’s Karisoke Research Center in Rwanda and described two new and one known Prototapirella species (Ito et al. 2016). In the same samples, a new species was found. To accommodate it, a new genus in the family Blepharocorythidae was established. This report will focus on the description of the morphology of Gorilloflasca africana n. g., n. sp.
Material and Methods
From August 2011 to August 2012, 2,601 fecal samples were obtained from 120 wild habituated Virunga mountain gorilla (Gorilla beringei beringei) in the Volcanoes National Park, Rwanda. All gorillas are individually known and monitored daily by staff of the Karisoke Research Center. These fecal contents were fixed in approximately two times the volume of 10% formalin solution and transported to Emory University for microscopic examination of gastrointestinal
4 parasites and commensals after their isolation using flotation and sedimentation techniques (Gillespie 2006). This initial examination revealed an unexpected high diversity of ciliates. A selection of 40 preserved fecal samples collected during four different seasons with individuals of all identified ciliate types were sent to the Laboratory of the Veterinary Pathology in Japan for further examination. The infraciliary bands were stained by the pyridinated silver carbonate impregnation method, following Ito and Imai (1998). The end of the cell where the vestibulum opens was termed the anterior and the opposite end was termed the posterior. The side from which the frontal lobe arises was termed the dorsal side and the opposite one was termed the ventral side, defining the right and left sides. Cell measurements were made from a sample of 20 fixed individuals using a calibrated micrometer. Body length was determined as the distance between the anterior and posterior ends of the cell. The term, polybrachykinety, refers to infraciliary
bands
composed
of
numerous,
short,
parallel
kineties
(Fernández-Galiano et al. 1985; Ito and Imai 1998).
Results
A new entodiniomorphid species was found in fecal samples of the mountain gorillas in Rwanda. It has the flask-shaped body composed of an anterior cylindrical region and a posterior spherical region, which is distinct from other entodiniomorphid ciliates. This new species has a frontal lobe, a tubular vestibulum, and a non-retractable adoral ciliary zone. Silver impregnation
5 revealed a vestibular ciliary zone constructed of longitudinal kinety rows. These morphological features indicate that the new species is a member of the family Blepharocorythidae. This new species also has a cavity with a ciliary zone similar to the cavity, “the left concavity”, of Troglocorys cava Tokiwa, Modrý, Ito, Pomajbíková, Petrželková, and Imai, 2010. However, this new species is not a member of the genus Troglocorys since it has two parallel longitudinal ciliary bands on the dorsal surface and lacks the dorsoadoral polybrachykinety and the dorsal polybrachykinety on the frontal lobe. Therefore, a new genus Gorilloflasca is proposed and Gorilloflasca africana n.g., n. sp. is described. G. africana was detected in five out of 40 fecal samples.
Gorilloflasca n. g.
Diagnosis.
Gorilloflasca
has
characteristics
of
the
family
Blepharocorythidae. The body is flask-shaped and composed of an anterior cylindrical region and a posterior spherical region. The vestibulum is long and tubular and extends posteriorly in the anterior cylindrical region of body. The frontal lobe arises from the anterior end of body. The large cavity opens at the posterior end of the body. The contractile vacuole is single. The macronucleus is ellipsoidal or peanut-shaped. The buccal ciliary zone consists of an adoral zone and a vestibular zone. Somatic zones are a cavity ciliary zone and two parallel longitudinal ciliary zones.
6 Type species. Gorilloflasca africana n. g., n. sp. Etymology. Gorilloflasca is named after the host animal and the body shape (Gorillo-, Gorilla; L. flasca, flask). Gender: female. Remarks. The genus Gorilloflasca is closely related to the genus Troglocorys Tokiwa, Modrý, Ito, Pomajbíková, Petrželková, and Imai, 2010 due to the presence of a large cavity with a ciliary zone, an ellipsoidal or peanut-shaped macronucleus, and a round frontal lobe. Troglocorys has a ciliary zone on the frontal lobe and a ciliary zone anterior to the vestibular longitudinal kineties. However, Gorilloflasca lacks these ciliary zones but has two parallel longitudinal ciliary zones on the dorsal surface of the posterior spherical region of the body.
Gorilloflasca africana n. g., n. sp.
Description (Figs. 1 - 5, 12, 13, Table 1). Cell measurements and morphometric ratios are shown in Table 1. The body is flask-shaped characterized by a slender and cylindrical anterior region and a large and spherical posterior region. The frontal lobe is round, arising from the anterior end of the body. The posterior cavity is large with a thick wall and opens widely posteriorly. A cytoproct is not present. The vestibulum is long and tubular, extending posteriorly in the anterior cylindrical region up to the posterior spherical region of the body. The adoral ciliary zone is non-retractable, extending along the vestibular opening except for the base of the frontal lobe. An adoral lip is present. The vestibular ciliary zone is distributed from the frontal lobe to the
7 vestibular bottom. The cavity ciliary zone lies in the posterior cavity, extending transversely along the ventral edge of the posterior cavity opening. Right and left longitudinal ciliary zones extend parallel on the dorsal surface of the body. The macronucleus is ellipsoidal or peanut-shaped, lying in the posterior spherical region of the body mostly near the posterior end of the vestibulum. The micronucleus is small and ellipsoidal, lying on various sides of the macronucleus. One contractile vacuoles occurs in the posterior spherical region of the body. Habitat, type host, locality. The intestine of the mountain gorillas (Gorilla beringei beringei) in the Volcanoes National Park, Rwanda. Etymology. Gorilloflasca africana is named after Africa. Type material. The slide containing the holotype specimen (accession number) is deposited in Meguro Parasitological Museum, Tokyo, Japan. Infraciliature (Figs. 6 -11, 13 - 20). Gorilloflasca africana has a buccal infraciliature composed of an adoral polybrachykinety (AP), a vestibular kinety band (VK), and paralabial kineties (PK). The AP is C-shaped along the vestibular opening, except for the base of the frontal lobe. The VK is stick-shaped with a bending anterior region, extending longitudinally from the frontal lobe to the vestibular bottom. The anterior bending region of the VK is composed of short kineties, ranging from eight to 11 in number (mode = 10). The remaining region of the VK is composed of long kineties, ranging from five to seven in number (mode = 6). Numerous bifurcate fibers arise from the dorsal side of the VK and stretch to the dorsal side turning to the ventral side. The PK are several rows of larger kinetids along the right ventral side of the AP. The kinetids in the PK are often
8 randomly disposed and do not form kinety rows. Gorilloflasca africana has three somatic infraciliary bands; a cavity polybrachykinety (CP), a left longitudinal polybrachykinety (LP) and a right longitudinal polybrachykinety (RP). The CP is crescent-shaped and incurved, extending transversely along the ventral edge of the posterior cavity opening. The LP and the RP are long wedge-shaped with tapering anterior ends, prolonging longitudinally on the dorsal surface near the cavity opening. Remarks. In this paper, since the side from which the frontal lobe arises is termed the dorsal side, two longitudinal ciliary zones are located on the dorsal surface of the body, while the cavity ciliary zone is at the ventral side. However, cells of G. africana are very deformable and the anterior cylindrical region is flexible relative to the posterior spherical region of the body. Therefore, it is often difficult to precisely apply the terminology of orientation to both the anterior and posterior regions of the body. The macronucleus is ellipsoidal or peanut-shaped found in 40% and 35 % of the examined cells (n = 20). However, 25% of the cells had a spherical macronucleus, which represent a long axis view of an ellipsoidal or peanut-shaped macronucleus. Troglocorys cava has a large cavity called “the left concavity”, which is analogous to the posterior cavity of G. africana. The cavity of T. cava has small projections and a groove and opens to the left side, whereas the cavity of G. africana does not have such structures and opens to the posterior side. The cytoproct was absent in G. africana, but exists in Troglocorys cava,
9 being slit like-shaped and located at the posterior dorsal side (Tokiwa et al. 2010).
Discussion
Gorilloflasca africana has a flask-shaped body which is distinct from other entodiniomorphid
species.
Initial
difficulties
with
the
morphological
interpretation of this species were resolved by applying the silver impregnation which revealed a new species and required the establishment of the new genus Gorilloflasca. The morphological features of the vestibular kinety band (VK) and the adoral ciliary zone (AC) are important when we discuss the taxonomical position of the genus Gorilloflasca. The VK of Gorilloflasca is made up of short kineties in its anterior region and long longitudinal kineties in its posterior region and the AC of Gorilloflasca is non-retractable. Such VK and AC are also found in Raabena and Pararaabena ciliates in the family Blepharocorythidae (Wolska 1967, 1968). In addition to the VK and the AC, the frontal lobe, the tubular vestibulum, and numerous lateral vestibular fibers of G. africana are morphological features characteristic of Blepharocorythidae. Therefore, we could describe the new genus Gorilloflasca as a member of the family Blepharocorythidae. Raabena and Pararaabena ciliates have the VK similar to that of Gorilloflasca although they have extremely different appearances; Raabena and Pararaabena have a laterally compressed ellipsoidal body. Wolska (1967, 1968, 1971) discussed the evolution within the family Blepharocorythidae based on the
10 development of the buccal and dorsal ciliary zones and showed that Raabena is the most primitive blepharocorythid form. The anterior end of the VK in Raabena extends dorsally to continue as the ciliary band on the frontal lobe, whereas the VK in Pararaabena is completely separated from its dorsal region which forms a ciliary zone on the frontal lobe. Wolska regarded Raabena as the primitive blepharocorythid because of a single ciliary band composed of the VK and the dorsal ciliary band. Following Wolska’s hypothesis that blepharocorythids with the VK combined with the dorsal ciliary zone are primitive, Gorilloflasca africana would be the most primitive blepharocorythid form rather than Raabena species because the VK of Gorilloflasca does not extend dorsally and lacks the dorsal ciliary zone on the frontal lobe. The VK composed of only longitudinal kineties are found in other ciliates in the families, Blepharocorythidae, Parentodiniidae and Pseudoentodiniidae (Ito et al 2002; Wolska 1971, 1986). Blepharocorys, Ochoterenaia and Troglocorys ciliates in the Blepharocorythidae have the VK composed of only longitudinal kineties, but they have a small ciliary zone lying anterior to the VK and a dorsal ciliary zone on the frontal lobe. Ciliates in the families Parentodiniidae and Pseudoentodiniidae also have the VK composed of only longitudinal kineties but their AC are retractable and ciliates in Parentodiniidae have a small ciliary zone anterior to the VK. Such ciliary zones and the retractable AC are not present in Gorilloflasca. The large posterior cavity with the ciliary zone of G. africana is a distinct morphological feature of this species. Raabena bella and Pararaabena dentata do
11 not have such cavity in spite of similarities in the buccal infraciliature (Wolska 1967, 1968). The cavity with the ciliary zone was also described in Troglocorys cava in the family Blepharocorythidae, inhabiting the intestine of chimpanzee (Tokiwa et al. 2010). T. cava has numerous round projections and a deep groove in its cavity, whereas G. africana lacks such structures in the posterior cavity but is characterized by many granules inside the posterior cavity. Granules are not structures of the posterior cavity but could derive from debris in the feces of the host gorilla or from excrements of G. africana. This cavity is an interesting structure and should be studied in the future to know the phylogenetic and morphological meaning of the space, ciliary zone, groove and projections in the cavity. T. cava has a slit-like cytoproct near the cavity (Tokiwa et al. 2010). However, we could not find the cytoproct in G. africana. If granules in the posterior cavity are excrements of G. africana, the cytoproct may be found inside the posterior cavity or may be the cavity itself. Goussard et al. (1983) reported “Entodiniomorph Type B” from lowland gorillas (Gorilla gorilla gorilla) in Gabon with two micrographs. The cell in one of their two microphotographs closely resembles Gorilloflasca africana in the flask-shaped body and the large cavity. Entodiniomorph Type B is much smaller than G. africana; the body width of the Entodiniomorph Type B and G. africana are 28.7 (23-34) μm and 51.1 (40.8 -84.2) μm, respectively. In addition, the Entodiniomorph Type B ciliate has only one ciliary zone (a tuft of cirri) in the anterior cytostome and lacks the ciliary zones as found in G. africana; the
12 vestibular ciliary zone and three somatic ciliary zones in the posterior cavity and on the dorsal surface of the body. Therefore, the Entodiniomorph Type B differs from G. africana and represents one or more new entodiniomorphid species which remain undescribed.
Acknowledgements
We would like to thank the Government of Rwanda and the Rwandan Development Board for permission to carry out this study with the mountain gorillas. Our sincere thank also goes to the Dian Fossey Gorilla Fund’s Karisoke Research Center for their immense help in collecting fecal samples and to G. Andrle, D. Ryu, and G. De La Torre for assistance in isolation and initial identification of ciliates. We also thank Mrs. Takako Ito for assistance in the silver impregnation. Funding for this study was provided by the Dian Fossey Gorilla Fund, Emory University, the Sasakawa Scientific Research Grant from The Japan Science Society.
13 References
Ashford, R. W., Reid, G. D. F., Wrangham, R. W., 2000. Intestinal parasites of the chimpanzee Pan troglodytes in Kibale Forest, Uganda. Ann. Trop. Med. Parasitol. 94, 173-179. Bonhomme, A., Grain, J., Collet, J. Y., 1989. Etude ultrastructurale de Troglodytella gorillae, Cilié de l’intestin des Gorilles. Eur. J. Protistol. 24, 225-237. Brumpt, E., Joyeux, C., 1912. Sur un infusoire nouveau parasite du Chimpanzé Troglodytella abrassarti, n. g. n. sp. Bull. Soc. Path. Exot. 5, 499-503. Dollinger, P., Rüedi, D., 1974. Endoparasiten im Affenbestand des Zoologoschen Gartens Basel und Behandlungsversuche mit Mebendazol und Levamisol. Zoologische Garten 44, 329-337. Fernández-Galiano, T., Serrano, S., Fernández-Galiano,D., 1985. General morphology and stomatogenesis of two species of the genus Entodinium (Ciliophora, Entodiniomorphida). Acta Protozool. 24, 181-186. Freeman, A. S., Kinsella, J. M., Cipolletta, C., Deem, S. L., Karesh, W. B., 2004. Endoparasites of western lowland gorillas (Gorilla gorilla gorilla) at Bai Hokou, Central African Republic. J. Wildl. Dis. 40, 775-781. Gillespie, T. R., 2006. Noninvasive assessment of gastrointestinal parasite infections in free-ranging primates. Int. J. Primatol. 27, 1129-1143. Gillespie, T. R., Lonsdorf, E. V., Canfield, E. P., Meyer, D. J., Nadler, Y., Raphael, J., Pusey, A. E., Pond, J., Pauley, J., Mlengeya, T., Travis, D. A., 2010.
14 Demographic and Ecological Effects on Patterns of Parasitism in Eastern Chimpanzees (Pan troglodytes schweinfurthii) in Gombe National Park, Tanzania. American Journal of Physical Anthropology 143, 534-544. Goussard, B., Collet, J. –Y., Garin, Y., Tutin, C. E. G., Fernandez, M., 1983. The intestinal entodiniomorph ciliates of wild lowland gorillas (Gorilla gorilla gorilla) in Gabon, West Africa. J. Med. Primatol. 12, 239-249. Hasegawa, H., Kano, T., Mulavwa, M., 1983. A parasitological survey on the feces of pygmy chimpanzees, Pan paniscus, at Wamba, Zaïre. Primates 24, 419-423. Howells, M. E., Pruetz, J., Gillespie, T. R., 2011. Patterns of gastro-intestinal commensals and parasites as an index of population and ecosystem health: the case of sympatric western chimpanzees (Pan troglodytes verus) and guinea baboons (Papio hamadryas papio) at Fongoli, Senegal. American Journal of Primatology 73, 173-179. Imai, S., Ikeda, S., Collet, J. Y., Bonhomme, A., 1991. Entodiniomorphid ciliates from the wild lowland gorilla with the description of a new genus and three new species. Eur. J. Protistol. 26, 270-278. Irbis, C., Garriga, R., Kabasawa, A., Usida, K., 2008. Phylogenic analysis of Troglodytella abrassarti isolated from Chimpanzees (Pan troglodytes verus) in wild and in captivity. J. Gen. Appl. Microbiol. 54, 409-413. Ito, A., Eckardt, W., Stoinski, T., Gillespie, T., Tokiwa, T., 2016. Prototapirella ciliates from wild habituated Virunga mountain gorillas (Gorilla beringei beringei) in Rwanda with the description of two new species. Eur. J.
15 Protistol. 54, 47-58. Ito, A., Imai, S., 1998. Infraciliary bands in the rumen ophryoscolecid ciliate Ostracodinium gracile (Dogiel, 1925), observed by light microscopy. J. Euk. Microbiol. 45, 628-636. Ito, A., Miyazaki, Y., Imai, S., 2002. Descriptions of new Parentodinium ciliates in the family Parentodiniidae n. fam. from Hippopotamus amphibius in comparison with some entodiniomorphs from horses and cattle. Eur. J. Protistol. 37, 405-426. Landsoud-Soukate, J., Tutin, C. E. G., Fernandez, M., 1995. Intestinal parasites of sympatric gorillas and chimpanzees in the Lope Reserve, Gabon. Ann. Trop. Med. Parasitol. 89, 73-79. Modrý, D. Petrželková, K. J., Pomajbíková, K., Tokiwa, T., Křížek, J., Imai, S., Vallo, P., Profousová, I., Šlapeta, J., 2009. The occurrence and ape-to-ape transmission of the entodiniomorphid ciliate Troglodytella abrassarti in captive gorillas. J. Eukaryot. Microbiol. 56, 83-87. Mortelmans, J., Vercruysse, J. Kaguruka, P., 1971. Three pathogenic intestinal protozoa of anthropoid apes: Entoamoeba histolytica, Balantidium coli and Troglodytella abrassarti. Proc. 3rd int. Congr. Primat., Zurich 1970 2, 187-191. Murray, S., Stem, C., Boudreau, B., Goodall, J., 2000. Intestinal parasites of baboons (Papio cynocephalus anubis) and chimpanzees (Pan troglodytes) in Gombe National Park. J. Zoo. Widl. Med. 31, 176-178. Myers, B. J., Kuntz, R. E., 1972. A checklist of parasites and commensals
16 reported for the Chimpanzee (Pan). Primates 13, 433-471. O’Donoghue, P.J., Gasser, R.B. Tribe, A., 1993. New host record for entodiniomorphid
ciliate,
Troglodytella
abrassarti,
from
siamangs
(Hylobates syndactylus). International Jour. Parasitol. 23, 415-418. Pomajbíková, K., Petrželková, K. J., Petrášová, J., Profousová, I, Kalousová, B., Jirků, M., Modrý, D., 2012. Distribution of the entodiniomorphid ciliate Troglocorys cava Tokiwa, Modrý, Ito, Pomajbíková, Petrzelková, & Imai, 2010, (Entodiniomorphida: Blepharocorythidae) in wild and captive cimpanzees. J. Eukaryot. Microbiol. 59, 97-99. Reichenow, E., 1917. Parasitos de la sangre y del intestino de los monos antropomorfos africanos. Bol. R. Soc. Esp. Hist. Nat. 17, 312-332. Reichenow, E., 1920. Den Wiederkäuer-Infusorien verwandte Formen aus Gorilla und Schimpanse. Arch. Protistenk. 41, 1-33. Tokiwa, T., Modrý, D., Ito, A., Pomajbíková, K., Petrželková, K. J., Imai, S., 2010. A new entodiniomorphid ciliate, Troglocorys cava n. g. n. sp., from the wild eastern chimpanzee (Pan troglodytes schweinfurthii) from Uganda. J. Eukaryot. Microbiol. 57, 115-120. Wolska, M., 1967. Studies on the family Blepharocorythidae Hsiung. III. Raabena bella gen. n., sp. n. from the intestine of the Indian elephant. Acta Protozool. 4, 285-290. Wolska, M., 1968. Studies on the family Blepharocorythidae Hsiung. IV. Pararabena dentata gen. n., sp. n. from the intestine of Indian elephant. Acta Protozool. 5, 219-224.
17 Wolska, M., 1971. Studies on the family Blepharocorythidae Hsiung. VI. Phylogenesis of the family and the description of the new genus Circodinium gen. n. with the species C. minimum (Gassovsky, 1918). Acta Protozool. 9, 171-194 Wolska, M., 1986. Pseudoentodinium gen. nov., sp. n. from the Order Entodiniomorphida. Proposition of the new family Pseudoentodiniidae. Acta Protozool. 25, 139-146.
18 Figs 1-5. Schematic figures of Gorilloflasca africana n. g., n. sp. 1. Cell from left side. 2. Cell from dorsal side. 3-5. Macronucleus and micronucleus. 3. Ellipsoidal macronucleus. 4. Peanut-shaped macronucleus. 5. Spherical macronucleus. AC, anterior cylindrical region; ACZ, adoral ciliary zone; CCZ, cavity ciliary zone; CO, cavity opening; CV, contractile vacuole; FL, frontal lobe; LCZ, left longitudinal ciliary zone; MA, macronucleus; MI, micronucleus; PC, posterior cavity; PS, posterior spherical region; RCZ, right longitudinal ciliary zone; VCZ, vestibular ciliary zone; VS, vestibulum. Bar = 10µm.
19 Figs 6-11. Gorilloflasca africana n. g., n. sp. after silver impregnation. 6. Cell from left side. 7. Vestibular kinety band from left side. 8. Anterior cylindrical region of body from right side. 9. Anterior cylindrical region of body from left side viewed diagonally frontal. 10. Posterior spherical region from dorsal side. 11. Cell from dorsal side viewed diagonally backward. AC, anterior cylindrical region; AL, adoral lip; AP, adoral polybrachykinety; CO, cavity opening; CP, cavity polybrachykinety; CV, contractile vacuole; FL, frontal lobe; LP, left longitudinal polybrachykinety; MA, macronucleus; MI, micronucleus; PC, posterior cavity; PK, paralabial kineties; PS, posterior spherical region; RP, right longitudinal polybrachykinety; VK, vestibular kinety band; VF, vestibular fiber; VS, vestibulum. Bar = 10µm.
20
21 Figs 12-20. Micrographs of Gorilloflasca africana n. g., n. sp. 12. Cell from right side. 13-20. Cells after silver impregnation. 13. Cell from left side. 14. Anterior cylindrical region from left side viewed diagonally forward. 15. Anterior cylindrical region from right side. 16-18. Anterior cylindrical region from left side. 19.
Right longitudinal polybrachykinety. 20. Posterior cavity. AC, anterior
clindrical region; AP, adoral polybrachykinety; CO, cavity opening; CP, cavity polybrachykinety; FL, frontal lobe; LCZ, left longitudinal ciliary zone; MA, macronucleus; MI, micronucleus; PC, posterior cavity; PK, paralabial kineties; PS, posterior spherical region; RCZ, right longitudinal ciliary zone; RP, right longitudinal polybrachykinety; VK, vestibular kinety band; VF, vestibular fiber; VS, vestibulum. Bar = 10µm.
22
23 Table 1. Cell measurements (μm) and morphometric ratios (n = 20) of Gorilloflasca africana. Mean
SD
Min
Max
Body length
80.3
15.6
63.8
137.7
Length of anterior cylindrical region of body
30.2
5.8
25.5
86.7
Length of posterior spherical region of body
50.2
10.3
38.3
86.7
Body width ( width of posterior spherical region)
51.1
9.2
40.8
84.2
Width of anterior cylindrical region of body
17.6
3.2
15.3
28.1
Body length / body width
1.58
0.23
1.33
2.44
Macronuclear length
12.1
2.7
7.7
17.9