Cotylophoron marajoensis n. sp. (Digenea: Paramphistomidae) a parasite of Bubalus bubalis on Marajó Island, Pará, Brazilian Amazon

author: Elane Guerreiro Giese. E-mail: lheaufra@gmail.com Abstract The genus Cotylophoron belongs to the Paramphistomidae family and its definitive hosts are ruminants in general. This work describes the presence of a new species of the gender, a parasite in the rumen and reticulum of Bubalus bubalis , on Marajó Island in the Eastern Brazilian Amazon, using of light microscopy, scanning electronic microscopy and molecular biology techniques. One hundred and ten animals were analyzed, of which 4.54% were parasitized by flukes in their adult forms. The helminths were found fixed to the ruminal mucosa and present Liorchis- type pharynx, Cotylophoron- type genital sucker, oblique testicles larger than the ovary, uterus in rings full of eggs and Cotylophoron- type acetabulum. These morphologic characters do not fit into any previously described species. Thus, it is proposed that this is a new species in the genus Cotylophoron . The present work expands the record of parasitism by helminths in Bubalus bubalis , this being the first record of trematoda from the genus Cotylophoron for


Introduction
The Bubalus bubalis species is known as the "buffalo" throughout Brazil and is bred in several regions of the world because of its sturdiness, milk and dairy products and good-quality meat, besides being used for work (Damasceno et al., 2010). In Brazil, buffalos have been imported from different countries such as Australia, Egypt, India, Italy, and Southwest Asia and were first introduced in the continent on Marajó Island in 1895, where the natural conditions of this region were excellent for their development (Marques, 2000). And although it is a domestic and economically important animal, little is known about its parasitic interactions.
The genus Cotylophoron (Stiles & Goldberger, 1910) belongs to the Paramphistomidae family (Fischoeder 1901). In general, they have ruminants as definite hosts, the rumen and reticulum being their sites of infection when adults. The immature forms inhabit the small intestine, where they perform the backward movement in their development process in the digestive tract, traveling from the small intestine to the rumen/reticulum (Forlano et al., 2001). This behavior is due to parasitic ability of genetic expression that allows migration through host's tissues while triggering it's immune responses (Pérez-Ponce de León & Hernández-Mena, 2019 (Martins & Pezzi, 1992).
This genus includes trematode parasites of rumen and reticulum in ruminants worldwide (Martínez & Velásquez, 2012). Alarcón & Velásquez (2009) described the C. cotylophorum species as also present in the rumen of Bos taurus in the region of Rio Negro, Colombia. Sánchez et al. (2009) described the same species as parasitizing the rumen of bovines in Loreto in Peru. Morales et al. (2015) reported the presence of C. fulleborni in the ruminal mucosa of bovines in Venezuela.
In Brazil, the genus Cotylophoron is reported as parasitizing different ruminants in different locations. Costa & Guimarães (1990) mention the first occurrence of C. bareilliensis parasitizing the rumen of sheep in the state of Pará and Costa & Guimarães (1992) describe the C. travassosi species in bovines in the state of Maranhão. Miranda & Costa (1999) report the incidence of C. panamensis parasitizing bovines of the state of Rondônia and C. fulleborni in bovines and goats in the states of Roraima, Pará and Maranhão and C. jacksoni also in bovines in the states of Pará, Roraima and Rondônia.
With this in mind, the present work describes the morphology, prevalence and molecular data of new species of Paramphistomidae that was found as a parasite of B. bubalis on Marajó Island, Brazil.

Preparation of parasites for light microscopy
The trematodes found were fixed and processed according to Giese et al. (2015). Ten trematodes were used for morphologic and morphometric analysis. The measures were obtained in millimeters and are presented in the form of average and amplitude between parentheses. The images in light microscopy were obtained by photomicroscope with connected clear vision camera (LEICA DM2500). For scanning electron microscopy, the helminths specimens were washed in distilled water, post-fixed in 1% osmium tetroxide, dehydrated to the critical point of CO 2 , metallized with gold+palladium, and analyzed using a TESCAN scanning electron microscope (VEGA 3) in the Scanning Electron Microscopy Laboratory, Universidade Federal Rural da Amazônia.

Molecular analysis
Molecular characterization was based on partial 18S small subunit ribosomal rRNA gene (18S SSU), which after DNA extraction through the Invisorb Spin Tissue Mini Kit (Stratec Molecular), was amplified using the primers cc18sf (5'-cggtgaaaccgcgaatggctc -3') and cc18sr (5'-gacgggcggtgtgtacaaagg -3'). The polymerase chain reactions (PCRs) were carried out in 25 µL final volume, containing 5-10 ng of DNA, 50 mM KCl, 2 mM MgCl 2 , 10 mM Tris-HCl, 50 µM of each DNTP, 0.5 µM of each oligonucleotide and one unit of Taq DNA polymerase (Invitrogen). The amplification reaction consisted of 35 cycles of 1 min at 95 °C, 1 min at 67 °C, and 1 min 30 sec at 72 °C, preceded by 5 min at 95 °C and followed by 10 min at 72 °C. Amplicons were enzymatically purified with Illustra ExoProStar (GE Healthcare). Nucleotide sequencing was performed in an ABI 3500 xL Genetic Analyzer (Thermo Fisher Scientific), according to the manufacturer's specifications. BioEdit software (Hall, 1999) was used to align forward and reverse sequences.

Results
Paramphistomidae ( [Based on 10 adult specimens: metrical data in Table 1]. Pear-shaped, smooth body, slightly recurved in the ventral direction, measuring 6.30 (3.42-754) × 2.98 (2.62-3.42) mm, maximum width to the testis level. Oral sucker, adorned with innumerable ciliate papillae in the anterior extremity of the trematode. Pharynx with well-defined muscular portion and evidencing papillae of varied sizes in its mucosa, characteristic of pharynx of Liorchis genus,

Morphological delimitation
The characteristics presented for the taxon studied here such as pear-shaped body, smooth tegument without papillae, oral sucker in the anterior extremity of the body, genital sucker with genital papilla in the anterior fraction of the body and sub-ventral acetabulum of average size, fit it into the Cotylophoron genus in accordance with the keys and descriptions of the genus (Eduardo, 1985). The morphologic characteristics of Cotylophoron, in the present work, are similar to those of the of genus Paramphistomum (Fischoeder 1901) and genus Balanorchis (Fischoeder 1901), but they are distinguished mainly in relation to the Paramphistomum because of the presence of genital papilla and, in relation to the Balanorchis, because of the absence of long papillae in the oral sucker (Jones et al., 2005).
Cotylophoron marajoensis n. sp. differs from C. cotylophorum in possessing a Liorchis pharynx, whereas in C. cotylophorum it is Calicophoron; still, absence of esophageal bulb in C. cotylophorum. Oblique and smaller testes in relation to the ovary presented by Cotylophoron marajoensis n. sp. distinguish it from C. cotylophorum, which presents larger in tandem testes in relation to the ovary (Alarcón & Velásquez, 2009). Associated to these morphologic characteristics, C. cotylophorum has not been found in B. bubalis until now, nor in Brazilian territory.
In Brazil, C. fulleborni e C. jacksoni had been only described in Bos indicus e Capra hircus (Miranda & Costa, 1999), without occurrence in B. bubalis. These species differ from Cotylophoron marajoensis n. sp. in possessing Calicophoron pharynx and larger testes in relation to the ovary (Morales et al., 2015). Around the world, C. panamensis has been described only in Bos indicus. It is shorter in length than Cotylophoron marajoensis n. sp., although larger in width. It possesses Calicophoron pharynx and, differently from Cotylophoron marajoensis n. sp. (whose testes are oblique and smaller than the ovary), it possesses horizontally parallel and larger testes in relation to the ovary, with its vitellarium exceeding the edge of the caeca, also differing from Cotylophoron marajoensis n. sp. because these do not exceed the caecal edges (Martínez & Velásquez, 2012).
The species Cotylophoron bareilliense occurs in B. bubalis in the Phillipines. In Brazil, however, it is mentioned only as parasitizing Bos indicus, Capra hircus and Ovis aries (Miranda & Costa, 1999). Moreover, it is longer in length than Cotylophoron marajoensis n. sp., and it possesses a Calicophoron pharynx (Eduardo, 1985), differing from the Liorchis pharynx of the new species here being described.
Cotylophoron macrosphinctris is parasitic on buffalo, but of the species Syncerus caffer, the African buffalo; it has not been described in B. bubalis, nor in the Brazilian territory it is differentiated from the Cotylophoron marajoensis n. sp. by its Calicophoron pharynx, larger esophageal bulb and testicles in relation to the ovary. Furthermore, it possesses a Schistocotyle genital sucker, whereas Cotylophoron marajoensis n. sp. possesses a Cotylophoron genital sucker (Eduardo, 1985).
Besides having B. bubalis as host and the same type of pharynx (Liorchis) of the C. xiangjiangense species, Cotylophoron marajoensis n. sp. differs by the description of its biome, found in Amazon, while for C. xiangjiangense has only been described as occurring in China. In addition, Cotylophoron marajoensis n. sp. is longer in length and it does not possess an esophageal bulb, which present in the C. xiangjiangense. To strengthen this difference, the testis of Cotylophoron marajoensis n. sp. has a lobe format, whereas C. xiangjiangense possesses an oval testis format (Eduardo, 1985).
The Cotylophoron travassosi species has not been described in B. bubalis up until now, being found, in Brazil, only in Bos indicus, Capra hircus and Ovis aries. It is shorter in length than Cotylophoron marajoensis n. sp., and it possesses a Paramphistomum pharynx (Miranda & Costa, 1999). Additional morphometric comparisons between Cotylophoron marajoensis n. sp. and the other species of the genus Cotylophoron are presented in Table 1.
When analyzed by the SEM, the external morphology of Cotylophoron marajoensis n. sp. disclosed a smooth body, presence of genital papilla, oral sucker and acetabulum, these structures being common to the Cotylophoron (Miranda & Costa, 1999). The internal morphology visualized by SEM has supplied data that have guaranteed diagnosis of the species, disclosing more details about the structures for type of pharynx and acetabulum.

Conclusion
In view of the above, the morphological evidence presented here for the new species falls within the genus Cotylophoron and the absence of all its valid species. Thus, we have added a species to this genus, named Cotylophoron marajoensis n. sp. Additionally we present its partial 18S small subunit ribosomal rRNA gene sequence which may contribute to future phylogenetics studies.