Choledocystus elegans ( Digenea : Plagiorchiidae ) of Leptodactylus paraensis ( Amphibia : Leptodactylidae ) from the Brazilian Amazon Choledocystus elegans ( Digenea : Plagiorchiidae ) de Leptodactylus paraensis ( Amphibia : Leptodactylidae ) da Amazônia brasileira Tássia

The trematodes are parasites of the several vertebrates including amphibians, however the knowledge about of the taxonomy these parasites is still confuse. The trematode Choledocystus elegans was found in the small intestine of the Leptodactylus paraensis in eastern Amazon and presents the following characteristics: several pointed tegumentary spines, papillae on the outer and inner edges of the oral and ventral suckers, a round, well-developed cirrus sac, a well-developed cirrus, oblique testicles, a ovary right side, uterine loops extending between the testicles, follicular vitellaria distributed throughout the body, starting at the genital pore region and caeca close the end of the body. For the first time, this study identified C. elegans parasitizing L. paraensis and describes morphological aspects never characterized using light and scanning electron microscopy.


Leptodactylus paraensis
is popularly known as the Pará thin-toed frog and can be found in eastern Amazon, from the state of Pará to northern state of Mato Grosso, Brazil.This amphibian has a terrestrial habitat and is found in both the wild and the peridomicile urban environment (HEYER, 2005;SILVANO & SEGALLA, 2005).
The genus Choledocystus Pereira & Cuocolo, 1941 was established to determine the species Choledocystus eucharis Pereira & Cuocolo, 1941 in Brazil, a Trematoda e of the bile ducts of Leptodactylus ocellatus Linnaeus, 1758, is composed by liver, gall blader and bile ducts parasites of amphibian of distribution in South America (RAZO-MENDIVIL & PÉREZ-PONCE DE LEÓN, 2008;TKACH, 2008).The characteristics that considered Choledocystus as valid genus in family Plagiorchiidae: the presence of a sucker-like structure surrounding the genital atrium, absence of a seminal receptacle and the shape of uterine loops along the body (RAZO-MENDIVIL & PÉREZ-PONCE DE LEÓN, 2008;TKACH, 2008).
Four specimens of L. paraensis were collected in peridomicile areas (01º28'03"S, 48º20'18"W) in the municipality of Belém, state of Pará, eastern Amazon (Brazil).The collected specimens were taken to the Laboratory of Cell Biology and Helminthology "Profa.Dra.Reinalda Marisa Lanfredi" of the Institute of Biological Sciences located at the Federal University of Pará (UFPA), anesthetised and euthanised with sodium thiopental.Digestive tubes were isolated in phosphate-saline buffer (PBS) (pH 7.4), and the presence of helminths was evaluated.
Light microscopy analysis were proceed from 10 specimens (one holotype and nine vouchers).The trematodes were fixed under coverslip pressure with AFA (2% glacial acetic acid, 3% formaldehyde and 95% ethanol 70° GL), stained chloridric carmine, dehydrated in a graded alcohol series and clarified with methyl salicylate (GIESE et al., 2015).The morphometric data were obtained with the aid of an Olympus BX 41 microscope attached to a drawing tube and the measurements are in millimetres, otherwise stated and are shown as: the mean ± standard deviation the mean + standard deviation followed by range in parenthesis.
Scanning electron microscopy, were proceed from 10 specimens were fixed in AFA, post-fixed in 1% OsO 4 , dried at the critical point of CO 2 , coated with gold and observed in SEM LEO-1450 microscope of the Laboratory of Electron Microscopy at the Institute of Geosciences of UFPA (GIESE et al., 2015).

Choledocystus elegans (Travassos, 1926) Ruiz, 1949 (Description Based/ Lower Case on 10 Vouchers: Using Light and Scanning Electron Microscopy)
Body elongated 2.77 ± 0.68 (1.73-3.86)× 0.87 ± 0.13 (0.62 -1.10); ratio body width/body length 1:3 (Figures 1 and 2a).Tegument covered with triangular spines (Figures 3a, b).Oral sucker subterminal, rounded, ventral 0.22 ± 0.03 (0.18 -0.31) × 0.23 ± 0.02 (0.20 -0.27) and surrounded by an elevation of the tegument at the anterior end (Figures 1 and 2a, b).Papillae distributed on the internal contour of the oral sucker with the following pattern: three pairs of papillae juxtaposed in the apical region and two pairs of lateral papillae (Figure 2b).Papillae of the outer edge of the oral sucker with the following pattern: three pairs of papillae juxtaposed on the anterior edge of the opening and four pairs of papillae lining the perimeter of the oral sucker (Figures 1b and 2b and c).Six tegumental papillae below the oral sucker, round and small (Figure 2b).Prepharynx present (Figures 1a, b).
Vitellaria distributed in the extracaecal and caecal region, follicular, starting before the genital pore extending up to half of the posterior third and just before the end of the caecum (Figure 1a).Uterine loops distributed in the intercaecal and caecal regions, descendent to the genital pore and then ascendant, ending in a well-developed metraterm that open at the atrium (Figures 1a and d).Eggs operculate elliptical, smooth and numerous 0.02 ± 0.00 (0.02-0.027) × 0.01 ± 0.000 (0.012-0.015) (Figures 1 and 3e, f )."Y"-shaped excretory vesicle.Excretory pore terminal, spherical opening with the presence of pleats of varied size, lack of spines or papillae on the outer edge (Figures 1a and 2f, g).
The morphological characteristics of the genus Choledocystus are similar to genus Glypthelmins, but is are distinguished mainly by the absence of a seminal receptacle in Choledocystus (RUIZ & LEÃO, 1942).Razo-Mendivil et al. (2006) described other characteristics that differ the two genera, the presence of a muscular structure (sucker-like) surrounding the genital pore is characteristic of Choledocystus and absent in Glypthelmins, the tegumental spines are triangular shows typical structure of Choledocystus, in Glypthelmins the spines are serrated as per the most recent revision of the genus.
Choledocystus hepaticus Lutz, 1928, parasite of liver in R. marina of the Venezuela, was considered synonymous of C. intermedius; however, as redescribed C. hepaticus is a different species, considered size of the sucker ratios, egg size and position of vitellaria different from other species (SULLIVAN, 1977).Therefore, there are three species considered valid to genus Choledocystus: C. intermedius, C. hepaticus and the type-species C. elegans.
In this study was observed for the first time in L. paraensis the occurrence of parasites of the genus Choledocystus.The species C. elegans in the Brazilian Amazon, parasites of the L. paraensis.Analysis the three species by description articles of the genus Choledocystus and the keys of identification to trematodes, were important for the identification of the parasite of this study (POJMANSKA, 2008).
In this study, C. elegans was found parasitizing small intestine of L. paraensis, the parasites were adhered to the intestinal mucosa.The ultrastructure of the tegument of C. elegans revealed the presence of tegumental papillae near the oral sucker and on the outer and inner edges of this sucker.Papillae on the inner edge of the ventral sucker never have been observed in C. elegans.Papillae interspersed with spines on the outer edge of the ventral sucker and tegumental papillae below the oral sucker are characteristics never described for this species.The present study is the first to illustrate the ultrastructural details of the papillae and their distribution pattern in tegument and suckers in trematodes of the genus Choledocystus, adding new characteristics to the genus.
Amphibians of the genus Leptodactylus are commonly parasitized by several groups of helminths, in L. paraensis the first and only record of parasite was made by Kuzmin et al. (2016) in a study who described two new species of nematodes of the genus Rhabdias Stiles et Hassall, 1905 and one of these species was parasitizing L. paraensis, the nematode Rhabdias stenocephala Kuzmin et al. (2016).
Thus, the present study is the second record of parasitism by helminths in L. paraensis, the first record of trematodes of the genus Choledocystus for the Brazilian Amazon and was added details of the ultrastructure of the tegument of C. elegans never been observed in parasites of the genus Choledocystus reinforcing characteristics inherent to the genus.

Figure 2 .
Figure 2. Scanning electron micrographs of Choledocystus elegans: (a) General view of the trematode ventral surface.Oral sucker (OS), everted cirrus (Ci), lightly concave ventral sucker.(VS) and excretory pore (EP).Bar = 100 μm; (b) Oral sucker, showing the apical elevation of the tegument (*) at the anterior border of the sucker and the internal distribution of papillae: two groups of three adjacent papillae (keys) and two lateral (arrowhead); in the tegument below oral sucker, are observed six papillae divided into two groups of three (arrows).Bar = 20 μm; (c) Details of the oral sucker, showing six pairs of small rounded (dotted line).Bar = 20 μm; (d) Detail of the ventral sucker outlined by papillae distributed around the perimeter of the opening (arrows), interspersed with a large number of spines (arrowhead).Bar = 20 μm; (e) In the inner region of the ventral sucker, two pairs of domed papillae were observed (arrows).Bar = 10 μm; (f, g) Detail of the terminal excretory pore (EP) showing its spherical aperture, lack of spines or papillae and the presence of little pleats.Bar = 20 μm and 4 μm, respectively.

Figure 3 .
Figure 3. Scanning electron micrographs of Choledocystus elegans: (a) Detail of the ventrolateral region of the first-third of the body, showing numerous spines (S).Bar = 10 μm; (b) Detail of tegumentary showing showing the cuneiform shape of spines (S) base and thin, delicate tip.Bar = 4 μm; (c, d) Detail of the genital pore (GP), showing the presence of the anterior extremity of the cirrus (Ci) and spermatozoids (S).Bar = 20 μm; (e, f ) Eggs (E) located at the opening of the genital pore next to the cirrus (Ci) and a small elevation (arrow) outlining the perimeter of the operculum.Bar = 10 μm and 4 μm, respectively.
The genus Choledocystus includes trematodes parasites of liver, gall bladder and bile ducts in amphibians from the North, Central and South Americas (RUIZ & LEÃO, 1942; RAZO-MENDIVIL & PÉREZ-PONCE DE LEÓN, 2008).Lunaschi & Drago (2010) described the genus Choledocystus living in small intestine in Leptodactylus latrans Steffen, 1815 from the Argentina.Fernandes & Kohn (2014) related Choledocystus parasitizing small intestine of the anurans Pseudis paradoxa Linnaeus, 1758 and R. marina from the Venezuela, Pseudis minuta Gunther, 1858 and L.latrans from the Uruguay and Hypsiboas raniceps Cope, 1862 from the Brazil.This is work are first record of C. elegans in Brazilian Amazon and site of infection small intestine in L. paraensis.