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The Genus Rhytidodoides Price, 1939 (Digenea: Rhytidodidae) in Brazil: New Geographic Occurrence and Report of Pathology in the Gallbladder

Summary The present note describes the occurrence of Rhytidodoides intestinalis and Rhytidodoides similis (Digenea: Rhytidodidae) in the gallbladder of two juvenile green turtles (Chelonia mydas - Testudines, Cheloniidae) found on the coast of Brazil. Both were detected in gallbladder and intestine of green turtles: Rhytidodoides similis (United States, Panama, Costa Rica and Brazil) and R. intestinais (United States, Panama and Costa Rica). This note is the first report of R. intestinalis in Brazil and South-West Atlantic Ocean. Also the histological lesions caused by the parasites in one gallbladder are described.


Introduction
Parasites of the family Rhytidodidae Odner, 1926, are found in the intestine and gallbladder of sea turtles. Only two genera are currently accepted for the family: Rhytidodes Looss, 1901[R. gelatinosus (Rudolphi, 1819) Looss, 1901 and Rhytidodoides Price, 1939(R. intestinalis Price, 1939, R. similis 1939and R. pricei Mehrotra & Gupta, 1978 (Price, 1939;Mehrotra & Gupta, 1978;Blair, 2005). In the Neotropical region (Central and South America), R. gelatinosus has been reported in the loggerhead turtle (Caretta caretta) in Brazil, the green turtle (Chelonia mydas) in Panama and Brazil and the hawksbill turtle (Eretmochelys imbricata) in Puerto Rico and Cuba. Rhytidodoides similis has been reported in the green turtle in Panama and Brazil. R. intestinalis has been reported in the green turtle in Panama and Costa Rica (Werneck and Silva, 2016). This note describes the occurrence of R. similis and R. intestinalis in green turtles found on the coast of Brazil. The injuries attributed to these parasites in the gallbladder of one host are also described.

Material and Methods
In October 2017, a green turtle was found stranded on Rasa Beach (22°43`39.324``S; 41° 58' 40.476''W) in the municipality of Búzios in the State of Rio de Janeiro, Brazil, and was taken to a rehabilitation center. The turtle (83 cm in curvilinear carapace length-CCL and weighing 28.45 kg) was thin (with bones evident in the plastron region) and lethargic. Treatment involved rehydration with saline solution, preventive antibiotic therapy (enrofl oxacin) and dietary supplementation through an esophageal tube. The animal died on the eleventh day of rehabilitation and was immediately submitted to necropsy. The examination revealed atrophy of the pectoral muscles, which had a gelatinous appearance. Moreover, the entire carcass was   pale in color. Samples were taken of the spleen, brain, cerebellum, heart, digestive tract, liver, ocular globe, salt gland, testis, pancreas, respiratory tract, kidneys, urinary tract and gallbladder.
The samples were fixed in 10% formalin solution, embedding in paraffin, sliced (thickness: 5 μm), stained with hematoxylin and eosin (HE) and analyzed with a light microscope. The second host was found dead in July 2018 caught in a fishing net near Ilha Grande (23°7'12.549''S; 44° 8' 3.124''W) in the municipality of Angra dos Reis in the State of Rio de Janeiro, Brazil. The turtle had 68.1 cm CCL and weighing 24.6 kg, tissue samples in this host were not histologically analyzed due to the autolytic state. During the analysis of the gallbladders, seven specimens of the family Rhytidodidae [three specimens of R. intestinalis and four specimens of R. similis were found in the first host and only eight R. similis were found in the second host]. The parasites were fixed in 70% alcohol, stained with hydrochloric carmine and cleared in a eugenol solution. The specimens were photographed and measured (data expressed as minimum-maximum values in µm) under a microscope (Nikon Eclipse 80i, Kurobane Nikon Co., Ltd., Otawara, Tochigi, Japan) with the aid of the NIS-Elements BR software. Analyses of the parasites were authorized by federal licenses for activities with scientific purposes (SISBIO 30600-1 and 9329-1). The helminths were deposited in the Helminthological Collection of the Oswaldo Cruz Institute (R. similis = CHIOC 38590; R. intestinalis = CHIOC 38589) in the State of Rio de Janeiro, Brazil.
The identification of the parasites was performed using the genus taxonomic key proposed by Blair (2005), the original description by Price (1939) as well as descriptions by Smith et al. (1941), Caballero 1954, Mehrotra & Gupta (1978) and Werneck et al. (2015). Descriptions by Price (1939), Smith et al. (1941), Caballero (1954) and Werneck et al. (2015) were used for the morphometric comparisons of R. similis (Table 1) and descriptions by Price (1939), Caballero (1954) and Mehrotra & Gupta (1978) were used for the comparison of R. intestinalis (   Table 2): body small, thin; oral sucker terminal; pharynx present; esophagus long, rectilinear; testes in tandem, rounded, located in posterior region of body; cirrus sac rounded, near cecal bifurcation; ovary spherical; Vitellaria formed by two groups of follicles, one on each side of the body, from the initial third of the esophagus to the posterior extremity of the body; Mehlis gland posterior to ovary eggs (18 measured) large, elliptical. Histological description: The histological analysis revealed a giant-cell inflammatory response due to spirorchiids eggs (type 1 and type 3 eggs) in the spleen, brain, cerebellum, heart, large intestine, salt gland and kidneys. In the gallbladder, a mild papil-lary pattern was found, with multifocal inflammatory infiltrate that reached the submucosal lamina propria and was limited to the basal membrane of the mucosa and bile ducts. The inflammation was composed of lymphocytes, plasmocytes as well as scarce heterophils and eonsinophils and was associated with mildly proliferated (mild fibrosis), reactive, loose connective tissue and moderate hydropic accumulation. The epithelial cells of the mucosa were moderately degenerated, with evident nucleoli and randomly pyknotic nuclei. An amorphous eosinophilic material covered the mucous layer, with cell debris formed by an increase in mucus secretion and necrotic epithelial cells (Fig. 5).

Ethical Approval and/or Informed Consent
For this study formal concern is not required

Remarks
The genus Rhytidodoides was created by Price (1939) to group two species: R. intestinalis (from the small intestine) and R. similis (from the gallbladder). The two species were described based on two specimens collected from a green sea turtle in captivity in the National Zoological Park, Washington, DC, USA. Nigrelli (1940) reported the occurrence of R. similis collected from the gallbladder of green turtles kept at the New York Aquarium, NY, USA. The author offered few morphometric data (i.e. mean body measuring 2.2 mm in length by 1.97 mm in width among 50 specimens analyzed) and some morphological data: "In larger specimens the vitellaria are definitely separated into two distinct follicular groupings at level of esophagus and two larger masses extending from level of cecal bifurcation to the extreme posterior end, where they unite"). Smith et al. (1941) described papillomatous disease in the gallbladder of green turtles associated with the presence of R. similis, reporting papillary hyperplasia of the mucosa, fibroplasia, lymphocytic inflammatory infiltrate, dilation of blood and lymphatic vessels, mucous exudate and the presence of trematode eggs enveloped by multinucleated giant cells in the liver. However, the authors did not describe the eggs.  described the parasitological analysis of 50 green turtles, which revealed 17 species distributed among seven families of trematodes, among which the authors included R. intestinalis, R. similis and "...a new species of Rhytidodoides Price" -all found in the gallbladder. The prevalence of R. similis was 65% and papillomatous disease associated with infection by R. similis was found in four gallbladders. Caballero (1954) examined 12 green turtles from Chepillo Island in the Panama Bay and reported the occurrence of nine species of trematodes, including one specimen of R. intestinalis collected from the duodenum as well as 17 specimens of R. similis collected from the liver. The author described papilloma formation in the bile ducts, leukocytic infiltrate and the proliferation of connective tissue (findings similar to Smith et al. in 1941) as well as the occurrence of inflammatory infiltrate composed of plasmocytes and eosinophils. Mehrotra & Gupta (1978) described R. pricei and listed the differences that established the new species (based mainly on the relationship between the oral sucker and acetabulum). Mehrotra & Gupta also stated that the specimen found by Caballero (1954) (identified by Caballero as R. intestinalis) was actually R. pricei. Unfortunately, the authors did not perform a comparative analysis of the specimens collected by Price (1939) and Caballero (1954) and did not state where the parasite had been deposited. Thus P. pricei is a synonym of R. intestinalis in our opinion. Santoro et al. (2006)  More recently, Werneck et al. (2015) described the occurrence of four specimens of R. similis in a juvenile green turtle on the coast of Brazil. However, no histological analysis was performed on the gallbladder. Both R. similis and R. intetinalis can be considered specialists (see Santoro et al., 2006), as these parasites have only been found in green turtles. Rhytidodoides similis has been reported in the United States (Price, 1939;Nigrelli, 1940Smith et al., 1941;Greiner 2013), Panama (Caballero, 1954), Costa Rica (Santoro et al., 2006;Santoro et al., 2007) and Brazil (Werneck et al., 2015 and present report). In addition, R. intestinalis has been reported in the United States (Price, 1939), Panama (Caballero, 1954), Costa Rica (Santoro et al., 2006) and Brazil (present report).
In the present study, the gallbladder exhibited a mild papillary pattern, multifocal accentuated inflammatory infiltrate that reached the submucosal lamina propria and was limited to the basal membrane of the mucosa. The moderate inflammation composed of lymphocytes, plasmocytes as well as scarce heterophils and eosinophils was associated with mildly proliferated (mild fibrosis), reactive, loose connective tissue and moderate hydropic accu- mulation. The epithelial cells of the mucosa were moderately degenerated, with evident nucleoli and randomly pyknotic nuclei. An amorphous eosinophilic material covered the mucous layer, with cellular debris formed by an increase in mucus secretion and necrotic epithelial cells (FIG. 5).
The microscopic findings associated with infection by R. similis are papillomatous formation, plasmocytic and eosinophilic inflammatory infiltrate, fibroplasia and mucous exudate (Smith et al., 1941;Caballero, 1954;Santoro et al., 2006). All these findings are compatible with the results described herein. However, the present report also describes edema in the submucosal lamina propria, the degeneration of epithelial cells of the mucosa, evident nucleoli and mild necrosis of epithelial cells of the mucosa. The edema around the ducts and hydropic degeneration of the mucosa can diminish the lumen and cause biliary stasis. The mild necrosis of the mucosa facilitates infection by infectious agents and makes the bile thicker, which hinders its excretion. Evident nucleoli occurred due to the aggression caused by the inflammation. The morphological findings of the specimens were compatible with data previously described by Price (1939), Caballero (1954) and Blair (2005). The morphometric analysis of the specimens of R. similis revealed wider ranges for total body length, acetabulum width, esophagus length and width as well as larger testes and Mehlis glands in comparison to previously published data ( Table 1). The analysis of R. intestinalis revealed wider ranges regarding the length and width of the oral sucker, acetabulum, anterior testis and cirrus sac as well as larger widths of the ovary and Mehlis gland in comparison to previously published data, whereas the length of the Mehlis gland was smaller compared to data published by other authors (Table 2). Although the morphometrics of both species demonstrated differences for some organs, these differences are believed to be due to the natural variation among individuals and do not compromise the correct identification of the species. The helminth fauna of green turtle of the coast of Brazil is composed of 36 species of trematodes distributed among ten families and three species of nematodes distributed between two families (Werneck and Silva, 2016, present report).