Occurrence of Rhytidodoides similis Price, 1939 (Digenea: Rhytidodidae) and Lesions Due to Spirorchiid Eggs in a Green Turtle, Chelonia mydas

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1 Occurrence of Rhytidodoides similis Price, 1939 (Digenea: Rhytidodidae) and Lesions Due to Spirorchiid Eggs in a Green Turtle, Chelonia mydas Linnaeus, 1758 (Testudines, Cheloniidae), from Brazil Author(s): Max R. Werneck, Érika Binoti, Isabella V. F. Martins, Antonio De Calais Junior, Moara C. Gomes, Jankerle N. Boeloni, Alessandro Trazzi, and Bruno Berger Source: Comparative Parasitology, 82(2): Published By: The Helminthological Society of Washington DOI: URL: BioOne ( is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne s Terms of Use, available at terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.

2 Comp. Parasitol. 82(2), 2015, pp Research Note Occurrence of Rhytidodoides similis Price, 1939 (Digenea: Rhytidodidae) and Lesions Due to Spirorchiid Eggs in a Green Turtle, Chelonia mydas Linnaeus, 1758 (Testudines, Cheloniidae), from Brazil MAX R. WERNECK, 1,5 ÉRIKA BINOTI, 2 ISABELLA V. F. MARTINS, 3 ANTONIO DE CALAIS JUNIOR, 2 MOARA C. GOMES, 2 JANKERLE N. BOELONI, 3 ALESSANDRO TRAZZI, 4 AND BRUNO BERGER 4 1 BW Consultoria Veterinária, Rua Ponciano Eugênio Duarte no. 203, Centro, Ubatuba, SP, CEP , Brazil ( max@bwvet.com.br), 2 Mestrando em Ciências Veterinárias da Universidade Federal do Espírito Santo (UFES) ( erikabinoti.mv@ hotmail.com; vetcalais@gmail.com; moaracgomes@yahoo.com.br), 3 Professora Adjunta do Departamento de Medicina Veterinária e do programa de Pós Graduação em Ciências Veterinárias da Universidade Federal do Espírito Santo (UFES) ( isabella.martins@ufes.br), and 4 Serviços em Meio Ambiente, CTA, Rua Saturnino Rangel Mauro 283, Pontal de Camburi, Vitória, ES, CEP , Brazil ( alessandro@cta-es.com.br; bruno.berger@cta-es.com.br) ABSTRACT: This paper describes the first occurrence of Rhytidodoides similis Price, 1939, parasitizing a juvenile green turtle (Chelonia mydas Linnaeus, 1758) found on the coast of Brazil. This parasite is known only from C. mydas, with previous reports of occurrences in the liver and gall bladder of green sea turtles found in the United States, Panama, and Costa Rica. In this case, no gross or microscopic lesions were found on the liver associated with R. similis. However, microscopic analysis of the liver revealed granulomas of trematode eggs consistent with spirorchiids. KEY WORDS: Chelonia mydas, Helminth fauna, hepatic lesions, marine turtle, parasitology, Rhytidodoides similis, Rhytidodidae, Spirorchiidae, Trematoda. The family Rhytidodidae Odhner, 1926, is represented only by the genera Rhytidodes and Rhytidodoides, members of which have been found in the intestines and gall bladders of sea turtles (Blair, 2005). The genus Rhytidodoides has only 3 known species: R. intestinalis Price, 1939, and R. similis Price, 1939, described from a green turtle, Chelonia mydas Linnaeus, 1758, found in the United States, and R. pricei Mehrotra and Gupta, 1978, described from a green turtle found in the Gulf of Mannar, India. Spirorchiids (family Spirorchiidae Stunkard 1921) are a group of trematodes that inhabit the circulatory, lymphatic, and nervous system of turtles. The family has approximately 100 species grouped into 19 genera (Platt, 2002), 10 of which are parasites of sea turtles described from Chelonia mydas Linnaeus, 1758, Caretta caretta Linnaeus, 1758, and Eretmochelys imbricata Linnaeus, 1766 (Smith, 1997). 5 Corresponding author. Infection with members of the family Spirorchiidae results in inflammatory lesions due to egg deposition in the blood stream, their accumulation in tissues, and embolism causing or contributing to host death (Gordon et al., 1998; Santoro et al., 2007; Stacy et al., 2010; Chen et al., 2012). The present note reports the occurrence of R. similis in a green turtle from Brazilian coast and the occurrence of hepatic lesions caused by spirorchiid eggs in the same host. A moribund green turtle was found on Guarapari Beach in the state of Espírito Santo, Brazil (20u S; 40u W) in September 2013, measuring 43.1 cm in curved carapace length and weighing 8.9 kg. The turtle was emaciated and lethargic and died on the first day of treatment. Necropsy was performed immediately. Liver samples were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned (5 mm), and stained with hematoxylin and eosin and Masson s trichrome stain for subsequent microscopic analysis. Histopatological analysis of the gall bladder was not made because it was inadvertently discarded after liver collection. Four specimens of R. similis (Fig. 1) were found in the gall bladder. The parasites were placed in saline solution, and 2 were fixed in a 10% formalin solution and stained with eosin. The other 2 were fixed in 70% alcohol, stained with carmine, and cleared with eugenol. Morphometric data were made with the aid of an image analysis program (ImageJ, National Institutes of Health). Drawings were made using a drawing tube. The specimens were deposited in the Helminth Collection of the Institute of Biosciences of the Paulista State University, of São Paulo State, Brazil (CHIBB number 7460). A taxonomic key 291

3 292 COMPARATIVE PARASITOLOGY, 82(2), JULY 2015 Figure 1. Rhytidodoides similis Price, 1939 (Digenea: Rhytidodidae) from Chelonia mydas Linnaeus 1758 (Testudines, Cheloniidae) from Brazil. (Blair, 2005), the original description (Price, 1939), comments by Smith et al. (1941), and the redescription by Caballero y Caballero (1954) were used for both morphological (Fig. 1) and morphometric comparisons (Table 1). Analyses of the parasites were authorized by federal licenses for activities with scientific purposes (SISBIO) and Macroscopic analysis of the liver revealed no abnormalities on the shape, color, or size (see Wyneken, 2001). However, microscopic analysis revealed granulomas containing a moderate number of trematode eggs with birefringent walls and a brownish coloration. These eggs were completely surrounded by multinucleated foreign-body giant cells (Fig. 2). The genus Rhytidodoides was originally described by Price (1939) to house R. intestinalis and R. similis, which were collected from the intestine and gall bladder, respectively, of a green turtle found in the United States. The descriptions were based on only 2 specimens of each species. After the original description (Price, 1939), R. similis was found in the gall bladder of C. mydas in the New York Aquarium, but little morphometric information was reported (Nigrelli, 1940). Later the author mentioned that R. similis occurred in 65% of the turtle gall bladders analyzed, but no additional information was offered (Nigrelli, 1941). Smith et al. (1941) reported the occurrence of R. similis and lesions in gall bladders associated with these flukes. These authors noted that the walls of infected gall bladders were thickened with papillary hyperplasia of the mucous membrane, an evident increase in the amount of connective tissue, infiltration of lymphoid cells, dilation of capillary and lymphatic vessels, and an increase in mucus production and the presence of trematodes eggs in the liver. However, the authors did not clearly specify the characteristics of these eggs, reporting only that they were surrounded by epithelioid cells and/or giant cells. In the same host species found in Panama, Caballero y Caballero (1954) reported one specimen of R. intestinalis and redescribed R. similis based on 16 specimens collected from the biliary ducts. He also provided a histopathological analysis of liver samples and reported the formation of papillomas and the proliferation of connective tissue evidencing leukocyte infiltration with the migration of plasmocytes and eosinphils. Mehrotra and Gupta (1978) found a specimen of the genus Rhytidodoides in a green turtle from the Gulf of Mannar, India, and described R. pricei. Based on an analysis of manuscripts by Price (1939) and Caballero y Caballero (1954), as well as morphometric variations, Mehrotra and Gupta (1978) determined that the species found by Caballero y Caballero (1954) (regarded by him as representing R. intestinalis) was actually identical to R. pricei. Thus to date only R. intestinalis, R. similis, and R. pricei are reported for the genus. Santoro et al. (2006) reported the occurrence of R. similis in 25% of nesting green sea turtles in Costa Rica, with a mean infection intensity of 10.6 (range: 1 to 34 specimens). These authors reported inflammation of the gall bladder, abnormalities in the mucous layer, and a small number of leukocytes associated with these lesions (Santoro et al., 2007). Analyzing 74 individuals of C. mydas from the coast of the state of Florida (United States) between 1991 and 2006, Greiner (2013) reported the occurrence of

4 RESEARCH NOTE 293 Table 1. Morphometric data of Rhytidodoides similis, Price, 1939 (Digenea: Rhytidodidae) from Chelonia mydas (Testudines, Cheloniidae). Data are presented as range (mean), and measurements are in millimeters. Price (1939) Smith et al. (1941) Caballero (1954) Present report Site Gall bladder Gall bladder Biliary ducts Gall bladder Locality United States United States Panama Brazil No. 2? 16 2 Body length Body width Oral sucker length * (0.293)*{ Oral sucker width Pharynx length Pharynx width Ventral sucker length (0.30)*{ Ventral sucker width Esophagus length Esophagus width Anterior testes length * Anterior testes width Posterior testes length * Posterior testes width Cirrus sac length Cirrus sac width Ovary length * * Ovary width Mehlis gland length Mehlis gland width Eggs length (0.045){ Eggs width (0.029){ * Diameter. { Range (mean) 304 specimens of R. similis in the liver and gall bladder of 16 hosts (prevalence: 21.6%) with a mean intensity of 19.0 (range: 1 to 98 specimens). The genus Rhytidodoides is considered restricted to C. mydas, and R. similis has been reported in sea turtles in the United States (Price, 1939; Nigrelli, 1940 and 1941; Greiner, 2013), Panama Figure 2. Liver with granulomatous lesions with foreign-body giant cells (arrow) surrounding trematode eggs compatible with spirorchiids. (Caballero y Caballero, 1954), Costa Rica (Santoro et al., 2006; Santoro et al., 2007), and Brazil (present report). Despite the lack of macroscopic indications of infection in the liver, the microscopic analysis revealed granulomatous lesions with foreign-body giant cells surrounding trematode eggs (Fig. 2) consistent with the family Spirorchiidae (Santoro et al., 2007). The eggs were classified as type I (see Wolke et al., 1982), known to be produced by members of the genera Learedius, Monticellius, and Hapalotrema, each of which has been reported in C. mydas (Smith, 1997). These 3 genera have also been described in Brazil: Learedius learedi Price 1934 has been found in C. mydas (Werneck et al., 2006); Monticellius indicum Mehra 1939 was found in both C. mydas (Werneck et al., 2008) and E. imbricata (Werneck, Souza, Trazzi et al., 2015); and Hapalotrema postorchis Rao 1976 has been found in both E. imbricata (Werneck et al., 2014) and C. mydas (Werneck, Souza, Berguer et al., 2015). Pathological changes induced by spirorchiid eggs have been reported by other authors (Gordon et al., 1998; Santoro et al., 2007; Stacy et al., 2010; Chen

5 294 COMPARATIVE PARASITOLOGY, 82(2), JULY 2015 et al., 2012) and are potentially debilitating or fatal. We considered this occurrence as an incidental finding (see appendix 1 in Stacy et al., 2010), and in our case we cannot affirm that the eggs are responsible for the host s death. This note is the first report of spirorchiid eggs lesions in C. mydas from Brazil. The morphometric analysis (Table 1) revealed a greater pharynx length and width, a greater anterior testes length, a greater cirrus sac length and width, and both lower Mehlis gland widths and egg widths in comparison to the specimens analyzed by Price (1939), Smith et al. (1941), and Caballero y Caballero (1954). However, we regard these differences merely as morphometric variations within the species. The little information on these helminthes and species descriptions based on a small number of specimens (see Price, 1939; Mehrotra and Gupta, 1978) underscores the need for further comparative work to clarify the validity of species in the genus Rhytidodoides. This note report the first occurrence of R. similis in green turtle from Brazil, and it is the first evidence of lesions by spirorchiids eggs in this host and region. These findings represent an important information about sea turtle parasites. The authors wish to express their special thanks to Dr. Paula Baldassin and Dr. David Blair for critical reading and suggestions to improve the manuscript, the Olé Web Designer staff, Marcella Baldassin, and José Carlos Barroso. The biological samples were obtained through the Beach Monitoring Project of the state of Espírito Santo and the northern portion of the Campos Basin, which is a requirement established by the federal environmental licensing division of the Brazilian environmental agency: Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis. E.B. had financial support from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. LITERATURE CITED Blair, D Family Rhytidodidae Odner, Pages in A. Jones, R. A. Bray, and D. I. Gibson, eds. Key to the trematodes. Vol. 2. CABI Publishing, London, U.K. Caballero y Caballero, E Helmintos de la Republica de Panamá. X. Alguns Tremátodos de Chelone mydas (L.) tortuga marina comestible Del Océano Pacífico Del Norte. Anales del Instituto de Biologia Universidad Nacional Autónoma de México 8: Chen, H., R. J. Kuo, T. C. Chang, C. K. Hus, R. A. Bray, and I. J. Cheng Fluke (Spirorchiidae) infections in sea turtles stranded on Taiwan: prevalence and pathology. Journal of Parasitology 98: Gordon, A. N., W. R. Kelly, and T. H. Cribb Lesions caused by cardiovascular flukes (Digenea: Spirorchiidae) in stranded green turtles (Chelonia mydas). Veterinary Pathology 35: Greiner, E. C Parasites of marine turtles. Pages in J. Wyneken, K. J. Lohmann, and J. A. Lutz, eds. The Biology of Sea Turtles. Vol. 3. CRC Press, Boca Raton, Florida. 475 pp. Mehrotra, V., and N. K. Gupta On the status of Rhytidodoides intestinalis Price, 1939 of Caballero 1954 (Trematoda, Rhytidodidae). Acta Parasitological Polonica 25: Nigrelli, R. F Observations on trematodes of the marine turtle, Chelonia mydas. Anatomical Record Philadelphia 78:178. Nigrelli, R. F Parasites of the Green turtle, Chelonia mydas (L.) with special references to the rediscovery of trematodes described by Looss from this host species. Journal of Parasitology 27: Platt, T. R Family Spirorchiidae Stunkard. Pages in D. I. Gibson, A. Jones, and R.A. Bray, eds. Keys to the Trematoda. Vol. 1. CABI Publishing, London, U.K. Price, E. W A new genus and two new species of digenetc trematodes from a marine turtle. Proceedings of the Helminthological Society of Washington 6: Santoro, M., E. C. Greiner, J. A. Morales, and B. Rodrígues-Ortiz Digenetic trematode community in nesting green sea turtles (Chelonia mydas) from Tortuguero Nacional Park, Costa Rica. Journal of Parasitology 92: Santoro, M., J. A. Morales, and B. Rodrígues-Ortiz Spirorchiidiosis (Digenea: Spirorchiidae) and lesions associated with parasites in Caribbean green turtles (Chelonia mydas). Veterinary Record 161: Smith, G. M., C. W. Coates, and R. F. Nigrelli A papillomatous disease of the gallbladder associated with infection by flukes, occurring in the marine turtles, Chelonia mydas (Linnaeus). Zoologica 26: Smith, J. W The blood flukes (Digenea: Sanguinicolidae and Spirorchidae) of cold-blooded vertebrates: part 2. Helminthological Abstracts 66: Stacy, B. A., A. M. Foley, E. Greiner, L. H. Herbst, A. Bolten, P. Klein, C. A. Manire, and E. R. Jacobson Spirorchiidiasis in stranded loggerhead Caretta caretta and green turtles Chelonia mydas in Florida (U.S.A.): host pathology and significance. Diseases of Aquatic Organisms 89: Werneck, M. R., P. Baldassin, F. T. D Azeredo, A. Trazi, and B. Berguer The Hawksbill sea turtle Eretmochelys imbricata Linnaeus 1758 (Testudines, Cheloniidae) as new host of Hapalotrema postorchis Rao, 1976 (Digenea: Spirorchiidae). Comparative Parasitology 81: Werneck, M. R., J. H. Becker, B. G. Gallo, and R. J. Silva Learedius learedi Price 1934 (Digenea, Spirorchiidae) in Chelonia mydas Linnaeus 1758 (Testudines, Chelonidae) in Brazil: case report. Arquivo Brasileiro de Medicina Veterinária e Zootecnia 58: Werneck, M. R., B. Gallo, and R. J. Silva First report of Monticellius indicum Mehra, 1939 (Digenea:

6 RESEARCH NOTE 295 Spirorchiidae) infecting Chelonia mydas Linnaeus, 1758 (Testudines: Chelonidae) from Brazil. Brazilian Journal of Biology 68: Werneck, M. R., G. Souza, B. Berguer, A. Trazzi, R. Ribeiro, M. A. Silva, H. J. Leandro, and E. C. Q. Carvalho Pathological changes by Hapalotrema postorchis Rao 1976 (Digenea: Spirorchiidae) in a green turtle Chelonia mydas Linnaeus 1758 (Testudines, Cheloniidae) from Brazil. Helminthologia 52: Werneck, M. R., V. R. Souza, A. Trazzi, and B. Berguer Monticellius indicum Mehra, 1939 (Digenea: Spirorchiidae) in a Hawksbill Turtle, Eretmochelys imbricata Linnaeus 1758 (Testudines, Cheloniidae) from Brazil. Comparative Parasitology 82: Wolke, R. E., R. Brooks, and A. George Spirorchidiasis in loggerhead sea turtles (Caretta caretta): pathology. Journal of Wildlife Diseases 18: Wyneken, J The anatomy of sea turtles. U.S. Department of Commerce NOAA Technical Memorandum NMFS-SEFSC-470, Washington, D.C. 172 pp.