249 Gastrointestinal Parasitism in Rescued Wild Birds Neotropical Helminthology (Lima). Vol. 18, Nº2, jul - dec 2024 Neotropical Helminthology Neotropical Helminthology, 2024, vol. 18 (2), 249-258 ORIGINAL ARTICLE / ARTÍCULO ORIGINAL FIRST REGISTER OF CONTRACAECUM SP. AND EUSTRONGYLIDES SP. (NEMATODA) IN KINOSTERNON SCORPIOIDES (LINNAEUS, 1766) (TESTUDINES: KINOSTERNIDAE) FROM NORTHEASTERN, BRAZILPRIMER REGISTRO DE CONTRACAECUM SP. Y EUSTRONGYLIDES SP. (NEMATODA) EN KINOSTERNON SCORPIOIDES (LINNAEUS, 1766) (TESTUDINES: KINOSTERNIDAE) DEL NORESTE DE BRASIL Alana Lislea de Sousa 1 , Almerinda Macieira Medeiros 2 , Júlia Boáis Almeida 2 , Silmara Cristina Silva de Aquino 1 , Elias Costa Ferreira Junior 1 , Diego Carvalho Viana 3 , Victor Puicón-Niño de Guzmán 4 & Germán Augusto Murrieta-Morey 1,5* ISSN Versión Impresa 2218-6425 ISSN Versión Electrónica 1995-1403 DOI: https://dx.doi.org/10.62429/rnh20242181853 Este artículo es publicado por la revista Neotropical Helminthology de la Facultad de Ciencias Naturales y Matemática, Universidad Nacional Federico Villarreal, Lima, Perú auspiciado por la Asociación Peruana de Helmintología e Invertebrados Af nes (APHIA). Este es un artículo de acceso abierto, distribuido bajo los términos de la licencia Creative Commons Atribución 4.0 Internacional (CC BY 4.0) [https:// creativecommons.org/licenses/by/4.0/deed.es] que permite el uso, distribución y reproducción en cualquier medio, siempre que la obra original sea debidamente citada de su fuente original. ABSTRACT Kinosternon scorpioides (Linnaeus, 1766) popularly known in Brazil as “Jurará” is a species of chelon that serves as a food resource and source of income for some families in Northeastern Brazil. In that sense, the present study aimed to analyze the metazoan endoparasites infecting K. scorpioides from the Baixada Maranhense, Brazil. Fifteen adult specimens 1 Universidade Estadual do Maranhão (UEMA). Programa de Pós-graduação em Ciência Animal (PPGCA), Cidade Universitária Paulo IV, São Luís, 65055-310, Maranhão-Brasil. 2 Universidade Estadual do Maranhão (UEMA). Programa de Pós-graduação em Biotecnologia e Biodiversidade da Amazônia Legal (Rede Bionorte), Cidade Universitária Paulo IV, São Luis, 65055-310, Maranhão-Brasil. 3 Universidade Estadual da Regiao Tocantina do Maranhão (UEMASUL). Programa de Pós-graduação em Ciência Animal (PPGCA), Cidade Universitária Paulo IV, São Luís, 65055-310, Maranhão-Brasil. 4 Grupo de Investigación Parasitología Veterinaria y Zoonosis Parasitaria, Laboratorio de Histopatología animal, Facultad de Medicina Veterinaria, Escuela Profesional de Medicina Veterinaria, Universidad Nacional de San Martín. Tarapoto, Perú. 5 Instituto de Investigaciones de la Amazonia Peruana (IIAP), Laboratorio de Parasitologia y Sanidad Acuicola, Carretera Iquitos-Nauta, Km 4.5 – San Juan Bautista, Iquitos, 0784, Loreto, Perú. * Corresponding author:germantiss1106@gmail.comAlana Lislea de Sousa: https://orcid.org/0000-0002-0920-2560Almerinda Macieira Medeiros: https://orcid.org/0000-0002-3808-2035Júlia Boáis Almeida: https://orcid.org/0009-0000-0230-732XSilmara Cristina Silva de Aquino: https://orcid.org/0000-0001-7680-9699Elias Costa Ferreira Junior: https://orcid.org/0000-0002-5925-6372Diego Carvalho Viana: https://orcid.org/0000-0002-3302-9892Victor Puicón Niño de Guzmán: https://orcid.org/0000-0003-2532-2551Germán Augusto Murrieta Morey: https://orcid.org/0000-0001-6244-2654
Lislea de Sousa et al. 250 Neotropical Helminthology (Lima). Vol. 18, Nº2, jul - dec 2024 were captured using funnel traps in fooded felds around the Aurá River, close to the city of São Bento-Maranhão. Samples were transported to the Postgraduate Multi-User Research Laboratory- Laboratories in Postgraduate Research/Postgraduate in Animal Science located at the State University of Maranhão- UEMA for parasitological analyses. Te results revealed the presence of larvae of Contracaecum sp. infecting the stomach, intestine, and liver, while Eustrongylides sp. was found encysted in the musculature, stomach, intestine, and inside an egg of a gravid female mud turtle. Te fndings of the present study constitute the frst records of these nematodes parasitizing K. scorpioides . Since K. scorpioides is consumed as food for some families in the State of Maranhão, Brazil, and the presence of both parasites with hygienic-sanitary importance, complementary studies should be designed to allow us to understand the relationship between host parasites and whether they could have zoonotic relevance. Keywords: Baixada Maranhense – endoparasites – helminths – Jurará – Nematoda - scorpion mud turtle RESUMEN Kinosternon scorpioides (Linnaeus, 1766), conocido popularmente en Brasil como “Jurará”, es una especie de quelonio que sirve como recurso alimenticio y fuente de ingresos para algunas familias del Nordeste de Brasil. En ese sentido, el presente estudio tuvo como objetivo analizar los endoparásitos metazoarios que infectan a K. scorpioides de la Baixada Maranhense, Brasil. Quince especímenes adultos fueron capturados utilizando trampas de embudo en campos inundados alrededor del río Aurá, cerca de la ciudad de São Bento-Maranhão. Las muestras fueron transportadas al Postgraduate Multi-User Research Laboratory- Laboratories in Postgraduate Research/Postgraduate in Animal Science ubicado en la Universidad Estatal de Maranhão - UEMA para análisis parasitológicos. Los resultados revelaron la presencia de larvas de Contracaecum sp. infectando el estómago, intestino e hígado, mientras que Eustrongylides sp. Se encontró enquistado en la musculatura, estómago, intestino y en el interior de un huevo de una tortuga de lodo hembra grávida. Los hallazgos del presente estudio constituyen los primeros registros de estos nematodos parasitando a K. scorpioides . Dado que K. scorpioides es consumido como alimento por algunas familias en el Estado de Maranhão, Brasil, y la presencia de ambos parásitos con importancia higiénico-sanitaria, se deben diseñar estudios complementarios que nos permitan comprender la relación entre los parásitos hospedadores y si pudieran tener relevancia zoonótica. Palabras clave: Baixada Maranhense – endoparásitos – helmintos – Jurará – Nematoda – tortuga de lodo escorpión RESUMO Kinosternon scorpioides (Linnaeus, 1766) popularmente conhecido no Brasil como “Jurará” é uma espécie de quelônio que serve como recurso alimentar e fonte de renda para algumas famílias no Nordeste do Brasil. Nesse sentido, o presente estudo teve como objetivo analisar os endoparasitas metazoários que infectam K. scorpioides da Baixada Maranhense, Brasil. Quinze espécimes adultos foram capturados usando armadilhas de funil em campos alagados ao redor do Rio Aurá, próximo à cidade de São Bento-Maranhão. As amostras foram transportadas para o Laboratório de Pesquisa Multiusuário de Pós-Graduação - Laboratórios de Pesquisa de Pós-Graduação/Pós-Graduação em Zootecnia localizado na Universidade Estadual do Maranhão - UEMA para análises parasitológicas. Os resultados revelaram a presença de larvas de Contracaecum sp. infectando o estômago, intestino e fígado, enquanto Eustrongylides sp. foi encontrado encistado na musculatura, estômago, intestino e dentro de um ovo de uma tartaruga fêmea grávida. Os achados do presente estudo constituem os primeiros registros desses nematoides parasitando K. scorpioides . Como K. scorpioides é consumido como alimento por algumas famílias no estado do Maranhão, Brasil, e a presença de ambos os parasitas com importância higiênico-sanitária, estudos complementares devem ser delineados para nos permitir entender a relação entre os parasitas hospedeiros e se eles podem ter relevância zoonótica. Palavras-chave: Baixada Maranhense – endoparasitas – helmintos – Jurará – Nematoda – tartaruga-escorpião
Contracaecum sp. and Eustrongylides sp. in Kinosternon scorpioides 251 Neotropical Helminthology (Lima). Vol. 18, Nº2, jul - dec 2024 INTRODUCTION Maranhão is a State that encompasses three of the main Brazilian morphoclimatic domains: Amazon, Caatinga, and Cerrado. Te State represents an ecotone zone between these biomes, with 14 distinct vegetation types (IBGE, 2004), refecting the transition between humid forests and semi-arid habitats (Da Silva, 2011). Te Baixada Maranhense region, placed in Maranhão, is located on the easternmost limit of the Amazon biome, forming part of the Brazilian Legal Amazon. Tis region is characterized by a landscape made up of lakes, felds, foodplains, igapós, landflls, river-marine systems, and dry land. Te foodable natural felds of Baixada Maranhense are extremely complex environments from an ecological point of view, with very diverse structures and functioning (Costa-Neto et al ., 2002; Pinheiro, 2013). Due to the recognized importance of the region’s foodable natural felds, the Government of the State of Maranhão established the Baixada Maranhense Environmental Protection Area (APA) with Decree No. 11,900 of June 11, 1991 (State of Maranhão, 1991). In 2000 it was designated by the National Wetlands Committee (CNZU) as a Ramsar Site: regions that provide fundamental ecological services - meeting water and food needs - for species of fauna and fora and human populations, rural and urban. In addition to regulating the water regime of vast regions, they function as a source of biodiversity at all levels. It has the largest set of lake basins in the Northeast, which contribute to high fshing productivity, the main basis of food and income support for its population (Costa-Neto et al ., 2002).Chelonians are animals that perform important ecosystem functions and are intrinsically linked in many traditional cultures, from the consumption and seed dispersal of various tree species to the important social role of communities that use river systems as a means of subsistence (Alves et al ., 2008; Rhodin et al., 2018). Kinosternon scorpioides (Linnaeus, 1766) popularly known as the scorpion mud turtle and commonly named in Brazil as “Jurará” is a species of chelon widely distributed in the Neotropical region, occurring from Panama, along the Caribbean mountain range to Brazil, covering the entire Amazon forest, eastern mountain ranges of Colombia, Ecuador, and Peru. Historically, many species of chelonians in diferent parts of the world have great nutritional, economic, and cultural importance, with their eggs, meat, viscera, fat, and shell being used intensively by humans (Van Dijk et al ., 2014). In Baixada Maranhense, species such as Phrynops geofroanus (Schweigger, 1812) and mainly Kinosternon scorpioides serve as a food resource and source of income for some families, being captured illegally, in adult and sub-adult form alive and hanging, and sold mainly from May to August, when females appear ovate. Wild populations are used for consumption as a regional delicacy, both for subsistence and recreational consumption (Machado Júnior et al ., 2006; Medeiros, 2016).According to Giari et al. (2022) in aquatic environments there exist metazoan parasites with zoonotic potential that can cause strong negative impacts on the safety and quality of the meat. Tese parasites can be of public health interest and can compromise the health of the consumers. Humans can be infected accidentally with zoonotic parasites by the ingestion of raw or poorly cooked meat (Giari et al ., 2022).Due to the consumption of K. scorpioides meat by the Brazilian population and the presence of parasites with zoonotic potential in aquatic organisms, the present study aimed to analyze the metazoan endoparasites infecting K. scorpioides from the Baixada Maranhense, Brazil. MATERIAL AND METHODS Te study was carried out around the Aurá River, located in the Baixada Maranhense Environmental Protection Area (APA), in the Northeast of Brazil, on the eastern edge of the Amazon (Figure 1). Tis area represents a wetland of international importance (Ramsar Site), bringing together a set of river basins and fooded felds, and seasonal periods with great contrasts in temperature, humidity, and rainfall (Costa-Neto et al ., 2002; Mendes, 2018). Te collections were carried out in fooded felds around the Aurá River, city of São Bento-Maranhão (2°42’08,1”S, 44°51’40,3”W and 2°40’60,0”S, 44°49’00,0”W) (Fig.1). Te animals were captured using funnel traps (Secco et al ., 2013; Balestra et al ., 2016). Captured animals presented 13,73 cm. mean carapace length, 9,17 cm. mean carapace width, 12,60 cm. mean plaston length, 7,97 cm. mean plaston width, 4,87 cm. mean height and 342,40 g. mean weight. Samples were transported at the Postgraduate Multi-User Research Laboratory- Postgraduate Multi-User Research Laboratory- Laboratories in Postgraduate Research/Postgraduate in Animal Science located at the State University of Maranhão- UEMA Campus Paulo VI. Te samples were collected in the region of the Municipality of São Bento-MA, belonging to the APA of Baixada Maranhense, Sítio Ramsar.
Lislea de Sousa et al. 252 Neotropical Helminthology (Lima). Vol. 18, Nº2, jul - dec 2024 Te musculature was analyzed and carefully observed. Internal organs were analyzed using a stereoscope Leica EZ4. Organs were placed in Petri dishes with distilled water, and with the aid of tweezers and needles, the tissue was examined by making fne cuts that allowed to observe inside them. In the presence of any parasites, these were removed with tweezers and preserved in ethanol 70% for posterior taxonomic identifcation. For taxonomic identifcation, cysts with nematodes were placed in Petri dishes, and then, with the aid of needles, the cysts were broken, liberating the larvae, and then preserved in 70% ethanol for posterior analyses. For the identifcation of nematodes, they were placed into glass slides with Lactic Acid, which is used for the clarifcation of parasite tissue and consequent visualization of external structures and internal organs. All parasites mounted in slides were observed under an optical microscope Leica DM750. Based on the morphological characteristics of the parasites, the identifcation was made using the information of Moraveck (1998) and Morey et al. (2022). Parasitological indices were calculated according to Bush et al. (1997). Figure 1 . Study area. Identifcation of collection points distributed along the Aurá River (blue) A-Forest areas; B- Flooded felds; C- Water areas (River). Source: IBGE (2018); ZEE (2019); MapBiomas (2021). Ethic aspects : Te collection of samples was carried out under the permissions granted by the Biodiversity Authorization and Information System (SISBIO), number 85805-1. RESULTS Te nematodes Contracaecum sp. and Eustrongylides sp. were found parasitizing K. scorpioides . Contracaecum sp. was found in the stomach, intestine, and liver (Fig. 2, while Eustrongylides sp. was found encysted in the musculature (Fig. 3) stomach, intestine and inside the egg of a gravid female K. scorpioides (Fig. 4). Both parasites were found encysted, being necessary to break the cyst to liberate the larvae. Parasitological indices are presented in Table 1. Contracaecum sp. medium-sized nematodes, opaque-white when alive. Transversely striated cuticles present and more distinct at the extremities of the body, with the anterior region interrupted by a short, lateral line. Te cephalic extremity is rounded with a small, ventral cuticular tooth. Te oesophageal muscle is narrow, the ventricle is small and rounded, and the ventricular appendix is a short, long intestinal caecum, extending anteriorly to the nerve ring. Tail conical without mucron (Fig. 5A-C).
Contracaecum sp. and Eustrongylides sp. in Kinosternon scorpioides 253 Neotropical Helminthology (Lima). Vol. 18, Nº2, jul - dec 2024 Figure 2 . A. Encysted Contracaecum sp. in internal organs of Kinosternon scorpioides . Black circles present the cyst of the parasites. Scale bar: 2 cm. Figure 3 . A. Eustrongylides sp. in the musculature of Kinosternon scorpioides . B. Cyst. C. Eustrongylides sp. being released from a cyst. D. Free Eustrongylides sp. Scale bar: A, B = 2 cm. C-E = 0.5 cm
Lislea de Sousa et al. 254 Neotropical Helminthology (Lima). Vol. 18, Nº2, jul - dec 2024 Eustrongylides sp. presented a cephalic extremity with a small oral cavity surrounded by 12 cephalic papillae of similar size arranged in two concentric rings, and genital primordia in the posterior part of the body (Fig. 5D-F).According to the results of the present study, it can be assumed that in the life cycle of Eustrongylides sp. and Contracaecum sp., diferent organisms are involved: invertebrates play the role of intermediate hosts, while fsh and K. scorpioides act as paratenic hosts. Finally, given that K. scorpioides are preyed upon by Amazonian alligators, it can be inferred that they act as fnal hosts for these nematodes in territories of the Baixada Maranhense, Maranhão, Brazil (Fig. 6). DISCUSSION Studies focusing on South American turtles are still recent and scarce (Bursey & Brooks, 2011; Mascarenhas et al ., 2013). Few studies have examined the nematodes parasites of K. scorpioides in Brazil: Serpinema magathi from specimens collected in Pará (Alho, 1965); Freitas and Dobbin Jr. (1971) from Pernambuco; S. monospiculatus from specimens collected in a semiarid region in the Northeast (Pereira et al ., 2018): S. pelliculatus was found from the gastrointestinal tract of specimens collected in Baía do Capim (municipality of Abaetetuba) and Ilha do Marajó (municipality of Soure), Pará, Brazil (Silva et al ., 2023). Tese parasites are commonly found in freshwater turtles worldwide (Hidalgo-Vila et al ., 2009; Bursey & Brooks, 2011; Moraga et al ., 2012; Mascarenhas et al ., 2013; Mascarenhas & Muller, 2015). Figure 4. A. Eustrongylides sp. in an egg of Kinosternon scorpioides . Te black arrow indicates the egg with the parasite. B. Broken egg showing the nematode inside. Scale bar: 2 cm. Table 1 . Parasitological indices of endoparasites reported in Kinosternon scorpioides. AS = Analyzed specimen. PS = Parasitized specimen. P% = prevalence. TNP = total number of parasites. mI = mean intensity of infection. mA = mean abundance of infection. ParasiteASPSP%TNPmImA Contracaecum sp.151173.33191.720.73 Eustrongylides sp.151280322.662.13
Contracaecum sp. and Eustrongylides sp. in Kinosternon scorpioides 255 Neotropical Helminthology (Lima). Vol. 18, Nº2, jul - dec 2024 Figure 5. A. Microphotography of the anterior part of Contracaecum sp. Dark circle showing intestinal cecum. B. Microphotography of larval tooth (dark circle). C. Microphotography of the tail of Contracaecum sp. D. Encysted Eustrongylides sp. E. Microphotography of the anterior part of Eustrongylides sp. F. Microphotography of the tail of Figu re 6 . T e life cycle of Nematoda larvae of Eustrongylides sp. and Contracaecum sp. 1. Nematoda eggs are liberated from the alligator. 2. Eggs with infective L1 larvae. 3. Eggs are consumed by an annelid (for Eustrongylides sp.) or for a copepod (for Contracaecum sp.). Nematoda larvae develop inside the invertebrates from L1 into L2 and L3. Invertebrates act as intermediate hosts. 4. Fish consume the invertebrates with Nematoda larvae. Fish are paratenic hosts. 5. Fish are consumed by Kinosternum scorpioides. T e mud turtle acts as a paratenic host. 6. Kinosternon scorpioides are consumed by an alligator. T e alligator acts as the f nal host of Nematoda larvae of Eustrongylides sp. and Contracaecum sp.
Lislea de Sousa et al. 256 Neotropical Helminthology (Lima). Vol. 18, Nº2, jul - dec 2024 For northeastern Brazil, the nematodes Serpinema magathi and Spiroxys fgueiredoi have been found in the digestive tract of K. scorpioides (Viana et al ., 2016). In the present study, none of these nematodes were found on specimens captured in the municipality of São Bento, Maranhão, Brazil. In the present study, the fndings of Contracaecum sp. and Eustrongylides sp. are the frst records of K. scorpioides . Since K. scorpioides is an omnivorous species that feeds on algae, plants, seeds, and a variety of insects, mollusks, fsh, and small amphibians (Berry & Ivwerson, 2001; Berry & Iverson, 2011), its feeding habits facilitate the infection of this freshwater turtle by diferent parasitic nematode larvae present in aquatic environments, such as Contracaecum spp. and Eustrongylides spp. Contracaecum larvae show low specifcity, are often found in abnormal hosts, and do not develop into adults in turtles (Shubber et al ., 2020). In this sense, it can be assumed that K. scorpioides acquired the nematodes by feeding on fshes in their ecosystems, a fact that was supported by a personal observation made in the feld during the capture of the animals in this study. In the life cycle of Eustrongylides spp. freshwater oligochaetes act as intermediate hosts; fsh as paratenic hosts and birds as fn7al hosts (Spalding, 2008). Eustrongylides spp. can infect poikilothermous hosts, as was demonstrated by experimental infections with piscine, amphibians, and reptilian hosts (Cooper et al ., 1978). Te presence of nematode larvae in K. scorpioides indicates that this turtle acts as the paratenic host of Contracaecum sp. and Eustrongylides sp. Natural predators of this mud turtle are documented as jaguars (Savage, 2002), owls (Pereira et al ., 2007), vultures, green iguanas (Acuña-Mesén, 1998), and alligators (Savage, 2002). Te presence of Eustrongylides sp. inside the egg of a gravid female K. scorpioides can be assumed as a strategy of the parasite to reach easily the fnal host, since eggs deposited by females are more vulnerable to predators, being predated by birds, terrestrial mammals, and reptiles, facilitating the contact of the nematode with the fnal host. In that way, due to the feeding habitat of this chelonian, it is assumed that invertebrates are the intermediate hosts, fsh, and K. scorpioides paratenic hosts, and alligators are the fnal hosts. Kinosternum scorpioides from the Baixada Maranhense are parasitized by the nematodes Contracaecum sp. and Eustrongylides sp. Since K. scorpioides is consumed as food for some families in the State of Maranhão, Brazil, and the presence of both parasites with hygienic-sanitary importance, complementary studies should be designed to allow us to understand the relationship between host parasites and whether they could have zoonotic relevance. Author contributions: CRediT (Contributor Roles Taxonomy)ALS = Alana Lislea de Sousa AMM = Almerinda Macieira Medeiros JBA = Júlia Boáis Almeida SCSDA = Silmara Cristina Silva de Aquino ECFJ = Elias Costa Ferreira Junior DCV = Diego Carvalho Viana VPN = Victor Puicón N. GAMM = Germán Augusto Murrieta Morey Conceptualization : ALS, AMM, JBA, ECF, DCV, ECF, DCV, GAMM Data curation : ALS, GAMM Formal Analysis : ALS, GAMM Funding acquisition : AMM, JBA, SCSDA, GAMM Investigation : SCSDA, ECF, DCV, VPN, GAMM Methodology : ALS, AMM, JBA, SCSDA, ECF, DCV, VPN, GAMM Project administration : ALS, AMM, JBA, GAMM Resources : AMM, JBA, ECF, DCV, GAMM Software : SCSDA, GAMM Supervision : AMM, JBA, GAMM Va lidation : ALS, AMM, JBA, SCSDA, ECF, DCV, GAMM Visualization : ALS, AMM, JBA, SCSDA, ECF, DCV, VPN, GAMM Writing – original draft : ALS, AMM, JBA, SCSDA, ECF, DCV, VPN, GAMM Writing – review & editing : ALS, AMM, JBA, SCSDA, ECF, DCV, VPN, GAMM ACKNOWLEDGMENTS We wish to thank the Maranhão State Foundation for Research Development (FAPEMA) for the fellowship granted to SCSDA (002832/2023) and to GAMM (000448/2023).
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