79 Zoonotic infections in soil of recreational areas Neotropical Helminthology, Vol. 18, Nº1, jan - jun 2024 Neotropical Helminthology Neotropical Helminthology, 2024, vol. 18 (1), 79-92 ISSN Versión Impresa 2218-6425 ISSN Versión Electrónica 1995-1403 DOI: https://dx.doi.org/10.62429/rnh2024 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 Brazil is the country with the highest diversity and description rate of amphibian species in the world. T e genus Rhinella has 100 species of small, medium and large animals, insectivorous, terrestrial or semi-aquatic and nocturnal. Rhinella diptycha Cope, 1862 and Rhinella granulosa Spix,1824 are widely distributed throughout the Brazilian Northeast. Despite their diversity and wide distribution, infectious diseases caused by helminths have caused a reduction in the populations of these animals. In this study, we determined the parasitic fauna of R. diptycha and R. granulosa recorded in the states of Piauí and Maranhão, Northeast Brazil. We collected 60 specimens, 30 of R. diptycha and 30 of R. granulosa , of which 78% of the total (n = 47 individuals) were infected with helminths. T e helminths found were Aplectana membranosa ORIGINAL ARTICLE / ARTÍCULO ORIGINAL HELMINTHS INFECTING RHINELLA DIPTYCHA COPE, 1862 AND RHINELLA GRANULOSA SPIX,1824 FROM NORTHEASTERN BRAZILHELMINTOS QUE INFECTAN A RHINELLA DIPTYCHA COPE, 1862 Y RHINELLA GRANULOSA SPIX,1824 DEL NORESTE DE BRASILHELMINTOS INFECTANDO RHINELLA DIPTYCHA COPE, 1862 E RHINELLA GRANULOSA SPIX,1824 DO NORDESTE DO BRASIL Leonardo Fernando da Silva Sousa 1 ; Sarah de Moura Pires 1 ; Tayná Rafaelle Coêlho de Carvalho 1 ; João Pedro de Sousa Rodrigues 1 ; Érica Vitória dos Santos Lima 1 ; Mariluce Gonçalves Fonseca 2 ; Ronildo Alves Benício 2 & Simone Mousinho Freire 1, * 1 Departamento de Biologia, Laboratório de Zoologia e Biologia Parasitária, Universidade Estadual do Piauí, Teresina, Piauí, Brasil. 2 Departamento de Biologia, Laboratório de Herpetologia e Parasitologia de Animais Silvestres, Universidade Federal do Piauí, Picos, Piauí, Brasil.* Corresponding author: simonemousinho@ccn.uespi.brLeonardo Fernando da Silva Sousa: https://orcid.org/0000-0002-3427-6174Sarah de Moura Pires: https://orcid.org/0000-0002-3086-9685Tayná Rafaelle Coêlho de Carvalho: https://orcid.org/0009-0005-3238-7291João Pedro de Sousa Rodrigues: https://orcid.org/0009-0006-3880-4874Érica Vitória dos Santos Lima: https://orcid.org/0009-0005-1085-0572Mariluce Gonçalves Fonseca: https://orcid.org/0000-0003-2135-7204Ronildo Alves Benício: https://orcid.org/0000-0002-7928-2172Simone Mousinho Freire: https://orcid.org/0000-0001-6417-3144
80 da Silva Sousa et al. Neotropical Helminthology, Vol. 18, Nº1, jan - jun 2024 Schneider, 1866, Cosmocerca sp. Diesing, 1861, Rhabdias sp Stiles & Hassal, 1905, Oswaldocruzia sp. Travassos, 1917, Physaloptera sp. Rudolphi, 1819, and Cylindrotaenia americana Jewell, 1916. Cosmocerca sp. represents the frst record of this taxon parasitizing R. diptycha . Our fndings contribute to expanding knowledge about the diversity of helminths parasitizing bufonids in the Northeast region of Brazil. More studies are still needed to understand the mechanisms associated with this parasite-host relationship. Keywords: Anurans – Bufonidae – Caatinga – Diversity – New record – Parasites RESUMEN Brasil es el país con mayor diversidad y tasa de descripción de especies de anfbios en el mundo. El género Rhinella cuenta con 100 especies de animales pequeños, medianos y grandes, insectívoros, terrestres o semiacuáticos y nocturnos. Rhinella diptycha Cope, 1862 y Rhinella granulosa Spix,1824 están ampliamente distribuidas por todo el Nordeste brasileño. A pesar de su diversidad y amplia distribución, las enfermedades infecciosas provocadas por helmintos han ocasionado una reducción de las poblaciones de estos animales. En este estudio, determinamos la fauna parasitaria de R. diptycha y R. granulosa registrada en los estados de Piauí y Maranhão, noreste de Brasil. Se recolectaron 60 ejemplares, 30 de R. diptycha y 30 de R. granulosa , de los cuales el 78% del total (n = 47 individuos) estaban infectados con helmintos. Los helmintos encontrados fueron Aplectana membranosa Schneider, 1866, Cosmocerca sp. Diesing,1861, Rhabdias sp. Stiles & Hassal,1905, Oswaldocruzia sp. Travassos, 1917, Physaloptera sp. Rudolphi, 1819, y Cylindrotaenia americana Jewell, 1916. Cosmocerca sp. representan el primer registro de este taxón parasitando a R. diptycha . Nuestros hallazgos contribuyen a ampliar el conocimiento sobre la diversidad de helmintos que parasitan a los bufónidos en la región Nordeste de Brasil. Aún se necesitan más estudios para comprender los mecanismos asociados con esta relación parásito-huésped. Palabras clave: Anuros – Bufonidae – Caatinga – Diversidad – Nuevo registro – Parásitos RESUMO O Brasil é o país com a maior diversidade e taxa de descrição de espécies de anfíbios do mundo. O gênero Rhinella possui 100 espécies de animais de pequeno, médio e grande porte, insetívoros, terrestres ou semiaquáticos e noturnos. Rhinella diptycha Cope, 1862 e Rhinella granulosa Spix, 1824 são amplamente distribuídas em todo o Nordeste brasileiro. Apesar da diversidade e ampla distribuição, as doenças infecciosas causadas por helmintos têm provocado uma redução nas populações destes animais. Neste estudo, nós determinamos a fauna parasitária de R. diptycha e R. granulosa registrados nos estados do Piauí e Maranhão, Nordeste do Brasil. Coletamos 60 exemplares, sendo 30 de R. diptycha e 30 de R . granulosa , dos quais 78% do total (n = 47 indivíduos) estavam infectados por helmintos. Os helmintos encontrados foram Aplectana membranosa Schneider, 1866, Cosmocerca sp. Diesing,1861, Rhabdias sp. Stiles & Hassal,1905, Oswaldocruzia sp. Travassos, 1917, Physaloptera sp. Rudolphi, 1819 e Cylindrotaenia americana Jewell, 1916. Cosmocerca sp. representa o primeiro registro deste taxon parasitando R. diptycha . Nossos achados contribuem para ampliar o conhecimento sobre a diversidade de helmintos parasitando bufonídeos na região Nordeste do Brasil. Mais estudos ainda são necessários para compreender os mecanismos associados nesta relação parasita-hospedeiro. Palavras-chave: Anuros – Bufonidae – Caatinga – Diversidade – Novo registro – Parasitos
81 Helminths infecting bufonids in Brazil Neotropical Helminthology, Vol. 18, Nº1, jan - jun 2024 INTRODUCTION Currently, around 8,722 species of amphibians are recognized in the world, belonging to three orders: Gymnophiona, with 222 species; Caudata, with 822 and Anura, with 7,678 species (Frost, 2024). Te most diverse order - Anura, comprises animals that have a life stage with larval morphology (tadpoles) inhabiting terrestrial environments and an adult stage in which they can be found in aquatic, terrestrial and arboreal environments. Tey are cosmopolitan, except for extreme latitudes in the North, Antarctica and most of oceanic islands (Stuart et al ., 2008; Jenkins et al ., 2013; Frost, 2024).Brazil is the country with the highest occurrence and description rate of amphibian species worldwide, with 1,188 species, the majority of which belong to the order Anura (Segalla et al ., 2021). In the country there are 21 families of anurans distributed in more than 100 genera. Among these families, one of the most representative is the Bufonidae family, with 646 described species, distributed in 53 genera, with one of the highlights being the genus Rhinella Fitzinger, 1826 (Frost, 2024).Te genus Rhinella has 100 species, represented by small, medium and large animals, insectivorous, terrestrial or semiaquatic, and nocturnal activity. Among these species, Rhinella diptycha Cope, 1862 and Rhinella granulosa Spix, 1824 are widely distributed throughout the Brazilian Northeast, they are large and small animals, respectively, and are nocturnal (Juncá, 2001; Rodrigues, 2003; Frost, 2024). Due to their diversity and wide distribution, occurrence in various types of habitats and sensitive skin, these species often sufer from various diseases and parasitism.Helminth fauna plays a crucial role in the ecosystem as it afects the ecology, evolution, growth and control of host populations and, consequently, all biodiversity (Marcogliese, 2023). Te study of parasitic fauna is extremely signifcant, contributing to the understanding of biodiversity and the parasite-host relationship (Toledo et al ., 2017). Furthermore, studies on anurans are essential, as they play an important role as environmental bioindicators (Prestes & Vincenci, 2019). Studies that describe parasitic species in anurans are essential for research in health and environmental conservation. Te helminth group is the most common of all invertebrates that parasitize amphibians. Among them, the most numerous are nematodes that are usually found in the digestive tract, lungs and blood vessels (Vieira et al ., 2021). In the Northeast, several studies have addressed the diversity of helminths parasitizing anurans (e.g., Lins et al ., 2017; Teles et al ., 2018; Oliveira et al ., 2019; Madelaire et al ., 2020; Vieira et al ., 2021; Machado et al ., 2022; Sampaio et al ., 2022; Oliveira et al ., 2023; Batista-Oliveira et al ., 2024).Several species of helminths have also been described parasitizing bufonids (e.g., Campião et al ., 2014; Teles et al ., 2018; Benício et al ., 2022). However, there is only one article in the literature on helminths parasitizing R. diptycha for the state of Piauí (Benício et al ., 2022) and, to date, no information on helminths parasitizing R. diptycha and R. granulosa for the state of Maranhão. Tus, the objective of this study was to determine the parasitic fauna of these species found in the states of Piauí and Maranhão, Northeast Brazil. MATERIAL AND METHODS Study area and collectionsTe study was carried out in the municipalities of Picos, in the state of Piauí, and in Timon, in the state of Maranhão, both located in the northeast of Brazil. Te location chosen for collections in the municipality of Picos has approximately 1,000 m 2 with an area of tree and shrub vegetation with an ephemeral stream and temporary water puddles. Te area is located on the outskirts of the Universidade Federal do Piauí, Picos campus (Fig. 1). Te municipality is within the Caatinga ecoregion, a semi-arid climate, with an average annual temperature of 27.2°C and an average annual precipitation of 684 millimeters, with greater precipitation between December and April (Oliveira-Filho et al ., 2021; Silva et al ., 2022). Te municipality of Timon, in turn, is located in the eastern region of the state (Silva, 2019). It has a transition area between the Caatinga and Cerrado biomes, a hot climate all year round, with an annual temperature of 27°C and an average annual precipitation of around 790 millimeters (Silva & Coelho, 2018). Te location chosen for collections in the municipality of Timon has approximately 1,000 m 2 composed of a vegetation area with dense forest formations with stretches of riparian vegetation with a stream and peridomiciliary area (Fig. 2). Te anurans were collected between February 2021 and March 2022 during the rainy season, at night between 6pm and 10pm, using the active search method, inspecting all available microhabitats (Crump & Scott Jr., 1994). Specimens of R. diptycha were collected at a site located in the rural area of Timon, Maranhão (5°01’39’’ S, 43°00’10’’ W), and the specimens of R. granulosa
82 da Silva Sousa et al. Neotropical Helminthology, Vol. 18, Nº1, jan - jun 2024 were captured surroundings the Universidade Federal do Piauí, Picos campus, Piauí, Northeast Brazil (7°04’54’’ S, 41°26’05’’ W).After collection, the anurans were transported alive to the Laboratório de Zoologia e Biologia Parasitária (ZOOBP) of the Universidade Estadual do Piauí (UESPI), on the Poeta Torquato Neto campus, in the city of Teresina, Piauí, Northeast Brazil for identifcation in accordance with specialized literature (Roberto et al ., 2013; Benício & Fonseca, 2014; Benício et al ., 2014; Benício et al ., 2021). Figure 1. Collection area for Rhinella granulosa specimens, Universidade Federal do Piauí, municipality of Picos, state of Piauí, Northeast Brazil. Figure 2. Collection area for Rhinella diptycha specimens, in the municipality of Timon, state of Maranhão, Northeast Brazil.
83 Helminths infecting bufonids in Brazil Neotropical Helminthology, Vol. 18, Nº1, jan - jun 2024 Laboratory proceduresAfter identifying the anurans, the individuals were measured with the aid of a caliper to obtain the SVL (snout-vent length), weighed on a digital scale, and were subsequently euthanized using the anesthetic compound 5% lidocaine when the anurans were considered small (weighing a maximum of 50 g) and 2% injectable lidocaine when the animal was large (when the animal weighed more than 50 g). Te ointment was applied to the entire ventral region of the individuals. After euthanasia, the organs were separated individually in petri dishes containing 0.9% NaCl saline and taken for analysis under a stereoscopic microscope in search of endoparasites. Te anurans were fxed in 10% formalin solution. After 24h in the fxative, the anurans were transferred to a 70% ethyl alcohol solution. Te collected specimens were deposited at the Laboratório de Zoologia e Biologia Parasitária (ZOOBP) of the Universidade Estadual do Piauí (UESPI), Poeta Torquato Neto campus, Teresina, Piauí. Te helminths are deposited in the Helminthological Collection of the Laboratório de Zoologia e Biologia Parasitária (ZOOBP) of the Universidade Estadual do Piauí (UESPI), Poeta Torquato Neto campus, Teresina, Piauí, under registration numbers CHZOOBP54, CHZOOBP55, CHZOOBP56, CHZOOBP57, CHZOOBP58, CHZOOBP59, CHZOOBP60.All helminths found were fxed in 70% hot ethyl alcohol, according to the protocol by Amato et al . (1991). For identifcation, the cestodes were stained in acetic carmine solution and then clarifed with beech creosote, the nematodes were clarifed using Amann’s Lactophenol solution (Andrade, 2000). Individually, the helminths were observed and measured using Olympus®. CX21 optical microscope, and photographed using the camera of a cell phone with the aid of a microscope adapter.Te taxonomic characters of the parasites were identifed using the following keys: Anderson et al . (2009), Vicente et al . (1990), and articles with specifc descriptions of species of the genera found. For statistical analysis, the defnitions of frequency (number of hosts infected by a given species of parasite), average intensity of infection (total quantity of a specifc parasite on infected hosts) and average abundance of parasites (total quantity of specifc parasite on the total number of anurans sampled) were analyzed according to Bush et al . (1997).Ethical proceduresTis project was submitted to the Sistema de Autorização e informação em Biodiversidade − SISBIO and to the Ethics Committee on the Use of Animals of the State University of Piauí − CEUA, having been approved through opinion no. 74248-1, 54745 (SISBIO) and 0509/ 2020, 006022/2021-93 (CEUA/UESPI). It was also registered in the National System of Genetic Heritage and Associated Traditional Knowledge − SisGen with number A1A6651. RESULTS We collected a total of 60 specimens, of which 30 individuals are R. diptycha , seven males and 14 females; and 30 are R. granulosa , eight males and 11 females. It was not possible to sex 20 anurans, as they were juveniles. Te weight of the animals varied between 38.7 and 758.5 g for R. diptycha specimens; and 5.62 to 21.6 g for specimens of R. granulosa . Te snout-vent length of the animals varied between 6.7 and 20 cm for specimens of R. diptycha ; and 3.5 to 5.8 cm for specimens of R. granulosa . Regarding positivity for parasites, 47 individuals were infected with helminths, around 78.3% of the total sampled, being 40% (n = 24) for R. granulosa and 38.3% (n = 23) for R. diptycha .We found seven helminths: Aplectana membranosa Schneider, 1866, Cylindrotaenia americana Jewell, 1916, Cosmocerca sp. Travassos, 1925, Cosmocercidae gen sp. Travassos, 1925, Oswaldocruzia sp. Travassos, 1917, Physaloptera sp. Rudolphi, 1819, and Rhabdias sp. Stiles & Hassall, 1905 (Fig. 3, 4, 5 and 6). Since A. membranosa , Rhabdias sp. and Oswaldocruzia sp. had the highest parasitic rates (Table 1).Te quantity and distribution of parasites varied in the two anuran species. For R. diptycha (n = 462): Oswaldocruzia sp. (n = 278), A. membranosa (n = 109), Rhabdias sp. (n = 52), Cosmocercidae gen sp. (n = 17), Physaloptera sp. (n = 5) and Cosmocerca sp. (n = 1); for R. granulosa (n = 364): A. membranosa (n = 204), Cosmocercidae gen sp. (n = 69), Oswaldocruzia sp. (n = 38), Rhabdias sp. (n = 35), Physaloptera sp. (n = 13) and C. americana (n = 5). Overall, the most abundant species were Oswaldocruzia sp. and A. membranosa , respectively (Figure 7).
84 da Silva Sousa et al. Neotropical Helminthology, Vol. 18, Nº1, jan - jun 2024 Table 1. Hosts examined and their associated parasites. P% = Prevalence; M.A = Mean Abundance; M.I = Mean Intensity; S.I = Site of Infection. S = Stomach; SI = Small Intestine; LI = Large Intestine; LIV = Liver; LUN = Lung. HospedeirosParasitosPM.AI.MS.I Rhinella diptychaOswaldocruzia sp.60%9,215,4S / SI / LI Rhabdias sp.36,6%1,74,7LUNCosmocercidae gen. sp.16,6%0,53,4SI/LI Aplectana membranosa 6,6%3,654,5SI/LI Physaloptera sp.6,6%0,12,5LIV Cosmocerca sp.3,3%0,01LI Rhinella granulosa Aplectana membranosa 33,3%6,820,4SI / LI Rhabdias sp.23,3%1,15LUN Oswaldocruzia sp.16,6%1,27,6SICosmocercidae gen. sp.16,6%2,313,8SI / LI Physaloptera sp.10%0,44,3S / LIV Cylindrotaenia americana 6,6%0,12,5SI Figure 3. Helminths found in Rhinella diptycha and R. granulosa , seen by optical microscopy (10x objective). A) Anterior region of female Rhabdias sp. B) Median part of the female Rhabdias sp., showing the vulva in detail (arrow). C) Anterior region of Cosmocercidae larvae. D) Anterior region of the larva of Physaloptera sp., showing a cephalic collar (arrow).
85 Helminths infecting bufonids in Brazil Neotropical Helminthology, Vol. 18, Nº1, jan - jun 2024 Figure 4. Morphology of the Aplectana membranosa parasite found in Rhinella diptycha and R. granulosa , seen by optical microscopy (A and D – 40x objective; B and C – 10x objective). A) Anterior region of the female, showing the pre-bulbar excretory pore (arrow 1) and bulb posterior to the esophagus (arrow 2) . B) Anterior region showing intestine immediately in continuation of the medulla oblongata, with the anterior part wider (arrow). C) Vulva just below the middle of the body (arrow). D) Posterior region of the male, showing in detail a pair of approximately equal spicules (arrow).
86 da Silva Sousa et al. Neotropical Helminthology, Vol. 18, Nº1, jan - jun 2024 Figure 5. Morphology of the parasite Oswaldocruzia sp. found in Rhinella diptycha and R. granulosa , seen by optical microscopy (A and C – 40x objective; B – 4x objective and D – 10x objective). A) Anterior region of the female, showing cephalic dilation in detail (arrow). B) General view of the male. C) Posterior region of the male, showing a pair of spicules (arrow 1) and the copulatory sac (arrow 2). D) Vulva in the median part of the female’s body (arrow).
87 Helminths infecting bufonids in Brazil Neotropical Helminthology, Vol. 18, Nº1, jan - jun 2024 Figure 6. Helminths found in Rhinella diptycha and R. granulosa , seen by optical microscopy (A – 4x objective; B – 40x objective; C, D and E – 10x objective). A) General view of the male Cosmocerca sp. B) Posterior region of male Cosmocerca sp. with plectanas, (arrow 1) and rudimentary spicules (arrow 2). C) Anterior region, D) Mature proglottids, E) Pre-pregnancy proglottids ( C. americana ).
88 da Silva Sousa et al. Neotropical Helminthology, Vol. 18, Nº1, jan - jun 2024 Figure 7. Total parasite load in Rhinella diptycha and R. granulosa , from the municipalities of Picos and Timon, states of Piauí and Maranhão, respectively, Northeast Brazil. DISCUSSION Nematodes from the Cosmocercidae, Rhabdiasidae and Strongyloididae families have been recorded parasitizing several species of amphibians in South America, being the most common parasites found in amphibians on this continent (Campião et al ., 2014). Among the anuran species commonly found in anthropic environments in Brazil, Rhinella diptycha and R. granulosa are ecologically considered generalists and opportunists, having a diet composed mainly of arthropods (Pereira-Junior et al ., 2013; Barbosa et al ., 2018). Tus, the exclusive infection by nematodes in these species may be related to the life cycle of these hosts and their foraging strategies, that is, the time spent in water or on land. In terrestrial habitats, most nematodes infect anurans by penetrating the skin (e.g., Rhabdias spp. and some cosmocercides) or by ingesting eggs (Anderson, 2000; Pinhão et al ., 2009; Teles et al ., 2018; Amorim et al ., 2019). Te Cosmocercidae family is made up of viviparous and oviparous nematodes (Felix-Nascimento et al ., 2020). Tey are frequently recorded infecting reptiles and amphibians and have been found parasitizing frogs of the genus Rhinella (Campião et al ., 2014; Teles et al ., 2018). Despite this, in this study we found only one individual of Cosmocerca sp. parasitizing R. granulosa . Tis, however, represents the frst record of this parasite for the host Rhinella granulosa in the state of Maranhão, northeastern Brazil. Rhabdias spp. are frequently seen infecting the lungs of anurans (Teles et al ., 2018). Infections in anurans can occur via direct penetration of the host’s skin, however, the life cycle of these parasites varies between a free-living and a parasitic phase, with only females acting as parasites (Anderson, 2000; Amorim et al ., 2019). Tis explains the fact that we found only females parasitizing the two anuran species in this study. Some species of the genus Rhinella (e.g., R. schineideri , R. crucifer , R. icterica , R. marina , R. ornata ) have already been reported to be parasitized by Rhabdias sp. (Graça et al ., 2017), including R. diptycha (Benício et al ., 2022).Te genus Physaloptera has nematodes with a heteroxenous life cycle, that is, they are parasites that require one or more intermediate hosts. Specimens of the genus Physaloptera are frequently found in the stomachs of vertebrates such as reptiles, mammals, birds, fsh and amphibians, including frogs of the family Bufonidae, where this parasite is commonly seen in the larval stage (Anderson, 2000; Campião et al ., 2014; Teles et al ., 2018; Amorim et al ., 2019). In this study, we found larvae of Physaloptera
89 Helminths infecting bufonids in Brazil Neotropical Helminthology, Vol. 18, Nº1, jan - jun 2024 sp. encysted in the liver membrane of both frog species and in the stomach of R. granulosa . Tis infection can be caused by the ingestion of larvae in the intermediate host (insect) (Vieira et al ., 2021). Aplectana membranosa is known to only infect anurans. Tis parasite occurs in the large intestine and rectum of its hosts (Teles et al ., 2018). Vieira et al . (2021) found a high prevalence of A. membranosa parasitizing the small intestine of Leptodactylus macrosternum . We found specimens of this nematode parasitizing both species of frogs, both in the small and large intestines. Tis fact can be justifed by the high parasitic load found in animals, causing helminths to migrate to other sites of infection. Nematodes of the genus Oswaldocruzia are known to infect anurans (Teles et al ., 2018). During the reproductive period of these anurans there is a greater intensity of infection by Oswaldocruzia sp. Tis parasite has a direct life cycle with active larvae penetrating the host’s skin. It is possible that during the rainy months, when the soil is very moist, these larvae have lower desiccation rates, higher survival rates and, consequently, greater ease in infecting their hosts (Madelaire et al ., 2012). Oswaldocruzia sp. has already been recorded parasitizing R. diptycha and R. granulosa (Teles et al ., 2018; Benício et al ., 2022). Cylindrotaenia americana belongs to the Nematotaennidae family and was found in our study parasitizing R. granulosa . Tis family is made up of parasites that infect the small intestine of amphibians and reptiles and are transmitted by ingestion of pregnant proglottids without the need for an intermediate host. Self-infection is common, and it is possible to fnd hosts with a large parasite load (Melo et al ., 2011), although this was not observed in our study. C. americana has a direct life cycle and is widely reported to infect anurans from the Bufonidae, Hylidae and Leptodactylidae families (Martins et al ., 2018), including R. diptycha and R. granulosa (Madelaire et al ., 2020; Benício et al ., 2022). Cylindrotaenia sp. has already been recorded parasitizing species of the genus Rhinella (e.g., R. fernandezae , R. icterica , R. schneideri ) (Justo et al ., 2017; Martins, 2018), including R. diptycha (Benício et al ., 2022). However, this is the frst record of Cylindrotaenia sp. parasitizing Rhinella granulosa in the state of Maranhão, northeastern Brazil.Te richness and species composition of parasites recorded in this study varied little between diferent populations of the two species (Teles et al ., 2018; Amorim et al ., 2019; Madelaire et al ., 2020; Neta et al ., 2020; Benício et al ., 2022). For example, for both R. diptycha and R. granulosa , species richness ranged from six to seven. Regarding species composition, there was a large number of shared species, with some taxa occurring in almost all populations studied. Te most commonly shared parasites were: Aplectana membranosa , Cylindrotaenia americana , Physaloptera sp. and Rhabdias sp. Tis is only the second article in the literature on helminths parasitizing R. diptycha for the state of Piauí (i.e., Benício et al ., 2022; this study) and the frst for both species in the state of Maranhão.In this study, the high prevalence of parasitized hosts (~80%) and the high abundance of parasites recorded (n = 826) in just two species draw attention to the health of these anuran populations, possible associated diseases and inherent declines, as well as the environmental quality of the ecosystems where these species were collected (in general, highly anthropized locations). Several studies have demonstrated how anthropogenic activities, pathogens, agrochemicals and changes in the landscape, for example, can enhance the efect of parasitism (such as coinfections and increased diseases) on amphibian populations around the world (e.g., Carrasco et al ., 2021; Herczeg et al ., 2021; Jacinto-Maldonado et al ., 2022; Oliveira et al ., 2024). Tus, studies like this – which map the diversity and distribution of parasites in amphibians, can help us better understand the complex parasite-host relationship, as well as propose more efective measures for the conservation of amphibian species and local ecosystems.Our fndings contribute to expanding knowledge about the parasitic fauna of bufonids in the Northeast region of Brazil. We found a high prevalence (78%) and abundance (n = 826) of helminths parasitizing the two frog species ( R. diptycha and R. granulosa ) in the states of Piauí and Maranhão, including new records of helminths. However, more studies are needed to understand the mechanisms associated with this parasite-host relationship, since these anurans are considered to be reservoirs of a great diversity of helminths, and their relationships are not yet fully understood. ACKNOWLEDGEMENTS Te authors thank Conselho Nacional de Desenvolvimento Científco e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado do Piauí (FAPEPI) for providing a research fellowship to (R.A.B., 301239/2022-3) and State University of Piaui by research fellowship
90 da Silva Sousa et al. Neotropical Helminthology, Vol. 18, Nº1, jan - jun 2024 PIBIC-CNPq to (L.F.S.S, 2020-1). To Instituto Chico Mendes de Conservação da Biodiversidade – ICMBio for collecting permits (SISBIO 54745). Author contributions: CRediT (Contributor Roles Taxonomy)LFSS = Leonardo Fernando da Silva Sousa SMP = Sarah de Moura Pires TRCC = Tayná Rafaelle Co ê lho de Carvalho JPSR = João Pedro de Sousa Rodrigues EVSL = Érica Vitória dos Santos Lima MGF = Mariluce Gonçalves Fonseca RAB = Ronildo Alves Benício SMF = Simone Mousinho Freire Conceptualization: LFSS, RAB, SMF, SMP, TRCC, JPSR, EVSL, MGF Data curation: LFSS, SMP, TRCC, JPSR, EVSL Formal Analysis: LFSS, RAB, SMF Funding acquisition: SMF Investigation: LFSS, SMP, TRCC, JPSR, EVSL Methodology: LFSS, RAB, SMF Project administration: LFSS Resources : LFSS, RAB, SMF, SMP, TRCC, JPSR, EVSL, MGF Software : LFSS, RAB, SMF, SMP, TRCC, JPSR, EVSL, MGF Supervision: SMF Validation : LFSS, RAB, SMF, SMP, TRCC, JPSR, EVSL, MGF Visualization : LFSS, RAB, SMF, SMP, TRCC, JPSR, EVSL, MGF Writing – original draft: LFSS, RAB, MGF, SMF Writing – review & editing: RAB, MGF, SMF BIBLIOGRAPHIC REFERENCES Amato, J.F.R., Boeger, W.A., & Amato, S.B. (1991). Protocolos para laboratório–coleta e procedimento de parasitos do pescado . Imprensa Universitária.Amorim, D.M., Oliveira, R.H., Dyna, C.S., Sousa, D.M., Lima, L.S., Pinto, L.C., & Ávila, R.W. (2019). Nemátodos parásitos de Rhinella jimi (Stevaux, 2002) (Anura: Bufonidae) en áreas de caatinga, nordeste de Brasil. Neotropical Helminthology, 13, 265-271.Anderson, R.C. (2000). Nematode parasites of vertebrates: their development and transmission . Cabi Digital Library. Anderson, R.C., Chabaud, A.G., & Willmott, S. (2009). Keys to the nematode parasites of vertebrates: archival volume . Cabi Digital Library.Andrade, C.M. (2000). Meios e soluções comumente empregados em laboratórios . Editora Universidade Federal Rural do Rio de Janeiro.Barbosa, V.N., Amaral, J.M.S., Alcântara, E.N.P., & Santos, E.M. (2018). Herpetofauna de uma área de Caatinga em Taquaritinga do Norte, Agreste de Pernambuco, Brasil. Cuadernos de Herpetología, 32, 109-115. Batista-Oliveira, D., Castro, K.A., De Oliveira, C.R., Cassiano-Lima, D., & Ávila, R.W. (2024). Nematodes Infecting the Shield Frog Adelophryne baturitensis in Rainforest Enclaves of Brazilian Semiarid. Caldasia, 46, 1-23.Benício, R.A., Dos Santos, R.S., Freire, S.M., Ávila, R.W., Silva, R.J., & Fonseca, M.G. (2022). Diversity of helminth parasites in amphibians from northeastern Brazil. Biologia, 77, 2571-2579.Benício, R.A, Ávila, R.W., & Fonseca, M.G. (2021). Guia ilustrativo dos anfíbios e répteis de Barras-Piauí . EDUFPI. Benício, R.A., & Fonseca, M.G. (2014). Guia ilustrado de anfíbios e répteis de Picos-Piauí . EDUFPI.Benício, R.A., Silva, G.R., & Fonseca, M.G. (2014). Comunidade de anuros em uma área de ecótono no nordeste do Brasil. Boletim do Museu Paraense Emílio Goeldi-Ciências Naturais, 9, 511-517.Bush, A.O., Laferty, K.D., Lotz, J.M., & Shostak, A.W. (1997). Parasitology meets ecology on its own terms: Margolis et al. Te Journal of Parasitology, 83, 575-583.Campião, K.M., Morais, D.H., Dias, O.T., Aguiar, A., Toledo, G., Tavares, L.E.R., & Silva, R.J. (2014). Checklist of helminth parasites of amphibians from South America. Zootaxa, 3843, 1-93.
91 Helminths infecting bufonids in Brazil Neotropical Helminthology, Vol. 18, Nº1, jan - jun 2024 Carrasco, G. H., de Souza, M. B., & de Souza Santos, L. R. (2021). Efect of multiple stressors and population decline of frogs. Environmental Science and Pollution Research, 28, 59519–59527.Crump, M.L., & Scott Jr., N.J. (1994). Visual encounter surveys . In: Heyer, W.R., Donnely, M.A., Mcdiarmid, R.W., Hayek, L.A., Foster, M. Standard methods for amphibians . Smithsonian Institution Press.Felix-Nascimento, G., Vieira, F.M., Muniz-Pereira, L.C., De Moura, G.J.B., Ribeiro, L.B. & Oliveira, J.B. (2020). Two new species of Cosmocercidae (Nematoda: Cosmocercoidea) of Leptodactylus macrosternum Miranda-Ribeiro (Anura: Leptodactylidae) from Caatinga Biome, Brazil. Zootaxa, 4877, 274-290.Frost, D.R. (2024). Amphibian Species of the World: An Online Reference . Version 6.2. Electronic Database. American Museum of Natural History. https://amphibiansoftheworld.amnh.org/index.phpGraça, R.J., Oda, F.H., Lima, F.S., Batista, V.G., Gambale, P., & Takemoto, R.M. (2017). Metazoan endoparasites of 18 anuran species from the mesophytic semideciduous Atlantic Forest in southern Brazil. Journal of Natural History, 51, 705-729.Herczeg, D., Ujszegi, J., Kásler, A., Holly, D., & Hettyey, A. (2021). Host–multiparasite interactions in amphibians: a review. Parasites & Vectors, 14, 296. Jacinto-Maldonado, M., García-Peña, G. E., Lesbarrères, D., Meza-Figueroa, D., Robles-Morúa, A., Salgado-Maldonado, G., & Suzán, G. (2022). Urbanization impacts parasite diversity in the cane toad Rhinella horribilis (Anura: Bufonidae). Global Ecology and Conservation, 38, e02275.Jenkins, C.N., Pimm, S.L., & Joppa, L.N. (2013). Global Patterns of Terrestrial Vertebrate Diversity and Conservation. PNAS, 110, E2602-E2610. Juncá, F.A. (2001). Declínio mundial das populações de anfíbios. Sitientibus, Série Ciências Biológicas, 1, 84-87.Justo, M.C.N., Fernandes, B.M.M., Knof, M., Cardenas, M.Q., & Cohen, S. (2017). Lista de cestoda do Brasil. Neotropical Helminthology, 11, 187-282.Lins, A.G.S., Aguiar, A., Morais, D.H., Da Silva, L.A.F., Ávila, R.W., & Da Silva, R.J. (2017). Helminth fauna of Leptodactylus syphax (Anura: Leptodactylidae) from Caatinga biome, northeastern Brazil. Revista Brasileira de Parasitologia Veterinária, 26, 74-80.Machado, H.T.S, De Oliveira, S.S., Benício, R.A., Castro, K.A., & Ávila, R.W. (2022). Helminths Infecting Sympatric Congeneric Treefrogs in Northeastern Brazil. Acta Parasitologica, 67, 658-667.Madelaire, C.B., Franceschini, L., Morais, D.H., Gomes, F.R., & Da Silva, R.J. (2020). Helminth parasites of three anuran species during reproduction and drought in the Brazilian Semiarid Caatinga region. Journal of parasitology, 106, 334-340.Madelaire, C.B., Gomes, F.R., & Da Silva, R.J. (2012) Helminth parasites of Hypsiboas prasinus (Anura: Hylidae) from two Atlantic forest fragments, São Paulo State, Brazil. Journal of Parasitology, 98, 560-564. Marcogliese, D.J. (2023). Major drivers of biodiversity loss and their impacts on helminth parasite populations and communities. Journal of Helminthology, 97, 1-20. Martins, A.C.J.S. (2018). Padrões de associação e distribuição de helmintos em anuros no Brasil. Tese (Doutorado em Ecologia e Evolução). Universidade do Estado do Rio de Janeiro, Rio de Janeiro.Melo, F.T.V., Giese, E.G , Furtado, A.P., Soares, M.J., Goncalves, E.C., Vallinoto, A.C.R., & Santos, J.N . (2011). Lanfrediella amphicirrus gen. nov. sp. nov. Nematotaeniidae (Cestoda: Cyclophyllidea), a tapeworm parasite of Rhinella marina (Linnaeus, 1758) (Amphibia: Bufonidae). Memórias do Instituto Oswaldo Cruz, 106, 670-677.Neta, A.F.D.S., de Alcantara, E.P., de Oliveira, C.R., de Carvalhoł, E.F.F., Morais, D.H., da Silva, R.J., & Ávila, R.W. (2020). Helminths associated with 15 species of anurans from the Ibiapaba Plateau, northeastern Brazil. Neotropical Helminthology, 14, 207-216.Oliveira, J.B., Felix-Nascimento, G., Berenguer, L.K.A.R., da Silva e Souza, D., Bernal-Valle, S., & de Souza, V.C. (2024). Internal Parasites and Teir Impact on the Health and Conservation of Neotropical Vertebrates . In: Acosta-Jamett, G., & Chaves, A. (eds). Ecology of Wildlife Diseases in the Neotropics . Springer, Cham.
92 da Silva Sousa et al. Neotropical Helminthology, Vol. 18, Nº1, jan - jun 2024 Oliveira, C.R., Gonçalves-Sousa, J.G., De Carvalho, E.F.F., Ávila, R.W., & Borges-Nojosa, D.M. (2023). Efect of altitude and spatial heterogeneity on the host-parasite relationship in anurans from a remnant humid forest in the Brazilian semiarid. Parasitology Research, 122, 2651-2666.Oliveira, C.R., Ávila, R.W., & Morais, D.H. (2019). Helminths associated with three Physalaemus species (Anura: Leptodactylidae) from Caatinga biome, Brazil. Acta Parasitologica, 64, 205-212.Oliveira-Filho, M.C., Nascimento, R.M.S., Silva, A.V., Vieira, F.J., & Vaz, M.A. (2021). Levantamento de espécies de abelhas nativas em fragmentos antropizados de caatinga no semiárido piauiense. Jornal Interdisciplinar de Biociências, 6, 1-6.Pereira-Junior, A.P., Campos, C.E.C., & Araújo, A.S. (2013). Composição e diversidade de anfíbios anuros do campus da Universidade Federal do Amapá. Biota Amazônia, 3, 13-21.Pinhão, R., Wunderlich, A.C., Dos Anjos, L.A., & Da Silva, R.J. (2009). Helminths of toad Rhinella icterica (Bufonidae), from the municipality of Botucatu, São Paulo State, Brazil. Neotropical Helminthology, 3, 35-40.Prestes, R., & Vincenci, K.L. (2019). Bioindicadores como avaliação de impacto ambiental. Brazilian Journal of Animal and Environmental Research, 2, 1473-1493.Roberto, I.J., Ribeiro, S.C., & Loebmann, D. (2013). Amphibians of the state of Piauí, Northeastern Brazil: a preliminary assessment. Biota Neotropica, 13, 322-330.Rodrigues, M.T. (2003). Herpetofauna da caatinga . In: Leal, I.R., Tabarelli, M., & Silva, J.M.C. Ecologia e conservação da Caatinga. Editora Universitária, Universidade Federal de Pernambuco.Sampaio, N.K.S., Teixeira, A.A.M., Do Nascimento, J.M., Ribeiro, S.C., Almeida, W.O., & Brito, S.V. (2022). Endoparasite community structure of an anuran assemblage in the Caatinga, Northeastern Neotropical Region. Journal of Helminthology, 96, 1-8.Segalla, M., Berneck, B.V.M., Canedo, C., Caramaschi, C., Cruz, C.A.G., Garcia, P.C.A., Grant, T., Haddad, C., Lourenço, A.C.C., Mângia, S., Mott, T., Nascimento, L.B., Toledo, L.F., Werneck, F.P., & Langone, J.A. (2021). List of Brazilian amphibians. Herpetologia Brasileira, 10, 121-216.Silva, T.R. (2019). Caracterização e análise do sistema de abastecimento de água do perímetro urbano do município de Timon, MA, Brasil. Acta Geografca, 13, 1-19.Silva, T.R., & Coelho, C.J.C. (2018). Análise de aspectos hidrológicos das sub-bacias do parnaíba e itapecuru em Timon, MA. Revista Geonorte, 9, 95-110.Silva, B.M.B., Vieira, L.L.S., Vieira, F.J., Pacheco, A.C.L., & Abreu, M.C. (2022). Diversidade de frutos em uma área de caatinga no município de Picos-PI. Pesquisas, Botânica, 76, 219-230.Stuart, S.N., Hofmann, M., Chanson, J.S., Cox, N.A., Berridge, R.J., Ramani, P., & Young, B.E. (2008). Treatened Amphibians of the World. Lynx Edicions, IUCN, and Conservation International.Teles, D.A., Brito, S., De Araújo Filho, S.A., Ribeiro, S.C., Teixeira, A.A.M., Mesquita, D., & Almeida, W. (2018). Nematodes of the Rhinella granulosa Spix, 1824 (Anura: Bufonidae) from the semiarid northeastern Caatinga Region of Brazil. Comparative parasitology, 85, 208-211. Toledo, G.M., Fonseca, M.G., Iannacone, J.A., Callirgos, J.M.C., Vidaurre, C.U.M., & Silva, R.J. (2017). Helminth parasites of Rhinella marina (Linnaeus, 1758) (Anura: Bufonidae) from Tarapoto, Peru. Te Biologist (Lima), 15, 459-468. Vicente, J.J., Rodrigues, H.O., Gomes, D.R., & Pinto, R.M. (1990). Nematoides do Brasil 2ª parte: nematoides de anfíbios. Revista Brasileira de Zoologia, 7, 549-626.Vieira, E.F., Lima, V.D., Félix, A.J.S., Costa, M.A.T, Pires, S.M., Santos, B.M.R., Andrade, E.B.A., & Freire, S.M. (2021). Fauna parasitária de Leptodactylus macrosternum (Anura: Leptodactylidae) no município de União–PI. Brazilian Journal of Development, 7, 49679-49692.Received March 14, 2024.Accepted May 21, 2024.