127 Gastrointestinal Parasitism in Rescued Wild Birds Neotropical Helminthology (Lima). Vol. 18, Nº2, jul - dec 2024 Neotropical Helminthology Neotropical Helminthology, 2024, vol. 18 (2), 127-142 ORIGINAL ARTICLE / ARTÍCULO ORIGINAL REDESCRIPTION OF PHYSALOPTERA LIOPHIS VICENTE & SANTOS, 1974 (NEMATODA: PHYSALOPTERIDAE) A PARASITE OF ANURA FROM PAMPA BIOMA, BRAZILREDESCRIPCIÓN DE PHYSALOPTERA LIOPHIS VICENTE & SANTOS, 1974 (NEMATODA: PHYSALOPTERIDAE) UN PARÁSITO DE ANURA DEL BIOMA PAMPA, BRASIL Marco Antonio Afonso Coimbra 1 , Carolina Silveira Mascarenhas 2 * & Gertrud Müller 3 ISSN Versión Impresa 2218-6425 ISSN Versión Electrónica 1995-1403 DOI: https://dx.doi.org/10.62429/rnh20242181807 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. 1 Núcleo de Reabilitação da Fauna Silvestre e Centro de Triagem de Animais Silvestres, Universidade Federal de Pelotas (NURFS-CETAS/UFPel), Campus Universitário, s/n., Capão do Leão, CEP 96160-000, RS, Brazil. coimbra.nurfs@gmail.com 2 Instituto Federal Sul-rio-grandense (IFSul), Campus Pelotas, Praça Vinte de Setembro, 455, Centro, Pelotas, CEP 96015-360, RS, Brazil. 3 Independent researcher. Pelotas, Brazil. gertrudmuller40@gmail.com* Corresponding author: phrybio@hotmail.comMarco Antonio A. Coimbra: https://orcid.org/0000-0003-3902-706XCarolina Silveira Mascarenhas: https://orcid.org/0000-0002-7045-0926Gertrud Müller: https://orcid.org/0000-0002-2213-6721 ABSTRACT Physaloptera liophis Vicente & Santos, 1974 was described as a parasiteofsnakes in southeastern Brazil; however, some morphological aspects need updating. In the study of helminths associated with Rhinella dorbignyi (Duméril & Bibron, 1841) (Anura: Bufonidae) in the south of the country, specimens of P. liophis were found and used for the redescription presented by this paper. T irteen out of 100 anurans under analysis were parasitized by P. liophis . Redescription provides new information on the number of papillae of males and morphological characteristics of the reproductive system of females. Keywords: Bufonidae – Dorbigny’s Toad – Nematoda – Physaloptera – Rhinella dorbignyi – Southern Brazil
128 Neotropical Helminthology (Lima). Vol. 18, Nº2, jul - dec 2024 Afonso Coimbra et al. RESUMEN Physaloptera liophis Vicente & Santos, 1974 fue descrito como un parásito de serpiente en el sudeste de Brasil; sin embargo, algunos aspectos morfológicos necesitan actualización. En el estudio de los helmintos asociados a Rhinella dorbignyi (Duméril & Bibron, 1841) (Anura: Bufonidae) en el sur del país, fueron encontrados especímenes de P. liophis y utilizados para la redescripción presentada por este trabajo. Trece de los 100 anuros analizados fueron parasitados por P. liophis . La redescripción aporta nueva información sobre el número de papilas de los machos y sobre las características morfológicas del aparato reproductor de las hembras. Palabras clave: Bufonidae – Nematoda – Physaloptera – Rhinella dorbignyi – Sapito de jardin de D’Orbigny– Sur de Brasil INTRODUCTION Physaloptera Rudolphi, 1819 species parasitize the gastrointestinal tract of vertebrates, mainly in the stomach of mammals, birds, reptiles and, rarely, amphibians and fsh (Anderson, 2000; Pereira et al., 2012). Te genus has more than 105 named species distributed in all continents (Ortlepp, 1922; Ortlepp, 1937; Baker, 1987; Pereira et al. , 2012; Pereira et al., 2014; Luiz et al., 2015; Maldonado Jr. et al., 2019; Matias et al., 2020, Alves et al ., 2022). In Brazil, eight species have been recorded in reptiles (Pereira et al., 2014; Matias et al., 2020). Physaloptera liophis Vicente & Santos, 1974 was described as parasite of the snake Erythrolamprus miliaris Linnaeus, 1758 (= Liophis miliaris ) (Dipsadidae) in Rio de Janeiro state, in southeastern Brazil (Vicente & Santos, 1974), being also recorded in Bothrops neuwiedi Wagler, 1824 (Gouveia et al ., 2012) and in Erythrolamprus viridis (Günther, 1862) (Quirino et al. , 2018) in Minas Gerais and Ceará, respectively. Physaloptera larvae have been frequently recorded in several amphibian species (González & Hamann, 2007; Hamann et al ., 2013a; Campião et al ., 2014; Velarde-Aguilar et al., 2014; Aguiar et al ., 2015; Toledo et al., 2015; Santos et al ., 2016; Campião et al ., 2016; Lins et al ., 2017; Toledo et al ., 2017ab). However, only three species have been recorded worldwide; two were described in anurans while one was described in lizards. Physaloptera amphibia Linstow, 1899 was described in Limnonectes macrodon (Duméril & Bibron, 1841) (= Rana macrodon ) in the Philippines and Physaloptera tigrinae Ali & Farooqui, 1969 in Hoplobatrachus tigerinus (Daudin, 1802) (= Rana tigrina ) in India (Baker, 1987; Pereira et al., 2012). Baker (1987) also listed four Ranidae species as hosts of P. amphibia in Europe, but he cast doubt on the records. Regarding P. tigrinae , there are no records in the literature other than the species description in 1969, which is cited by Baker (1987), and Pereira et al. (2012). Physaloptera retusa (Rudolphi, 1819) was described in Squamata (Ortlepp, 1922), and recorded in northern Brazil parasitizing Rhinella granulosa (Spxi, 1824) (= Bufo granulosus ), and Rhinella margaritifera (Laurenti, 1768) (= Bufo thyfonius ), and Physaloptera sp. in Rhinella marina (Linnaeus, 1758) (= Bufo marinus ) in Amazonas state (Gonçalves et al ., 2002). In a study of helminths associated with Rhinella dorbignyi (Duméril & Bibron, 1841) (= Rhinella fernandezae ) (Anura: Bufonidae) in the southern of Brazil, specimens of P. liophis were found and used for the redescription presented by this paper, i.e., it redescribes some characteristics previously described by Vicente & Santos (1974). Rhinella dorbignyi occurs in Argentina, Uruguay, and southern Brazil and Paraguay (Frost, 2024), where it inhabits grasslands, wetlands, agroecosystems and areas close to human households (Loebmann, 2005; Maneyro et al., 2017). In R. dorbignyi species of Nematoda, Digenea, Cestoda and Acanthocephala have been reported in Paraguay (Lent et al ., 1946; Smales, 2007), Uruguay (Lent & Freitas, 1948), Brazil (Santos & Amato, 2010; Henzel et al ., 2020; Coimbra et al ., 2023), and Argentina (González & Hamann, 2007; Hamann et al ., 2013a, Hamann et al ., 2013b; Draghi et al ., 2020). Before Coimbra et al. (2023) (material used in this study), there were only records of Physaloptera larvae parasitizing this bufonid (González & Hamann 2007; Hamann et al . 2013a). Terefore, this study redescribes P. liophis associated with Rhinella dorbignyi from Pampa biome, southern Brazil.
129 Redescription of Physaloptera liophis parasites of anura from Brazil Neotropical Helminthology (Lima). Vol. 18, Nº2, jul - dec 2024 MATERIAL AND METHODS A hundred specimens of R. dorbignyi were collected in Pelotas (31°46’38.0”S - 52°13’57.2”W) and Capão do Leão (31°48’5.79”S - 52°24’53.39”W), Rio Grande do Sul (RS), southern Brazil, from August 2017 to October 2020. Anurans were manually collected and individually taken to the Laboratório de Parasitologia de Animais Silvestres (LAPASIL/UFPel), where they were weighed and measured. Fifty-four anurans were necropsied after freezing while the others were either examined immediately after death or refrigerated for no more than 24 hours. During the necropsy, all organs were individualized and examined. Nematodes were fxed in AFA (ethanol 70°GL - 93 parts; formalin 37% - 5 parts; glacial acetic acid - 2 parts), preserved in glycerinated ethanol (5% glycerin in 70°GL ethanol), and clarifed in Amann’s lactophenol (Amato & Amato, 2010). Tree male specimens were prepared for scanning electron microscopy (SEM) analysis at the Central Laboratory of Microscopy and Microanalysis of the Pontifcia Universidade Católica do Rio Grande do Sul (LabCEMM/PUCRS), Brazil. Specimens preserved in 70% ethanol was dehydrated in 90% ethanol for 20 minutes and then in 100% ethanol for 20 minutes. Subsequently, the critical point procedure was performed in a BALZERS CPD30 equipment for fnal drying of the sample. In this process liquid CO 2 is injected into the chamber to make the replacements until the ethanol is completely removed. Next, the samples were metallized with Au. Te metallization was done in a Quorum Q 150R ES plus metallizer in order to make the samples conductive to be visualized on the SEM. Te images were taken on an SEM-FEG from FEI, model Inspect F50. Measures (mean, standard deviation and range) are expressed as micrometers (µm), unless otherwise indicated. Minimum and maximum values (range) are shown between parentheses. Parasitological indices were calculated in agreement with Bush et al. (1997). Photomicrographs were prepared on an Olympus BX 41 microscope with a camera system and plates were made with Adobe Photoshop CS5. Vouchers were deposited in the “Coleção Helmintológica do Instituto Oswaldo Cruz” (CHIOC), Rio de Janeiro, Brazil, and in the “Coleção de Helmintos do Laboratório de Parasitologia de Animais Silvestres” at the Universidade Federal de Pelotas (CHLAPASIL-UFPel), RS, Brazil. Images of holotype, allotype and paratype (CHIOC 31034a-c) from the Coleção Helmintológica at the Instituto Oswaldo Cruz were examined. Ethic aspects: Te study was licensed by the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio no. 47397) and approved by the Ethics Committee on Animal Experimentation (CEEA/UFPel no.1859/2015). Euthanasia was performed in agreement with Resolution no. 1000/2012 issued by the Conselho Federal de Medicina Veterinária (CFMV, 2012). RESULTSRedescription Physaloptera liophis Vicente & Santos,1974 (Figs. 1 – 6) Host: Rhinella dorbignyi (Duméril & Bibron, 1841), Dorbigny´s Toad. Site of infection: stomach. Locality : Laranjal, Pelotas (31°46’38.0”S - 52°13’57.2”W), and UFPel Campus, Capão do Leão (31°48’5.79”S - 52°24’53.39”W), Rio Grande do Sul, Brazil. Prevalence and mean intensity of infection: 13%; 13.69 helminths/host (1-89 nematodes). Specimens deposited: CHLAPASIL-UFPel (904-918), and CHIOC (39171, 39172).Description based on 15 specimens: Body fliform with anterior extremity more tapered than posterior extremity. Cuticle with transverse striations forming well marked annulations on the frst and last third of the body. Cuticle at the anterior end has dilatations that form the cephalic collar. Oral opening surrounded by two well-developed lateral pseudolips, convex and semicircular in shape. Each pseudolip has a pair of cephalic papillae dorsoventrally located, a lateral small amphids, and a well-developed triangular tooth. Absence of buccal capsule. Deirids located at the same level. Excretory pore just below the deirids. Long esophagus divided into a muscular anterior part and a shorter glandular posterior part. Nervous ring surrounding the muscular esophagus.
130 Neotropical Helminthology (Lima). Vol. 18, Nº2, jul - dec 2024 Afonso Coimbra et al. Figure 1. Physaloptera liophis Vicente & Santos, 1974 a parasite of Rhinella dorbignyi (Duméril & Bibron, 1841) (Anura: Bufonidae) in southern Brazil. A-B : Dorsoventral view of the anterior extremity (g - glandular esophagus; r - nerve ring; m - muscular esophagus; arrows indicate the deirids) (bar – 60µm). C-D : Lateral view, the arrow indicates the excretory pore located near the junction of the glandular and muscular esophagus (bar – 60µm). E : Dorsoventral view, the arrow indicates the deirid (bar – 60µm).
131 Redescription of Physaloptera liophis parasites of anura from Brazil Neotropical Helminthology (Lima). Vol. 18, Nº2, jul - dec 2024 Figure 2. Ventral view of the tail of a Physaloptera liophis Vicente & Santos, 1974 male parasite of Rhinella dorbignyi (Duméril & Bibron, 1841) (Anura: Bufonidae) in southern Brazil. A : General view of the tail, emphasis is given to the position of the four pairs of pedunculated papillae (p) and the tip of the left spicule exiting through the cloaca (arrow) (bar – 75µm). B : Detail of phasmids (ph), between the two last pairs of sessile papillae (s), and the dome-shape protuberance (asterisk) between the papillae of the last pair (bar – 75µm). C – D: Detail of the sessile papillae (s) (bar – 50µm).
132 Neotropical Helminthology (Lima). Vol. 18, Nº2, jul - dec 2024 Afonso Coimbra et al. Figure 3. Lateral view of the middle third of a Physaloptera liophis Vicente & Santos, 1974 female parasite of Rhinella dorbignyi (Duméril & Bibron, 1841) (Anura: Bufonidae) in southern Brazil. A : Detail of vulvar opening (arrow), ovojector (ov) and beginning of egg chamber (ch) (bar – 100µm). B : Detail of the vulvar opening and muscular vagina (v) (bar– 25µm). C: Detail of the didelph uterus (arrows) directed towards the posterior body region (bar – 75µm). D: Detail of the junction of the ovojector (ov) and egg chamber (ch) (bar – 100µm).
133 Redescription of Physaloptera liophis parasites of anura from Brazil Neotropical Helminthology (Lima). Vol. 18, Nº2, jul - dec 2024 Figure 4. Physaloptera liophis Vicente & Santos, 1974 a parasite of Rhinella dorbignyi (Duméril & Bibron, 1841) (Anura: Bufonidae) in southern Brazil. A : Detail of the vulvar opening and eggs in uterus, lateral view (bar – 55µm). B : Embryonated eggs (bar – 55µm). C : Spicules of the male (bar – 33µm).
134 Neotropical Helminthology (Lima). Vol. 18, Nº2, jul - dec 2024 Afonso Coimbra et al. Figure 5. Physaloptera liophis Vicente & Santos, 1974 a parasite of Rhinella dorbignyi (Duméril & Bibron, 1841) (Anura: Bufonidae) in southern Brazil. A: Dorsoventral view of the anterior extremity (bar – 60µm). B: Left spicule (bar – 35µm). C: Right spicule (bar – 35µm). D: Genital tract from vagina to uterus didelph (bar– 520µm). E: Lateral view of the posterior extremity of the female (bar – 250µm). F: Ventral view of the tail of male (bar – 75µm).
135 Redescription of Physaloptera liophis parasites of anura from Brazil Neotropical Helminthology (Lima). Vol. 18, Nº2, jul - dec 2024 Figure 6. Physaloptera liophis Vicente & Santos, 1974 male parasite of Rhinella dorbignyi (Duméril & Bibron, 1841) (Anura: Bufonidae) in southern Brazil. A - B: Anterior extremity (cp - cephalic papillae; arrow indicate the triangular tooth. C: Detail of cephalic papillae (cp) and amphid (arrow). D: Posterior extremity (s - sessile papillae; phasmids (arrow). E: Detail of the 5th pair of sessile papillae (s), phasmids (ph) and dome-shaped protuberance (asterisk). F: Detail of the mammiliform sessile papillae (s) on the lower margin of the cloacal opening, and of the tip of the right spicule (sp).
136 Neotropical Helminthology (Lima). Vol. 18, Nº2, jul - dec 2024 Afonso Coimbra et al. Male based on seven specimens: overall body length 7.61 ± 1.11 (6.20–9.50) mm; width (at the level of the esophagus-intestine junction) 334 ± 46.50 (280–410). Distance from nerve ring to anterior extremity of the body 238.93 ± 24.57 (202.50–282.50). Distance from excretory pore to anterior extremity 317 ± 26.28 (300–360). Distance from deirid to anterior extremity 276 ± 35.52 (240–330). Esophagus length 1935 ± 273.5 (1550–2330), representing 25.43% ± 2.57 % (21.47–29.64%) of total body length. Muscular esophagus 267 ± 17.99 (240–290) long, and 61 ± 9.00 (50–70) width; glandular esophagus 1668 ± 259.69 (1300–2050) long, and 120 ± 16.33 (100–140) width. Curved tail with well-developed two caudal alae fused posteriorly. Caudal ala 714 ± 89.97 (610–850) long, right portion 116 ± 24.40 (80–150) wide, and left portion 111 ± 32.88 (50–150) wide. Ventral face of alae ornamented with small tubercles arranged in longitudinal rows, less evident near posterior extremity of tail. Twenty-one caudal papillae: 4 pedunculated pairs, 6 sessile pairs and 1 large precloacal unpaired papillae. Pedunculated papillae: 2 subventral precloacal pairs, 1 pair aligned with the cloaca and 1 postcloacal pair. Precloacal sessile papillae: 1 small median pair on the same line followed by 1 large unpaired papilla near the cloacal opening. Post-cloacal sessile papillae: 2 pairs of median mammiliform papillae on the lower margin of the cloacal opening; 3rd pair just below the cloaca, followed by the 4th pair, both paired diagonally (in some specimens, pairs are aligned); 5th pair apart from the others and aligned near the tip of the tail. Between the papillae of the 5th pair, there is a dome-shape protuberance. Spicules of diferent shape, and sub-equal size, slightly sclerotized, surrounded by a small membranous sheath. Spear-shaped left spicule, with rounded base and widening in the second half, ending in a thin tip, 181.43 ± 24.70 (162.5–220) long. Needle-shaped right spicule with rounded base and fne tip, 172.1 ± 10.04 (155–187.5) long. Distance from cloaca to tail end 387 ± 62.91 (320–500). Gubernacle absent. A pair of phasmids is located between the 4th and 5th pairs of postcloacal sessile papillae.Female based on eight specimens (six non-gravid females and two gravid with few eggs): overall body length 7.79 ± 1.32 (6.00–9.75) mm; width (at the level of the esophagus-intestine junction) 357.5 ± 39.55 (290–400). Distance from nerve ring to anterior extremity of the body 232.50 ± 21.51 (187.5–255). Distance from excretory pore to anterior extremity 362.5 ± 41.32 (290–410). Distance from deirides to anterior extremity 315.63 ± 31.42 (267.5–370). Esophagus length 1805.63 ± 320.75 (1310 – 2285), representing 23.47% ± 3.87% (15.73–27.50%) of the total body length. Muscular esophagus 290 ± 41.40 (230–360) long, and 67.50 ± 8.86 (50–80) width; glandular esophagus 1515.63 ± 306.24 (980–1925) long, and 125 ± 27.26 (90–150) width. Vulvar opening located in the middle third of the body, distant 3277.14 ± 657.57 (2450–4125) from anterior extremity (seven specimens were measured). Vulvar lips not very prominent. Muscular short vagina 72.50 ± 20.54 (50–100) (fve specimens were measured), directed posteriorly followed by muscular ovojector of 420.83 ± 81.83 (337.50–500) (three specimens were measured); egg chamber 563.33 ± 79.67 (470–660) long, posteriorly directed didelph uterus, with the two uterine loops beginning after the egg chamber, and no common trunk. Smooth-shelled eggs 40 (40–40) long and 22.5 (22.5–22.50) width (three eggs were measured), fully developed, containing larvae. Posterior end rounded; lateral phasmidial pores evident 93.75 ± 18.90 (62.5–112.50) distant from tail tip. Tail length 221.50 ± 26.96 (200–280). Remarks : Specimens of P. liophis that parasitize the anuran R. dorbignyi are proportionally smaller than those described while parasitizing the snake E. miliaris (Table 1). Vicente & Santos (1974) described the presence of 23 caudal papillae in P. liophis male; however, the penultimate pair of post-cloacal papillae are redescribed by this study as phasmids. Furthermore, the authors did not mention dome-shape protuberance between the last pair of post-cloacal papillae. Such characteristics were observed in the holotype (CHIOC 31034a) and also in the specimens collected from R. dorbignyi . Redescription of the female provides information on the morphology and morphometry of the vagina, ovojector and egg chamber, which were not described by Vicente & Santos (1974), possibly because they examined only a female, which was considered oviparous. Females collected from anurans by this study exhibited larval eggs. Regarding Physaloptera species described in amphibians, P. amphibia and P. trigrinae , there is no information on the uterus morphology (Ortlepp, 1922; Pereira et al ., 2012). However, the P. liophis male difers from the P. amphibia one, since the latter has 16 caudal papillae (Ortlepp, 1922) while the former has 21 papillae. Physaloptera liophis could not be compared with P. trigrinae , since the species description could not be accessed; it was listed by Baker (1987) and Pereira et al. (2012) and there no other records of the species after its des cription in 1969 were found.
137 Redescription of Physaloptera liophis parasites of anura from Brazil Neotropical Helminthology (Lima). Vol. 18, Nº2, jul - dec 2024 Among the species recorded in reptiles in Brazil, P. liophis difers from P. retusa , P. obtusissima Molin, 1860, P. lutzi Cristofaro, Guimarães & Rodrigues, 1976, P. tupinambae Pereira, Alves, Rocha, Lima & Luque, 2012, P. bainae Pereira, Alves, Rocha, Lima & Luque, 2014 and P. nordestina Matias, Moraes & Ávila, 2020 due to the position of the vulvar opening, which is located near the anus in P. lutzi , and in the frst third of the body in the other species. However, in P. liophis , the vulva is located in the middle third of the body. Physaloptera liophis and P. bonnei Ortlepp, 1922 have the same number of caudal papillae, but difer in the shape and size of the spicules, which measure 455µm in P. bonnei . Te female of P. bonnei has a vagina, egg chamber, and the bifurcation of the uterus directed forward, unlike P. liophis . In addition, P. bonnei has two pairs of teeth on each pseudolabial (the outer is conical and obtuse, whereas the inner is membranous and tripartite), unlike P. liophis which has only one pair of triangular teeth. DISCUSSION Even though transmission and development of some Physaloptera species in mammals have been intensively studied, there is limited information on species in reptiles. Physalopterinae species are usually found frmly adhered to the gastric mucosa of their defnitive hosts; however, studies of species that occur in mammals suggest that the helminths do not feed on their mucosa but on contents found in their stomach, a fact that infuences development of the third-stage larvae into adult forms. Infection of the defnitive hosts involves prey-predator interactions, since Table 1 . Morphometry of Physaloptera liophis as parasites of snakes and anuran in Brazil . Vicente & Santos (1974)Tis studyHost: Erythrolamprus miliaris Linnaeus (Serpentes) Host: Rhinella dorbignyi (Duméril & Bibron) (Anura) Locality : Volta Redonda, Rio de Janeiro Locality : Pelotas and Capão do Leão, Rio Grande do Sul Male Body length (mm)12.18 – 13.8 6.2 – 9.5 Body width500 – 520280 – 410Length of muscular esophagus310 – 350240 – 290Length of glandular esophagus1860 – 22701300 – 2050Distance of the deirids from the anterior extremity340 – 380240 – 330Distance of the nerve ring from the anterior extremity260202.5 – 282.5Spicules length250 – 260162.5 – 220 (left) 155 – 187.5 (right) Female Body length (mm)12.78 6.0 – 9.75 Body width510290 – 400Length of muscular esophagus360230 – 360Length of glandular esophagus2380980 – 1925Distance of the deirids from the anterior extremity390267.5 – 370Distance of the nerve ring from the anterior extremity310187.5 – 255Distance from vulva to anterior end70702450 – 4125Egg length x width50 x 2040 x 22.5Tail length390200 – 280
138 Neotropical Helminthology (Lima). Vol. 18, Nº2, jul - dec 2024 Afonso Coimbra et al. vertebrates become infected by ingesting arthropods (e. g . , cockroaches and crickets) that act as intermediate hosts in which the infective third-stage larvae develop (Anderson, 2000). Te role of anurans in the life cycle of Physaloptera species is not sufciently known. Tere are several records of larval forms parasitizing these vertebrates in the Neotropics (González & Hamann, 2007; Hamann et al ., 2013a; Campião et al ., 2014; Velarde-Aguilar et al. , 2014; Aguiar et al ., 2015; Toledo et al ., 2015; Santos et al ., 2016; Campião et al ., 2016; Lins et al ., 2017), suggesting that anurans may act as paratenic hosts which transmit infective forms through the trophic chain. On the other hand, the few records of adult forms of Physaloptera in anurans suggest that these parasites, throughout their evolution, have not had the same success of infection and establishment in these vertebrates, by comparison with mammals, birds, and reptiles. Four Physaloptera species have been recorded as stomach parasites of anurans; two of them have been described in reptiles, P. retusa and P. liophis , and recorded in anurans belonging to the Rhinella Fitzinger (Ortlepp, 1922; Cristofaro et al. , 1976; Baker, 1987; Gonçalves et al ., 2002; Pereira et al. , 2012; Pereira et al. , 2014; Coimbra et al ., 2023; present study). González et al. (2021) reported that anurans can be defnitive hosts of Physaloptera venancioi Lent, Freitas & Proença, 1946; however, this species, described in Rhinella diptycha (Cope, 1862) (= Bufo paracnemis ) in Paraguay (Lent et al ., 1946), was transferred to Physalopteroides Wu & Liu, 1940 (Bursey & Goldberg, 1994).In general, the association between Physaloptera species (adults and/or larvae) and anurans belonging to Rhinella is characterized by low infection indices (Gonçalves et al ., 2002; González & Hamann, 2007; Hamann et al ., 2013a; Toledo et al. , 2017ab; Teles et al. , 2018; Coimbra et al ., 2023) possibly refecting the diet of these anurans. Several species of Rhinella , such as R. dorbignyi , use ants as their main food resource, and may also consume other arthropods (e.g., coleopterans, isopterans, arachnids, hemipterans, dipterans, cockroaches, orthopterans and lepidopterans), which are usually little important in the diet of species that belong to this genus (Sabagh et al. , 2008; Quiroga et al. , 2009; Batista et al. , 2011; Maragno et al. , 2011; Piatti et al. , 2011; Isacch & Barg, 2002; Maia-Carneiro et al. , 2013; Oliveira et al. , 2014). Studies of the feeding ecology of R. dorbignyi were carried out in Argentina, where the species was found to consume mainly Formicidae (Isacch & Barg, 2002; Da Rosa et al ., 2002; Duré et al ., 2009; Peltzer et al ., 2010). Te helminth fauna of R. dorbignyi is well known in its area of occurrence, since studies have been developed in Paraguay (Lent et al ., 1946; Smales, 2007), Uruguay (Lent & Freitas, 1948), Brazil (Santos & Amato, 2010; Henzel et al ., 2020; Coimbra et al. , 2023), and Argentina (González & Hamann, 2007; Hamann et al ., 2013a; Hamann et al ., 2013b; Draghi et al ., 2020). González & Hamann (2007) and Hamann et al . (2013a) recorded Physaloptera larvae, whose prevalence ranged from 3.1% (2/65) to 4% (1/25) and mean intensity of infection was one to six helminths/host. Terefore, the feeding preference of Rhinella species for ants supports the hypothesis that infections by Physaloptera species are infrequent or occasional. Tis study introduced new information on the morphology of males and females of P. liophis , a parasite of anurans in the Pampa biome, and provided ecological information for future studies on this anuran species. ACKNOWLEDGMENTS Special thanks to Marcelo Knof (FIOCRUZ-RJ) and Alessandra Gomes (FIOCRUZ-RJ) for their help in searching and collecting images of material deposited in the Coleção de Helmintos do Instituto Oswaldo Cruz, to Sr. Wagner Prates (LabCEMM/PUC-RS) for his help with the Scanning Eletron Microscopy analysis, to Felipe Bisaggio Pereira (UFMG) for his bibliographic support, to colleagues Frank Lira, Ana Beatriz D. Henzel, Juliana H. Wolter, and Ricardo R. C. Silva for their collaboration in the collections and necropsies, to CAPES (Coordenação de Aperfeiçoamento do Pessoal de Nível Superior) for the fnancial support (process no. 32/2010) and post-doctoral fellowship (2014-2019) PNPD to CSM. Author contributions: CRediT (Contributor Roles Taxonomy)MAAC = Marco Antonio Afonso Coimbra CSM = Carolina Silveira Mascarenhas GM = Gertrud Müller Conceptualization: MAAC, CSM Data curation: MAAC, CSM Formal Analysis: MAAC, CSM, GM Funding acquisition: GM Investigation: MAAC, CSM Methodology: MAAC, CSM
139 Redescription of Physaloptera liophis parasites of anura from Brazil Neotropical Helminthology (Lima). Vol. 18, Nº2, jul - dec 2024 Project administration: CSM Resources: MAAC, CSM, GM Software: CSM Supervision: GM Validation: MAAC, CSM, GM Visualization: MAAC, CSM, GM Writing – original draft: MAAC, CSM Writing – review & editing: MAAC, CSM, GM BIBLIOGRAPHIC REFERENCES Aguiar, A., Toledo, G.M., Anjos, L.A., & Silva, R.J. (2015). Helminth parasite communities of two Physalaemus cuvieri Fitzinger, 1826 (Anura: Leiuperidae) populations under diferent conditions of habitat integrity in the Atlantic Rain Forest of Brazil. Brazilian Journal of Biology, 75 , 963-968Alves, P.V., Couto, J.V., & Pereira, F.B. (2022). Redescription of the two most recorded Physaloptera (Nematoda: Physalopteridae) parasitizing lizards in the Americas: frst step towards a robust species identifcation framework. Systematic Parasitology , 99 , 63-81. Amato, J.F.R., & Amato, S.B. (2010). Técnicas gerais para coleta e preparação de helmintos endoparasitos de aves . In: Von Matter, S., Straude, F. C., Accordi, I., Piacentini, V., Cândio-Jr, J. F. (eds). Ornitologia e conservação ciência aplicada técnicas de pesquisa e levantamento . Rio de Janeiro: Technical Books.Anderson, R.C. (2000). Nematode Parasites of Vertebrates: Teir Development and Transmission . 2nd ed. CABI Publishing.Baker, M.R. (1987). Synopsis of the Nematoda parasitic in amphibians and reptiles. Memorial University of Newfoundland Occasional Papers in Biology, 11 , 1-325.Batista, R.C., De-Carvalho, C.M., Freitas, E.B., Franco, S.C., Batista, C.C., Coelho, W.A., & Faria, R.G. (2011). Diet of Rhinella schneideri (Werner, 1894) (Anura: Bufonidae) in the Cerrado, Central Brazil. Herpetology Notes, 4 , 17-21.Bursey, C.R., & Goldberg, S.R. (1994). Physalopteroides bahamensis n. sp. (Nematoda: Spiruroidea) from the Cuban Treefrog Osteopilus septentrionalis (Hylidae) from San Salvador Island, Bahamas. Transactions of the American Microscopical Society, 113 , 169-176.Bush, A.O., Laferty, K.D., Lotz, J.M., & Shostak, A.W. (1997). Parasitology meets ecology on its own terms: Margolis et al. revisited. 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.Campião, K.M., Silva, I.C.O., Dalazen, G.T., Paiva, F., & Tavares, L.E.R. (2016). Helminth parasites of 11 anuran species from the Pantanal Wetland, Brazil. Comparative Parasitology , 83 , 92-100. Coimbra, M.A.A., Mascarenhas, C.S., Henzel, A.B.D, Wolter, J.H., Silva, R.R.C, Silveira, F.L. & Müller, G. (2023). Parasite-host relations and new reports of helminths for Rhinella dorbignyi (Duméril & Bibron, 1841) (Anura: Bufonidae) from Neotropical region. Parasitology International 96 , 102766. CFMV - Conselho Federal de Medicina Veterinária (2012). Resolução n°1000: Dispõe sobre procedimentos e métodos de eutanásia em animais, e dá outras providências . http://ts.cfmv.gov.br/manual/arquivos/resolucao/1000.pdf Cristofaro, R., Guimarães, J.F., & Rodrigues, H.O. (1976). Alguns nematódeos de Tropidurus torquatus (Wied) e Ameiva ameiva (L.) – Fauna helmintológica de Salvador, Bahia. Atas da Sociedade de Biologia do Rio de Janeiro , 18 , 65-70.Da Rosa, I., Canavero, A., Maneyro, R., Naya, D. E. & Camargo, A. (2002). Diet of four sympatric anuran species in a temperate environment. Boletín de la Sociedad Zoológica del Uruguay , 13 : 12–20.
140 Neotropical Helminthology (Lima). Vol. 18, Nº2, jul - dec 2024 Afonso Coimbra et al. Draghi, R., Drago, F.B., Saibene, P.E., & Agostini, M.G. (2020). Helminth communities from amphibians inhabiting agroecosystems in the Pampean Region (Argentina). Revue Suisse de Zoologie , 127 , 261-274. Duré, M.I., Kehr, A.I., & Schaefer, E.F. (2009). Niche overlap and resource partitioning among fve sympatric bufonids (Anura, Bufonidae) from northeastern Argentina. Phyllomedusa , 9 , 27-39.Fernandes, B.M.M., & Kohn, A. (2014). South American trematodes parasites of amphibians and reptiles . Ofcina de Livros.Frost, D. R. (2024). Amphibian Species of the World: an Online Reference. Version 6.1 Electronic Database. https://amphibiansoftheworld.amnh.org/ American Museum of Natural History.Gonçalves, A.G., Vicente, J.J., & Pinto, R. M. (2002). Nematodes of Amazonian vertebrates deposited in the Helminthological Collection of the Oswaldo Cruz Institute with new records. Revista Brasileira de Zoologia , 19 , 453-465.González, C.E., & Hamann, M.I. (2007). Nematode parasites of two species of Chaunus (Anura: Bufonidae) from Corrientes, Argentina. Zootaxa , 1393 , 27-34.González, C.E., Hamann, M.I., & Duré, M.I. (2021). Nematodes of amphibians from the South American Chaco: distribution, host specifcity and ecological aspects. Diversity , 13 , 321. Gouveia, R.V., Silva, D.A.N., Novelli, I.A., & Vieira, F.M. (2012). Bothropoides neuwiedi (Neuwied’s Lancehead). Endoparasites. Herpetological Review , 43 , 340. Hamann, M.I., Kehr, A.I., & González, C.E. (2013a). Helminth communities in the burrowing toad, Rhinella fernandezae , from Northeastern Argentina. Biologia , 68 , 1155-1162.Hamann, M.I., Kehr, A.I., & Gonzalez, C.E. (2013b). Biodiversity of trematodes associated with amphibians from a variety of habitats in Corrientes Province, Argentina. Journal of Helminthology , 87, 286-300.Henzel, A.B.D., Mascarenhas, C.S., Silveira, F.L., & Müller, G. (2020). Digenetic helminths of Leptodactylus latrans (Anura: Leptodactylidae) and Rhinella dorbignyi (Anura: Bufonidae) in southern Brazil. Revista Brasileira de Zoociências , 21 , 1-10.Isacch, J.P., & Barg, M. (2002). Are bufonid toads specialized ant-feeders? A case test from the Argentinian fooding pampa. Journal of Natural History , 36 , 2005-2012.Lent, H., & Freitas, J.F.T. (1948). Uma coleção de nematódeos, parasitos de vertebrados do Museu de História Natural de Montevideo. Memórias do Instituto Oswaldo Cruz , 46 , 1-71. Lent, H., Freitas, J.F.T., & Proença, M.C. (1946). Alguns helmintos de batráquios colecionados no Paraguai. Memórias do Instituto Oswaldo Cruz , 44 , 195-214.Lins, A.G.S., Aguiar, A., Morais, D.H., Silva, L.A.F., Ávila, R.W., & Silva, R.J. (2017). Helminth fauna of Leptodactylus syphax (Anura: Leptodactylidae) from Caatinga biome, northeastern Brazil. Brazilian Journal Veterinary Parasitology , 26 , 74-80. Loebmann, D. (2005). Os Anfíbios da Região Costeira do Extremo Sul do Brasil . Pelotas, USEB. Luiz, J.S, Simões, R.O., Torres, E.L., Barbosa, H.S., Santos, J.N., Giese, E.G.; Rocha, F.L., & Maldonado Jr., A. (2015). A new species of Physaloptera (Nematoda: Physalopteridae) from Cerradomys subfavus (Rodentia: Sigmodontinae) in the Cerrado Biome, Brazil. Neotropical Helminthology , 9 , 301-312. Maia-Carneiro, T., Kiefer, M.C., Van Sluys, M., & Rocha, C.F.D. (2013). Feeding habits, micro-habitat use, and daily activity period of Rhinella ornata (Anura, Bufonidae) from three Atlantic rainforest remnants in southeastern Brazil. North-Western Journal of Zoology , 9 , 135-140.
141 Redescription of Physaloptera liophis parasites of anura from Brazil Neotropical Helminthology (Lima). Vol. 18, Nº2, jul - dec 2024 Maldonado Jr., A., Simões, R.O., Luiz, J.S., Costa-Neto, S.F., & Vilela, R.V. (2019). A new species of Physaloptera (Nematoda: Spirurida) from Proechimys gardneri (Rodentia: Echimyidae) from the Amazon rainforest and molecular phylogenetic analyses of the genus. Journal of Helminthology , 94 , 1-11. Maneyro, R., Loebmann, D., Tozetti, A., & Fonte, L.F.M. (2017). Anfíbios das planícies costeiras do extremo sul do Brasil e Uruguai . Anolisbooks. Maragno, F.P., & Souza, F.L. (2011). Diet of Rhinella scitula (Anura, Bufonidae) in the Cerrado, Brazil: Te importance of seasons and body size. Revista Mexicana de Biodiversidad , 82 , 879-886.Matias, C.S.L, Morais, D.H., & Ávila, R.W. (2020). Physaloptera nordestina n. sp . (Nematoda: Physalopteridae) parasitizing snakes from Northeastern Brazil. Zootaxa , 4766 , 173-180. Oliveira, J.C.D., Sousa, A.P.M., & Dyego, F. (2014). Frequency of occurrence of vegetables in the diet Rhinella jimi (Anura, Bufonidae) Cuité, Paraíba, Brazil. Agropecuária Científca no Semiárido , 10 , 90-95. Ortlepp, R.J. (1922). Te nematode genus Physaloptera Rudolphi. Proceedings of the Zoological Society of London , 4 , 999-1107. Ortlepp, R.J. (1937). Some undescribed species of the nematode genus Physaloptera together with key to the sufciently known forms. Onderstepoort Journal of Veterinary Science and Animal Industry , 9 , 71-84.Peltzer, P.M., Attademo, A.M., Lajmanovich, R.C., Junges, C.M., Beltzer, A.H., & Sanchez, L.C. (2010). Trofc dynamics of three sympatric anuran species in a soybean agroecosystem from Santa Fe Province, Argentina. Te Herpetological Journal , 20 , 261-269.Pereira, F.B., Alves, P.V., Rocha, B.M., Souza Lima, S., & Luque, J.L. (2012). A new Physaloptera (Nematoda: Physalopteridae) parasite of Tupinambis merianae (Squamata: Teiidae) from southeastern Brazil. Journal of Parasitology , 98 , 1227-1235. Pereira, F.B., Alves, P.V., Rocha, B.M., Lima, S.S. & Luque, J.L. (2014). Physaloptera bainae n. sp. (Nematoda: Physalopteridae) parasitic in Salvator merianae (Squamata: Teiidae), with a key to Physaloptera species parasitizing reptiles from Brazil. Journal of Parasitology , 100 , 221-227. Piatti, L., & Souza, F. (2011). Diet and resource partitioning among anurans in irrigated rice felds in Pantanal, Brazil. Brazilian Journal of Biology , 71 , 653-661.Quirino, T.F., Ferreira, A.J.M.G., Silva, M.C., Silva, R.J., Morais, D.H. & Ávila, R.W. (2018). New records of Helminths in Reptiles from Five states of Brazil. Brazilian Journal of Biology , 78 , 750-754. Quiroga, L.B., Sanabria, E.A., & Acosta, J.C. (2009). Size-and Sex-dependent variation in diet of Rhinella arenarum (Anura: Bufonidae) in a Wetland of San Juan, Argentina. Journal of Herpetology , 43 , 311-317.Sabagh, L.T., & Carvalho-e-Silva, A.M.P.T. (2008). Feeding overlap in two sympatric species of Rhinella (Anura: Bufonidae) of the Atlantic Rain Forest. Revista Brasileira de Zoologia , 25 , 247-253.Santos, V.G.T., & Amato, S.B. (2010). Helminth fauna of Rhinella fernandezae (Anura: Bufonidae) from the Rio Grande do Sul coastland, Brazil: analysis of the parasite community. Journal of Parasitology , 96 , 823-826. Santos, V.G.T., Martins, M.B., & Amato, S.B. (2016). Community structure of parasites of the tree frog Scinax fuscovarius (Anura, Hylidae) from Campo Belo do Sul, Santa Catarina, Brazil. Neotropical Helminthology , 10 , 41-50.Smales, L.R. (2007). Acanthocephala in amphibians (Anura) and reptiles (Squamata) from Brazil and Paraguay with description of a new species. Journal of Parasitology , 93 , 392-398.Teles, D.A., Brito, S.V., Araújo-Filho, J.A., Ribeiro, S.C., Teixeira, A.A.M., Mesquita, D.O., & Almeida, W.O. (2018). Nematodes of the Rhinella granulosa Spix, 1824 (Anura: Bufonidae) from the Semiarid Northeastern Caatinga Region of Brazil. Comparative Parasitology , 85 , 208-211.
142 Neotropical Helminthology (Lima). Vol. 18, Nº2, jul - dec 2024 Afonso Coimbra et al. Toledo, G.M., Morais, D.H., Silva, R.J., & Anjos, L.A. (2015). Helminth communities of Leptodactylus latrans (Anura: Leptodactylidae) from the Atlantic rainforest, south-eastern Brazil. Journal of Helminthology , 89 , 250-254.Toledo, G.M., Fonseca, M.G., Iannacone, J., Callirgos, J.M.C., Vidaurre, C.U.M., & Silva, R.J. (2017a). Helminth parasites of Rhinella marina (Linnaues, 1758) (Anura: Bufonidae) from Tarapoto, Peru. Te Biologist (Lima) , 15 , 459-468.Toledo, G. M., Schwartz, H.O., Nomura, H.A.Q., Aguiar, A., Velota, R.A.M.V., Silva, R.J., & Anjos, L.A. (2017b). Helminth community structure of 13 species of anurans from Atlantic rainforest remnants, Brazil. Journal of Helminthology , 92 , 438-444.Velarde-Aguilar, M.G, Romero-Mayén, A.R., & León-Règagnon, V. (2014). First report of the genus Physaloptera (Nematoda: Physalopteridae) in Lithobates montezumae (Anura: Ranidae) from Mexico. Revista Mexicana de Biodiversidad , 85 , 304-307.Vicente, J.J., & Santos, E. (1974). Sobre um novo nematódeo do gênero Physaloptera Rudolphi, 1819 parasito de cobra d’água (Nematoda, Spiruroidea). Atas da Sociedade de Biologia do Rio de Janeiro , 17 , 69-71.Received June 24, 2024.Accepted August 5, 2024.