First report of helminth parasitizing Trachycephalus typhonius (Anura: Hylidae) from northeastern Argentina

Abstract

Two species of nematodes, Rhabdias cf. elegans (Rhabdiasidae) and Aplectana hylambatis (Cosmocercidae), were recovered from the lungs and intestine of Trachycephalus typhonius (Anura: Hylidae) captured in Formosa Province, Argentina. Morphological and morphometric data of both species are provided, and previous records of Rhabdias spp. in the Neotropical region are summarized. The finding of Rhabdias cf. elegans constitutes the first record of this genus in the Formosa Province, and the finding of A. hylambatis represents the first record of this species as a parasite of hylid frogs, and the first report in Formosa Province. Trachycephalus typhonius represents a new host for both parasites.

Resumen

Dos especies de nematodos, Rhabdias cf. elegans (Rhabdiasidae) y Aplectana hylambatis (Cosmocercidae), fueron recuperados de los pulmones e intestino de Trachycephalus typhonius (Anura: Hylidae) capturados en la provincia de Formosa, Argentina. Se proveen datos morfológicos y morfométricos de ambas especies y se resumen los registros previos de Rhabdias spp. en la región neotropical. El hallazgo de Rhabdias cf. elegans constituye el primer registro del género Rhabdias en la provincia de Formosa y el de A. hylambatis representa el primer registro de esta especie en hílidos, y el primer reporte en la provincia de Formosa. Trachycephalus typhonius es un nuevo hospedero para ambos parásitos.

The veined tree frog or common milk frog, Trachycephalus typhonius (Linnaeus, 1758) (Anura: Hylidae), is a very widespread frog from the lowlands of tropical Mexico, Central America (to 2,500 m asl in Guatemala), to Amazon Basin of Colombia, Ecuador, Peru, Venezuela, and Brazil, Guianas, and south to Brazil, Paraguay and northern Argentina (Frost, 2014; Soares, Iop, & Santos, 2012). In Argentina, this anuran species is distributed in Corrientes, Chaco, Entre Ríos, Formosa, Jujuy, Misiones, Salta, Santiago del Estero and Santa Fe Provinces (SIB, 2014). The veined tree frog is a nocturnal amphibian typically found perching on tree branches and vegetation while foraging at night. It preys on spiders, insects and bats, having a generalist diet (Duré & Kehr, 2006).

The helminth fauna of T. typhonius has been poorly studied, so far 6 species of helminths have been recorded parasitizing this species (cited as Phrynohyas venulosa and Trachycephalus venulosus): Polystoma lopezromaniCombes and Laurent, 1979 (Monogenea: Polystomatidae) from Argentina (Combes & Laurent, 1979); Batracholandros spectatus (Freitas and Ibáñez, 1962) Freitas and Ibáñez, 1965 (Nematoda: Pharyngodonidae), and larvae of Physaloptera sp. (Nematoda: Physalopteridae) from Peru (Bursey, Goldberg, & Parmalee, 2001); and Parapharyngodon duniaeBursey and Brooks, 2004 (Nematoda: Pharyngodonidae), Aplectana incerta Caballero, 1949 and Aplectana itzocanensis Bravo-Hollis, 1943 (Nematoda: Cosmocercidae) from Costa Rica (Bursey & Brooks, 2004, emsp 2010). The aim of this paper is to increase the knowledge of the diversity of helminth parasites from anurans from northeastern Argentina.

On September 17^th 2012, 2 specimens of T. typhonius were collected at La Marcela farm in Pirané, Formosa Province, Argentina (26°17’35” S, 59°08’38” W), with authorization of Ministerio de la Producción y Ambiente, Dirección de Fauna y Parques of Formosa Province. The frogs were dissected after an overdose with 20% benzocaine and all organs were examined using a Stemi 2000-C Zeiss stereoscope. The collected nematodes were counted and fixed in 5% formaline and cleared in Aman's lactophenol for light microscopic investigation, using a Standard 25 Zeiss optical microscope. For their identification were used taxonomic descriptions taken from specific literature (Anderson & Bain, 1982; Baker & Vaucher, 1986; González, Quiroga, Moreno, & Sanabria, 2013; Gutiérrez, 1945). All measurements are given in micrometres (μm) unless otherwise stated, as the range followed by the mean and standard deviation in parentheses. The nematodes were deposited in the Helminthological Collection of the Museo de La Plata (MLP), and the hosts in the Herpetological Collection of the Museo de La Plata, La Plata, Argentina (accession numbers: MLP A5642, MLP A5643).

Rhabdiasidae Railliet, 1915

Rhabdias Stiles and Hassall, 1905

Rhabdias cf. elegansGutiérrez, 1945 (Fig. 1)

Figure 1-4.

Nematodes from Trachycephalus typhonius. 1. Rhabdias cf. elegans, hermaphrodite gravid specimen. Scale bar= 500 μm. 2. Aplectana hylambatis, female, entire worm. Scale bar= 200 μm. 3. Aplectana hylambatis, male, posterior extremity. Scale bar= 50 μm. 4. Aplectana hylambatis, male, enlarged view of the distal articulation of spicules. Scale bar= 50 μm. Abbreviations: da, distal articulation of the spicules; g, gubernaculum; po, postcloacal papillae; pr, precloacal papillae; s, spicules; v, vulva.

Host: Trachycephalus typhonius (L.) (Anura: Hylidae).

New host record.

Site of infection: lungs.

Prevalence and intensity of infection: 50% (1 of 2); 2.

Voucher specimens deposited: MLP-He 6792.

Description

(Based on 2 hermaphrodite gravid specimens). Body cylindrical. Body length 5.9-6.4 (6.1±0.38) mm, maximum width 0.43-0.46 (0.44±0.02) mm. Anterior end rounded, posterior end conical. Body cuticle swollen, with irregular folds. Oral opening narrow, without labial structures. Buccal capsule infundibuliform, with sclerotized walls, 12-19 (15.6±5.3) deep, 16.5-19 (17.7±1.8) wide. Esophagus small, cylindrical, with a posterior dilatation. Esophagus length, 369-391 (380±15.71), 6-6.2 (6.1)% of the body length. Esophagus width 45-57 (51±8.32) at anterior region. Posterior bulb 67-114 (90±33.48) wide. Inconspicuous nerve ring at 166.6 from the anterior region of the body (2.8% of the body length). Inconspicuous excretory duct. Intestine wide and filled with black and brown contents. Vulva near to middle body, slightly post-equatorial, distance from the anterior region of the body to vulva 3.3 mm (55% of body length). Genital system amphidelphic. Tail short, conical, 328.4-348 (338±13.66) long, representing 5.3-5.43 (5.5)% of body length. Eggs numerous, oval, thin-walled, in different stages of development and containing developed larvae near vulva, 101-110 (106±6.66) long by 49-66 (57±11.8) wide.

Remarks

Adult nematodes in the family Rhabdiasidae are common lung parasites of amphibians and reptiles, and the infection occur by skin penetration (Anderson, 2000), orally, or potentially via transport hosts (Baker, 1979). So far, of the approximately 90 species of this genus distributed worldwide (Tkach, Kuzmin, & Snyder, 2014), 21 species have been reported in the Neotropical Region (Table 1), and 5 of them in amphibians from Argentina: Rhabdias fülleborniTravassos, 1926 in Rhinella schneideri (Werner) from Corrientes Province; Rhabdias elegansGutiérrez, 1945 in Rhinella arenarum (Hensel) from Buenos Aires and Salta Provinces and in R. schneideri, Leptodactylus bufonius Boulenger and Odontophrynus americanus (Duméril and Bibron) from Corrientes Province; Rhabdias mucronata Schuurmans Stekhoven, 1952 in Leptodactylus latrans (Steffen) from Corrientes Province; Rhabdias truncata Schuurmans Stekhoven, 1952 in Telmatobius schreiteri Vellard from Tucumán Province and Rhabdias aff. sphaerocephala in Rhinella fernandezae (Gallardo) from Corrientes Province. Moreover, specimens of Rhabdias sp. in Leptodactylus chaquensis Cei and Physalaemus biligonigerus (Cope) were reported from Corrientes and Córdoba Provinces, respectively (González & Hamann, 2007, emsp 2008, emsp 2009; Gutiérrez, 1945; Gutiérrez, Attademo, Guerrero, Peltzer, & Lajmanovich, 2005; Kloss, 1971; Hamann, Kehr, & González, 2012, 2013; Schuurmans Stekhoven, 1952; Sueldo & Ramírez, 1976).

Table 1.

List of host species for Neotropical Rhabdias spp.

Host group	Host species	Species of Rhabdias	Country	Reference
Amphibia
Bufonidae	Anaxyrus kelloggi (Taylor)	R. americanus Baker, 1978	Mexico	Goldberg & Bursey, 2002
	Incilius occidentalis (Camerano)	R. kuzmini Martinez-Salazar	Mexico	Martinez-Salazar & Leon-Regagnon, 2007
		& Leon-Regagnon, 2007
	Incilius marmoreus Wiegmann	R. fülleborni	Mexico	Galicia-Guerrero, Bursey, Goldberg, & Salgado-Maldonado, 2000
	Incilius mazatlanensis (Taylor)	R. americanus	Mexico	Goldberg & Bursey, 2002
	Incilius nebulifer (Girard)	R. fülleborni	Mexico	Galicia-Guerrero et al., 2000
	Rhinella arenarum	R. elegans	Argentina	Gutierrez, 1945
			Paraguay	Kloss, 1971
			Uruguay	Kloss, 1971
		R. fülleborni	Brazil	Travassos, 1926
	Rhinella bergi (Cespedez)	Rhabdias sp.	Argentina	Gonzälez & Hamann, 2007
	Rhinella crucifer (Wied)	R. hermafroditaKloss, 1971	Brazil	Kloss, 1974
	Rhinella fernandezae	R. aff. sphaerocephala	Argentina	Hamann et al., 2013
	Rhinella icterica (Spix)	R. fülleborni	Brazil	Travassos, 1926; Kloss, 1971; Vicente,
				Rodrigues, Gomes, & Pinto, 1991; Luque, Martins, & Tavares, 2005; Pinhäo, Wunderlich, Anjos, & Silva, 2009
	Rhinella marina L.	R. alabialisKuzmin, Tkach and Books, 2007	Costa Rica	Kuzmin, Tkach, & Books, 2007
		R. americanus	Mexico	Espinoza-Jimenez, Garcia-Prieto,
				Osorio-Sarabia, & Leon-Regagnon, 2007
		R. fülleborni	Brazil	Kloss, 1971; Kloss, 1974
			Mexico	Galicia-Guerrero et al., 2000; Goldberg, Bursey, Salgado-Maldonado, Baez,& Caneda, 2002; Espinoza-Jimenez et al.,2007
		R. paraensis Santos, Melo, Nascimento,	Brazil	Santos et al., 2011
		Nascimento, Giese & Furtado, 2011
		R. pseudosphaerocephalaKuzmin, Tkach & Brooks, 2007 (as Rhabdias sphaerocephala Goodey, 1924)	Brazil	Kloss, 1971, emsp 1974
			BermudaIsland	Kloss, 1974
		Rhabdias sp.	Mexico	Galicia-Guerrero et al., 2000; Guill en- Hernändez, 1992
			Nicaragua	Kuzmin et al., 2007
			Costa Rica	Kuzmin et al., 2007
	Rhinella margaritifera (Laurenti)	R. androgynaKloss, 1971	Brazil	Kloss, 1971
	Rhinella rubescens (Lutz)	R. elegans	Brazil	Kloss, 1971
	Rhinella schneideri	R. fülleborni	Brazil	Kloss,1971; Vicente et al., 1991
		R. elegans	Argentina	Kloss, 1971
Craugastoridae	Craugastor occidentalis (Taylor)	R. manantlanensisMartinez-Salazar, 2008	Mexico	Martinez-Salazar, 2008
Cycloramphidae	Thoropa miliaris (Spix)	R. fülleborni	Brazil	Travassos, 1926
Hylidae	Dendropsophus microcephalus(Cope)	Rhabdias sp.[= R. tobagoensis of Goldberg, Bursey, Salgado-Maldonado et al. (2002)]	Mexico	Martinez-Salazar et al., 2009
	Hypsiboas albopunctatus (Spix)	Rhabdias sp.	Brazil	Holmes, Bocchiglieri, Araujo, & Silva, 2008
	Pseudacris hypochondriaca	R. peninsularis Martinez-Salazar,	Mexico	Martinez-Salazar, Falcon-Ordaz,
	(Hallowell)	Falcon-Ordaz, Gonzälez-Bernal,		Gonzälez-Bernal, Parra-Olea, & Perez
		Parra-Olea & Perez Ponce de Leon, 2013		Ponce de Leon, 2013
	Pseudacris regilla (Baird and	R. peninsularis (as R. ranae)	Mexico	Goldberg, Bursey, & Gergus, 2001
	Girard)
	Pseudis platensis Gallardo	Rhabdias sp.	Brazil	Campiäo, da Silva, & Ferreira, 2010
	Scinax staufferi (Cope)	Rhabdias sp.	Mexico	Martinez-Salazar et al., 2009
	Smilisca baudinii Dumeril and	R. americanus	Mexico	Goldberg & Bursey, 2002
	Bibron	Rhabdias sp.	Mexico	Guillen-Hernändez, 1992
	Smilisca cyanosticta (Smith)	R. fülleborni	Mexico	Goldberg et al., 2002
	Trachycephalus typhonius	Rhabdias cf. elegans	Argentina	Present study
Leptodactylidae	Leptodactylus chaquensis	Rhabdias sp.	Brazil	Vicente et al., 1991
		Rhabdias sp.	Argentina	Hamann, Kehr, & Gonzälez, 2006
	Leptodactylus bufonius	R. elegans	Argentina	Hamann et al., 2012
Leptodactylidae	Leptodactylus labyrinthicus (Spix)	R. fülleborni	Brazil	Vicente et al., 1991
(cont.)	Leptodactylus latrans	R. mucronata	Argentina	Schuurmans-Stekhoven, 1952
	Leptodactylus macrosternum Miranda	R. breviensisNascimento, Goncalves, Melo, Giese, Furtado and Santos, 2013	Brazil	Nascimento et al., 2013
	Leptodactylus melanonotus	R. elegans	Mexico	Goldberg et al., 2002
	(Hallowell)	R. elegans	Mexico	Martinez-Salazar & Leon-Regagnon, 2007
		R. ranae Walton, 1929	Mexico	Goldberg & Bursey, 2002
		Rhabdias sp.	Mexico	Mata-Lopez, Leon-Regagnon,& Garcia-Prieto, 2013
	Leptodactylus podicipinus (Cope)	Rhabdias sp.	Brazil	Campiäo, da Silva, & Ferreira, 2009
	Leptodactylus syphax Bokermann	Rhabdias sp.	Brazil	Vicente et al., 1991
	Physalaemus biligonigerus	Rhabdias sp.	Argentina	Gutierrez et al., 2005
	Pristimantis terraebolivaris (Rivero)	R. tobagoensisMoravec & Kaiser, 1995	Tobago	Moravec & Kaiser, 1995
OdontophrynidaeRanidae	Odontophrynus americanus	R. elegans	Argentina	Gonzalez & Hamann, 2009
	Lithobates berlandieri (Baird)	R. fülleborni	Mexico	Leon-Regagnon, Martinez-Salazar, Lazcano-Villarreal, & Rosas-Valdez, 2005
		R. savageiBursey and Goldberg, 2005Rhabdias sp.	Mexico	Leon-Regagnon et al., 2005
			Mexico	Guillen-Hernandez, 1992
	Lithobates forreri (Boulenger)	R. savagei	Costa Rica	Bursey & Goldberg, 2005
	Lithobates cf. forreri	R. pseudosphaerocephala	Mexico	Cabrera-Guzman, Leon-Regagnon, & Garcia-Prieto, 2007
	Lithobates magnaocularis (Frost and Bagnara)	R. ranae	Mexico	Goldberg & Bursey, 2002
	Lithobates tarahumarae (Boulenger)	R. ranae	Mexico	Bursey & Goldberg, 2001
	Lithobates vaillanti (Brocchi)	R. fülleborni	Mexico	Goldberg et al., 2002
		R. pseudosphaerocephala	Mexico	Paredes-Calderon, Leon Regagnon, & Garcia-Prieto, 2004
		Rhabdias sp.	Mexico	Guillen-Hernandez, 1992
	Lithobates sp.	R. pseudosphaerocephala	Mexico	Martinez-Villarreal, 1969
Telmatobiidae	Telmatobius schreiteri	R. truncata	Argentina	Schuurmans-Stekhoven, 1952
Reptilia
Anguidae	Elgaria paucicarinata (Fitch)	Rhabdias sp.	Mexico	Goldberg & Bursey, 2004
Dactyloidae	Anolis frenatus (Cope)	R. anolisBursey, Goldberg & Telford, 2003	Panama	Bursey, Goldberg, & Telford, 2003
	Anolis megapholidotus Smith	R. leonaeMartinez-Salazar, 2006	Mexico	Martinez-Salazar, 2006
	Anolis capito Peters	R. nicaraguensisBursey, Goldberg & Vitt, 2007	Nicaragua	Bursey, Goldberg, & Vitt, 2007

The specimens here studied are closely related to the original description of R. elegans when considering the slightly postequatorial position of the vulva (3.3 vs. 2.4-4.4 mm from anterior region of the body), the body length (5.9-6.4 vs. 4.5-9.5 mm), the absence of lip structures in the anterior end, the cylindrical esophagus (369-391 vs. 314-490 long) and the eggs size (101-110 3 49-66 vs. 91-112 3 52-59). Given that have been found only 2 specimens and that has been stated by several researchers that the conservative morphology of the genus Rhabdias (uniform shape and size) and absence of males in the parasitic generation can result in the incorrect identification of some species (Kuzmin, Tkach, & Synder, 2003; Martínez-Salazar, Pérez-Ponce de León, & Parra-Olea, 2009) more specimens will be necessary for a proper morphological and molecular taxonomic determination.

Cosmocercidae Raillet, 1916

Aplectana Railliet and Henry, 1916

Aplectana hylambatis (Baylis, 1927) Travassos, 1931

(Figs. 2, 3, and 4)

Host: Trachycephalus typhonius (L.) (Anura: Hylidae).

New host record.

Site of infection: small and large intestine.

Prevalence and intensity of infection: 50% (1 of 2); 48.

Voucher specimens deposited: MLP-He 6793.

Description

(Based on 15 specimens). Small, slender nematodes. Cuticle bearing transverse striations, uniform along the body. Triangular mouth surrounded by 3 lips: 1 dorsal with 2 cephalic papillae and 2 ventral with 1 cephalic papilla each. Two amphids. Esophageal bulb well distinct and with chitinous valves. Excretory pore pre-bulbar.

Female

(Based on 10 gravid specimens). Body 2.7-3.6 (3.2±0.28) mm long by 210-280 (244±21.7) wide. Pharynx muscular, 36-60 (45±7.85) long by 26-33 (30±2.11) wide; esophagus muscular, 386-467 (424±74.9) long; valvulated esophageal bulb 95-170 (108±22.35) long by 100-124 (110±6.9) wide. Nerve ring and excretory pore at 131-241 (210±55.15) and 238-483 (230±113.15) from anterior extremity, respectively. Prodelphic uterus. Vulva postequatorial at 1.9-2.3 (2.1±0.16) mm from anterior extremity, with scalloped edges. Eggs thin shelled, 75-91 long by 38-46 (81 3 53) wide. Tail 214-243 (230±10.88) long.

Male

(Based on 5 specimens). Body 2-2.4 (2.2±0.17) mm long by 120-220 (156±34.18) wide. Pharynx muscular, 36-45 (41±3.27) long by 18-24 (23±2.4) wide; esophagus muscular 357-448 (408±33.16) long; valvulated esophageal bulb 71-95 (83±12) long by 79-102 (87±10.04) wide. Nerve ring and excretory pore at 167 and 362-410 (362±23) from anterior extremity, respectively. Spicules long, equal, 296-328 (311±13.6) with a distal articulation that represents, approximately, 1/8 of its total length. Gubernaculum 68-88 (76±8.8) long. Caudal papillae distributed as follow: 3-4 pairs of ventral precloacal papillae, 1 sublateral adcloacal pair, 3 pairs on anterior lip, 1 unpaired papilla on anterior lip, 2 pairs of ventrolateral postcloacal papillae in the median region of the tail and 1 pair next to the posterior end. Tail 121-214 (172±33.6) long.

Remarks

Considering the revision of the genus Aplectana performed by Baker (1980) and the posterior descriptions and synonymies (Baker & Vaucher, 1986; González et al., 2013), A. hylambatis shares with Aplectana chamaeleonis (Baylis, 1929) Travassos, 1931, the characteristic morphology of the spicules. However, the specimens here described are easily distinguished from A. chamaeleonis by the presence of 2 pairs of ventrolateral postcloacal papillae in the median region of the tail. In addition, the papillae disposition, the size of the spicules and gubernaculum in the male and the postequatorial vulva with scalloped edges in the females allowed the specific determination.

In general, the specimens here described are similar in size to those described by González and Hamann (2010) parasitizing Physalaemus santafecinus from Corrientes Province, but slightly smaller than the specimens described by Gutiérrez (1945) in R. arenarum from Buenos Aires Province, those described by Baker (1980) in Rhinella achalensis from Córdoba Province and those described by González et al. (2013) in R. arenarum from San Juan Province, Argentina.

Aplectana hylambatis is a generalist species parasitizing a wide range of anurans hosts. In Argentina, it was found parasitizing 2 families of frogs: Bufonidae and Leptodactylidae (subfamilies Leptodactylinae and Leiuperinae) from Buenos Aires, Córdoba, Corrientes, Salta and San Juan Provinces (González et al., 2013).

This note represents the first record of A. hylambatis as a parasite of hylid frogs from Argentina, and the findings of A. hylambatis and R. cf. elegans constitute the first records of both genera in the Formosa Province. Trachycephalus typhonius represents a new host for both parasites.

The authors thank Dr. Carlos Montoya for his help and hospitality during the stay in Formosa Province. We also thank Luis Pagano and Agustín Abba for their assistance in collecting the amphibian hosts, and the two anonymous reviewers for the helpful comments that greatly improved this manuscript. The authors, Lía Lunaschi and Regina Draghi, are members of the Comisión de Investigaciones Científicas de la provincia de Buenos Aires (CIC). The present study was funded by CIC (Res. N° 243/13) and UNLP (11/N603).