A new species of Cosmocercoides (Nematoda; Cosmocercidae) and other helminths in Leptodactylus latrans (Anura; Leptodactylidae) from Argentina.

Cosmocercoides latrans n. sp. (Cosmocercidae) from the small intestine of Leptodactylus latrans (Anura: Leptodactylidae) from Northeastern Province of Buenos Aires, Argentina is described. The new species can be distinguished from their congeners by a combination of the characters, among which stands out the number of rosette papillae, the lack of gubernaculum and the presence of lateral alae in both sexes. There are over 20 species in the genus Cosmocercoides, and Cosmocercoides latrans n. sp. represents the third species from the Neotropical realm and the second for Argentina. Additionally, seven previously known taxa are reported; Pseudoacanthocephalus cf. lutzi, Catadiscus uruguayensis, Rauschiella palmipedis, Aplectana hylambatis, Cosmocerca parva, Schrankiana sp. and Rhabdias elegans; providing literature records and information on distribution and host-parasite relationships.


INTRODUCTION
Previous reports of endoparasites in Leptodactylus latrans (Steffen, 1815) have been summarized in checklists from South America (Campião et al. 2014, Fernandes & Kohn 2014 and Argentina (González & Hamann 2015). These reports, mainly as isolated records of a taxonomic nature, include several species of acanthocephalans, digeneans, cestodes and nematodes parasitizing this large frog species. The criolla frog, as it is commonly known, inhabits flooded fields and temporary or permanent shallow ponds and is also common to fi nd in periurban places and crops. It is widely distributed in South America, being present in Trinidad and Tobago, Colombia, southern Baker, 1982and Neocosmocercella Baker & Vaucher, 1983(Bursey et al. 2011. The genus Cosmocercoides includes species that commonly parasitize amphibians and reptiles, but also occasionally occur in land snails and slugs (Chen et al. 2018). The aims of the present work are (1) to describe a new species of Cosmocercoides and (2) to increase the knowledge of the diversity of helminths parasites of the Criolla frog, L. latrans.

MATERIALS AND METHODS
Fourty-eight L. latrans (15 females, 23 males, 10 sexually undifferentiated) were captured by active search at night using the visual encounter survey technique (REV) (Crump & Scott 1994) between 2011 and 2012 in the surroundings of La Plata city, Buenos Aires Province, Argentina with authorization of the Dirección de Flora y Fauna, M.A.A.BA (Exp n° 225500-11319/10). The frogs were transported alive to the laboratory, then euthanized with an overdose of 20% benzocaine, and all organs were examined using a Stemi 2000-C Zeiss stereoscope.
Helminths were fixed in 5% formalin and preserved in 70% ethyl alcohol. Trematodes were stained with a 1:6 dilution in 96% ethanol of hydrochloric carmine, dehydrated, cleared in xilol and mounted in Canada balsam. Acanthocephalans and nematodes were cleared in Aman's lactophenol and examined as temporary mounts. The systematic determination of the helminths was carried out following specific keys (Yamaguti 1961, 1963, Petrochenko 1971a, b, Anderson et al. 1974, Gibson et al. 2002, Jones et al. 2005, Bray et al. 2008 and drawings were made with the aid of a drawing tube. Measurements are given in micrometres (μm) unless otherwise stated, as the mean followed by the standard deviation, and the range in parentheses. The values of prevalence, mean intensity and intensity of infection were calculated following the criteria given by Bush et al. (1997).

RESULTS
Of the 48 frogs examined, 34 (71 %) were infected with at least one parasite. Eight helminth taxa (one acanthocephalan, two digeneans and five nematodes) were found.

Description
General morphology: Small, stout nematodes. Sexual dimorphism evident, males half-length of females. Lateral alae present in both males and females, extending from the anterior esophageal region to the tail. Cuticle transversely striated, somatic papillae present. Mouth with three prominent lips, dorsal lip with two sessile papillae, each ventrolateral lip with one ventral sessile papilla and one lateral amphid. Shallow mouth cavity, short pharynx, cylindrical corpus, short isthmus and valved bulb present. Excretory pore anterior to esophageal bulb.
Male ( Gubernaculum absent. Caudal papillae in the following arrangement: 3-4 pairs of precloacal rosette papillae, ventral and arranged in two rows; 1 pair of simple adcloacal papillae and 1 unpaired papilla on the anterior border of the cloaca, 2 pairs of simple and ventral postcloacal papillae in the middle region of the tail and 2 pairs defining a long and acute tip-tale, ventrolateral in position (6-8:3:8). Each rosette papillae presented one circle of punctuations around the central papillae.
Female (  Other reported hosts and geographic range in South America: Aplectana hylambatis is a generalist species that parasitizes a wide range of amphibian hosts. In the Neotropical region, has been observed in different countries (Peru, Paraguay, Uruguay, Brazil) and anuran families, for example, Bufonidae, Ceratophrydae, Hylidae, Leptodactylidae, Leuperidae and Microhylidae (González et al. 2013, Campião et al. 2014, 2016, Aguiar et al. 2015. In Argentina, it is the species of parasitic nematodes of amphibians with the greatest geographical distribution and was found parasitizing Bufonidae, Leptodactylidae and Hylidae from Buenos Aires, Córdoba, Corrientes, Formosa, Salta and San Juan Provinces (González et al. 2013, González & Hamann 2015, Draghi et al. 2015. Remarks: For the specific identification we consider the review of the genus Aplectana carried out by Baker (1980)  Other reported hosts and geographic range in South America: Cosmocerca parva was originally described parasitizing Hylodes nasus (Lichtenstein, 1823) (Anura, Hylodidae) (cited as Elosia nasus) from Angra dos Reis, Rio de Janeiro, Brazil. Since its original description, it has been found parasitizing several amphibian species from Argentina, Brazil, Colombia, Guyana, Paraguay, and Peru (see Campião et al. 2014). In our country, has been found parasitizing 14 amphibian species included in Bufonidae, Odontophrynidae, Hylidae and Leptodactylidae, constituting one of the most common nematode species found parasitizing amphibians (González & Hamann 2015). All reports were made in the Province of Corrientes (Mordeglia & Digiani 1998, González & Hamann 2006, 2009, 2011, 2016, Hamann et al. 2006b, Schaefer et al. 2006. Remarks: The presence of the lateral alae, the arrangement of the plectanes and papillae (5-6 pairs of precloacal papillae, 2-4 pairs of adcloacal plus 1 unpaired papilla on the anterior lip of the cloaca and 5 pairs of post-cloacal) and the size of the spicules and gubernaculum (104 and 63, respectively) allowed the allocation of our specimens to the species C. parva. The rest of the morphometric characters are also consistent with those established for the species in other hosts (see Baker & Vaucher 1984, Vicente et al. 1991, Mordeglia & Digiani 1998, González & Hamann 2006, with some differences that are considered as intraspecific variations. For example, the variability in the number of plectanes (5-7) and cloacal papillae and the absence of the unpaired papilla anterior to the rows of plectanes reported by Mordeglia & Digiani (1998). This is the first record of this species from Buenos Aires Province, and constitutes the first time this species is found parasitizing L. latrans in Argentina, given that it was previously reported parasitizing this host species in Brazil (Santos & Amato 2013).

Atractidae (Railliet, 1917) Travassos, 1919
Schrankiana sp. (Fig. 4a,  Remarks: The specimens here studied were located in the genus Schrankiana owing to the monodelphic condition of the females, and the shape of spicules in the male (short and robust). Also, the esophagus is of cosmocercid type, with an entirely muscular corpus, a marked isthmus and a bulb with chitinous valves. Species of the genus Schrankiana are very similar, and can only be distinguished by metric characters such as the esophagus total length, the size of the vagina, cephalic morphological characters, lateral wing extension, vulva location, and male caudal characteristics (papillae and spicules) (Baker & Vaucher 1988).
More studies are needed to properly describe the cephalic morphology and the pattern of caudal papillae of males of the specimens here found. The present record is the first one of this genus parasitizing amphibians in Buenos Aires Province, Argentina.

Rhabdiasidae Railliet, 1915
Rhabdias elegans Gutiérrez, 1945 ( Other hosts and geographic range in South America: Rhabdias elegans was originally described by Gutiérrez (1945) from specimens collected from the lungs of R. arenarum from Buenos Aires Province, Argentina. It has subsequently been found in several amphibian hosts of the Neotropical realm (for a list of host species for Rhabdias spp. see Draghi et al. 2015    Remarks: Although the totality of the specimens recovered from L. latrans were females, characteristics such as the cylindrical trunk without spines, widened in the anterior region and rounded at its posterior end (6 x 1.05 mm); the cylindrical proboscis, with 16 longitudinal and alternating rows of 5-6 hooks each, progressively increasing in size towards the base; the cylindrical proboscis receptacle, with a double wall and cerebral ganglia near its base; digitiform to claviform lemnisci, not much longer than the receptacle; genital apparatus occupying an average of 9.9% of the total length of the trunk and composed of an uterine bell, uterus and vagina; ovoid eggs without polar extensions and the ventral and subterminal genital pore, allowed the identification.

DISCUSSION
The genus Cosmocercoides can be distinguished from other genera of Cosmocercinae by the morphology of the male´s caudal papillae, described as rosette papillae without plectanas. In contrast, in the genus Cosmocerca the rosette papillae arise prominently of the cuticular surface, and are accompanied by plectanes. Males of the genera Cosmocercella and Neocosmocercella present vesiculated papillae; and those of Paradollfusnema, Raillietnema, Aplectana, Oxyascaris and Oxysomatium have short and mamiliform papillae (Anderson et al. 1974, Bursey et al. 2011. The rosette papillae present in males of Cosmocercoides are formed by circles of punctuations which do not protrude from the cuticular surface (González et al. 2012).
In the specimens described in this study, this feature was confirmed by scanning electron microscope study.  18-19:0:10 (precloacal: adcloacal: postcloacal). Also, the unpaired papilla on the anterior lip of the cloaca is not present (Ramallo et al. 2007). The specimens of C. kumaoni and C. microhylae present lateral alae but can be easily distinguished from C. latrans n. sp. by their shorter tail (130-150 and 157) and rosette papillae pattern, 24:2:10 and 20:0:0, respectively (Wang et al. 1978, Arya 1991. Eleven Cosmocercoides spp. were reported in the Oriental Region, seven in the Sino-Japanese Region, three in the Nearctic Region, two in the Palaearctic Region and two in the Neotropical Region (Bursey et al. 2015, Chen et al. 2018, Tran et al. 2015. Of those in the Neotropical region Cosmocercoides sauria Ávila, Strussmann & da Silva, 2010 was found parasitizing the gymnophtalmid lizard Iphisa elegans Gray, 1851 in Brazil. Cosmocercoides sauria has 4 pairs of precloacal rosette papillae, but unlike the species here described, the males of C. sauria possesses gubernaculum and compared with the species here described are smaller (1.3 vs. 1.48-2.68) and have a shorter tail (54 vs. 178.5-364.14) (Ávila et al. 2010). The second species from this region, C. lilloi, was compared above.
The species here described present a pattern of caudal papillae of 6-8:3:8, which is not known for any species of the genus previously reported. This pattern allows us to conclude that it is a new species for science. Cosmocercoides latrans n. sp. represents the third species of the genus to be reported from Neotropical hosts, and the second one for Argentina.
The current identification of Cosmocercoides species remains mainly based on traditional methods, but, due to the similarity in their morphological characters, it is not easy to identify and distinguish some of them. To accomplish, recently authors as Tran et al. (2015) and Chen et al. (2018) have proposed the use of molecular techniques to sequence and analyze DNA. Unfortunately, it was not possible to apply these techniques in our material due to the specimens were originally fixed in formalin.
The life cycle of the new species is unknown, although according to studies carried out with Cosmocercoides varibilis (Harwood, 1930) , the female lay eggs which develop into first stage larvae in the external environment, and molt twice to the infective third stage larvae, before they infect the host via skin penetration (Anderson 2000). Regarding other helminth species found parasitizing the criolla frog in our study, they are generalist species with an aquatic and indirect life cycle (P. cf. lutzi, C. uruguayensis, R. palmipedis), as well as nematodes species with direct life cycle (C. latrans n. sp., A. hylambatis, C. parva, R. elegans and Schrankiana sp.). Habitat and mobility (water-ground) of the host, associated with the search for prey and parental care of the foam nest, could favor the penetration of the infective stages of the helminths, resulting in a richer and diverse parasitic community (Hamann et al. 2013).