Nematode parasites of two anuran species Rhinella schneideri ( Bufonidae ) and Scinax acuminatus ( Hylidae ) from Corrientes , Argentina

The nematological fauna of most anuran species from Corrientes province, north of Argentina; has not been studied. We report for the first time the nematode species found in Rhinella schneideri and Scinax acuminatus. Forty four amphibians representing two species (R. schneideri -six males, three females and two juvenilesand S. acuminatus –fifteen males and eighteen females) were collected near the city of Corrientes, between January 2002 and December 2003 and searched for nematodes. R. schneideri contained eight species of nematodes (adults: Rhabdias füelleborni, R. elegans, Oswaldocruzia proencai, Cosmocerca podicipinus, C. parva and Falcaustra mascula; larvae: Porrocaecum sp. and Physaloptera sp.), and S. acuminatus contained three (adults: Cosmocerca parva and Oxyascaris caudacutus; larvae: Physaloptera sp.). We present morphology (scanning electron microscope) and metric information, range extensions, and new host records for these nematode species. Rev. Biol. Trop. 56 (4): 2147-2161. Epub 2008 December 12.

Rhinella schneideri is distributed from the Atlantic coast of Brazil inland through Paraguay to central Bolivia; and southwest to northern and central Argentina and Uruguay, whereas to S. acuminatus is distributed in southern Mato Grosso and Mato Grosso do Sul (Brazil), Paraguay, Bolivia and northern Argentina (Frost 2007).Rhinella schneideri, like the majority species of Rhinella, is highly terrestrial and is an active predator with a generalist diet.On the other hand, S. acuminatus occurs in forests, shrublands, and grasslands, and is very well adapted to anthropogenic areas; it has a generalist diet that it can be considered intermediate between a sit-and-wait and an actively foraging predator (Duré 2004).
The purpose of this study is to report for the first time nematodes harboured by R. schneideri and S. acuminatus.We describe the nematodes found in both species and present new morphological and metric data.Amphibians were transported live to the laboratory, killed in a chloroform (CHCL 3 ) solution; and their snout-vent length (SvL) and body weight were recorded.At necropsy, hosts were sexed and the alimentary canal, lungs, liver, kidneys, urinary bladder, musculature and integument examined for parasites by dissection.Nematodes were observed in vivo, counted and killed in hot distilled water and preserved in 70% ethyl alcohol, cleared in glycerine or lactophenol and examined as temporary mounts.Some specimens were studied by scanning electron microscopy (SEM); these specimens were dehydrated in ethanol series, dried using the critical point technique, coated with gold, and examined with a JSM-5800 scanning electron microscope.Measurements are given in micrometers (µm) unless otherwise stated, as the mean ± SD followed by range in parentheses.Prevalence, mean intensity and mean abundance were calculated according to Bush et al. (1997).voucher specimens of all nematode species were deposited in the Helminthological Collection of the Centro de Ecología Aplicada del Litoral (CECOAL), Corrientes, Argentina.Amphibians were deposited in the Herpetological Collection of CECOAL (S. acuminatus: Cecoal 2405; R. schneideri: Cecoal 2663)
Compared with specimens studied by Gutierrez (1945) the female found in R. schneideri has eggs with a smaller length (Gutiérrez 1945: 91-112 µm); other characters (e.g., body size and esophagus length) are similar.
Family Molineidae Durette-Desset & Chabaud, 1977Genus Oswaldocruzia Travassos, 1917 Oswaldocruzia proencai Ben Slimane & Durette-Desset, 1995 Description: Based on 1 male specimen.Body 10.5 mm x 127.5, with maximum width at level of midbody.Claviform esophagus 540 length.Nerve ring 184 from anterior extremity.Excretory pore 282 from anterior extremity.Caudal bursa: rays 8 arising on root of the dorsal ray, and overlapped with rays 6 in half of its length only.Dorsal ray conical, rising from common base with ribs 8, tapering to pointed tip.Spicules 165.6 in length, distal third divided into 3 branches: blade, shoe and fork.Ribs 4 more short than ribs 5. Branches of the spicule with equivalent length.
Remarks: Ben Slimane et al. (1996) analyzed the morphology of the caudal bursa and identified 72 species of Oswaldocruzia.The species O. proencai is included within the groupe possessing type II bursa.The neotropical Oswaldocruzia have spicula divided in three main branches: blade, shoe and fork and the division of the fork always occurs before the distal third of its length (in holartic species occurs beyond the distal third).Gubernaculum is absent.On the other hand, Ben Slimane and Durette-Desset (1995) proposed the species O. proencai for the specimens collected in the amphibians Bufo paracnemis, Leptodactylus ocellatus and L. bufonius from Paraguay and that was described by Lent et al. (1946) like O. mazzai Travassos, 1935.This is the first report of O. proencai in an amphibian from Argentina.
Family Cosmocercidae Travassos, 1925 Genus Cosmocerca Diesing, 1861 Cosmocerca podicipinus Baker & vaucher, 1984 The caudal portion of the males of this species was extensively detailed for González and Hamann (2004), those that provided new morphologic information related to the structure of the plectanas and the adanal papillae.This species is readily distinguished from other species by the fusion of the underlying sclerotized plectane supports between the plectanes (Baker and vaucher 1984).The Table 2 shows the metric characters of the males and females of C. podicipinus found in R. schneideri.
Cosmocerca parva Travassos, 1925 (Fig. 1B, C, D) This species is distinguished from the Cosmocerca podicipinus in the morphology of the plectanes; in C. parva union between the plectanas of each row does not exist, whereas in C. podicipinus the plectanes on each side of body are fused by underlying sclerotized supports; in addition, the lateral alae in this species are very marked.
The metric characters of the males and females of these nematodes found in R. schneideri and S. acuminatus are present in Table 2.

Remarks:
In South America, the genus Cosmocerca is widely distributed in amphibians and reptiles (Baker 1987).
The general metric characteristics of the specimens of Cosmocerca podicipinus corresponds to others nematodes of the same species analyzed from others hosts of Corrientes, Argentina.Nevertheless, the body size of the females of this study is greater (3.0-7.0 mm) than the found ones in other hosts; for example, the maximum body size of females of C. podicipinus from P. falcipes: 6.0 mm (González and Hamann, 2004); from C. fernandezae: 4.3 mm; from C. bergi: 6.9 mm (González and Hamann, 2007a).The other metric characters, in both males and females, are similar to those found by the authors mentioned for this geographic region.The length of spicule of male collected from S. acuminatus is longer than found in R. schneideri.
With respect to Cosmocerca parva, we found that the female specimens collected from R. schneideri are longer than found in S. acuminatus and the other specimens collected from bufonids of the same area, i.e., from Chaunus granulosus major: 5.1 mm (González and Hamann 2006a); C. fernandezae: 6.7 mm and C. bergi: 5.6 mm (González and Hamann 2007a).
The males of C. parva can have from 5 to 7 pairs of plectanes (Baker and vaucher 1984).
In the present study, the most males of C. parva analyzed from R. schneideri had 5 pairs of plectanes, although some of them had 4 pairs.Each plectane was formed by one interior complete rosette of 12-15 punctations and one exterior complete rosette of 12-15 punctations, and, a relatively inconspicuous underlying sclerotized support which is not fused to other plectanes.Previously reports of plectanes with scanning electron microscopy realized by Mordeglia and Digiani (1998) showed 12-16 punctations in each rosette of these structures.The only male of C. parva found in S. acuminatus presented 6 pairs of plectanes.
The numbers of pairs of adanal papillae varied between 2 and 4 pairs in specimens from R. schneideri but were 3 pairs only in male collected from S. acuminatus.González andHamann (2006a, 2007a) found 2-4 pairs of adanal papillae in males of C. parva from C. granulosus major and C. fernandezae; and 3 pairs only in males from C. bergi.We observed the unpaired little papilla on the anterior lip of anus in males of C. parva in both hosts of this study.
Both, R. schneideri and S. acuminata, represent new host records for C. podicipinus and C. parva.Genus Oxyascaris Travassos, 1920 Oxyascaris caudacutus (Freitas, 1958) Baker & vaucher, 1984 (Fig. 2 A-G) Description: Nematodes with marked dimorphism in size; mature females more than twice as large as male.Oral opening triangular, three small lips present.Four large outer papillae and six minute inner labial papillae in the cephalic extremity.Lateral alae extending from just anterior to nerve ring in both sexes to the preanal region in males and to near midbody in females.Markedly wide and thick at anterior end and tapering rapidly posteriorly.Somatic papillae present, in two subventral and two subdorsal rows.
Remarks: This species was originally described from Hyla nasica (identification of host not definitive according to Freitas 1958), of Sao Paulo, Brazil.Then, it was found in Leptodactylus fuscus and L. mystacinus (Leptodactylidae) from Brazil, too (Fabio 1982) and, in Ololygon fuscovaria (Hylidae) from Paraguay (Baker and vaucher 1985).This is the first report of this species for Argentinean amphibians.
The general morphology of this specimens correspond with others descriptions (Freitas 1958, Baker andvaucher 1985) but we added with this study some metric characters, e.i., the range of body size of male specimens: of these study are greater than of those of the original description, and of the other host (Freitas 1958: 2.78-3.62 mm;Baker and vaucher 1985: 3.50-3.70 mm;present study: 2.65-3.90 mm); in these specimens they have been spicules of greater length than in the specimens of previous studies (Freitas 1958: 122-134 µm;Baker and vaucher 1985: 148-155 µm; present study: 110-161 µm); finally, with respect to gubernaculum, Freitas (1958) does not provide any measurement of this masculine genital structure; on the other hand, Baker and vaucher (1985) found in this structure a length between 36 and 40 µm.The lateral alae got to measure 35 µm in specimens studied by Baker and vaucher (1985), whereas in the present study they reached 39 µm.
The measurements of these specimens are something greater than those found by Freitas and Lent (1941) and by vicente and dos Santos (1976); for example, the total length of body (Freitas and Lent 1941: 8.66-9.78 mm;vicente  and dos Santos 1976: 7.23 mm; present study: 9.37-12.12mm), the length of excretory pore to anterior end (Freitas and Lent 1941: 1.07-1.20 mm;vicente and dos Santos 1976: 1.07 mm;present study: 0.93-1.25 mm) and the distance of vulva to the anterior end (vicente and dos Santos 1976: 5.67 mm;present study: 6.47-8.92 mm).This is the first record of F. mascula in an amphibian from Argentina.

Remarks:
In South America, Porrocaecum larvae were recorded in Pipa pipa (Pipidae) from Peru (Bursey et al. 2001) and in L. chaquensis (Leptodactylidae) from Argentina (Hamann et al. 2006b).These larvae undoubtedly belong to a Porrocaecum species parasitizing predatory birds, with amphibians and reptiles serving as paratenic hosts.
The measures of the larvae found in the bufonid were, in general, greater than the found ones in the hilids.These measurements correspond to the larvae found in other bufonids studied in the same area (González and Hamann 2006a).

DISCUSSION
Four of nine species of nematodes found in amphibians of this study were new records for Argentina: Rhabdias füelleborni, Oswaldocruzia proencai, Falcaustra mascula and Oxyascaris caudacutus, whereas Rhabdias elegans, Cosmocerca parva, C. podicipinus, Porrocaecum sp. and Physaloptera sp. have previously been reported from Argentinean amphibians.All these nematodes have been found in hylids and bufonids of South America, except Porrocaecum sp. that until now had been found in leptodactilids and pipids amphibians only from Argentina (Hamann et al. 2006b) and Peru (Bursey et al. 2001).In the present study no rhabditids were found in S. acuminatus.Besides, this host has less than half species of nematodes that R. schneideri.
All nematode species found in this study were classified as generalist because they were not restricted to a single host species.
In general, the bufonids present high predominance of nematodes in their parasitofauna; the highest species richness of nematodes in South America was reported by Luque et al. (2005) in Bufo ictericus from Brazil with thirteen nematodes species.For other bufonids the reports were the following: six species for B. typhonius, four species for B. marinus and Atelopus bomolochus, and two species for B. glaberrimus from Peru (Bursey et al. 2001, Iannacone 2003), three species for Atelopus spurelli from Colombia (Goldberg and Bursey 2003), and four species for Chaunus granulosus major and C. fernandezae from Argentina (González andHamann 2006a, 2007); lowest species richness of nematodes in Argentinean bufonids were two species for C. bergi (González and Hamann 2007a,b).
In the genus Scinax nematode species richness is, in general, low.In Peruvian anurans only one species was found in S. pedromedinai, two in S. garbei and S. ruba, and three species in S. icterica (Bursey et al. 2001);in Brazil, Azevedo-Ramos et al. (1998) found two species of nematodes in S. trilineata and in S. nebulosa, and, and Goldberg et al. (2007) found two species in S. fuscomarginatus.
In agreement with Bursey et al. (2001), we found different nematode species between R. schneideri (terrestrial habitat), and S. acuminatus (mostly arboreal habitat), suggesting host behaviour and parasites biology are an important factor in the infection of parasites, but, in contraposition of these authors we found the most important intestinal nematodes in terrestrial amphibia were those that infect host by skin penetration (i.e., Rhabdias spp.and Cosmocerca spp.) this result could be explained by the type of habitat in our studies.

ACKNOWLEDGMENTS
This work was partially supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) from Argentina, through grant PIP 2945 to M. I. Hamann.The suggestions made by referees have greatly improved the manuscript.