Digenetic helminths of Leptodactylus latrans (Anura: Leptodactylidae) and Rhinella dorbignyi (Anura: Bufonidae) in southern Brazil

Even though anurans have been hosts to an array of helminths, data on the helminth fauna of anurans in Brazil are scarce. This study aims at reporting digenetic helminths on Leptodactylus latrans (Steffen, 1815) and Rhinella dorbignyi (Duméril & Bibron, 1841) in southern of Brazil. Sixty specimens of anurans L. latrans (n= 30) and R. dorbignyi (n= 30) were collected between August 2017 and August 2018. Then, necropsy was performed and helminths were collected, fixed and dyed to be identified in agreement with specific bibliography. Estimated infection indices were prevalence (P%), mean intensity of infection (MII) and mean abundance (MA). Twenty-eight anurans (46.66%) exhibited digenetic helminths, totalizing 255 parasite specimens. Leptodactylus latrans was infected with Gorgoderina megacysta Mañé-Garzón & González, 1978 (Gorgoderidae) (P = 40%), Haematoloechus freitasi Mané Garzón & Solares, 1959 (Haematoloechidae) (P = 23.33%), Catadiscus spp. (Diplodiscidae) (P = 30%), Plagiorchioideae fam. gen. spp. (P = 63.33%) and Halipegus sp. (Derogenidae) (P = 3.33%), whereas R. dorbignyi was infected with Gorgoderina sp. (P = 3.33%), Haematoloechus sp. (P = 3.33%), Catadiscus sp. (P = 3.33%), Plagiorchioideae fam. gen. sp. (P = 3.33%) and Diplostomidae gen. spp. (metacercariae) (P = 6.66%).

Anurans, which have been reported as hosts to a rich diversity of digenetic helminths, play important roles as definitive, intermediate and paratenic hosts. In many cases, they are fundamental to the development of the life cycle of these parasites (Aho, 1990;Campião et al., 2014;Fernandes & Kohn, 2014).
Rhinella dorbignyi (Duméril & Bibron, 1841) (Bufonidae) has been found in the area that stretches from eastern Rio Grande do Sul (RS) state to northern Argentina. It measures from 36 to 68 mm, finds shelter in excavated galleries in flooded areas with grass, where it preys on arthropods (Loebmann, 2005). However, little is known about helminths associated with R. dorbignyi, even though Yamaguti (1958)   Before the necropsy, an external inspection, which included eyes, oral cavity and cloaca, was carried out. Afterwards, trachea, esophagus, stomach, intestine, cloaca, gall bladder, lungs, liver, kidneys, bladder, spleen, heart and ovary/testicles were examined separately. Digenetic helminths were processed for identification in agreement with techniques recommended by .
When they were collected alive, they were kept for 24 hours under cold action in a physiological solution to relax their muscles and later compressed between microscope slide and micro cover glasses and fixed in AFA (93% 70° ethanol, 5% commercial formalin and 2% glacial acetic) por 48h, kept in ethanol 70° GL and dyed with Langeron carmine, i. e., the so-called regressive process method was applied. After  Table 1.
Prevalence of Haematoloechus freitasi was 23% in L. latrans, whereas, in R. dorbignyi, its low rates and the quality of the material did not enable the digeneans of this group to be identified. About 50 species of Haematoloechus have been described worldwide. These parasites are found in frog lungs, as well as in other amphibians (León-Règagnon, 2017). Mollusks and Odonata nymphs act as first and second intermediate hosts, respectively (Olsen, 1974 Adult amphibians are infected when they ingest dragon-fly larvae with metacercariae of the parasite (Krull, 1935;Zelmer & Esch, 1998).
Catadiscus is represented by species that act as parasites in the intestine of amphibians and reptiles; it has also been reported on anurans in South America (Campião et al., 2014). The study reported by this paper found three morphotypes of Catadiscus; two of them in L. latrans and one in R.
dorbignyi is the host of helminths that belong to Catadiscus. Little has been known about the life cycle of Catadiscus species, however representatives of this genus were found to use mollusks as intermediate hosts; young ones are released to the environment, where tadpoles are infected by ingesting metacercariae encysted in aquatic plants (Kehr & Hamann, 2003;Hamann, 2004).
Species that belong Plagiorchioideae use an array of vertebrates as definite hosts (Olsen, 1974). Pereira & Cuocolo, 1941, Plagiorchis Lühe, 1899and Glypthelmins Stafford, 1905. When they are adults, they infect organs of the digestive system of amphibians (Campião et al., 2014;Gomes, et al., 2017). Plagiorchioideae have a quite polemic classification, since researchers have not reached a consensus about morphological differences that differentiate their families, genera and species. To define Plagiorchioideae families, there are two classifications: the one based on morphological characteristics and aspects of their life cycles and the most recent one that based family classification on knowledge of phylogeny that results from molecular studies (Bray, 2008). In this study, specimens found on anurans have characteristics that are similar to the ones of Choledocystus, Plagiorchis (Plagiorchiidae) and Glypthelmins (Glypthelminthidae). However, in order to identify these genera, helminths must be observed by electronic microscopy and molecular studies must be used (Razo-Mendivil & Ponce de León, 2008;Gomes et al., 2017). Therefore, these studies introduce and broadly discusses digenetic helminths that belong to this group. Visible morphological differences were observed in the collected specimens, a fact that suggests the occurrence of more than one species of Plagiorchioideae on L. latrans. Martin (1969) studied the life cycle of Glypthelmins hyloreus (Martin, 1969) and There have been reports of Diplostomidae on anurans in South America (Campião et al., 2014).

Some of them are Choledocystus
According to Hamann & Gonzáles (2009), anurans may get the infection when they are tadpoles as the result of direct penetration of cercariae. Therefore, finding metacercariae of Diplostomidae, even at low infection indices, implies that R. dorbignyi acts as the second intermediate host of helminths of this group in the region. Transmission to definite hosts should occur through the trophic chain.
All helminths found on R. dorbignyi have been reported for the first time in this host, while the digenetic helminth G. megacysta has been reported, for the first time, in L. latrans, in Brazil. Data shown by this study highlight the need to keep carrying out parasitological studies of anurans in Rio Grande do Sul and to expand knowledge about the diversity of helminths associated with this group of vertebrates so as to generate information that may help understand parasite-host relations.