Introduction

Knowing the parasitological fauna of wild animals, such as snakes, provides fundamental ecological information of both parasite and host. Digenea species have a heteroxenic life cycle, that is, they need 2 or more hosts to reach their adult form and reproduce, and their cycles can occur in aquatic, semi-aquatic or terrestrial environments i^{Marcogliese, 2004}). Snakes are very important in food chains, since they are predators and prey for many different animals (^{Costa et al., 2014}; ^{Hartmann & Marques, 2005}); in addition, they occupy diversified habitats i^{Bernarde, 2012}; ^{Marques et al., 2001}). Such characteristics make these reptiles interesting hosts for digenetic trematodes, since they need to interact with the environment or other hosts to become infected and transport these helminths.

Brazil holds a total of 405 species of snakes (^{Costa & Bérnils, 2018}); however, the diversity of Digenea is poorly known, with 41 species having been recorded parasitizing these reptiles in the country (^{Fernandes & Kohn, 2014}; ^{Quirino et al., 2018}). In the extreme south of Brazil this number is even lower, with 4 Digenea species recorded in snakes of the Pampa Biome (^{Artigas et al., 1942}; ^{Ruiz & Leão, 1942a}, ^b). However, parasitological studies with snakes in other countries that belong to this biome, such as Uruguay and Argentina, have reported 13 and 23 Digenea species, respectively (^{Fernandes & Kohn, 2014}). The Pampa Biome is characterized by native grasslands with riparian forests, hillside forests, shrub formations, wetlands, and rocky outcrops (^{Overback et al., 2015}), which provide a rich snake fauna.

Therefore, the objective of this study was to record Digenea species and their infection indices in 11 snake species in the Pampa Biome, extreme south of Brazil, and contribute to the knowledge of the parasitological fauna of these animals in Brazilian territory.

Materials and methods

There were 42 specimens examined belonging to: Atractus reticulatus (Boulenger, 1885) (n = 1), Phalotris lemniscatus (Duméril, Bibron & Duméril, 1854) (n = 1), Philodryas aestiva (Duméril, Bibron & Duméril, 1854) (n = 1), Erythrolamprus jaegeri (Günther, 1858) (n = 2), Thamnodynastes strigatus (Günther, 1858) (n = 2), Dipsas ventrimaculatus (Boulenger, 1885) (n = 3), Philodryas olfersii (Lichtenstein, 1823) (n = 4), Pseudablabes patagoniensis (Girard, 1858) (n = 6), Helicops infrataeniatus Jan, 1865 (n = 6), Erythrolamprus poecilogyrus (Wied-Neuwied, 1825) (n = 10) (Dipsadidae) and Bothrops alternatus (Duméril, Bibron & Duméril, 1854) (n = 6) (Viperidae). The snakes came from the municipalities of Capão do Leão (31°45'48" S, 52°29'02" W), Pelotas (31°46'19" S, 52°20'33" W), Rio Grande (32°02'06" S, 52°05'55" W), Encruzilhada do Sul (30°32'38" S, 52°31'19" W) and Dom Pedrito (30°58'58" S, 54°40'23" W), Rio Grande do Sul, Brazil. Thirty-four were collected dead on roads from March 2017 to June 2019. The collections were licensed by Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio No. 38913). Four snakes were donated by the Núcleo de Reabilitação da Fauna Silvestre and Centro de Triagem de Animais Silvestres of the Federal University of Pelotas (NURFS-CETAS/UFPel), where they died after a rehabilitation attempt. Four E. poecilogyrus were donated by the Vertebrate Zoology Laboratory of the Federal University of Pelotas, where the specimens were fixed in formalin and conserved in 70°GL alcohol.

The hosts were necropsied for analysis of the infection sites. The helminths were compressed and fixed in AFA, conserved in 70% ethanol, stained with Langeron carmine or Delafield hematoxylin, cleared with creosote and mounted with Canada balsam (^{Amato et al., 1991}). The systematic determination of the helminths was carried out following the approaches given by ^{Artigas et al. (1942)}, ^{Mañé-Garzón and Gortari (1965)}, ^{Mañé-Garzón and Holcman-Spector (1967)}, ^{Artigas and Perez (1969)}, ^{Sey (1983)}, ^{Jones (2005)} and ^{Tkach (2008a}, ^b). The parasitological indices were calculated according to ^{Bush et al. (1997)}. The specimens were deposited in the helminth collection of the Laboratory of Wild Animal Parasitology (CHLAPASIL-UFPel), and the helminthological collection at the Instituto Oswaldo Cruz (CHIOC). The photomicrographs were done in the Olympus® BX 41 microscope with attached camera system and the images were prepared in Adobe Photoshop®CS5.

Results

A total of 178 specimens belonging to 9 taxa of Digenea were found parasitizing 50% of the snakes analyzed. Erythrolamprus jaegeri, D. ventrimaculatus, P. lemniscatus and A. reticulatus showed no association with these helminths. A taxonomic summary of results is provided below.

Opisthogonimidae Travassos, 1928

Opisthogonimus Lühe, 1900

Opisthogonimus megabothrium Pereira, 1928 (Fig. 1A)

Hosts: Erythrolamprus poecilogyrus (Wied-Neuwied, 1825).

Developmental stage: adult.

Site of infection: oral cavity.

Prevalence, mean abundance and mean intensity of infection: 10% (1/10), 0.1 and 1.0 helminth/host.

Locality: Pelotas, Rio Grande do Sul, Brazil.

Specimens deposited: 858 (CHLAPASIL-UFPel).

Opisthogonimus lecithonotus Lühe, 1900 (Fig. 1B)

Hosts: Thamnodynastes strigatus (Günther, 1858), Pseudablabes patagoniensis (Girard, 1858), Philodryas olfersii (Lichtenstein, 1823) and Helicops infrataeniatus Jan, 1865.

Developmental stage: adult.

Site of infection: oral cavity and esophagus.

Prevalence, mean abundance and mean intensity of infection: P. patagoniensis: 83.33% (5/6), 6.16 and 7.4 helminths/host; P. olfersii: 75% (3/4), 3.0 and 4.0 helminths/host; T. strigatus: 50% (1/2), 1.5 and 3.0 helminths/host; H. infrataeniatus: 16.66% (1/6), 0.66 and 4.0 helminths/host.

Locality: Capão do Leão, Pelotas and Dom Pedrito, Rio Grande do Sul, Brazil.

Specimens deposited: 859-866 (CHLAPASIL-UFPel); 39732a-b, 39733 (CHIOC).

Opisthogonimus sulina (^{Artigas, Ruiz & Leão, 1942}) (Fig. 1C)

Hosts: Philodryas aestiva (Duméril, Bibron & Duméril, 1854).

Developmental stage: adult.

Site of infection: oral cavity.

Prevalence, mean abundance and mean intensity of infection: 100% (1/1), 6.0 and 6.0 helminths/host.

Locality: Capão do Leão, Rio Grande do Sul, Brazil. Specimens deposited:856-857 (CHLAPASIL-UFPel).

Opisthogonimus sp.

Hosts: Erythrolamprus poecilogyrus (Wied-Neuwied, 1825), Helicops infrataeniatus Jan, 1865 and Bothrops alternatus (Duméril, Bibron & Duméril, 1854).

Developmental stage: adult.

Site of infection: oral cavity and esophagus.

Prevalence, mean abundance and mean intensity of infection: E. poecilogyrus: 20% (2/10), 0.2 and 1.0 helminth/host; H. infrataeniatus: 16.66% (1/6), 0.16 and 1.0 helminth/host; B. alternatus: 16.66% (1/6), 0.16 and 1.0 helminth/host.

Locality: Capão do Leão and Pelotas, Rio Grande do Sul, Brazil.

Specimens deposited: 961-963 (CHLAPASIL-UFPel).

Remarks

Opisthogonimus parasites the oral cavity and esophagus of snakes, with 12 species recorded in Brazil (^{Fernandes & Kohn, 2014}). Metacercariae of Opisthogonimus have been recorded in several species of anurans in Argentina (^{Hamann & González, 2009}; ^{Hamann et al., 2012}), indicating these vertebrates as secondary intermediate hosts in the helminth cycle. The specimens of Opisthogonimus sp. found in this study presented a large quantity of eggs, making it difficult to visualize the diagnostic structures. Opisthogonimus megabothrium (Fig. 1A) differs from the other species found because it has an acetabulum twice as large as its oral sucker. Opisthogonimus lecithonotus (Fig. 1B) and O. sulina (Fig. 1C) differ in the size of the caeca, being long and short, respectively, and position of the vitellaria, arranged in the same field and in 2 distinct fields, respectively (^{Artigas, 1942}; ^{Artigas & Perez, 1969)}. Despite some records for snakes in Brazil (^{Fernandes & Kohn, 2014}), the association between O. sulina with P. aestiva, O. megabothrium with E. poecilogyrus and O. lecithonotus with T. strigatus, P. patagoniensis, P. olfersii and H. infrataeniatus are reported for the first time in the country.

Plagiorchiidae Lühe, 1901

Styphlodora Looss, 1899

Styphlodora gili^{Mañé-Garzón & Holcman-Spector, 1967} (Fig. 1D)

Hosts: Bothrops alternatus (Duméril, Bibron & Duméril, 1854).

Developmental stage: adult.

Site of infection: kidney.

Prevalence, mean abundance and mean intensity of infection: 33.33% (2/6), 2.16 and 6.5 helminths/host.

Locality: Rio Grande and Capão do Leão, Rio Grande do Sul, Brazil.

Specimens deposited:848-849 (CHLAPASIL-UFPel); 39734a-b (CHIOC).

Styphlodora sp. 1 (Fig. 1E)

Hosts: Pseudablabes patagoniensis (Girard, 1858) and Erythrolamprus poecilogyrus (Wied-Neuwied, 1825).

Developmental stage: adult.

Site of infection: stomach, large intestine and coelomic cavity.

Prevalence, mean abundance and mean intensity of infection: P. patagoniensis: 16.66% (1/6), 12.0 and 72.0 helminths/host; E. poecilogyrus: 10% (1/10), 0.1 and 1.0 helminth/host.

Locality: Capão do Leão and Pelotas, Rio Grande do Sul, Brazil.

Specimens deposited: 850-851(CHLAPASIL-UFPel).

Styphlodora sp. 2 (Fig. 1F)

Hosts: Bothrops alternatus (Duméril, Bibron & Duméril, 1854).

Developmental stage: adult.

Site of infection: large intestine.

Prevalence, mean abundance and mean intensity of infection: 16.66% (1/6), 0.33 and 2.0 helminths/host.

Locality: Dom Pedrito, Rio Grande do Sul, Brazil.

Specimens deposited:852 (CHLAPASIL-UFPel).

Remarks

Styphlodora comprises species that can be found in the gastrointestinal and excretory systems of reptiles. Little is known about the biology of the species of this genus, the vast majority of papers are about taxonomy (^{Byrd et al., 1940}), however, in general, life cycles of the species of Plagiorchidae include 3 hosts (^{Bowman, 2014}). ^{Hamann and Gonzales (2009)} recorded larval forms of Styphlodora in tadpole, indicating amphibians as second intermediate hosts. The morphotypes collected in this study differed mainly by the shape, position and extension of the vitellariae in the body, as well as site of infection. Styphlodora gili was described in B. alternatus in Uruguay (^{Mañé-Garzón & Holcman-Spector, 1967}), and in the present study we also reported parasitizing this snake species, characterizing then the first record of this trematode in B. alternatus in Brazil. We also recorded Styphlodora spp. in P. patagoniensis and E. poecilogyrus and B. alternatus in Brazil.

Figure 1 Digenea associated to snakes in the Pampa Biome, southern Brazil. A. Opisthogonimus megabothrium Pereira, 1928 (Opisthogonimidae) found in Erythrolamprus poecilogyrus (Wied-Neuwied, 1825) (Dipsadidae) (scale bar = 310 μm). B. Opisthogonimus lecithonothus Lühe, 1900 found in Pseudablabes patagoniensis (Girard, 1858) (Dipsadidae) (scale bar = 520 μm). C. Opisthogonimus sulina (^{Artigas, Ruiz & Leão, 1942}) found in Philodryas aestiva (Duméril, Bibron & Duméril, 1854) (Dipsadidae) (scale bar = 650μm). D. Styphlodora gili^{Mañé-Garzon & Holcman-Spector, 1967} (Plagiorchiidae) found in Bothrops alternatus (Duméril, Bibron & Duméril, 1854) (Viperidae) (scale bar = 230 μm). E. Styphlodora sp. 1 found in P. patagoniensis (scale bar = 130 μm). F. Styphlodora sp. 2 found in B. alternatus (scale bar = 490 μm). 

Plagiorchiidae Lühe, 1901

Travtrema^{Pereira, 1929}

Travtrema stenocotyle (Cohn, 1902) (Fig. 2A)

Hosts: Erythrolamprus poecilogyrus (Wied-Neuwied, 1825).

Developmental stage: adult.

Site of infection: stomach, small intestine, large intestine and coelomic cavity.

Prevalence, mean abundance and mean intensity of infection: 40% (4/10), 2.2 and 5.5 helminths/host.

Locality: Pelotas and Capão do Leão, Rio Grande do Sul, Brazil.

Specimens deposited: 853-854 (CHLAPASIL-UFPel).

Remarks

Travtrema stenocotyle is a parasite of the gastrointestinal system of snakes. This species uses mollusks as intermediate hosts and amphibians as secondary intermediate hosts (^{Ostrowski-De Núñez, 1979}, in ^{Pinto et al., 2012}). Travtrema stenocotyle is common in snakes, existing several records in Brazil, including in E. poecilogyrus (^{Fernandes & Kohn, 2014}), and some in Rio Grande do Sul (RS) in Xenodon merremi (Wagler, 1824) , Thamnodynastes pallidus (Linnaeus, 1758) and P. patagoniensis (^{Ruiz & Leão, 1942b}); however, it is reported for the first time in E. poecilogyrus in RS.

Diplodiscidae Cohn, 1904

Catadiscus Cohn, 1904

Catadiscus sp. (Fig. 2B)

Hosts: Erythrolamprus poecilogyrus (Wied-Neuwied, 1825) .

Developmental stage: adult.

Site of infection: stomach.

Prevalence, mean abundance and mean intensity of infection: 10% (1/10), 0.9 and 9.0 helminths/host.

Locality: Pelotas, Rio Grande do Sul, Brazil.

Specimens deposited: 855 (CHLAPASIL-UFPel).

Remarks

Catadiscus Cohn, 1904 comprises species that parasitize the intestines of amphibians and rarely reptiles (^{Hamann, 1992}). Biology of Catadiscus species that parasitize snakes is little known, but the species of this genus in general are transmitted through the ingestion of metacercariae and have mollusks as first intermediate hosts (^{Kehr & Hamann, 2003}). Five species of Catadiscus have been recorded in snakes in South America (^{Fernandes & Kohn, 2014}). Catadiscus uruguayensis Freitas & Lent, 1939 has already been recorded in E. poecilogyrus in Argentina (^{Lunaschi & Drago, 2002}), therefore this trematode is reported for the first time in Brazil for this snake species.

Figure 2 Digenea associated to Erythrolamprus poecilogyrus (Wied-Neuwied, 1825) (Dipsadidae) in the Pampa Biome, southern Brazil. A. Travtrema stenocotyle (Cohn, 1902) (Plagiorchiidae) (scale bar = 340 μm). B. Catadiscus sp. (Diplodiscidae) (scale bar = 200 μm). 

Discussion

All snakes analyzed in this study have a restricted distribution to the South American continent (^{Uetz et al., 2021}). However, most records of Digenea in these hosts are in Argentina and the southeastern and central-western regions of Brazil (Table 1), which highlights the importance of studies in this area.

Even with only 1 individual of P. aestiva sampled, it was possible to report, for the first time, digenetic helminths in this species. On the other hand, there is still a large gap on the parasitic fauna of P. lemniscatus and A. reticulatus, which may be related to the fossorial habit of these species that implies difficulties for their sampling (^{Balestrin et al., 2007}; ^{Quintela & Loebmann, 2009}); therefore, in the present study only 1 individual of each species was sampled.

Habitats and host behavior may influence the parasite fauna of snakes, since those hosts that had higher abundance and richness of parasites are related to semi-aquatic and grassland environments, like E. poecilogyrus, P. patagoniensis, and B. alternatus. These environments are favorable for the development and maintenance of digenetic helminths and their networks of interactions, since parasites of this group use aquatic and semi-aquatic organisms as intermediate and paratenic hosts.

Many digeneans found in the snakes analyzed, such as Opisthogonimus spp., Styphlodora spp. and Travtrema stenocotyle, have as intermediate hosts amphibian anurans (^{Hamann & González, 2009}; ^{Hamann et al., 2012}; ^{Ostrowski-De Núñez, 1979} in ^{Pinto et al., 2012}), which are food resources for several snake species (^{Thaler et al., 2018}; ^{Vasconcelos-Filho et al., 2015}). Some of these parasites were present in B. alternatus, corroborating diet studies of the species, which, besides mammals, can also feed on amphibians, even if sporadically (^{Bellini et al., 2015}). Although the diet of D. ventrimaculatus is specialized in mollusks (^{Quintela & Loebmann, 2009}), which are intermediate hosts of digenetic trematodes, the 3 individuals of this snake analyzed did not present digeneans, what may be related to the low number of hosts sampled, since there are records in Argentina (^{Lunaschi & Sutton, 1985}; ^{Poumarau, 1968} in ^{Fernandes & Kohn, 2014}).

In this context, studies of diet and helminth fauna of snakes are complementary and contribute to the understanding of the biology of species and their role in ecosystems. Such studies generate important information that can be used in conservation programmes for the species and their habitats.