Introduction

Turvo State Park (Parque Estadual do Turvo, PST) is a natural reserve located in the northwest of the state of Rio Grande do Sul, southern Brazil, comprising an area of approximately 17,491 hectares. The park is covered almost entirely by subtropical broadleaf or broadleaf deciduous forest. The mammal fauna of the park consists of carnivores such as Galictis sp., Cerdocyon thous, Nasua nasua, Procyon cancrivorus, Eira barbara, wild cats, herbivorous like deer and tapirs, some species of lagomorphs, primates, didelphimorphia, and xenarthrans, many species of chiropterans and as well as numerous rodents (^{KASPER et al., 2007}; ^{MELO et al., 2011}).

One of the genera of Cricetidae rodents that is well represented in Brazil is Akodon Meyen, 1833, with 10 species recorded in this country (^{SILVEIRA et al., 2013}). Among these, Akodon montensis Thomas, 1913 is the only species of this genus found in this reserve (^{MELO et al., 2011}). This species occurs in Paraguay, northern Argentina and southeastern to southern Brazil, including the states of Minas Gerais, Rio de Janeiro, São Paulo, Paraná, Santa Catarina, and Rio Grande do Sul (^{MUSSER & CARLETON, 2005}).

Filarioids of the genus Litomosoides Chandler, 1931, are parasites of the abdominal and thoracic cavities of bats, marsupials and many rodents. Currently, this genus is represented by 43 known species (^{NOTARNICOLA et al., 2012}; ^{NOTARNICOLA & de la SANCHA, 2015}; ^{OVIEDO et al., 2016}). In Brazil, species of Litomosoides have been found parasitizing marsupials (^{BAIN et al., 1980}; ^{MULLER, 1980}), bats (LINS DE ALMEIDA, 1936 apud ^{NOTARNICOLA et al., 2010}; ^{REGO, 1961}; ^{GUERRERO et al., 2002}; ^{BAIN et al., 2003}) and rodents (TRAVASSOS, 1919 apud ^{PADILHA & FARIA, 1977}; ^{PADILHA & FARIA, 1977}; ^{BAIN et al., 1989}; ^{MORAES-NETO et al., 1997}; ^{SIMÕES et al., 2012}). Two species, Litomosoides silvai (^{PADILHA & FARIA, 1977}) and Litomosoides chagasfilhoi (^{MORAES-NETO et al., 1997}) have been recorded in the rodent Akodon cursor Winge, 1887, in Itaguaí and Rio Bonito, in the Atlantic Forest region of the state of Rio de Janeiro (^{PADILHA & FARIA, 1977}; ^{MORAES-NETO et al., 1996}, ¹⁹⁹⁷; ^{MORAES-NETO & LANFREDI 1997}; ^{SIMÕES et al., 2011}, ²⁰¹²).

The aim of the present study was to report on L. silvai parasitizing A. montensis in the Turvo State Park, state of Rio Grande do Sul, Brazil, which is in the Upper Paraná Atlantic Forest ecoregion, part of Atlantic Forest biome. Furthermore, this study provides new morphological data for some structures, such as the cephalic extremity, tail and vagina, spicules and microfilariae tail; and it presents parasitological indices (prevalence, mean intensity and mean abundance) and data on range and infection site.

Materials and Methods

During a survey carried out to study tick-borne diseases (^{KRAWCZAK, 2016}), 164 specimens of small mammals were caught: 117 Akodon montensis, 13 Brucepattersonius iheringi, 1 Cryptonanus guahybae, 7 Didelphis aurita, 1 Euryoryzomys russatus, 9 Oligoryzomys nigripes, 7 Oxymycterus judex, 4 Sooretamys angouya and 5 Thaptomys nigrita. They were caught in the Turvo State Park (latitude 27°00’ S to 27°20’ S; longitude 53°40’ W to 54°10’ W), in the northwest of the state of Rio Grande do Sul, Brazil, between June 2013 and January 2014. However, only some of the rodents, which died due to the procedures for anesthesia and blood collection, were used in the present study: 20 specimens of Akodon montensis Thomas, 1913; two Brucepattersonius iheringi Thomas, 1896; one Oxymycterus judex Thomas, 1909; and one Oligoryzomys nigripes Olfers, 1818. Some other rodents that died were preserved in absolute ethanol until arrival at the Parasitic Diseases Laboratory of the School of Veterinary Medicine and Animal Science, Universidade de São Paulo (USP). These were then analyzed for tick presence and were identified in accordance with ^{Bonvicino et al. (2008)} and ^{Melo et al. (2011)}. These animals were deposited in the Rodent Collection of the “Luiz de Queiroz” Higher Agriculture School, USP, state of São Paulo, Brazil.

The animals were caught using a total of 80 live traps (75 Sherman and five Tomahawk) baited with bacon, banana, apple and peanut butter. These were set up for four consecutive nights during each field campaign. Additionally, three pitfall stationary traps each consisting of five buckets of 42.5 cm in diameter and 60 cm in height, which were connected using a plastic fence (at least 30 m long and 50 cm high), were set up for the same period. The animals thus trapped were then anaesthetized using ketamine and xylazine.

Adult nematode specimens were recovered from the thoracic cavity of A. montensis, stored in Railliet & Henry liquid, and were sent to the Veterinary Parasitology Laboratory, Universidade Federal de Santa Maria, state of Rio Grande do Sul, Brazil, for identification. To study their morphological characteristics, they were then cleared in lactophenol, temporarily mounted, examined under a bright-field microscope (Olympus BX-51) and photographed using a Zeiss Axiophot. Microfilariae from the uterus of one fixed female were dissected and examined. Illustrations were made with the aid of a drawing tube. Measurements are given in micrometers unless otherwise stated. The parasite terms used are in accordance with ^{Bush et al. (1997)}.

Nematode specimens were deposited in the Helminth Collection of the Museum of La Plata (MLP-he). Moreover, specimens of L. silvai parasitizing A. cursor, from Rio Bonito, state of Rio de Janeiro (^{MORAES-NETO et al., 1996}) deposited in the Oswaldo Cruz Institute Helminthological Collection (CHIOC) under number 33518 (one female and one male) were studied to improve the diagnosis.

This study was approved by the Ethics Committee on Animal Use of the School of Veterinary Medicine and Animal Science, USP, under protocol number 2908/2013; by the Chico Mendes Institute for Biodiversity Conservation (ICMBio), under license number SISBIO 38502-1; and by the State Environmental Department of Rio Grande do Sul (SEMA, RS).

Results

Three female filarioid specimens, two anterior extremities and three posterior extremities were recovered from the thoracic cavity of two specimens of A. montensis. Morphometric data on the female and microfilarial specimens are showed in Table 1. The species identification was aided through studying male and female specimens deposited in CHIOC (number 33518).

Litomosoides silvai Padilha and Faria, 1977 (Figures 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11)

Figure 1-7 1-4. Female specimens of Litomosoides silvai parasitizing Akodon montensis from Turvo State Park, state of Rio Grande do Sul. 1. Anterior region of female, lateral view. 2. Anterior extremity, lateral view. 3. Detail of the vulva, lateral view. 4. Uterine microfilaria. 5-7. Male specimen of L. silvai parasitizing A. cursor, from Rio Bonito, state of Rio de Janeiro, CHIOC 33518. 5. Posterior extremity showing the spicules and the cloaca, lateral view. 6. Detail of the left spicule, lateral view. 7. Detail of the right spicule, lateral view. Scale bars: 1 = 200 µm; 2 and 4 = 20 µm; 3, 5-7 = 50 µm. 

Figure 8-11 Detail of anterior extremities of Litomosoides silvai. 8-9. Female and male specimens, respectively, of L. silvai parasitizing Akodon cursor, from Rio Bonito, state of Rio de Janeiro, CHIOC 33518. 10-11. Female specimen of L. silvai parasitizing A. montensis from Turvo State Park, state of Rio Grande do Sul. Scale bars: 8-9 = 25 µm; 10-11: 10 µm. 

Diagnosis: Adult worms small, males four times smaller than females. Cephalic extremity flattened. In apical view, 4 labial papillae distributed in a stretched dorsoventral rectangle; 2 dorsal cephalic papillae. Buccal capsule slender; anterior segment transparent and posterior chitinous portion with irregular external walls; buccal cavity with 2 short enlargements near the base. Female tail slender, with a constriction at tip of tail. Esophagus muscular, becoming slightly glandular in the posterior portion. Vulva posterior to the esophagus-intestine junction. Vagina “s” shaped; vagina perpendicular to the axis of the vulva with a flattened lumen consisting of a simple “s” shaped tube surrounded by muscular walls. Male tail slender; left spicule with handle longer than blade; blade consisting of a membranous ala that converts into a filament; right spicule is a twisted conduit with a heel that is not strongly cuticularized. Microfilariae slender, with sheath closely applied to the body; nuclei not extending to the tip of tail; tail becomes constricted.

Remarks

The specimens found in A. montensis correspond to the species L. silvai and are in accordance with the morphology and morphometrical characteristics given by ^{Padilha & Faria (1977)} and ^{Moraes-Neto et al. (1996)}. The identification was confirmed by the shape of the buccal capsule, flattened anterior extremity, vulva located posteriorly to the esophagus-intestine junction, non-globular shape of the vulva, tail tip with a constriction, shape of the spicules corresponding to the carinii species group, and constricted tail tip of the microfilariae. In the present study, female specimens were compared with previous specimens deposited in CHIOC, which confirmed the identity of the species.

Discussion

Litomosoides silvai was originally described by ^{Padilha & Faria (1977)}, as a parasite of A. cursor. Later on, ^{Moraes-Neto et al. (1996)} redescribed the species including new morphological data and scanning electron microphotographs. These authors showed the distribution of the head papillae, the rugose area, the tip of the tail and the structure of the cuticle at the vulva. However, the position of the head papillae was not stated.

Here, four labial papillae distributed in a stretched dorsoventral rectangle and two dorsal cephalic papillae were observed; amphids were located ventrally in relation to the cephalic papillae. Additionally, the vulva was “s” shaped and differed from the globular shape of other Litomosoides species (^{NOTARNICOLA et al., 2000}; ^{OVIEDO et al., 2016}).

Recently, ^{Simões et al. (2011}, ²⁰¹²) reported the presence of L. silvai parasitizing the sympatric rodents A. cursor and A. montensis in Teresópolis, state of Rio de Janeiro (RJ). All previous reports of L. silvai are from the Atlantic Forest ecoregion, in the state of Rio de Janeiro. Therefore, in the present study, the occurrence of L. silvai in A. montensis in southern Brazil is a new geographical record, in the Upper Paraná Atlantic Forest ecoregion of the northwest of the state of Rio Grande do Sul, which is part of the Atlantic Forest biome.

According to ^{Moraes-Neto & Lanfredi (1997)}, L. silvai parasitizing A. cursor in Rio Bonito, RJ (Serra do Mar Coastal Atlantic Forest ecoregion), presented P = 20.7% (12/58), MI = 18.6 (223/12) and MA = 3.8 (223/58); while ^{Simões et al. (2011)} reported P = 12%, MI = 2 and MA = 0.2, in Teresópolis, RJ, which is a nearby locality. Moreover, these last authors also found L. silvai parasitizing the sympatric rodent A. montensis (P = 2%, MI = 26 and MA = 0.5). The results from the present study show intermediate values that may be related to a different ecoregion or that may show that environmental factors are involved. Interestingly, in Rio Bonito A. cursor was also found to be parasitized with L. chagasfilhoi, with a prevalence of 12%, and both species of filarioids were concurrent in 4 out of the 58 hosts sampled (^{MORAES-NETO & LANFREDI, 1997}).

It seems that L. silvai always is located in the thoracic cavity when it parasitizes A. cursor or A. montensis, while L. chagasfilhoi is located in the abdominal cavity. Both species of filarioid are present in Rio Bonito, while only L. silvai is present in the other localities (Teresópolis and Turvo State Park). Thus, L. silvai is present in both Akodon species, but because of the different southern distribution of their hosts, the filarioid only follows A. montensis, which is the only species of this genus that has been trapped in the study area (^{MELO et al., 2011}). It is interesting to note that among more than 236 specimens of A. montensis that were sampled by one of the present authors (JN, unpublished data) in different localities in Misiones province (Argentina), which is close to Turvo State Park, no filarioid specimens were detected.