New morphological data of Litomosoides chagasfilhoi ( Nematoda : Filarioidea ) parasitizing Nectomys squamipes in Rio de Janeiro , Brazil

Litomosoides chagasfilhoi, originally described by Moraes Neto, Lanfredi & De Souza (1997) parasitizing the abdominal cavity of the wild rodent, Akodon cursor (Winge, 1887), was found in the abdominal cavity of Nectomys squamipes (Brants, 1827), from the municipality of Rio Bonito, Rio de Janeiro State, Brazil. This study led to addition of new morphological data and a new geographical distribution for this filarioid in Brazil. Several characters were detailed and emended to previous records of L. chagasfilhoi in N. squamipes, and confirming the original description in A. cursor: buccal capsule longer than wide with walls thinner than the lumen, right spicule slightly sclerotized, with membranous distal extremity slender, with a small tongue-like terminal portion, left spicule with handle longer than the blade, whose edges form large membranous wings folded longitudinally.

Litomosoides chagasfilhoi is known to parasitize the abdominal cavities of A. cursor in Rio Bonito (MORAES et al., 1997), andN. squamipes in Sumidouro (MALDONADO et al., 2006), Rio de Janeiro State, Brazil.The leading morphological characteristics of this species are as follows: the buccal capsule is higher than it is wide and has walls thinner than the lumen, and the left spicule presents a handle longer than the blade, whose edges form large membranous wings folded longitudinally.
This paper contributes with new morphological data and a new geographical distribution for this filarioid in Brazil.

Studied area and hosts captures
This study was carried out in Rio Bonito municipality (22°42'31"S, 42°36'35" W, with an altitude of 40 meters), Rio de Janeiro, Brazil, between 2007 and2011.This locality has small Atlantic Forest fragments on the top of the mountain.The rodents were live-caught in wire-mesh live-traps (Tomahawk  and Sherman  ) placed on the floor along the streams, which is the most important habitat of N. squamipes (GENTILE & FERNANDEZ, 1999).Traps were spaced 10 m apart, and baited with peanut butter mixed with banana, oat, and bacon on manioc pieces.Captures transects included 50 traps and were conducted during five consecutive days each trapping session.
Rodents were placed in polyethylene boxes measuring 30 × 18.5 cm, and then asphyxiated with CO 2 and necropsied at the local Center of Control of Zoonoses (CCZ), localized in Rio Bonito municipality.

Helminth collection
The adults of L. chagasfilhoi were collected from the abdominal cavity during necropsies of twenty naturally infected N. squamipes.
For the light microscopy studies, ten adult males and ten adult females of the Litomosoides sp. were washed in a 0.9% physiological solution and fixed in AFA (the solution contained glacial acetic acid, 37% formalin, and 70% ethanol) at 70 °C; they were then washed in distilled water and mounted in lactophenol.The samples were deposited in the CHIOC, preserved in vials with 70% ethanol.
The drawings were made using a draw tube linked to a Nikon Eclipse E200MV light microscope.The helminths' body length measurements were obtained using a Profile Projector (Nikon GC-2).The measurements are given in micrometers (μm), unless otherwise stated.
The adopted classification of the nematodes follows the criteria published by Anderson & Bain (1976) and Chabaud (1975).Confirmation of the taxonomic status of the host was achieved via karyotype analysis and morphology studies of the skull by Dr. Lena Geise from the Departamento de Genética, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro State, Brazil.

Imaging analysis
Images were acquired in a Zeiss Axio Imager.M2 (Carl Zeiss, Germany) equipped with a Plan-Neofluar 40×/1.30NA Oil DIC objective lens and Plan-Apochromat 63×/1.40NA Oil DIC objective lens.The AxioVision software (version 4.8.2,Carl Zeiss) was used to operate the microscope.The images were digitally captured with a charge-coupled device (CCD)-camera (AxioCam HR3, pixel size 6.45 μm × 6.45 μm, 1388 × 1040 pixels).DIC transmitted light images were acquired after setting Köhler illumination using standard DIC optics, and for DIC image acquisition the Nomarski prism bias was kept at the same position to minimize variations.In order to obtain optical sections some images were kept at 0.16 × 0.16 mm in the XY direction and 0.5 μm in the Z direction.The illumination source was Zeiss Colibri light-emitting diode (LED).This multi-user equipment is installed on the Cell Ultrastructure Laboratory of the IOC/FIOCRUZ (Laboratório de Ultraestrutura Celular, IOC/FIOCRUZ).
For the scanning electron microscopy (SEM), the nematodes were fixed in a solution containing 2.5% glutaraldehyde, 4% paraformaldehyde, and 5 mM of calcium chloride in 0.1 M sodium cacodylate buffer (pH 7.2) at room temperature.The samples were later washed three times with 0.1 M sodium cacodylate buffer (pH 7.2), postfixed in 1% osmium tetroxide in 0.1 M cacodylate buffer containing 0.8% potassium ferricyanide and 5 mM of calcium chloride, washed with cacodylate buffer, dehydrated in ethanol, processed in a Critical Point Dryer with CO 2 , sputter coated with gold and gold-palladium, and examined using a JSM-6510LV scanning electron microscope at an accelerating voltage of 15 kV at the Plataforma de Microscopia Eletrônica, IOC/FIOCRUZ.

Ethical aspects
The collection licenses for the rodents were obtained from the FIOCRUZ Ethical Committee on Animal Use (CEUA L-0015/07) and the Brazilian government's Institute for Wildlife and Natural Resources (Number: 24353-1).All animal procedures followed the guidelines for capture, handling and care of mammals of the American Society of Mammalogy and the bio-security procedures of the Brazilian Health Ministry (LEMOS & D'ANDREA, 2014).

Results
Litomosoides chagasfilhoi Moraes Neto, Lanfredi and De Souza, 1997 General -Bodies of both sexes white, long, slender, cylindrical, and slightly tapered at both ends (Figures 1, 5, 6 and 10).Cephalic end slightly rounded, bearing a simple oral opening, without lips, bounded by a thick rim.Four labial papillae, two cephalic ventral papillae, two well-developed amphids, and a pattern of cuticular striations that formed a labyrinth (Figure 17).Buccal capsule longer than wide, and with symmetric and irregular walls thinner than the lumen.Its posterior extremity is embedded into the bulbous anterior end of the muscular esophagus (Figures 2, 8 3 and 5).Right spicule not well chitinized, and distal region slender with a small tongue-like portion at the extremity (Figures 4 and 5).Cloacal opening ventrally located.Tail with one pair of adcloacal papillae, four to six pairs of postcloacal papillae, and one single papilla near the end of the tail with one single nipple shaped papillae (Figures 5 and 19).Area rugosa with bands of discoid cuticular prominences (Figure 18).

Discussion
The analysis of our results led to the conclusion that the species found parasitizing the abdominal cavity of N. squamipes from Rio Bonito, Rio de Janeiro, Brazil is indeed L. chagasfilhoi.The referred filarioid species, until now, known to parasitize the abdominal cavity of the rodent A. cursor from the same locality and from N. squamipes from Sumidouro, Rio de Janeiro (MORAES et al., 1997;MALDONADO et al., 2006).
The filarioids parasitizing N. squamipes showed morphological and morphometric similarities to those found in A. cursor and emended to previous records of L. chagasfilhoi in N. squamipes, when compared to original description.Same variations were observed to: tail length, spicule length, and buccal capsule length.However, the maximum body width differs, showing that the specimens of N. squamipes are wider than those of A. cursor.
Some species of Litomosoides exhibit low specificity for the vertebrate host, which can be due from a recent speciation among rodents (NOTARNICOLA & NAVONE 2002).In fact, previous studies stated that N. squamipes have been found infected by L. khonae and L. navonae (NOTARNICOLA, 2005).It reinforces our finds which remarks L. chagasfilhoi, originally described in A. cursor, as a parasite of the Sigmodontinae rodent, N. squamipes.
Therefore, two species of Litomosoides showed a close relationship to our material, but they differ in certain aspects.The first was L. kohnae (= L.carinii SENSU Vaz, 1931) Bain et al. (1989), which presents a right spicule that is not well esclerotized.The right spicule's distal region is cone shaped, sustained by two thin cuticular sticks, ending in a short membranous thong-like structure, generally folded dorsally in the spicule; the tail is often slightly projected.The other species, L. navonae Notarnicola (2005), differs from L. chagasfilhoi in the following aspects: the anterior region is generally robust, but it is also slender in some specimens.The amphids are slightly salient, featuring a conspicuous channel.There are four externo-labial papillae arranged in a rectangle, and they are aligned dorsoventrally; there is one ventral cephalic papilla.The esophagus is divided, with the glandular portion appearing to be slightly wider than the muscular portion.
One aspect that contributes to this finding is that Litomosoides uses an ectoparasite as vector (BAIN et al., 1989), and that both hosts could share ectoparasites.
In order to clarify the importance of the niche sharing between Litomosoides species, it is necessary additional studies once they can parasitize pleural and/or abdominal cavities.
Thus, the present study led to addition of new data to original description of L. chagasfilhoi and a new vertebrate host record, N. squamipes, in Rio de Janeiro, Brazil.