Detailed morphological description of Habronema clarki Foster & Chitwood, 1937, a nematode parasite of capybaras Hydrochoerus hydrochaeris (Linnaeus, 1766) in Brazil

The genus Habronema has four valid species, of which only two are properly known. The present study aimed to describe in detail the morphology of Habronema clarki through optical and scanning electron microscopy analyses. Our results showed that the labial morphology of this parasite is closer to H. muscae than to H. microstoma. Even so, the characteristic pseudolabia and the slightly convex border of the dorsal and ventral lips are sufficient to distinguish these nematodes. Additional morphological data are presented, thus contributing to the knowledge on this little known nematode. In addition, this study provides new locality records for this species.


Introduction
The Habronematoidea Chitwood & Wehr, 1932, are a highly diverse group of nematode parasites of birds and mammals. Contrary to the International Code of Zoological Nomenclature, the taxon used as the basis for the superfamily is not the oldest, but the most recognized species: Habronema muscae (Carter, 1861) Diesing, 1861 (CHABAUD, 2009). Oddly, although there are four valid species in the genus Habronema Diesing, 1861, which is the type genus of this group, only H. muscae and Habronema microstoma (Schneider, 1866) Ramson, 1911 have been properly studied regarding their biology (ANDERSON, 2000) and morphology (NAEM, 2007).
In Brazil, three species of this genus have been reported (VICENTE et al., 1997) and, of these, only Habronema clarki Foster & Chitwood, 1937, seems to be native, since H. muscae and H. microstoma were probably introduced along with their natural host, the domestic horse.
Despite a recent record of this nematode in capybaras Hydrochoerus hydrochaeris (Linnaeus, 1766) in Bolivia (CASAS et al., 1995), the morphology of this nematode was only discussed in the original description, based on specimens obtained from Hydrochoerus isthmius Goldman, 1912, from Panama (FOSTER;CHITWOOD, 1937). There are no other records relating to the morphology of *Corresponding author: Estevam Guilherme Lux Hoppe Rod. Prof. Paulo Donato Castelane, s/n, CEP 14884-900, Jaboticabal, SP, Brazil e-mail: hoppe@fcav.unesp.br this nematode, and, particularly, its labial morphology, which is essential for the group (CHABAUD, 1958), is scarcely known.
The present study aimed to describe in detail the morphology of Habronema clarki, a nematode parasite of Hydrochoeridae rodents.

Specimens
We analyzed 23 specimens deposited in the helminthological collection of the 'Prof. Dr. Orlando Ferrari' Animal Parasitic Diseases Laboratory, Department of Preventive Veterinary Medicine and Animal Reproduction, FCAV/UNESP, Jaboticabal, state of São Paulo, Brazil. These were obtained from a capybara in Bandeirantes, state of Paraná, Brazil. Specific identification of these nematodes was performed prior to this study, based on the original description of this species (FOSTER;CHITWOOD, 1937).
Three specimens were prepared for scanning electron microscopy analysis on the labial morphology and the others were processed using routine procedures for optical microscopy investigation.

Scanning electron microscopy
For scanning electron microscopy (SEM), the nematodes were post-fixed overnight with 2% osmium tetroxide at 23 °C for 12 h. After that, the specimens were dehydrated in a graded acetone series, critical-point dried with liquid CO 2 , and then mounted on SEM stubs and coated with gold. This study was conducted using a JEOL JSM-5410 scanning electron microscope.

Optical microscopy
For optical microscopy, the parasites were clarified in 80% acetic acid (v/v) and beechwood creosote. Measurements in millimeters, expressed as the mean ± standard deviation and range in parentheses, were obtained using an Olympus® BX-51 microscope equipped with QColor3® image system. The images obtained were processed using the Image ProPlus® v.5.0 software. All measurements on morphological characteristics were based on data from ten specimens of each sex, unless otherwise stated.

Habronema clarki Foster & Chitwood, 1937
General. Large, whitish nematodes in vivo. The dorsal and ventral lips bear two papillae each, with the anterior border slightly convex. The pseudolabia are deeply trilobed, with amphids at their bases. The outer lobes are a little longer than the inner ones, and all three lobes are rounded at the distal extremity ( Figure 1). Bilateral, symmetrical lateral alae may be observed along the body.

Discussion
Among the Habronema species, only H. muscae shows a morphological pattern similar to H. clarki, with well-developed trifurcated pseudolabia, while H. microstoma has simple trapezoid pseudolabia (NAEM, 2007). The characteristic pseudolabia and the slightly convex border of the dorsal and ventral lips are sufficient to differentiate H. clarki from H. muscae. The description of the labial morphology was based on the SEM findings. Nonetheless, analysis of en face preparations is sufficient to view the pseudolabia and papillae. Remarkably, the labial morphology of Habronema zebrae Theiler, 1923, the fourth species of the Habronema genus, is still unknown. Further studies are important in order to evaluate the classification of the latter species within this genus.
In comparison with the other Habronema species registered in Brazil, H. clarki shows a longer stoma, but the general structure of the caudal portion of the male is identical to H. muscae and H. microstoma. However, the left spicule length, which is slightly less than the size proposed in keys for the Habronema species (Chabaud, 1958) and also in the original description (FOSTER; CHITWOOD, 1937), may be used for specific identification.
In the original description, Foster and Chitwood (1937) used the vaginal musculature as characteristic in the specific key that they provided. Moreover, the key proposed by Chabaud (1958) focused specific identification only on male characteristics. In our study, it was found that observation of the vaginal musculature may be very difficult in mature females because of their thickened body. We suggest that the stoma should be used as the identification criterion for females, especially in situations of monosexual infections.
Although this study represents the first official record of H. clarki in Brazil, previous studies have reported occurrences of Habronema nematodes in Brazilian capybaras in the states of São Paulo and Mato Grosso do Sul (SINKOC et al., 2004;TRAVASSOS, 1945), thus suggesting that this species has widespread distribution.

Conclusion
Based on the present data, the trifurcated pseudolabia could be an important characteristic of this genus and, therefore, should be studied in detail. Also, these characteristics should be included in future taxonomic keys in an attempt to improve the beta-taxonomy of this group. Additionally, new locality data is given in our study.