Ochoterenella esslingeri n. sp. (Nematoda: Onchocercidae: Waltonellinae) from Bokermannohyla luctuosa (Anura: Hylidae) in Minas Gerais, Brazil, with notes on Paraochoterenella Purnomo & Bangs, 1999

The waltonelline Ochoterenella esslingeri n. sp., a filarial parasite of the anuran Bokermannohyla luctuosa in Minas Gerais, Brazil is described. Several characters distinguish this new species from the 15 species presently included in the genus: the cuticular ornamentation of the female that is restricted to the posterior region of the body, the irregular arrangement of the small, rounded bosses, the postoesophageal vulva, the short glandular oesophagus, the size and shape of the microfilariae, the long left spicule and high spicular ratio. Irregularly arranged, tiny, rounded bosses are common in the monotypic genus Paraochoterenella from an Indonesian ranid, which is not well defined but likely valid. In the Neotropical Realm, the type hosts of the species of Ochoterenella are Hylidae (O. esslingeri n. sp.), Leptodactylidae (two species) and the remaining 13 species were described from the giant toad Rhinella marina (Bufonidae).


INTRODUCTION
T he filarial Onchocercidae Leiper, 1911 from anurans belong to either the Waltonellinae Bain & Prod 'Hon, 1974or the Icosiellinae Anderson, 1958. The presence of a long tail and the absence of cephalic spines distinguish the Waltonellinae from the Icosiellinae, which have a very short tail and bear two pairs of submedian cephalic spines (Anderson & Bain, 1976). The present filariae from an anuran host in Brazil belong to the first subfamily. The Waltonellinae are represented by four genera that were redefined by Esslinger (1986a, b) namely Foleyel-lides Caballero, 1935(= Waltonella Schacher, 1975), Ochoterenella Caballero, 1944, Madochotera Bain & Brunhes, 1968, Paramadochotera Esslinger, 1986, and a fifth, less clearly defined genus, Paraochoterenella Purnomo & Bangs, 1999. Currently, Ochoterenella is the only genus reported from South and Central America. Several species are known only by the female and microfilariae. The genus is remarkably diverse in a bufonid, the giant toad Rhinella marina (Linnaeus, 1758), while only two of a total of 15 species were described from Leptodactylidae. The specimens described here comprise males and females, and possess the main characteristics of Ochoterenella. Intersestingly, the type host is a hylid.

MATERIAL AND METHODS
T he filariae were recovered from a single heavily infected specimen of Bokermannohyla luctuosa (Pombal & Hadad, 1993), captured in the Municipal Park Lajinha (21º 47' 45.3'' S -43º 22' 14.9'' W), Juiz de Fora, state of Minas Gerais, Brazil. Living nematodes could be observed through the host's skin and Original contribution Parasite, 2012, 19, 341-350 the anuran was euthanized. Subsequently, the body cavity was opened by a longitudinal ventral incision from the cloacal opening to the mouth. The filariae were removed from the body cavity and muscular aponeuroses of the thighs. They were fixed in AFA (95 parts 70 % ethanol, three parts 40 % formalin, and two parts glacial acetic acid), stored in 70 % ethanol and cleared in lactophenol for examination. The anterior extremity was studied in apical view, after the head was cut with a razor blade.
In Waltonellinae the cuticular ornamentation is of taxonomic importance (Bain & Prod'Hon, 1974;Esslinger, 1986aEsslinger, , 1989. The presence of cuticular bosses and their arrangement was analyzed, the diameter of these bosses and distances between them were measured at levels defined by Esslinger (1986a): at mid-body of the females and at three times the length of the oesophagus from the apex of the males. The width of the lateral chords in lateral view were measured or illustrated at several levels. Samples of microfilariae were extracted from the uterus near the ovijector for detailed study, and the ovijector was dissected out in one specimen. The ratio of the oesophagus length/body length is given as a percentage, and the vulvar ratio is distance of vulva from anterior extremity/body length, also given as a percentage. The tail ratio is the tail length/body length, expressed as a percentage. The spicular ratio is the length of left/right spicule. Specimens were drawn using a microscope equipped with a camera lucida. Measurements were made on drawings and are given in micrometres, except where otherwise stated. Authority names and dates of the species of Ochoterenella are listed in Table III, as well as the type host and family and its geographic origin. The nomenclature of anuran hosts follows that of Frost (2009).  Tables I, II).

• Male
Anatomy of head and oesophagus as in female, but processes of cephalic papillae shorter. Laterally elongated rectangular cephalic plate 54 × 30. Posterior region helically coiled with three-five turns (Fig.  2B). Rounded cuticular bosses present on the ventral surface of the body from the glandular oesophageal region to the caudal region; bosses initially large, not numerous and irregularly arranged (Fig. 2F), becoming more numerous and organized along the body, to gradually form transverse bands; distance between bosses within a band, measured at 3,700-3,900 from tail tip, about three times shorter than between bands (Fig. 2G). In the area rugosa, the start of which is indistinguishable from the ventral ornamentation of the body, the bosses are smaller and more numerous (Fig. 2H). In the caudal region, the area rugosa is made of smaller bosses and again irregularly arranged (Fig. 2C). Caudal papillae: a single large precloacal papilla (or plaque, according to Esslinger, 1986aEsslinger, , 1987Esslinger, , 1988 with an internal transverse furrow, and four pairs of large sessile papillae; the latter arranged symmetrically in two groups: one precloacal pair; three postcloacal pairs, equidistant (20 to 30 apart), the last pair located about 50 from end of tail (Fig. 2B, C). Spicules distinctly unequal and dissimilar, spicular ratio 3.7-4.1; right spicule simple, distal end tapered and rounded, proximal end expanded and strongly cuticularized for the insertion of the retractor muscle (Fig. 2E); left spicule slender, ventrally curved, with narrowing at the transition between handle and blade; blade about two thirds of the spicule length, lined with narrow alae, slightly widening distally; attennuated membranous tip (Fig. 2D). Original contribution Sheath present, exceeding the length of the larva to a larger or lesser extent at the anterior and posterior extremities (Fig. 1J); tiny refractile granules seen along its entire length. At dissection, microfilariae adhered to each other and to uterine wall. Anterior extremity wider and rounded, body gradually tapering to posterior region; very small cephalic hook (Fig. 1K); short cephalic space, oesophageal axis often conspicuous in anterior end (Fig. 1J); rounded tail tip with terminal nucleus (Fig. 1I, J). Measurements (n = 25, from paratype): body 112 ± 24 (97-132) long, 4.5 ± 0.7 (4-6) wide; cephalic space 2.5 long. Immature microfilariae folded in sheath (Fig. 1I).   T he filariae described in this paper present the main characters of the genus Ochoterenella as redefined by Esslinger (1986a, b): cuticularized parastomal structures, distinct buccal capsule, no lateral or caudal alae. The single discrepancy found is that the "bands of longitudinally oriented bosses in mid-region" which, according to Esslinger (1986a, b), are present in both sexes, are absent in the current females. However, bosses were not entirely absent in the females studied by us, but they were restricted to the posterior region; they are rounded and irregularly arranged. Using the key proposed by Esslinger (1989) for Ochoterenella, which is mainly based on the cuticular ornamentation of females, as males are often unknown, the studied specimens are clearly different from the 15 species described to date.
The oesophagus was not measured in the remaining three species, O. convoluta, O. scalaris and O. vellardi, but detailed descriptions of their cuticular ornamentation, in which they are distinct from the present material, were provided. No illustrations but some measurements (Travassos, 1929) are available for the following two species: O. vellardi females have a long tail (1,000), and males have a shorter left and longer right spicule; two precloacal pairs of papillae are reported, as also in O. convoluta, but this might be an erroneous interpretation, the unpaired papilla being as large and salient as the paired papillae.
The single species of Paraochoterenella must be considered as well, since the definition of the genus does not appear clearly distinct from that of Ochoterenella when comparing Esslinger (1986b) and Purnomo & Bangs (1999). Paraochoterenella javanensis Purnomo & Bangs, 1999, a parasite of the dicroglossid Fejervarya cancrivora (Gravenhorst, 1829) (= Rana cancrivora) in Indonesia, was described as "cuticular bosses minute (< 2-3), non bacillary in appearance, with irregular distribution". Therefore it is rather similar to the present material, but the bosses are not restricted to the posterior region. Moreover, in P. javanensis, both sexes are smaller, and the male differs in the absence of an unpaired precoloacal papilla. In addition, the male is distinct in having two precloacal and four postcloacal pairs of papillae (instead of one and three, respectively), and the area rugosa is organized in transverse bands anterior and posterior to the cloacal aperture.  1929Travassos, 1929Travassos, 1929Esslinger, 1986 Bain & Prod 'Hon, 1974Bain et al., 1979Bain et al., 1979Bain et al., 1979Bain et al., 1979Esslinger, 1987Esslinger, 1987Esslinger, 1988Esslinger, 1988Esslinger, 1988Esslinger, 1989 97  We therefore conclude that the material described herein represents a new species, Ochoterenella esslingeri n. sp.

DISCUSSION
O choterenella esslingeri n. sp. expands the host range of the genus to the Hylidae. Some representatives of this anuran family have been listed as hosts of a few Ochoterenella species that were described from other type hosts (Vicente et al., 1990;Azevedo-Ramos et al., 1998;Goldberg & Bursey, 2008). However, in cases where filarial identifications were not based on detailed morphological studies, these data ought to be considered with caution, since the works of Esslinger (1986aEsslinger ( , 1987Esslinger ( , 1988  Ochoterenella esslingeri n. sp. presents the main generic characters of Ochoterenella, and the slight particularities that were seen in the new species (female ornamentation and position of the vulva) do not deserve a higher taxomic rank than specific. The two species parasitic in Leptodactylidae are too poorly known to draw any conclusions. What remains is an incredibly high diversity of Ochoterenella in the giant toad, R. marina (Travassos, 1929;Caballero, 1944;Bain & Prod'Hon, 1974;Bain et al., 1979;Esslinger 1986aEsslinger , 1987Esslinger , 1988aEsslinger , b, 1989. This poses the question of the origin of this diversity. The geographic range of the giant toad is large, extending from Colombia to Brazil in South America, to Guatemala and Mexico in Central America. The vectors of these filariae are culicids (see review in Bain & Chabaud, 1986). Either diversification might have occurred from a single ancestral species, but this should be supported by some distinctive traits, or the giant toad is parasitized by species from co-occurring anuran hosts in the surrounding environment, or a mixed evolutionary process took place. Paraochoterenella, although not clearly different from Ochoterenella in the original definition, very likely represents a distinct genus with the main character being its caudal papillae: the two pairs of precloacal papillae, distinctly anterior to the cloacal aperture as stressed by Purnomo & Bangs (1999), and the absence of an unpaired papilla. In addition, the area rugosa forms transverse bands near the cloacal aperture. Finally, the cuticular bosses of P. javanensis are strangely drawn and do not seem to be salient (Purnomo & Bangs, 1999, Figs 11-17). In contrast, the absence of a sheath in the microfilaria is not decisive because  , 1929Travassos, 1929Travassos, 1929Esslinger, 1986 Bain & Prod' Hon, 1974Bain et al., 1979Bain et al., 1979Esslinger, 1988a 14. this delicate character is often very difficult to observe, particularly in Giemsa stained blood smears, where the sheath often remains unstained. It is expected that more species will be described in the Oriental Realm and will support this interpretation of a particular lineage of Waltonellinae. The present references on Waltonelinae from this region do not allow a generic assignation (Johnston, 1967;Moravec & Sey, 1985), except that of Petit & Yen (1979) in Malaysia, but it concerns a species of Foleyellides according to Esslinger (1986b). Interesting materials from anurans were reported more recently in India (Sarkar & Manna, 2008;Oinam & Gambbir, 2011), but descriptions were not accurate and the generic assignation to Ochoterenella was not supported.