Ultrastructural redescription of Chordodes moraisi (Carvalho, 1942) and Chordodes straviarskii Carvalho and Feio, 1950, and re‐interpretation of Chordodes gestri Camerano, 1904 and Pseudochordodes griffinii (Camerano, 1898) (Gordiida, Nematomorpha)

The Gordiida species Chordodes moraisi (Carvalho, 1942) and C. staviarskii Carvalho and Feio, 1950 are re‐described using scanning electron microscopy (SEM). Chordodes moraisi is characterized by four areole types, two of which correspond to tubercle areoles, the crown areoles appear as single structures and occur on both sides along the ventral midline. Chordodes staviarskii is characterized by a polymorphism of crowned areoles which appear as two types with distinct distribution patterns. Two species, Chordodes gestri Camerano, 1904 and Pseudochordodes griffinii (Camerano, 1898), are synonymized with Neochordodes occidentalis (Montgomery, 1898) and Chordodes peraccae (Camerano, 1894).


Introduction
The Gordiida (Nematomorpha) possess either a smooth cuticle or one with cuticular structures, called areoles. The cuticle of genus Chordodes Creplin, 1847 displays a high diversity of areole types but is characterized by the existence of a specialized type of areole called crowned areoles (De Villalobos and Zanca 2001;Schmidt-Rhaesa 2002a). Chordodes is mainly distributed throughout tropical and subtropical regions, with a few exceptions such as Chordodes morgani Montgomery, 1898 which is only found in North America (Schmidt-Rhaesa and Ehrmann 2001;Schmidt-Rhaesa 2002b). Twenty-five species of Chordodes are recorded in the literature for South America. The majority of the original descriptions were based upon light microscopy of small areas of the cuticle and give scarce information on the differences in shape and distribution of the areoles along the whole body. At present, the scanning electron microscope allows a perfect documentation of the cuticular features. This technique has been applied only to seven species of Chordodes from South America: C. carmelitanus (Miralles, 1989), C. lotus , C. matensis , C. cornuta (De Villalobos and Camino, 1999), C. festae Camerano, 1897 andC. peraccae (Camerano, 1894) (De Villalobos and Zanca 2001), and C. balzani Camerano, 1896(De Villalobos et al. 2004. The aim of this paper is: (1) to redescribe by scanning electron microscopy (SEM) two South American species, Chordodes moraisi and Chordodes staviarskii, and (2) to re-interpret the taxonomic position of Chorodes gestri and Pseudochordodes griffinii.

Material and methods
We investigated the holotypes of Chordodes moraisi, Chordodes staviarskii (Museu Nacional of Rio de Janeiro, Brazil) and the holotypes and the paratypes of Chordodes gestri (Museum Regionale di Science Naturali, Torino, Italy; MZUT G34) and Pseudochordodes griffinii (Museum Regionale di Science Naturali, Torino, Italy; MZUT G39). Body measurements of Chordodes moraisi and Chordodes staviarskii were made with outstretched worms using a ruler. Diameters were measured under dissecting microscope using a calliper ruler. Fragments of all the worms studied were dehydrated in an increasing ethanol series, critical point-dried, mounted on bronze blocks and gold sputter-coated. Observations were performed using a JEOL SLM 1000 scanning electron microscope.

Description
Body length 208 mm, diameter in the middle region 2 mm. Anterior end tapered, without dark ring. Mouth terminal. Terminal end with 484 mm maximum width, with a slightly pronounced furrow in the ventral surface ( Figure 1A). The cloacal opening is situated terminally at the posterior end and is rounded. The cuticle surrounding the cloacal opening is free of areoles but with small bristles. The body colour is uniformly yellowish brown. Dorsal and ventral grooves are hardly visible.
The cuticle shows four types of areoles which are numbered consecutively for the purpose of description and orientation. Types 1, 2 and 3 are distributed all over the body surface while the fourth areolar type is found only in the longitudinal ventral groove. Type 1 areoles are the most abundant ( Figure 1B, C), they are rounded (15-23 mm in diameter) with a smooth surface or a surface partially traversed by superficial furrows. Scattered among these areoles, there are small areoles (type 2, Figure 1B) (6.5-8.9 mm), rounded and with a long (9.3 mm), thin (1.3 mm) and eccentrically situated tubercle (tubercle areole). Type 3 areoles are very similar to type 1 ( Figure 1B, D) in shape and size (21 mm) but different in their short (6.6 mm) and wide (5.3 mm) tubercle with a lateral position (also tubercle areole) and they are scarce or rarely found. In the interareolar groove there are scattered tubercles (13 mm) with pointed ends ( Figure 1C). Type 4 areoles, crowned areoles ( Figure 1D, E), appear as single structures, they are short and sometimes difficult to distinguish, rounded (8.7 mm diameter) with long filiform projections arising from their centre (125 mm) covering the longitudinal ventral groove ( Figure 1D, E).

Comments
Carvalho (1942), based on a female collected from a cockroach, Blatta orientalis, described a new species as Neochordodes moraisi with one type of areole. In 1944, Carvalho studied a male specimen collected from Miniblatta sp. and when comparing it with the female specimen of N. moraisi he noticed that both specimens possessed areoles with filiform projections (crowned areoles) which had not previously been observed in the female, and transferred them to the genus Chordodes. Carvalho (1944), when describing the cuticle of C. moraisi, considered that it displayed three areolar types distributed on the whole surface of the body. Our ultrastructural study allows us to increase the number of areolar types for C. moraisi to four, two of which correspond to tubercle areoles. Carvalho (1944) described crowned areoles as distributed in pairs between or on which filiform projections emerge, shorter in the male and longer in the female. We observed that in the female, crowned areoles never appear in pairs and are distributed only in the longitudinal ventral groove, the projections arising at the areolar apex and not between areoles.
Material examined. SEM posterior end and midbody.

Description
Body length is 324 mm with a diameter of 2.1 mm. The anterior end is tapered, a white tip or dark collar is not present. The posterior end (Figure 2A) is rounded. The cloacal opening is circular, terminal and surrounded by radiating grooves. There are scattered fine bristles in the area between the radiating grooves and the cuticle of the terminal end. The body colour is dark brown with scattered white patches. The whole body is covered with protruding structures, the areoles. Six types of areole can be distinguished. Type 1 areoles ( Figure 2B, C) are the most abundant and are slightly oval or circular in shape, they are structured apically (like a blackberry) with diameters of 8-11 mm and about 5 mm high.
Among these are scattered areoles with the same shape, but with a tubercle on the top (tubercle areole, type 2; Figure 2B, C). This tubercle is about 7 mm long and bears a distinct finger-like projection. Type 3 areoles are more elevated than the first two (14 mm), have a rounded apex and occur in clusters of two, three or more over the whole cuticle ( Figure 2B, C). The areolar types 4 and 5 have filaments on the top and are termed crowned areoles. These crowned areoles occur in clusters of two and are surrounded by 10 or 12 elevated areoles (12-15 mm) of type 6 ( Figure 2C, D). Type 4 crowned areoles bear moderately short filaments (20 mm) and are distributed over the whole body cuticle ( Figure  2C). Type 5 crowned areoles have long filaments (108 mm), and occur only on each side along the dorsal and ventral midline ( Figure 2D). Type 6 areoles have a slightly curved apex and a thin tubercle can be observed in some of them ( Figure 2C). In the interareolar furrow separating areolar types 1, 2 and 3 there are scattered conical tubercles of up to 9 mm length ( Figure 2B, C).

Comments
Chordodes staviarskii resembles C. brasiliensis Janda, 1894, but differs in the areolar groups formed by type 4 and 6 areoles distributed over the cuticle. Carvalho and Feio (1950) described the cuticle of C. staviarskii with four areolar types. Analysis using SEM shows with more precision the differences between areoles, which enables us to increase to six the number of recognizable areolar types.
[Chordodes gestri Camerano, 1904] ( Host. Unknown. Camerano (1904) described a male and a female from Guatemala with three types of areole. One type of short areole of variable dimensions; a second type of large areole arranged in groups of 7-10 between which a tubercle emerges, and a third type of areole distributed among type 1 areoles and with a small tubercle (see also Camerano 1915). These characters are not sufficient for the description of a new species belonging to the genus Chordodes, because the diagnostic feature of Chordodes is the existence of a specialized type of areole, called crowned areoles, which were not described for this species (De Villalobos and Zanca 2001;Schmidt-Rhaesa 2002a;De Villalobos et al. 2004). The reason why Camerano (1904) included this species in the genus Chordodes is seen to be unknown. In a SEM investigation the posterior end of the holotype is undivided, and a ventral groove is present. The cloacal opening is slit-like and surrounded by unbranched or apically branched circumcloacal spines ( Figure 3A, B). The cuticle of the holotype and the paratype contains areoles of one type, which vary in size ( Figure 3C). A megareolar pattern is present mainly in the midbody region. Among some of these large areoles a rounded apex tubercle emerges ( Figure 3C). All the areoles are very close together. In the mid-anterior body region this areolar pattern changes and the areoles are of similar sizes, some with superficial furrows. The interareolar furrow is large with scattered small tubercles ( Figure 3D).
The features observed in the cuticle along the body allow us to relate these two specimens of Chordodes gestri with the holotype and with the specimen from Nebraska of Neochordodes occidentalis (Montgomery, 1898) which were studied by Schmidt-Rhaesa et al. (2003, Figure 13A, F). Therefore, we regard Chordodes gestri as a synonym of Neochordodes occidentalis. Type locality. Altoyac, Vera Cruz Mexico.
Host. Unknown. Camerano (1898) described the new species Chordodes griffinii from a male and a female from Mexico, having two types of areole. Miralles and De Villalobos (1994), basing their decision on Figure 5 and 5a of Camerano (1899), transferred this species to the genus Pseudochordodes Carvalho, 1942. In SEM investigation of the holotype ( Figure 4A, B) and the paratype, the cuticle shows the same characteristics that were observed for Chordodes peraccae (see De Villalobos and Zanca 2001).
It is important to emphasize that although the male specimen shows crowned areoles with short filaments that occur in clusters of two and are surrounded by 8-10 elevated areoles ( Figure 4B), it does not have crowned areoles with very long filaments distributed on both sides along the ventral midline. These types of crowned areole occur only in the females (De Villalobos and Zanca 2001) so their presence should be considered as a sexual dimorphism for this species.
On the other hand, the only possible explanation for the fact that Camerano (1898, 1899) did not notice the presence of crowned areoles in these specimens is that he probably based his analysis on a small portion of the cuticle. Therefore, we regard Chordodes griffinii as a synonym of Chordodes peraccae.

Conclusion
From this investigation we can confirm that Chordodes moraisi and C. staviarskii are valid species. Chordodes staviarskii shows two types of crowned areoles. This polymorphism of crowned areoles has been noted in Chordodes queenslandi Schmidt-Rhaesa, 2002 from Australia and in Chordodes balzani from Trinidad (De Villalobos et al. 2004). Likewise, this study by SEM allows us to confirm the observations previously made by Carvalho (1944) for C. moraisi and Carvalho and Feio (1950) for C. staviarskii: in females the crowned areoles with long filaments are limited to the ventral and/or dorsal and ventral groove. This particular feature in females has been mentioned for other South American species such as C. brasiliensis (Janda, 1894) (Camerano 1897;Carvalho 1946), C. carmelitanus Carvalho and Feio, 1950, C. peraccae (Camerano, 1894) (De Villalobos and Zanca 2001 and C. balzani Camerano, 1896 (Carvalho andFeio 1950;De Villalobos et al. 2004).
Although more studies are necessary on both sexes of species in the genus Chordodes in South America, there seems to be sexual dimorphism, as was considered by Carvalho (1946) for the species C. brasiliensis and by Camerano (1987), Carvalho and Feio (1950) and De Villalobos et al. (2004) for C. balzani, and in the present study for C. peraccae.
The ultrastructural studies of the holotypes and of the paratypes of Chordodes gestri and Pseudochordodes griffinii allow us to consider C. gestri as a synonym of Neochordodes occidentalis, enlarging its distribution to Guatemala, and C. griffinii as a synonym of C. peraccae, enlarging its distribution to Mexico.