(Kathlaniidae), Including Redescription of the Species and Amendment of Genus Diagnosis Author(S): Felipe B

A Morphological and Molecular Study of Spectatus spectatus (Kathlaniidae), Including Redescription of the Species and Amendment of Genus Diagnosis Author(s): Felipe B. Pereira, Luiz E. R. Tavares, Fernando Paiva, Tomáš Scholz, and José L. Luque Source: Journal of Parasitology, 101(4):468-475. Published By: American Society of Parasitologists DOI: http://dx.doi.org/10.1645/15-764.1 URL: http://www.bioone.org/doi/full/10.1645/15-764.1

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A MORPHOLOGICAL AND MOLECULAR STUDY OF SPECTATUS SPECTATUS (KATHLANIIDAE), INCLUDING REDESCRIPTION OF THE SPECIES AND AMENDMENT OF GENUS DIAGNOSIS

Felipe B. Pereira, Luiz E. R. Tavares*, Fernando Paiva*, Toma´sˇ Scholz†, and Jose´ L. Luque‡ Programa de Pos-Gradua¸´ caõ em Ciências Veterina´rias, Universidade Federal Rural do Rio de Janeiro, Caixa Postal 74.540, 23851-970, Seropédica, RJ, Brazil. Correspondence should be sent to: [email protected]

ABSTRACT: Spectatus spectatus Travassos, 1923 (Nematoda: Kathlaniidae) found in the intestine of Piaractus mesopotamicus (Holmberg, 1887) (Characiformes: Serrasalmidae) from the River Miranda, Mato Grosso do Sul, Brazil is redescribed based on morphological evaluation of newly collected material and examination of type and voucher specimens from the Coleca¸õ Helmintologica´ do Instituto Oswaldo Cruz. The following characteristics previously unreported or insufficiently described were observed: morphology of lips, presence of lamellae-like supplementary lips, presence of pharynx and cuticular ring surrounding the oral opening associated with a complex cuticular apparatus anterior to the pharynx, the number and arrangement of caudal papillae (13 pairs plus 1 unpaired), and the position of nerve ring. Since S. spectatus is the type species of Spectatus, the diagnosis of this Neotropical genus is amended. Synonymy of Chabaudinema Dıáz-Ungrıá, 1968 with Spectatus, first proposed in 1980 by Baker, is supported by the present data. Molecular data that include the first sequence of the SSU rDNA for any species of Spectatus indicate a basal position of S. spectatus within Cosmocercoidea, forming a distant lineage from that comprising 2 species of Falcaustra Lane, 1915. This separate position of S. spectatus supports validity of the genus.

The nematode genus Spectatus Travassos, 1923 (Nematoda: MATERIALS AND METHODS Kathlaniidae) includes only 2 species, Spectatus spectatus One specimen of Piaractus mesopotamicus (Holmberg, 1887) (Char- Travassos, 1923 and Spectatus cichlasomae Moravec and Barusˇ, aciformes: Serrasalmidae) (total body length of 40.5 cm) was collected by 1971, parasites of the posterior intestine of freshwater fishes from hook in the River Miranda, Mato Grosso do Sul, Brazil (208S, 568W), on 8 Central and South America (Moravec, 1998). Its type species, June 2013. Host nomenclature and classification follows Froese and Pauly Spectatus spectatus, has been reported mainly from serrasalmid (2014). Nematodes found in the intestine were washed in saline and 4 fishes (Serrasalmidae) and, less frequently, parasitizing fishes of specimens were fixed in hot (almost boiling) 4% formaldehyde solution the families Bryconidae, Doradidae, and Pimelodidae in the and then preserved in 70% ethanol. For morphological observations, nematodes were cleared in glycerine. The middle body part of another Parana´River basin in Brazil and Argentina (Travassos, 1923; specimen (male) individual was fixed in molecular-graded 96–99% ethanol Travassos and Kohn, 1965; Hamann, 1982a, 1982b; Masi for genetic studies (see below). Drawings were made using a drawing tube Palla´res, 1990; Parra et al., 1997; Moravec, 1998; Santos et al., attached to a microscope Olympus BX51 (Olympus America Inc., Center 2003; Campos et al., 2009). Despite its widespread occurrence, the Valley, Pennsylvania). Measurements are given in range and micrometers morphology of S. spectatus is not sufficiently known, which has (lm), unless otherwise stated. Some specimens (1 male and 1 female) for SEM were dehydrated through a graded ethanol series, dried in generated problems in separating Spectatus from 2 other hexamethyldisilazane, coated with gold, and examined in a JEOL JSM- kathlaniid genera, Falcaustra Lane, 1915 and Chabaudinema 740 1F SEM (Jeol, Tokyo, Japan) operating at 4 kV. The type material Dıáz-Ungrıá, 1968 (see Baker, 1980). Thus, obtaining new and voucher specimens of S. spectatus and Spectatus sp. deposited in the morphological and molecular data on the species of this poorly- CHIOC were observed (see Table I for list of specimens and Table II for known genus is needed. measurements). Newly collected specimens studied were deposited in the Here we provided the emended diagnosis of Spectatus based on CHIOC (Coll. No. 36729a). The systematic classification of the parasites follows Moravec (1998) and Anderson et al. (2009). a redescription of S. spectatus that also includes scanning electron The anterior and posterior ends from the male specimen used for microscopy (SEM) observations using newly collected material molecular analysis, i.e., hologenophores (see Astrin et al., 2013), were and examination of type and voucher specimens of S. spectatus identified based on their morphology (CHIOC 36729b). Taxa for which from the Coleca¸õ Helmintologica´ do Instituto Oswaldo Cruz sequences have been retrieved from GenBank are listed in Table III, (CHIOC). In addition, we assess the phylogenetic relationships of including species used as outgroups; they were chosen based on the phylogenetic analyses of Cernotıˇ ´kovaét al. (2011). Genomic DNA was S. spectatus with other cosmocercoid nematodes for the first time isolated using a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) using the newly generated sequence of the SSU rDNA. following the manufacturer’s instructions. The SSU rDNA gene (18S) was amplified using the protocol and primers PhilonemaF þ PhilonemaPCRr described in Cernotıˇ ´kovaét al. (2011). PCR products were purified through an enzymatic treatment with exonuclease I and shrimp alkaline phosphatase (Werle et al., 1994) and Sanger-sequenced at GATC Biotech (Konstanz, Germany) using the PCR primers and 2 internal primers WF760 and WR800 (Cernotıˇ ´kovaét al., 2011). Contiguous sequences Received 15 March 2015; revised 20 April 2015; accepted 27 April 2015. were assembled in Geneious (Geneious ver. 7 created by Biomatters, * Laboratorio´ de Parasitologia Veterina´ria, Centro de Cienciasˆ available from http://www.geneious.com/) and deposited in the GenBank Biologicas´ e da Saude,´ Universidade Federal do Mato Grosso do database under the accession no. KR139827 (SSU rDNA). Sul, Cidade Universita´ria S/N, Campo Grande, 7907-900, Mato The SSU rDNA datasets were aligned separately using the E-INS-i Grosso do Sul, MS, Brazil. algorithm of the program MAFFT (Katoh et al., 2002) implemented in † Institute of Parasitology, Biology Centre of the Czech Academy of Geneious software, and ambiguously aligned positions were excluded. Sciences, Branisˇovska´31, Ceskˇ eBud´ ejovice,ˇ 370 05, Czech Republic. Gene alignments were then subjected to maximum likelihood (ML) and ‡ Departamento de Parasitologia Animal, Universidade Federal Rural Bayesian inference (BI) analyses. ML and BI trees were calculated under do Rio de Janeiro, Seropedica,´ Caixa Postal 74.540, 23851-970, Rio the GTRþIþG model using PHYML (Guindon and Gascuel, 2003) and de Janeiro, RJ, Brazil. MrBayes (Huelsenbeck and Ronquist, 2001) Geneious plugins, respec- DOI: 10.1645/15-764.1 tively. BI analysis was run for 2 3 106 generations, sampling every 500th

468 PEREIRA ET AL.—REDESCRIPTION OF SPECTATUS SPECTATUS 469

TABLE I. List of specimens of Spectatus Travassos, 1923 deposited in the Coleca¸˜o Helmintologica´ do Instituto Oswaldo Cruz (CHIOC) and analyzed in the present study.

CHIOC accession no. Species identification Specimens status Remarks

8626, 1654, 1656, 20737, 20746 Spectatus sp. Vouchers Poorly preserved; impossible to identify the specific status 1655 Spectatus sp. Vouchers Poorly preserved; only buccal capsule recognizable indicating that the material belongs to a Camallanus sp. 4049, 4051–53, 4060, 4078, 11102–11104 Spectatus spectatus Vouchers Poorly preserved; impossible to identify the specific status 14724–27, 14744, 14745, 14749, 14752, 16054– S. spectatus Vouchers* Poorly preserved; possible to perform some 56, 20507, 20735–36, 20738–45, 20750–53 measurements and observe some features (see remarks and discussion) 37498a, b S. spectatus Vouchers* Well preserved; possible to measure and observe most features 20747 S. spectatus Type males* 4 specimens on 1 slide; poorly preserved but possible to identify and observe caudal papillae 20748 S. spectatus Type females* 4 specimens on 1 slide; possible to identify 20749 S. spectatus Paratype* 1 female poorly preserved; impossible to identify the specific status; lacking the anterior end and broken in middle body

* Specimens used in the present study for taking measurements and for comparisons. tree and discarding a burn-in fraction of 5 3 105 trees. ML nodal support (Figs. 1J, L, 2D, F, G). One ventral unpaired papilla right anterior to was estimated by 100 non-parametric bootstrap replications. cloacal opening (Figs. 1J, L, 2D–H). Region anterior to cloacal opening elevated, internally supported by sclerotized structure (Fig. REDESCRIPTION 1L). Region posterior to cloacal opening delimited by smooth cuticular lines, forming a roughly T-shaped area (Figs. 1J, 2F–H). Gubernac- Spectatus spectatus Travassos, 1923 ulum V-shaped consisting of 2 fused pieces, 1 dorsal and 1 ventral (Fig. (Figs. 1, 2) 1H), 231–272 long in ventral view. Horn-shaped sclerotized structure (Fig. 1I), ventrally adjacent to gubernaculum, visible only in ventral General: Whitish nematodes, medium- to large-sized, with smooth view (Fig. 1J). Spicules equal, with rounded proximal end and pointed cuticle. Cephalic end somewhat inflated (Figs. 1A, C, D, 2B). Cephalic distal tip (Fig. 1K), 751–906 long, representing 4.2–5.3% of total body apex provided with 6 ill-defined lips, like membranous folds with length. Tail conical, ending in tapering tip (Figs. 1J–L, 2D–G), 556–563 undulated edges; 2 dorsal and 2 ventral lips bearing 1 small cephalic long. papillae each; lateral lips bearing amphid papilla with adjacent, tube-like Female (based on 2 adult specimens): Body 18.4–19.3 mm long, structure (Figs. 1B, 2A–C). Eight cuticularized, lamellae-like formations maximum width 903–939; lips 24–29 long. Cephalic end inflation 200–215 originating at lips base protruding anteriorly (supplementary lips), wide. Pharynx 59–92 long. Length of entire esophagus 2.2–2.3 mm, of demarcating circular oral opening (Figs. 1B, 2A). Oral cavity surrounded corpus 1.6–1.7 mm, of isthmus 341–395. Bulb 259–270 long, 328–333 wide. by hexagonal sclerotized ring composed of 6 plates, associated to rather- Entire length of esophagus representing 11.9–12.1% of total body length. complex cuticular apparatus anterior to pharynx, composed of articulated Nerve ring and excretory pore located at 423–503 and 1.7 mm from plates W-shaped in dorso–ventral view (Figs. 1B–D, 2A). Muscular anterior end, respectively. Vagina anteriorly directed from vulva, with esophagus composed of short pharynx, long corpus, slightly dilated well-developed musculature, followed by short muscular ovijector (Fig. isthmus, and posterior pear-shaped bulb, with central fingerprint-like 1F). Amphidelphic uterus full of eggs; ovaries narrow and tubular (Fig. valve (Fig. 1A). Esophagus–intestinal valve well-developed (Fig. 1A). 1F). Vulva 11.2–12.2 mm from anterior end, at 61.2–63.3% of body Nerve ring narrow, encircling esophagus at 2/3 of its length (Fig. 1A). length. Eggs oval, thin-walled, containing large cells (Fig. 1G), measuring Deirids not observed. Excretory pore conspicuous, located somewhat 85–98 3 51–63. Phasmidial pores inconspicuous, difficult to observe, posterior to corpus–isthmus junction (Fig. 1A). Intestine brown, straight, located near tail tip (Figs. 1E, 2I). Tail conical, ending in tapering tip narrow, bulbously inflated on its anterior end. Males with ventral oblique (Figs. 1E, 2I), 418–527 long. muscular bands, precloacal sucker, gubernaculum, and 2 equal spicules (Fig. 1K). Tail conical. Taxonomic summary Male (based on 2 adult specimens): Body 17.1–17.7 mm long, maximum width 829–928; lips 25–27 long. Cephalic end inflation Type host: Piaractus brachypomus (Cuvier, 1818) (Characiformes: 180–190 wide. Pharynx 57–90 long. Length of entire esophagus 2.0–2.1 Serrasalmidae), common name ‘pirapitinga.’ mm, of corpus 1.4–1.5 mm, of isthmus 373–390. Bulb 223–266 long, Other hosts: Piaractus mesopotamicus (Holmberg, 1887) (Characi- 292–320 wide. Entire length of esophagus representing 11.6–11.9% of formes: Serrasalmidae), common name ‘pacu’, Pseudoplatystoma sp. total body length. Nerve ring and excretory pore located at 512 and (Siluriformes: Pimelodidae), Pterodoras granulosus (Valenciennes, 1821) 1.5–1.6 mm from anterior end, respectively. Posterior end of body (Siluriformes: Doradidae), common name granulated catfish, and ventrally bent. Precloacal sucker well-developed, 2.5–2.6 mm from Salminus brasiliensis (Cuvier, 1816) (Characiformes: Bryconidae), com- posterior end. Thirteen pairs of weakly-developed caudal papillae; 7 mon name ‘dorado.’ subventral precloacal pairs, of which 2 are anterior to precloacal Site of infection: Posterior intestine. sucker; 2 adcloacal pairs, 1 subventral and 1 lateral; 4 pairs postcloacal, Type locality: River Chiba´, Porto Saõ Joaõ, Mato Grosso, Brazil. first and second pairs subventral, followed by 1 lateral, last 1 ventral Other localities: River Parana´drainage system in Brazil (State of Mato and near tail tip (Figs. 1J–L, 2D–H). Small subventral phasmidial Grosso: Rivers Saõ Lourenco¸ and Porto Esperanca;¸ State of Mato Grosso pores, located between second and third postcloacal pairs of papillae do Sul: Rivers Aquidauana, Miranda and Salobra; State of Saõ Paulo: 470 THE JOURNAL OF PARASITOLOGY, VOL. 101, NO. 4, AUGUST 2015

FIGURE 1. Spectatus spectatus Travassos, 1923. (A) Anterior end, dorso–ventral view. (B) Cephalic end, apical view. (C, D) Cephalic end, dorso– ventral and lateral views. (E) Posterior end of female, lateral view. (F) Vulval region, lateral view. (G) Egg. (H) Sclerotized horn-shaped structure, ventral view. (I) Gubernaculum, ventral view. (J) Tail of male, ventral view. (K) Posterior end of male, lateral view. (L) Tail of male, lateral view. s, supplementary lip. PEREIRA ET AL.—REDESCRIPTION OF SPECTATUS SPECTATUS 471

FIGURE 2. Spectatus spectatus Travassos, 1923, scanning electron micrographs. (A) Cephalic end, apical view (asterisks indicate cuticular plates forming cuticular ring). (B) Cephalic end, sublateral view. (C) Detail of amphid. (D) Caudal region of male, lateral view. (E) Posterior end of male, sublateral view (arrowheads indicate caudal papillae). (F–H) Caudal and cloacal region of male, ventral and subventrals views, respectively (star indicates T-shaped area formed by smooth cuticular lines posterior to cloacal opening). (I) Tail of female, lateral view. a, amphid; c, cephalic papilla; g, gubernaculum; p, phasmid; s, supplementary lip; u, unpaired papilla; vs, ventral sucker. 472 THE JOURNAL OF PARASITOLOGY, VOL. 101, NO. 4, AUGUST 2015

Pirassununga, Porto Cabral, River Parana´); and Argentina (River stone, 1930, Nemhelix bakeri (Morand and Petter, 1986), and Parana´). Cosmocercoides dukae (Holl, 1928) (95.1, 94.9, 94.8, and 94.4% Remarks of sequence identity, respectively). In the cladogram construct- ed using ML and BI, S. spectatus differed from all the Nematodes found in P. mesopotamicus resemble more or less those of Spectatus spectatus Travassos, 1923, described from the congeneric host P. cosmocercoid genera, forming a separate branch (Fig. 3). The brachypomus (Cuvier, 1818) from Pantanal region, Mato Grosso do Sul, topography of the phylogenetic trees generated using ML and Brazil. To confirm species identification, all specimens of S. spectatus and BI was the same. Spectatus sp. deposited in the CHIOC (including type material of the Comparison of the new sequence of S. spectatus with those of former species) were observed. Most of this material was in bad condition and some of these specimens were completely destroyed, thus making it cosmocercoid parasites available in GenBank has shown that this impossible to observe their morphology and confirm species identification species forms a separate, well-supported lineage which is basal (see Table I). and a sister group of the clade that includes all genera of The main differences observed between the present material and that Cosmocercoidea (Fig. 3). The genus Falcaustra, represented by 2 described by Travassos (1923, 1956) were in the presence of a pharynx and species, forms a monophyletic well-supported lineage among cuticular ring associated with a complex, sclerotized apparatus in the oral region (not described by Travassos 1923, 1956) and in the number and cosmocercoid nematodes (Fig. 3). arrangement of caudal papillae in males. However, all these features not originally reported (or in the case of caudal papillae, incompletely DISCUSSION described) in the species description by Travassos (1923) were now observed in the type and voucher specimens, including the number and The present observation of a pharynx and a cuticular ring arrangement of caudal papillae, which was stable in all males studied. associated with a complex sclerotized apparatus in the oral Another slight difference represents the position of nerve ring. Whereas Travassos (1956) reported this structure to be situated at the level of the region of S. spectatus is important for resolving the validity of excretory pore, it is actually located much anterior and does not vary in its the genus Chabaudinema. This monotypic genus was proposed position (Fig. 1A). by Dıáz-Ungrıá (1968) to accommodate Chabaudinema amer- Light and SEM observations have also revealed several features not icanum Dıáz-Ungrıá, 1968 from Colossoma macropomum described by Travassos (1923) and Travassos (1956), e.g., detailed structure of lips, cephalic papillae, and amphids, the presence of (Cuvier, 1816) (Characiformes: Serrasalmidae) found in Vene- supplementary lips, the position of phasmids in males and females, the zuela. This genus was differentiated from Spectatus by its detailed structure and morphology of the gubernaculum, the presence of a possession of a pharynx and a complex cuticular ring anterior horn-shaped sclerotized structure ventrally adjacent to the gubernaculum, to it. However, Baker (1980) considered Chabaudinema as and smooth cuticular lines forming a roughly T-shaped area just posterior to the cloacal opening (Figs. 1B, H–J, 2A, C, F–I). junior synonym of Spectatus whereas Moravec (1998) and It is worth noting that the referred horn-shaped structure (Fig. 1H) is Anderson et al. (2009) retained the former genus as valid. The quite similar to the telamon present in strongylid nematodes and may only character used to separate these genera was the presence work analogously to it. Because the telamon is considered a typical feature of parasites from the superfamily Strongyloidea Baird, 1853 (sensu or absence of the features referred above. Thus, the present Hodda, 2011), we decided to describe this ‘‘accessory piece’’ as just a observations support the synonymy of Chabaudinema from ‘‘horn-shaped structure’’ in order to avoid confusion. Spectatus, first proposed by Baker (1980). Besides S. spectatus, only 1 nominal species has been assigned to the In fact, Spectatus americanum (syn. C. americanum)isquite genus, namely S. cichlasomae Moravec and Barusˇ, 1979, a parasite of Nandopsis tetracanthus (Valenciennes, 1831) (Perciformes: Cichlidae) from similar to S. spectatus. The only differences between these Havana, Cuba (Moravec and Barusˇ, 1971). This species was described species are in the body size, i.e., S. americanum is larger than based on only 1 female, is pending revision (Moravec, 1998), and requires S. spectatus (total length in males 30 mm vs. 12.7–23 mm; 34 redescription. Furthermore, genus assignation of S. cichlasomae in mm vs. 13.9–23.7 mm in females), and the former species Spectatus is questionable because the morphology of this species does not largely correspond to that in the generic diagnosis of Spectatus. possesses only 10 pairs of caudal papillae (instead of 13 as in However, S. cichlasomae is tentatively retained in Spectatus until new S. spectatus). In addition, both species occur in separate river morphological and molecular data are available. basins (Amazon vs. Parana´). In other morphological charac- Based on the new data, genus diagnosis of Spectatus is amended. teristics, such as the shape of gubernaculum and spicules, Spectatus Travassos, 1923 pattern of caudal papillae, the shape of the cuticular ring surrounding the oral opening, and the sclerotized apparatus in Diagnosis: Buccal opening surrounded by six external lips. Four cephalic papillae, 2 dorsal and 2 ventral, plus 2 lateral amphids, located in the anterior region, both taxa are indistinguishable. However, edge of lips. Supplementary lips present or not. Buccal cavity surrounded as we had no access to the type material of S. americanum,we by hexagonal sclerotized ring associated with rather-complex cuticular tentatively retain S. americanum as a valid species, but its apparatus anterior to pharynx. Esophagus with short anterior pharynx, validity (or synonymy with S. spectatus) has to be confirmed followed by corpus, isthmus, and posterior pear-shaped bulb; pre-bulb absent. Male with 2 equal spicules and precloacal sucker. Gubernaculum by a study of newly collected material. present, V-shaped, consisting of 2 pieces, 1 dorsal and 1 ventral. Didelphic Moravec et al. (1997) reported a single, young female of and amphidelphic uterus. Vulva near mid-length of body or slightly Spectatus sp. from P. mesopotamicus in Parana´River and, posterior. Eggs oval not containing larvae. Tail conical in both sexes. according to the authors, the specimen was quite similar to S. Intestinal parasites of freshwater fishes. spectatus. However, Moravec et al. (1997) did not identify this young female as S. spectatus because, in contrast to the original RESULTS OF MOLECULAR EXAMINATION description by Travassos (1923), their specimen possessed a The partial sequence of the SSU rDNA (1,745 base pairs distinct pharynx. Now, there is no doubt that this young female long) of S. spectatus was obtained. This sequence was most belongs to S. spectatus, because they occur in the same host similar to those of Raillietnema sp., Cruzia americana Maple- species and river basins. TABLE II. Selected comparative measurements of Spectatus spectatus Travassos, 1923 deposited in the Coleca¸õ Helmintologica´ do Instituto Oswaldo Cruz (CHIOC) and newly collected by the present authors with the appropriate accession number inside parentheses. NM ¼ not measured.

Host and locality

Piaractus brachypomus (Cuvier, 1818)* Piaractus mesopotamicus (Holmberg, 1887)† Pacu´ (vernacular name; host not identified)‡

Parque Nacional River SaõJoaõ, River Salobra, do Pantanal, River Miranda, River Parana´, River Salobra, River Parana´, Mato Grosso§ Mato Grosso Mato Grosso Mato Grosso do Sul|| Saõ Paulo Mato Grosso Saõ Paulo

Male n (CHICO) 5 (4052) 5 (20507) 1 (37498a) 2 (36729a) 5 5 2 (20750, 20751) (14724, 14727, 14744) (20735, 20736a–d) Total body length (mm) 12.7–13.1 16.3–18.8 17.2 17.1–17.7 21.7–23.0 15.9–17.5 20.4–21.6 Maximum body width 580–766 NM 790 829–928 917–1000 540–610 662 Pharynx length 60–63 54–72 43 57–90 67–78 47–68 69 Corpus length (mm) 13.1–13.7 1.6–1.7 1.3 1.4–1.5 1.6–1.7 1.6–1.8 1.1–2.0 Isthmus length 270–341 306–388 314 373–390 281–363 223–420 308–383 Bulb length 251–275 172–260 212 223–266 241–283 236–341 228–397 Bulb width 295–316 230–289 231 292–320 296–341 290–319 353–362 Esophagus/body length ratio (%) 14.1–15.3 11.9–13.1 10.8 11.6–11.9 10.0–10.8 13.0–14.0 10.0–13.0 Nerve ring to anterior end 445–472 484–541 420 512 482–569 486–555 NM Excretory pore to anterior end (mm) 1.4–1.4 1.6–1.7 1.4 1.5–1.6 1.7–1.9 1.6–1.8 1.9–2.0 Gubernaculum length 218–279 241–278 235 231–272 220–294 245–273 266–275 Spicule length 832–879 831–887 955 751–906 867–1000 788–860 909–934 Spicule/body length ratio (%) 6.6–6.7 4.5–5.1 5.6 4.2–5.3 3.8–4.6 4.5–5.1 4.3–4.4 Tail length 383–525 507–592 515 556–563 442–631 380–573 446–577 Female n (CHICO) 6 (4052) 5 (20507) 1 (37498b) 2 (36729a) 5 5 1 (20752)

(14724, 14744, 14745) (20739a–c, 20740, 20743) OF AL.—REDESCRIPTION ET PEREIRA Total body length (mm) 13.9–21.1 16.6–20.7 24.1 18.4–19.3 21.7–23.1 16.5–17.5 23.7 Maximum body width 924–947 NM NM 903–939 NM 609–709 721 Pharynx length 74–85 50–82 70 59–92 62–82 65–80 NM Corpus length (mm) 1.6–1.7 1.6–1.7 1,76 1.6–1.7 1.7–1.8 NM NM Isthmus length 379–382 291–386 226 341–395 281–455 371 NM Bulb length 250–291 235–276 318 259–270 232–374 285–333 NM Bulb width 309–325 279–310 346 328–333 315–337 293–379 NM Esophagus/body length ratio (%) 11.4–15.7 10.9–12.8 9.5 11.9–12.1 10.3–11.5 14.1–14.9 NM Nerve ring to anterior end 521–565 490–557 500 423–503 538–620 503 NM Excretory pore to anterior end (mm) 1.6–1.9 1.7–1.8 1.7 1.7 1.8–1.9 1.6–1.8 NM

Vulva to anterior end (mm) 9.1–13.3 10.1–12.8 14.5 11.3–12.2 14.0–14.3 10.3–11.7 15.5 SPECTATUS SPECTATUS Relative position of vulva (%) 63.1–65.6 60.9–64.9 60 61.2–63.3 61.9–64.3 62.7–66.6 65.1 Tail length 399–546 368–383 NM 418–527 520–527 393–501 NM

* Type host. † Host from the present study. ‡ Probably Myleus pacu. § Type locality. || Locality from the present study. 473 474 THE JOURNAL OF PARASITOLOGY, VOL. 101, NO. 4, AUGUST 2015

TABLE III. List of cosmocercoid nematodes (Cosmocercoidea) and two outgroups whose sequences of the small subunit (SSU) rDNA were used for analysis; associated with geographical origin of each sample.

GenBank Parasite Host species Host group and habitat Geographical origin accession no.

Atractidae Rondonia rondoni (Travassos, Pterodoras granulosus Freshwater fish Peru DQ442679 1920) (Valenciennes, 1821) Labeonema sp. Synodontis ocellifer Boullenger, Freshwater fish West Africa EF375487 1900 Cosmocercidae Cosmocercoides dukae (Holl, Deroceras parnomitanum (Lessona Terrestrial gastropod USA FJ516753 1928) and Pollonera, 1882) Nemhelix bakeri (Morand and Helix aspersa Muller,¨ 1774 Terrestrial gastropod UK DQ118537 Petter, 1986) Raillietnema sp. Ceratobatrachus guentheri Anuran amphibian Canada DQ503461 Boullenger, 1884 Kathlaniidae Cruzia americana Maplestone, Didelphis virginiana (Kerr, 1792) Terrestrial mammal Not specified (probably United U94371 1930 States) Falcaustra araxiana (Massino, Emys orbicularis (Linnaeus, 1758) Freshwater turtle Iran KM200716 1924) Falcaustra catesbeianae Walton, Lithobates catesbeianus (Shaw, Bullfrog amphibian Japan AB818380 1929 1802) Myolaimidae Myolaimus sp.* No information available Not specified MSU81585 Rhabditidae Teratorhabditis synpapillata* No information available Not specified AF083015 Sudhaus, 1985

* Outgroups.

Moravec et al. (1997) also pointed out the morphological and Maplestone (1926), who considered them synonymous, i.e. similarity of Spectatus with Desmognathinema Baker, Goater and Spectatus as a junior synonym of Falcaustra. Esch, 1987 (syn. Wakubitinema Hasegawa, 1998) (Quimperiidae) Even though a very few cosmocercoid taxa have been and deduced that Spectatus may be a representative of sequenced, molecular data indicated that some families may be Seuratoidea rather than Cosmocercoidea. However, during our non-monophyletic (Fig. 3). Interrelationships of members of this preliminary phylogenetic examinations, we performed some diverse group of parasitic nematodes thus remain unresolved and analyses including several representatives of Ascaridida and all pending future intensive research. results indicated the genetic proximity of Spectatus with cosmocercoid nematodes, forming a similar clade as in Figure 3. ACKNOWLEDGMENTS In addition, Spectatus did not cluster together with Seuratoidea; This study was supported by the ‘‘Cienciaˆ sem fronteiras’’ Brazilian thus, it is prudent to retain the genus in Cosmocercoidea until new program-visiting researcher modality (135/2012; T.S. accomodations at molecular data is available. the Universidade Federal Rural de Rio de Janeiro) and Conselho Among the kathlaniid genera, Spectatus appears to be Nacional de Desenvolvimento Cientı´fico e Tecnologico´ (CNPq) grants morphologically close to Myleusnema Moravec and Thatcher, to J.L.L. (474077/2011-0, 304254/2011-8, 402665/2012-0) and to L.E.R.T. (311567/2013-4). A fellowship of the Coodernaca¸õ de Aperfeicoamento¸ de 1996. Nevertheless, both are easily discernible by the unusual Pessoal de Nı´vel Superior (CAPES) supported F.B.P. for a 5-mo-stay at morphology of Myleusnema, which has the cephalic portion the Institute of Parasitology, Biology Centre of the Czech Academy of narrower and separated from rest of the body by a marked Sciences, Ceskˇ e´ Budejoviceˇ (IPCAS). T.S. was also supported by the cuticular fold and a conspicuous postcloacal, lobe-like formation IPCAS (RVO: 60077344) and the Czech Science Foundation (P505/12/ armed with 2 horns (Moravec and Thatcher, 1996). Unfortunate- G112). The authors’ thanks are also due to the staff of the Laboratory of Electron Microscopy, IPCAS, for their technical assistance, Blanka ly, there are no molecular data on Myleusnema available for Skorıˇ ´kova´for help with providing scientific literature and Jan Brabec genetic comparisons. (both from IPCAS) for help with molecular analyses, and David Based on the phylogenetic analysis, the genus Spectatus Gonza´lez-Solı´s from El Colegio de la Frontera Sur, Chetumal, Mexico represented by its type-species is basal among other cosmocercoid for valuable advice. Thanks are also due to Philippe Vieira Alves and genera, and it forms a well-supported basal branch separated Juliana Moreira, Universidade Federal Rural do Rio de Janeiro, Seropedica,´ for help with collecting fish and their parasitological from other lineages with cosmocercoid nematodes. A separate examination. We are thankful to Marcelo Knoff, curator of the position of Spectatus from that of 2 species of Falcaustra supports Helminthological Collection of the Instituto Oswaldo Cruz (CHIOC), the validity of both genera, contrary to the classification of Yorke for lending types and vouchers of S. spectatus. PEREIRA ET AL.—REDESCRIPTION OF SPECTATUS SPECTATUS 475

FIGURE 3. Bayesian tree from phylogenetic analysis of the sequences of SSU rDNA from cosmocerdoid nematodes (families Atractidae, Cosmocercidae, and Kathlaniidae). First number represents branch support of Bayesian posterior probability (for 2 3 106 generations; burn-in ¼ 1 3 103); the second number shows the maximum likelihood (ML) bootstrap value (from 100 replications).

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