(Kathlaniidae), Including Redescription of the Species and Amendment of Genus Diagnosis Author(S): Felipe B
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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 BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. J. Parasitol., 101(4), 2015, pp. 468–475 Ó American Society of Parasitologists 2015 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¸´ ca˜o em Ciˆencias Veterina´rias, Universidade Federal Rural do Rio de Janeiro, Caixa Postal 74.540, 23851-970, Serop´edica, 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¸˜o 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 Dı´az-Ungrı´a, 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 Dı´az-Ungrı´a, 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¸˜o 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 ´kova´et 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 ´kova´et 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 ´kova´et 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