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Strongyluris amazonicus n. sp. (Nematoda: Heterakidae): A parasite of Tropidurus oreadicus from the Brazilian Amazon
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ARTICLE IN PRESS Acta Tropica xxx (2013) xxx–xxx
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Strongyluris amazonicus n. sp. (Nematoda: Heterakidae): A parasite of Tropidurus oreadicus from the Brazilian Amazon Jeannie Nascimento dos Santos a,∗ , Francisco Tiago de Vasconcelos Melo a , Lodney Coelho Nazaré a , Adriano Penha Furtado a , Elane Guerreiro Giese b a Laboratório de Biologia Celular e Helmintologia “Profa. Dra. Reinalda Marisa Lanfredi”, Universidade Federal do Pará, Instituto de Ciências Biológicas, Rua Augusto Corrêa, 01, Guamá, Belém 66075-110, Pará, Brazil b Laboratório de Histologia e Embriologia Animal, Instituto de Saúde e Produc¸ão Animal, Universidade Federal Rural da Amazônia, Av. Perimetral, 2501 Montese, Belém 66077-901, Pará, Brazil
a r t i c l e
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Article history: Received 12 April 2013 Received in revised form 20 June 2013 Accepted 28 June 2013 Available online xxx Keywords: Nematoda Heterakidae Strongyluris Tropidurus oreadicus SEM Brazil
a b s t r a c t Tropidurus oreadicus Rodrigues 1987 is a recently described species, thus there are no reports of helminthes parasites for this species. In this study, a morphological characterisation was performed of a nematode species parasite of the large intestine of T. oreadicus captured in an urban area. This urban area is similar to the estuarine dale from Guamá river and the “igarapés” of Belém, Pará State, Brazil. Morphological analysis suggested that the parasite is a new species of nematode, based mainly on the number and distribution pattern of the caudal papillae in males, which is unique for this species. In the present work, we describe for the first time the structure, number, and disposition of the cervical papillae. The morphological data were supported by scanning electron microscopy, which served as an important tool for distinguishing these nematodes from other species of the genus. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Tropidurus oreadicus (Squamata: Tropiduridae) Rodrigues, 1987 is an endemic species from Brazil that is frequently observed in open vegetation areas; it is common in the central Brazilian “Cerrado” areas and is also found in open enclaves in the Amazonian forest, south of the Amazon River (Colli et al., 1992; Faria and Araujo, 2004; Frost et al., 2001). Despite the extensive literature on the helminthes of lizards in Brazil, including studies of hosts of the genus Tropidurus, only 13 species of nematodes have been described for these hosts, and there are no data on helminthes parasites of T. oreadicus (Ávila and Silva, 2010; Vicente et al., 1993). A total of 31 species are recognised within the genus Strongyluris Müeller, 1894 that parasitise the intestines of amphibian and reptiles: 29 species from reptiles and 2 from amphibians. In the
∗ Corresponding author. Tel.: +55 91 3201 7890; fax: +55 91 3201 7601. E-mail addresses: [email protected], jeannie [email protected] (J.N. dos Santos).
Neotropical region, 4 species have been described: S. acaudata Caballero, 1941; S. oscari Travassos, 1923; S. panamaensis Bursey et al., 2003, and S. similis Caballero, 1934 (Bursey et al., 2003). S. oscari is the only species that has been described in Brazil. The present work describes a new species of Strongyluris from the intestinal tract of T. oreadicus from Belém, Pará State, Brazil.
2. Materials and methods A total of 26 hosts were caught in an urban area of Federal University of Pará, Campus Belém, situated on the eastern margin of the Guamá River (48◦ 32 W to 48◦ 29 W, 1◦ 15 S to 1◦ 29 S) between January 2007 and March 2008. The lizards were transported alive to the laboratory. The animals were anaesthetised with sodium thiopental, weighed, and euthanised by exsanguination [Comitê de Ética em Pesquisa Animal da Universidade Federal do Pará (CEPAE-UFPA) license BIO 010-10]. Worms were then collected. Subsequently, the nematodes in the large intestine were rinsed with phosphate-buffered saline
0001-706X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.actatropica.2013.06.017
Please cite this article in press as: dos Santos, J.N., et al., Strongyluris amazonicus n. sp. (Nematoda: Heterakidae): A parasite of Tropidurus oreadicus from the Brazilian Amazon. Acta Trop. (2013), http://dx.doi.org/10.1016/j.actatropica.2013.06.017
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Fig. 1. Light microscopy analysis of a male holotype specimen of Strongyluris amazonicus n. sp. (a) General view of the male; bar: 800 m. (b) Transverse section of the anterior end; bar: 100 m. (c) Transverse section of the middle of the body; bar: 50 m. (d) Ventral view of the posterior end; bar: 100 m. (e) Ventral view of the spicules; bar: 100 m. (f) Scheme of caudal papilla distribution and the location of the amphids.
(PBS) at pH 7.4, fixed in alcohol–formalin–acetic acid (AFA) at 60◦ C, and stored in alcohol 70◦ GL. For light microscopy, the helminthes were diaphanized in Aman lactophenol and examined with an Olympus BX41 microscope with a drawing tube attached. For scanning electron microscopy (SEM), the worms were processed following Giese et al. (2010). The specimens were examined and photographed with a LEO 1459 VP scanning electron microscope. Measurements are given in millimetres, unless otherwise noted, and they are represented as mean and standard deviation with the range in parenthesis; caudal papillae are represented as total (precloacal:adcloacal: postcloacal).
3. Results 3.1. Strongyluris amazonicus n. sp. (Figs. 1–5) General description from light microscopy and SEM: Heterakidae Railliet and Henry, 1912; Spinicaudinae Travassos, 1920. The anterior end is similar in both sexes. Three triangular lips (1 dorsal and 2 ventro-lateral); each lip has 1 tooth. Each of the lateral lips
has amphid and 1 cephalic papilla, whereas the dorsal lip features 2 papillae. The limitations of the lips present a characteristic groove. Twenty pairs of caudal pedunculated papillae, distributed in 4 rows of 5 papillae (2 latero-dorsal and 2 latero-ventral), with a prominent button central structure located in a circular depression. Cuticular transversal ridges along the body. Oesophagus with a triangular lumen, divided into the pharynx, corpus, and valved bulb. Excretory pore located on the anterior third of the body, anterior to the oesophagus bulb and presenting a longitudinal elliptical aperture. Intestine rectilinear with anterior dilatation.
3.1.1. Males (1 holotype and 9 paratypes) Total length 8.05 ± 0.36 (7.52–8.41) and width 0.42 ± 0.04 (0.35–0.48) at bulb level. Oesophagus total length 1.50 ± 0.40 (1.44–1.56) × 0.08 ± 0.01 (0.06–0.07). Pharynx 0.22 ± 0.01 (0.2–0.23) and corpus 1.24 ± 0.04 (1.16–1.27). Bulb 0.26 ± 0.20 (0.22–0.28) with a maximum width of 0.25 ± 0.20 (0.2–0.26). Nerve ring 0.43 ± 0.03 (0.37–0.47) and excretory pore 1.04 ± 0.07 (0.9–1.12) from the anterior end. Posterior end with a truncated copulatory bursa, with a pre-cloacal chitinous sucker
Please cite this article in press as: dos Santos, J.N., et al., Strongyluris amazonicus n. sp. (Nematoda: Heterakidae): A parasite of Tropidurus oreadicus from the Brazilian Amazon. Acta Trop. (2013), http://dx.doi.org/10.1016/j.actatropica.2013.06.017
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Fig. 2. Light microscopy analysis of a female allotype specimen of Strongyluris amazonicus n. sp. (a) General view of the female; bar: 800 m. (b) Anterior end; bar: 200 m. (c) Transverse section of the anterior end; bar: 100 m. (d) Transverse section of the middle of the body; bar: 50 m. (e) Vulva, vaginal vestibule, and sphincter; bar: 100 m. (f) Unembryonated egg; bar: 30 m. (g) Embryonated egg; bar: 30 m. (h) Lateral view of the posterior end; bar: 100 m.
at 0.21 ± 0.020 (0.18–0.26) from the tail tip and 95.9 m ± 10 (80–100) diameter. One pair of sub-equal spicules: left 0.71 ± 0.04 (0.62–0.77) and right 0.75 ± 0.04 (0.69–0.81). Caudal pedunculated papillae with the following pattern: 3 pre-cloacal pairs (1 anterior pair and 2 pairs adjacent to the sucker) and an unpaired micro-papilla located medially along the inferior border of the sucker; 3 ad-cloacal pairs (1 ventral pair, 1 ventro-lateral pair, and 1 lateral pair); 3 post-cloacal pairs (two median pairs and 1 lateral pair) and a phasmid pair located lateral to the caudal appendix. Conical and short caudal appendix ventrally curveted and needle shaped. 3.1.2. Females (1 allotype and 9 paratypes) Total length 8.53 ± 0.33 (8.04–9.31) and width at bulb level 0.5 ± 0.03 (0.43–0.54). Oesophagus total length 1.49 ± 0.05 (70–90). Pharynx 0.22 ± 0.01 (1.53–1.68) × 80 m ± 10 (0.21–0.24/0.2). Corpus 1.30 ± 0.05 (1.21–1.36). Bulb 0.28 ± 0.02 (0.26–0. 32) × 0.28 ± 0.01 (0.26–0.3). Nerve ring 0.48 ± 0.06 (0.43–0.6) and excretory pore 1.11 ± 0.07 (1.0–1.12) from the anterior end. Vulva 5.5 ± 0.27 (5.24–6.19) from the anterior end. Tail
0.24 ± 0.03 (0.21–0.27). Elliptical eggs with thick shell 60 m ± 1 (61–68) × 40 m ± 1.5 (40–42).
3.2. Taxonomic summary Type host: T. oreadicus (Squamata: Tropiduridae) Rodrigues, 1987 Site of infection: Large intestine Type locality: Belém, Pará, Brazil (48◦ 27 W to 41◦ 82 W, 1◦ 27 S to 18◦ 04 S) Data type and depository: The holotype (No. MPEG 0017), allotype (No. MPEG 0018) and paratypes (No. MPEG 0019 to MPEG 0022) were deposited at the Colec¸ão Helmintológica do Museu Paraense Emílio Goeldi (MPEG). material examined: Strongyluris panaComparative mensis HWML No. 16970 and Strongyluris similis HWML No. 67113 and 67114 at the Harold W. Manter Laboratory of Parasitology, University of Nebraska, Lincoln, NE, USA.
Please cite this article in press as: dos Santos, J.N., et al., Strongyluris amazonicus n. sp. (Nematoda: Heterakidae): A parasite of Tropidurus oreadicus from the Brazilian Amazon. Acta Trop. (2013), http://dx.doi.org/10.1016/j.actatropica.2013.06.017
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Fig. 3. Scanning electron microscopy analysis of the cephalic region of Strongyluris amazonicus n. sp. (a) Anterior extremity view showing the distribution of the 5 pairs of cephalic papillae: (aa) anterior pair, (mp) medial pair, and (pp) posterior pair; bar: 20 m. (b) Mouth details, with cephalic structures, 3 lips with the cephalic papillae (cp) and the amphids (am) in the ventro-lateral lips; bar 10 m. (c) Detail of the lips (l), in which it is possible to observe 1 tooth at each lip; bar: 5 m. (d) Superior view of the mouth showing the teeth (t) in 2 ventro-lateral lips and the dorsal lip (dl); bar: 6 m. (e) Detail of the cephalic papillae; bar: 4 m.
Etymology: The species has been named after the biome in which it was found. Host-parasite data: Prevalence, 42.3%; range of intensity 1–7. 4. Discussion This new species, S. amazonicus n. sp., has the general characteristics of the genus (Anderson et al., 2009). A total of 31 species of Strongyluris have been described as intestinal parasites of reptiles and, occasionally, amphibians (Baker, 1984; Bursey et al., 2003). There have been few reports of species of Strongyluris that exhibit papillae in the cervical region. Among the species described, only Caballero (1938) and Caballero (1968) reported sessile papillae in the cervical region of S. similis Caballero, 1938 and S. skarjibini Caballero, 1968, respectively. In a review of the family Heterakidae, Inglis (1957) highlighted the presence of sessile papillae as a common characteristic of this genus. However, there have
been no reports of the number, distribution, and characteristics of these papillae, and analysis of the specimens Strongyluris panamensis HWML n◦ 16970 and Strongyluris similis HWML n◦ 67113 and 67114 deposited in the Harold W. Manter Laboratory of Parasitology revealed cervical papillae in S. similis; however, it was not possible to identify the number and distribution pattern. For S. amazonicus n. sp., we used light microscopy to observe the papillae with an intracuticular peduncle and scanning electron microscopy analysis to confirm the number and disposition of these papillae (20 papillae distributed in 4 rows of 5 papillae each: 2 latero-ventral and 2 latero-dorsal rows). This is the first report of this characteristic pattern and number of cervical papillae, which is unique among the species of the genus. Based on the number of papillae in the male caudal region, S. amazonicus n. sp. belongs to the group of species with unpaired papillae close to the caudal sucker; this group includes
Please cite this article in press as: dos Santos, J.N., et al., Strongyluris amazonicus n. sp. (Nematoda: Heterakidae): A parasite of Tropidurus oreadicus from the Brazilian Amazon. Acta Trop. (2013), http://dx.doi.org/10.1016/j.actatropica.2013.06.017
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Fig. 4. Scanning electron microscopy analysis of the structures from females of Strongyluris amazonicus n. sp. (a) General view of the anterior third of the body showing the position of the excretory pore (ep); bar: 50 m. (b) Detail of the excretory pore (ep) aperture; bar: 5 m. (c) Detail of the vulva (v) aperture, showing the transversal elliptical shape and the alterations in the pattern of cuticular ridges around this region; bar: 5 m. (d) Lateral view of the phasmids; bar: 3 m. (e) General view of the posterior extremity of the female, showing the sub-terminal position of the anus and the anal aperture (an); bar: 20 m.
S. oscari Travassos, 1923; S. elegans Railliet and Henry, 1914; S. media Harwood, 1935; S. riojai Caballero, 1968; S. similis Caballero, 1938; S. skrjabini Caballero, 1968; S. winteri Caballero, 1968; and S. panamaensis Bursey et al., 2003. However, among the species listed above, only 4 species of Strongyluris have been described from the Neotropical region: S. oscari Travassos, 1923; S. similis Caballero, 1938; S. acaudata Caballero, 1941; and S. panamaensis Bursey et al., 2003 (Bursey et al., 2003). Among the Neotropical species, S. oscari has the greatest total length (11.3–14.8 in males and 12.5–17.4 mm in females), and the smaller species are S. panamaensis (4.9–6.5 in males and 5.8–7.7 in females) and S. acaudata (5.3–5.9 in males and unknown in females). These values are different from those observed for S. amazonicus n. sp., but they indicate that the new taxon is
more similar to S. similis (8.9–9.4 in males and 9.8–10.4 in females). Based on the number and distribution of the caudal papillae, S. amazonicus n. sp. is easily distinguishable from Neotropical species because it possesses the unique characteristics of 19 caudal papillae distributed in 3 pre-cloacal pairs, an unpaired papilla situated centrally in the inferior border of the caudal sucker, 3 ad-cloacal pairs and 3 post-cloacal pairs (7:6:6), as well as a pair of phasmids lateral to the caudal end (Table 1). S. amazonicus n. sp. is the second species described from lizards of the genus Tropidurus in Brazil; the other species is S. oscari, which was described for T. torquatus (Weid, 1820) by Travassos (1923). S. media has 21 papillae, but it was described from Chamaeleon multituberculata (a different host) in an Ethiopian region and has a different pattern of papillae (7:4:10).
Table 1 Comparison of Strongyluris amazonicus n. sp. and his cogeneric species from Neotropical region, using selected characters. Species Strongyluris amazonicus n. sp. S. oscari Travassos, 1923 S. acaudata Caballero, 1941 S. panamensis Bursey et al., 2003 S. similis Caballero, 1938 a
Male length (mm) 7.52–8.41 8.2–8.8 5.3–5.9 4.9–6.5 8.9–9.4
Spicule length
Papillae patterna
Female length (mm)
Vulva length from anterior end (mm)
Host
0.71/0.75 0.8/0.9 0.75/0.8 0.5/0.7 0.72/0.8
21 (7:6:8) 21 (11:4:6) 20 (12:0:8) 21 (9:2:10) 21 (9:2:10)
7.52–8.41 8.3–9.0 Female unknown 5.8–7.7 9.8–10.4
5.5 5.5–5.8
Tropidurus oreadicus Tropidurus torquatus Sceloporus ferrariperezi Anolis biporcatus Sceloporus torquatus
3.0–4.7 6.35
Total (precloacal:adcloacal:postcloacal).
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Fig. 5. Scanning electron microscopy of the male posterior end of Strongyluris amazonicus n. sp. (a) General aspects of male copulatory bursa (cb) in a ventral view, showing the chitinous sucker (cs), the cloacal aperture, and the disposition of caudal papillae: 3 pre cloacal pairs (prc), 3 ad-clocal pairs (adp), and 2 post-cloacal pairs (ptc) and the phasmids (ph); bar: 40 m. (b) General view of copulatory bursa, showing details of the ventral sucker, the lateral pre-clocal papillae, and the 2 ad-clocal pairs, situated ventrally; bar: 25 m. (c) Detail of the intermediary region between the ventral sucker and the cloacae, showing the unpaired micropapillae (mp); bar: 20 m. (d) Superior view of the cloacal aperture, showing the exteriorisation of the membrane that covers the spicules (mem); bar: 12 m. (e) Detail of the cloacal aperture, showing the distal extremity of the spicules (sp) and the terminal aperture of the digestive tract. Observe the details of the medial pair of ad cloacal papillae (adp) and the first medial pair post cloacal papillae (ptc); bar: 10 m. (f) Dorsal view of the male copulatory bursa detached from the caudal appendix (ca); bar: 20 m. (g) Lateral view of the male posterior extremity, showing the caudal appendix (ca) and the position of the phasmids (ph); bar: 10 m.
5. Conclusions The morphological analysis revealed similarities between the new taxon and S. similis with respect to the number of caudal papillae and the presence of cervical papillae. However, the two are easily distinguishable by the number and distribution of the papillae. SEM analysis revealed morphological details of the features and added new data to the morphology of the genus. The number, disposition, and characteristics of the cephalic papillae, in addition to those of the caudal papillae and pre-cloacal sucker, were clearly observed by SEM and were corroborated by light microscopy analysis. SEM is an important tool because it permits the observation of surface details that occasionally cannot be adequately visualised by light microscopy analysis. This technique adds new details to the morphology of helminthes parasites, which enriches taxonomic
keys and facilitates the description of new species by alpha taxonomy. The present paper proposes the 32nd species in the genus Strongyluris, the fifth for the Neotropical region, the second species of the genus of parasites of lizards of the genus Tropidurus and from Brazil, and the first for the host T. oreadicus and the Amazon region. Acknowledgements The authors thank Dr. Hilton Túlio Costi of the Institutional Laboratory of Scanning Electron Microscopy at the Paraense Emílio Goeldi Museum (Museu Paraense Emílio Goeldi, MPEG) for technical support, as well as Dr. Tereza Cristina Sauer de Avila Pires and Marinus Steven Hoogmoed for identifying the reptile specimens. Financial support was provided by PROCADNF 2009 [Programa Nacional de Cooperac¸ão Acadêmica – Ac¸ão Novas Fronteiras (National Programme of Academic Cooperation
Please cite this article in press as: dos Santos, J.N., et al., Strongyluris amazonicus n. sp. (Nematoda: Heterakidae): A parasite of Tropidurus oreadicus from the Brazilian Amazon. Acta Trop. (2013), http://dx.doi.org/10.1016/j.actatropica.2013.06.017
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– New Frontiers)], a special programme of CAPES [Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (Coordination for the Improvement of Higher Education Personnel)]; SISBiota 2010 [Sistema Nacional de Pesquisa em Biodiversidade (National Research System in Biodiversity)], a programme of CNPq [Conselho Nacional de Desenvolvimento Científico e Tecnológico (National Counsel for Scientific and Technological Development)]; PROPESP [Pró-Reitoria de Pesquisa e Pós-Graduac¸ão (Dean of Research and Postgraduate Studies)]; FADESP [Fundac¸ão de Amparo e Desenvolvimento da Pesquisa (Foundation for Research Development)], a division of UFPA; and Parasitologia Básica 2010 (Basic Parasitology), a special programme of CAPES. References Anderson, R.C., Chabaud, A.G., Willmott, S., 2009. Keys to the Nematode Parasites of Vertebrates. Archival Volume. CAB International, Wallingford, UK, 463 pp. Ávila, R.W., Silva, R.J., 2010. Checklist of helminths from lizards and amphisbaenians (Reptilia, Squamata) of South America. J. Venom. Anim. Toxins Incl. Trop. Dis. 16 (4), 543–572.
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Baker, M.R., 1984. The systematics and zoogeography of Spinicaudinae and Meteterakinae (Heterakoidea: Nematoda) parasitic in reptiles and amphibians. Syst. Parasitol. 6, 275–287. Bursey, C.R., Goldberg, S.R., Telford Jr., S.R., 2003. Strongyluris panamaensis n. sp. (Nematoda: Heterakidae) and other helminths from the lizard, Anolis biporcatus (Sauria: Polychrotidae), from Panama. J. Parasitol. 89 (1), 118–123. Caballero, E., 1938. Nematodes of the reptiles of Mexico II. Ann. Trop. Med. Parasitol. 32, 225–229. Colli, G.R., Araújo, A.F.B., Silveira, R., Roma, F., 1992. Niche partioning and morphology of two syntopic Tropidurus (Sauria: Tropiduridae) in Mato Grosso. Brazil. J. Herpetol. 26, 66–69. Faria, R.G., Araujo, A.F.B., 2004. Sintopy of two Tropidurus lizard species (Squamata: Tropiduridae) in a rocky cerrado habitat in central Brazil. Braz. J. Biol. 64 (4), 775–786. Frost, D.L.R., Rodrigues, M.T., Grant, T.E., Titus, T.A., 2001. Phylogenetics of the lizard Genus Tropidurus (Squamata: Tropiduridae: Tropidurinae): direct optimization, descriptive efficiency, and sensitivity analysis of congruence between molecular data and morphology. Mol. Phylogenet. Evol. 21 (3), 352–371. Giese, E.G., Furtado, A.P., Lanfredi, R.M., Santos, J.N., 2010. A new cucullanid species (Nematoda) from Ageneiosus ucayalensis Castelnau, 1855 (Pisces: Auchenipteridae) from Pará, Brazil. J. Parasitol. 96 (2), 389–394. Inglis, W.G., 1957. A review of the nematode superfamily Heterakoidea. Ann. Mag. Nat. Hist. 12 (10), 905–912. Vicente, J.J., Rodrigues, H.O., Gomes, D.C., Pinto, R.M., 1993. Nematóides do Brasil, Parte III: nematóides de répteis. Rev. Bras. de Zool. 10 (1), 19–168.
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Strongyluris amazonicus n. sp. (Nematoda: Heterakidae): A parasite of Tropidurus oreadicus from the Brazilian Amazon Jeannie Nascimento dos Santos a,∗ , Francisco Tiago de Vasconcelos Melo a , Lodney Coelho Nazaré a , Adriano Penha Furtado a , Elane Guerreiro Giese b a Laboratório de Biologia Celular e Helmintologia “Profa. Dra. Reinalda Marisa Lanfredi”, Universidade Federal do Pará, Instituto de Ciências Biológicas, Rua Augusto Corrêa, 01, Guamá, Belém 66075-110, Pará, Brazil b Laboratório de Histologia e Embriologia Animal, Instituto de Saúde e Produc¸ão Animal, Universidade Federal Rural da Amazônia, Av. Perimetral, 2501 Montese, Belém 66077-901, Pará, Brazil
a r t i c l e
i n f o
Article history: Received 12 April 2013 Received in revised form 20 June 2013 Accepted 28 June 2013 Available online xxx Keywords: Nematoda Heterakidae Strongyluris Tropidurus oreadicus SEM Brazil
a b s t r a c t Tropidurus oreadicus Rodrigues 1987 is a recently described species, thus there are no reports of helminthes parasites for this species. In this study, a morphological characterisation was performed of a nematode species parasite of the large intestine of T. oreadicus captured in an urban area. This urban area is similar to the estuarine dale from Guamá river and the “igarapés” of Belém, Pará State, Brazil. Morphological analysis suggested that the parasite is a new species of nematode, based mainly on the number and distribution pattern of the caudal papillae in males, which is unique for this species. In the present work, we describe for the first time the structure, number, and disposition of the cervical papillae. The morphological data were supported by scanning electron microscopy, which served as an important tool for distinguishing these nematodes from other species of the genus. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Tropidurus oreadicus (Squamata: Tropiduridae) Rodrigues, 1987 is an endemic species from Brazil that is frequently observed in open vegetation areas; it is common in the central Brazilian “Cerrado” areas and is also found in open enclaves in the Amazonian forest, south of the Amazon River (Colli et al., 1992; Faria and Araujo, 2004; Frost et al., 2001). Despite the extensive literature on the helminthes of lizards in Brazil, including studies of hosts of the genus Tropidurus, only 13 species of nematodes have been described for these hosts, and there are no data on helminthes parasites of T. oreadicus (Ávila and Silva, 2010; Vicente et al., 1993). A total of 31 species are recognised within the genus Strongyluris Müeller, 1894 that parasitise the intestines of amphibian and reptiles: 29 species from reptiles and 2 from amphibians. In the
∗ Corresponding author. Tel.: +55 91 3201 7890; fax: +55 91 3201 7601. E-mail addresses: [email protected], jeannie [email protected] (J.N. dos Santos).
Neotropical region, 4 species have been described: S. acaudata Caballero, 1941; S. oscari Travassos, 1923; S. panamaensis Bursey et al., 2003, and S. similis Caballero, 1934 (Bursey et al., 2003). S. oscari is the only species that has been described in Brazil. The present work describes a new species of Strongyluris from the intestinal tract of T. oreadicus from Belém, Pará State, Brazil.
2. Materials and methods A total of 26 hosts were caught in an urban area of Federal University of Pará, Campus Belém, situated on the eastern margin of the Guamá River (48◦ 32 W to 48◦ 29 W, 1◦ 15 S to 1◦ 29 S) between January 2007 and March 2008. The lizards were transported alive to the laboratory. The animals were anaesthetised with sodium thiopental, weighed, and euthanised by exsanguination [Comitê de Ética em Pesquisa Animal da Universidade Federal do Pará (CEPAE-UFPA) license BIO 010-10]. Worms were then collected. Subsequently, the nematodes in the large intestine were rinsed with phosphate-buffered saline
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Fig. 1. Light microscopy analysis of a male holotype specimen of Strongyluris amazonicus n. sp. (a) General view of the male; bar: 800 m. (b) Transverse section of the anterior end; bar: 100 m. (c) Transverse section of the middle of the body; bar: 50 m. (d) Ventral view of the posterior end; bar: 100 m. (e) Ventral view of the spicules; bar: 100 m. (f) Scheme of caudal papilla distribution and the location of the amphids.
(PBS) at pH 7.4, fixed in alcohol–formalin–acetic acid (AFA) at 60◦ C, and stored in alcohol 70◦ GL. For light microscopy, the helminthes were diaphanized in Aman lactophenol and examined with an Olympus BX41 microscope with a drawing tube attached. For scanning electron microscopy (SEM), the worms were processed following Giese et al. (2010). The specimens were examined and photographed with a LEO 1459 VP scanning electron microscope. Measurements are given in millimetres, unless otherwise noted, and they are represented as mean and standard deviation with the range in parenthesis; caudal papillae are represented as total (precloacal:adcloacal: postcloacal).
3. Results 3.1. Strongyluris amazonicus n. sp. (Figs. 1–5) General description from light microscopy and SEM: Heterakidae Railliet and Henry, 1912; Spinicaudinae Travassos, 1920. The anterior end is similar in both sexes. Three triangular lips (1 dorsal and 2 ventro-lateral); each lip has 1 tooth. Each of the lateral lips
has amphid and 1 cephalic papilla, whereas the dorsal lip features 2 papillae. The limitations of the lips present a characteristic groove. Twenty pairs of caudal pedunculated papillae, distributed in 4 rows of 5 papillae (2 latero-dorsal and 2 latero-ventral), with a prominent button central structure located in a circular depression. Cuticular transversal ridges along the body. Oesophagus with a triangular lumen, divided into the pharynx, corpus, and valved bulb. Excretory pore located on the anterior third of the body, anterior to the oesophagus bulb and presenting a longitudinal elliptical aperture. Intestine rectilinear with anterior dilatation.
3.1.1. Males (1 holotype and 9 paratypes) Total length 8.05 ± 0.36 (7.52–8.41) and width 0.42 ± 0.04 (0.35–0.48) at bulb level. Oesophagus total length 1.50 ± 0.40 (1.44–1.56) × 0.08 ± 0.01 (0.06–0.07). Pharynx 0.22 ± 0.01 (0.2–0.23) and corpus 1.24 ± 0.04 (1.16–1.27). Bulb 0.26 ± 0.20 (0.22–0.28) with a maximum width of 0.25 ± 0.20 (0.2–0.26). Nerve ring 0.43 ± 0.03 (0.37–0.47) and excretory pore 1.04 ± 0.07 (0.9–1.12) from the anterior end. Posterior end with a truncated copulatory bursa, with a pre-cloacal chitinous sucker
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Fig. 2. Light microscopy analysis of a female allotype specimen of Strongyluris amazonicus n. sp. (a) General view of the female; bar: 800 m. (b) Anterior end; bar: 200 m. (c) Transverse section of the anterior end; bar: 100 m. (d) Transverse section of the middle of the body; bar: 50 m. (e) Vulva, vaginal vestibule, and sphincter; bar: 100 m. (f) Unembryonated egg; bar: 30 m. (g) Embryonated egg; bar: 30 m. (h) Lateral view of the posterior end; bar: 100 m.
at 0.21 ± 0.020 (0.18–0.26) from the tail tip and 95.9 m ± 10 (80–100) diameter. One pair of sub-equal spicules: left 0.71 ± 0.04 (0.62–0.77) and right 0.75 ± 0.04 (0.69–0.81). Caudal pedunculated papillae with the following pattern: 3 pre-cloacal pairs (1 anterior pair and 2 pairs adjacent to the sucker) and an unpaired micro-papilla located medially along the inferior border of the sucker; 3 ad-cloacal pairs (1 ventral pair, 1 ventro-lateral pair, and 1 lateral pair); 3 post-cloacal pairs (two median pairs and 1 lateral pair) and a phasmid pair located lateral to the caudal appendix. Conical and short caudal appendix ventrally curveted and needle shaped. 3.1.2. Females (1 allotype and 9 paratypes) Total length 8.53 ± 0.33 (8.04–9.31) and width at bulb level 0.5 ± 0.03 (0.43–0.54). Oesophagus total length 1.49 ± 0.05 (70–90). Pharynx 0.22 ± 0.01 (1.53–1.68) × 80 m ± 10 (0.21–0.24/0.2). Corpus 1.30 ± 0.05 (1.21–1.36). Bulb 0.28 ± 0.02 (0.26–0. 32) × 0.28 ± 0.01 (0.26–0.3). Nerve ring 0.48 ± 0.06 (0.43–0.6) and excretory pore 1.11 ± 0.07 (1.0–1.12) from the anterior end. Vulva 5.5 ± 0.27 (5.24–6.19) from the anterior end. Tail
0.24 ± 0.03 (0.21–0.27). Elliptical eggs with thick shell 60 m ± 1 (61–68) × 40 m ± 1.5 (40–42).
3.2. Taxonomic summary Type host: T. oreadicus (Squamata: Tropiduridae) Rodrigues, 1987 Site of infection: Large intestine Type locality: Belém, Pará, Brazil (48◦ 27 W to 41◦ 82 W, 1◦ 27 S to 18◦ 04 S) Data type and depository: The holotype (No. MPEG 0017), allotype (No. MPEG 0018) and paratypes (No. MPEG 0019 to MPEG 0022) were deposited at the Colec¸ão Helmintológica do Museu Paraense Emílio Goeldi (MPEG). material examined: Strongyluris panaComparative mensis HWML No. 16970 and Strongyluris similis HWML No. 67113 and 67114 at the Harold W. Manter Laboratory of Parasitology, University of Nebraska, Lincoln, NE, USA.
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Fig. 3. Scanning electron microscopy analysis of the cephalic region of Strongyluris amazonicus n. sp. (a) Anterior extremity view showing the distribution of the 5 pairs of cephalic papillae: (aa) anterior pair, (mp) medial pair, and (pp) posterior pair; bar: 20 m. (b) Mouth details, with cephalic structures, 3 lips with the cephalic papillae (cp) and the amphids (am) in the ventro-lateral lips; bar 10 m. (c) Detail of the lips (l), in which it is possible to observe 1 tooth at each lip; bar: 5 m. (d) Superior view of the mouth showing the teeth (t) in 2 ventro-lateral lips and the dorsal lip (dl); bar: 6 m. (e) Detail of the cephalic papillae; bar: 4 m.
Etymology: The species has been named after the biome in which it was found. Host-parasite data: Prevalence, 42.3%; range of intensity 1–7. 4. Discussion This new species, S. amazonicus n. sp., has the general characteristics of the genus (Anderson et al., 2009). A total of 31 species of Strongyluris have been described as intestinal parasites of reptiles and, occasionally, amphibians (Baker, 1984; Bursey et al., 2003). There have been few reports of species of Strongyluris that exhibit papillae in the cervical region. Among the species described, only Caballero (1938) and Caballero (1968) reported sessile papillae in the cervical region of S. similis Caballero, 1938 and S. skarjibini Caballero, 1968, respectively. In a review of the family Heterakidae, Inglis (1957) highlighted the presence of sessile papillae as a common characteristic of this genus. However, there have
been no reports of the number, distribution, and characteristics of these papillae, and analysis of the specimens Strongyluris panamensis HWML n◦ 16970 and Strongyluris similis HWML n◦ 67113 and 67114 deposited in the Harold W. Manter Laboratory of Parasitology revealed cervical papillae in S. similis; however, it was not possible to identify the number and distribution pattern. For S. amazonicus n. sp., we used light microscopy to observe the papillae with an intracuticular peduncle and scanning electron microscopy analysis to confirm the number and disposition of these papillae (20 papillae distributed in 4 rows of 5 papillae each: 2 latero-ventral and 2 latero-dorsal rows). This is the first report of this characteristic pattern and number of cervical papillae, which is unique among the species of the genus. Based on the number of papillae in the male caudal region, S. amazonicus n. sp. belongs to the group of species with unpaired papillae close to the caudal sucker; this group includes
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Fig. 4. Scanning electron microscopy analysis of the structures from females of Strongyluris amazonicus n. sp. (a) General view of the anterior third of the body showing the position of the excretory pore (ep); bar: 50 m. (b) Detail of the excretory pore (ep) aperture; bar: 5 m. (c) Detail of the vulva (v) aperture, showing the transversal elliptical shape and the alterations in the pattern of cuticular ridges around this region; bar: 5 m. (d) Lateral view of the phasmids; bar: 3 m. (e) General view of the posterior extremity of the female, showing the sub-terminal position of the anus and the anal aperture (an); bar: 20 m.
S. oscari Travassos, 1923; S. elegans Railliet and Henry, 1914; S. media Harwood, 1935; S. riojai Caballero, 1968; S. similis Caballero, 1938; S. skrjabini Caballero, 1968; S. winteri Caballero, 1968; and S. panamaensis Bursey et al., 2003. However, among the species listed above, only 4 species of Strongyluris have been described from the Neotropical region: S. oscari Travassos, 1923; S. similis Caballero, 1938; S. acaudata Caballero, 1941; and S. panamaensis Bursey et al., 2003 (Bursey et al., 2003). Among the Neotropical species, S. oscari has the greatest total length (11.3–14.8 in males and 12.5–17.4 mm in females), and the smaller species are S. panamaensis (4.9–6.5 in males and 5.8–7.7 in females) and S. acaudata (5.3–5.9 in males and unknown in females). These values are different from those observed for S. amazonicus n. sp., but they indicate that the new taxon is
more similar to S. similis (8.9–9.4 in males and 9.8–10.4 in females). Based on the number and distribution of the caudal papillae, S. amazonicus n. sp. is easily distinguishable from Neotropical species because it possesses the unique characteristics of 19 caudal papillae distributed in 3 pre-cloacal pairs, an unpaired papilla situated centrally in the inferior border of the caudal sucker, 3 ad-cloacal pairs and 3 post-cloacal pairs (7:6:6), as well as a pair of phasmids lateral to the caudal end (Table 1). S. amazonicus n. sp. is the second species described from lizards of the genus Tropidurus in Brazil; the other species is S. oscari, which was described for T. torquatus (Weid, 1820) by Travassos (1923). S. media has 21 papillae, but it was described from Chamaeleon multituberculata (a different host) in an Ethiopian region and has a different pattern of papillae (7:4:10).
Table 1 Comparison of Strongyluris amazonicus n. sp. and his cogeneric species from Neotropical region, using selected characters. Species Strongyluris amazonicus n. sp. S. oscari Travassos, 1923 S. acaudata Caballero, 1941 S. panamensis Bursey et al., 2003 S. similis Caballero, 1938 a
Male length (mm) 7.52–8.41 8.2–8.8 5.3–5.9 4.9–6.5 8.9–9.4
Spicule length
Papillae patterna
Female length (mm)
Vulva length from anterior end (mm)
Host
0.71/0.75 0.8/0.9 0.75/0.8 0.5/0.7 0.72/0.8
21 (7:6:8) 21 (11:4:6) 20 (12:0:8) 21 (9:2:10) 21 (9:2:10)
7.52–8.41 8.3–9.0 Female unknown 5.8–7.7 9.8–10.4
5.5 5.5–5.8
Tropidurus oreadicus Tropidurus torquatus Sceloporus ferrariperezi Anolis biporcatus Sceloporus torquatus
3.0–4.7 6.35
Total (precloacal:adcloacal:postcloacal).
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Fig. 5. Scanning electron microscopy of the male posterior end of Strongyluris amazonicus n. sp. (a) General aspects of male copulatory bursa (cb) in a ventral view, showing the chitinous sucker (cs), the cloacal aperture, and the disposition of caudal papillae: 3 pre cloacal pairs (prc), 3 ad-clocal pairs (adp), and 2 post-cloacal pairs (ptc) and the phasmids (ph); bar: 40 m. (b) General view of copulatory bursa, showing details of the ventral sucker, the lateral pre-clocal papillae, and the 2 ad-clocal pairs, situated ventrally; bar: 25 m. (c) Detail of the intermediary region between the ventral sucker and the cloacae, showing the unpaired micropapillae (mp); bar: 20 m. (d) Superior view of the cloacal aperture, showing the exteriorisation of the membrane that covers the spicules (mem); bar: 12 m. (e) Detail of the cloacal aperture, showing the distal extremity of the spicules (sp) and the terminal aperture of the digestive tract. Observe the details of the medial pair of ad cloacal papillae (adp) and the first medial pair post cloacal papillae (ptc); bar: 10 m. (f) Dorsal view of the male copulatory bursa detached from the caudal appendix (ca); bar: 20 m. (g) Lateral view of the male posterior extremity, showing the caudal appendix (ca) and the position of the phasmids (ph); bar: 10 m.
5. Conclusions The morphological analysis revealed similarities between the new taxon and S. similis with respect to the number of caudal papillae and the presence of cervical papillae. However, the two are easily distinguishable by the number and distribution of the papillae. SEM analysis revealed morphological details of the features and added new data to the morphology of the genus. The number, disposition, and characteristics of the cephalic papillae, in addition to those of the caudal papillae and pre-cloacal sucker, were clearly observed by SEM and were corroborated by light microscopy analysis. SEM is an important tool because it permits the observation of surface details that occasionally cannot be adequately visualised by light microscopy analysis. This technique adds new details to the morphology of helminthes parasites, which enriches taxonomic
keys and facilitates the description of new species by alpha taxonomy. The present paper proposes the 32nd species in the genus Strongyluris, the fifth for the Neotropical region, the second species of the genus of parasites of lizards of the genus Tropidurus and from Brazil, and the first for the host T. oreadicus and the Amazon region. Acknowledgements The authors thank Dr. Hilton Túlio Costi of the Institutional Laboratory of Scanning Electron Microscopy at the Paraense Emílio Goeldi Museum (Museu Paraense Emílio Goeldi, MPEG) for technical support, as well as Dr. Tereza Cristina Sauer de Avila Pires and Marinus Steven Hoogmoed for identifying the reptile specimens. Financial support was provided by PROCADNF 2009 [Programa Nacional de Cooperac¸ão Acadêmica – Ac¸ão Novas Fronteiras (National Programme of Academic Cooperation
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– New Frontiers)], a special programme of CAPES [Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (Coordination for the Improvement of Higher Education Personnel)]; SISBiota 2010 [Sistema Nacional de Pesquisa em Biodiversidade (National Research System in Biodiversity)], a programme of CNPq [Conselho Nacional de Desenvolvimento Científico e Tecnológico (National Counsel for Scientific and Technological Development)]; PROPESP [Pró-Reitoria de Pesquisa e Pós-Graduac¸ão (Dean of Research and Postgraduate Studies)]; FADESP [Fundac¸ão de Amparo e Desenvolvimento da Pesquisa (Foundation for Research Development)], a division of UFPA; and Parasitologia Básica 2010 (Basic Parasitology), a special programme of CAPES. References Anderson, R.C., Chabaud, A.G., Willmott, S., 2009. Keys to the Nematode Parasites of Vertebrates. Archival Volume. CAB International, Wallingford, UK, 463 pp. Ávila, R.W., Silva, R.J., 2010. Checklist of helminths from lizards and amphisbaenians (Reptilia, Squamata) of South America. J. Venom. Anim. Toxins Incl. Trop. Dis. 16 (4), 543–572.
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Please cite this article in press as: dos Santos, J.N., et al., Strongyluris amazonicus n. sp. (Nematoda: Heterakidae): A parasite of Tropidurus oreadicus from the Brazilian Amazon. Acta Trop. (2013), http://dx.doi.org/10.1016/j.actatropica.2013.06.017