Acta Tropica 130 (2014) 162–166
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
New Aspidoderidae species parasite of Didelphis aurita (Mammalia: Didelphidae): A light and scanning electron microscopy approach V.A. Chagas-Moutinho a,b,∗ , V. Sant’Anna a,b , A. Oliveira-Menezes a,c , W. De Souza b a
Laboratório de Biologia de Helmintos Otto Wucherer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Bloco G, Ilha do Fundão, 21941-902 Rio de Janeiro, RJ, Brazil Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Bloco G, Ilha do Fundão, 21941-902 Rio de Janeiro, RJ, Brazil c Pólo Avanc¸ado Universidade Federal do Rio de Janeiro, Macaé, Brazil b
a r t i c l e
i n f o
Article history: Received 22 November 2011 Received in revised form 25 September 2013 Accepted 4 October 2013 Available online 12 October 2013 Keywords: Taxonomy Morphology Scanning electron microscopy Aspidoderidae Aspidodera
a b s t r a c t Nematodes of the family Aspidoderidae (Nematoda: Heterakoidea) Skrjabin and Schikobalova, 1947, are widely distributed in the Americas. The family Aspidoderidae includes the subfamilies Aspidoderinae Skrjabin and Schikobalova, 1947, and Lauroiinae Skrjabin and Schikobalova, 1951. These two subfamilies are delineated by the presence or absence of cephalic cordons at the anterior region. The nematodes in the subfamily Aspidoderinae, which includes the genus Aspidodera Railliet and Henry, 1912, are represented by nematodes with anterior cephalic cordons at the anterior end. The nematodes of the genus Aspidodera Railliet and Henry, 1912, are found in the cecum and large intestine of mammals of the orders Edentata, Marsupialia and Rodentia. Species within this genus have many morphological similarities. The use of scanning electron microscopy allows the specific characterization of the species within this genus. In the present work, we describe a new species of Aspidodera parasite of the large intestine of Didelphis aurita (Mammalia: Didelphidae) Wied-Neuwied, 1826, collected from Cachoeiras de Macacu, Rio de Janeiro. The combination of light and scanning electron microscopy allowed us a detailed analysis of this nematode. © 2013 Published by Elsevier B.V.
1. Introduction Nematodes of the family Aspidoderidae (Nematoda: Heterakoidea) Skrjabin and Schikobalova, 1947, are widely distributed throughout the Americas, occurring in Brazil, Mexico, Panama, Paraguay, Trinidad, Argentina, the United States of America, Peru, Bolivia, Guatemala, Venezuela and Suriname (Pinto and Gomes, 1980; Santos et al., 1990). The family Aspidoderidae includes the subfamilies Aspidoderinae Skrjabin and Schikobalova, 1947, and Lauroiinae Skrjabin and Schikobalova, 1951 (Jiménez-Ruiz et al., 2008). Nematodes are separated into these subfamilies according to the presence or absence of cephalic cordons. The subfamily Aspidoderinae, which includes the genus Aspidodera Railliet and Henry, 1912, is composed of nematodes with anterior cephalic cordons. The nematodes of the genus Aspidodera Railliet and Henry, 1912, are parasites of mammals of the orders Edentata, Marsupialia and Rodentia (Santos et al., 1990). These nematodes parasitize the ceca
∗ Corresponding author at: Laboratório de Biologia de Helmintos Otto Wucherer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS, Av. Carlos Chagas Filho Bloco G, Ilha do Fundão, CEP: 21949-902 Rio de Janeiro, RJ, Brazil. Tel.: +55 21 22606963. E-mail address: [email protected] (V.A. Chagas-Moutinho). 0001-706X/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.actatropica.2013.10.005
and large intestines of their hosts (Jimenez-Ruiz et al., 2006). The main morphological characteristic used to identify species of the genus Aspidodera is an anterior end adorned with cephalic cordons with six longitudinal loops and three lips, each of which have a pair of papillae; an esophagus with a terminal bulb; a ventral sucker on males, a pair of spiculae and one gubernaculum; and a posterior region that ends in a digitiform projection (Santos et al., 1990; Chagas-Moutinho et al., 2007). Currently, the genus Aspidodera is composed of the species A. scoleciformes (Diesing, 1851) Railliet and Henry, 1912, A. subulata (Molin, 1860) Railliet and Henry, 1912, A. fasciata (Schneider, 1866) Railliet and Henry, 1913, A. binansata Railliet and Henry, 1913, A. raillieti Travassos, 1913, A. ansirupta Proenc¸a, 1937, A. vazi Proenc¸a, 1937, A. lacombae Vicente, 1964, and A. sogandaresi Jiménez-Ruiz, Gardner and Varela-Stokes, 2006. Species within this genus possess many morphological similarities, and light microscopy is sometimes insufficient to establish a correct classification of the nematodes of this genus. Scanning electron microscopy is a useful tool for identifying and characterizing species within this genus. The aim of this study was to record the Aspidoderidae parasites of D. aurita. A nematode was analyzed by light microscopy and scanning electron microscopy and proposed as a new species named Aspidodera lanfredi.
V.A. Chagas-Moutinho et al. / Acta Tropica 130 (2014) 162–166
2. Materials and methods Two D. aurita specimens were captured in Rio de Janeiro (22◦ 17 52.8 S, 41◦ 41 53.5 W), Brazil, by staff from the Laboratório de Vertebrados–Instituto de Biologia–Universidade Federal do Rio de Janeiro (capture license no. 099/06-RJ of the Instituto Brasileiro de Meio Ambiente e dos Recursos Naturais Renováveis). The marsupials were necropsied, and adult nematodes were collected from the large intestine. The nematodes were rinsed in 0.9% NaCl solution, fixed and stored in AFA solution at 60 ◦ C (Mafra and Lanfredi, 1998).
163
For morphometric studies, the nematodes were clarified in 50% phenol and visualized with the aid of a Zeiss Standard 20 microscope. Drawings were made with the aid of a Lucida camera. Measurements are in millimeters, except when indicated. For scanning electron microscopy (SEM), the nematodes were washed in 0.1 M cacodylate buffer pH 7.2, post-fixed in 1% OsO4 and 0.8 K3 Fe(CN)6 for 1 h, dehydrated in a graded series of ethanol solutions (30–100%) for 1 h for each step, critical point-dried in CO2 , sputter-coated with gold and examined using a Jeol JSM-5310 scanning electron microscope.
Fig. 1. Light microscopy of Aspidodera lanfredi n.sp. A – General view of anterior region showing the cephalic cap, the excretory pore, the esophagus with a terminal bulb, and the intestine with a pyriform dilatation of the anterior portion (Bar: 0.1 mm). B – Detail of the cephalic cap showing the lips (Bar: 0.1). C – Detail of the cephalic cap showing the cordons (Bar: 0.1 mm). D – Ventral view of the posterior region of the male showing the sucker, the cloaca, the spiculae, the gubernaculum, the caudal projection, and the caudal papillae (Bar: 0.1 mm). D – Lateral view of the posterior region of the male showing the sucker, the cloaca, the spiculae, the gubernaculum, the caudal projection, and the caudal papillae (Bar: 0.1 mm).
164
V.A. Chagas-Moutinho et al. / Acta Tropica 130 (2014) 162–166
3. Results 3.1. Description 3.1.1. Aspidodera lanfredi n. sp Diagnosis: Nematodes with slender bodies, tapered ends and white when alive. A cephalic cap with cephalic cordons (Fig. 1A–C) and oral aperture surrounded by three lips, one dorsal and two lateroventral, were present (Fig. 1A–C). The esophagus is long and has a terminal bulb (Fig. 1A). The excretory pore is ventrally located at the anterior third of the body (Fig. 1A). The intestine has a pyriform projection on the anterior portion (Fig. 1A). By SEM, we observed that each lip emits two slender cuticular projections toward the posterior region (Fig. 2A and C). A pair of papillae and one amphid are present on the apex of the lateroventral lips (Fig. 2A and C). A papillae-like nodule is located near the amphid on the lateroventral lips (Fig. 2C). The left lateroventral lip contains a membranous cuticular projection that fits into the right lateroventral lip (Fig. 2A and C). The right lateroventral lip contains two acute projections that fit into the left lateroventral lip and the dorsal lip, respectively (Fig. 2A). The dorsal lip possesses
two lateral projections (Fig. 2A). Membranous cuticular projections coating the oral vestibule are present on the internal margin of each lip. These projections seem to be able to close the oral aperture (Fig. 2A and C). The interlabial projections are robust and have a wide base that is tapered toward the apex (Fig. 2A and B). A pore-like structure is present on the apex of the interlabial projections (Fig. 2B). The oral aperture is triangular (Fig. 2A and C). The cuticle on the cephalic region is smooth and transversally striated along the body (Figs. 2A, C–E and 3A–D). Lateral alae are absent. The posterior region ends in a digitiform projection (Figs. 1D, E and 2A). In females, the posterior region becomes tapers gradually, ending in a digitiform projection. The vulval aperture contains a flap structure and three pairs of papillae nearby (Fig. 2D). Elliptical eggs were observed in the uterus. The anus is located in the caudal extremity and forms a transversal fissure (Fig. 2E). In males, the posterior region is ventrally bent (Figs. 1D, E and 3A). A ventral sucker with a chitinous ring (Figs. 1D, E and 3A and C) is present. There is a papilla on the internal margin of the sucker (Fig. 3C). A pair of spicules (Figs. 1D, E and 3A), with the apex spear-shaped (Fig. 3A), and
Fig. 2. Scanning electron microscopy of Aspidodera lanfredi n.sp. A – General view of the cephalic cap showing the lips with two slender projections (p), interlabial projections (i); a pair o papillae (thin arrow) and one amphid (empty head arrow) on latero-ventral lips; dorsal lip with two rounded projections (a) on lateral margin; right latero-ventral lip with two sharp projections (Pa); left latero-ventral lip with a membranous cuticular projection (large arrow); and oral vestibule covered by a cuticular membrane (asterisk) (Bar: 10 m). B – Detail of the pore-like structure (Pl) on the apex of interlabium (Bar: 5 m). C – Detail of the apical region of the lips showing the pair of papillae (thin arrow), the amphid (empty head arrow) and the papillae-like nodule (full head arrow) present on latero-ventral lips; membranous cuticular projection on left latero-ventral lip and the oral vestibule (asterisk) covered by a cuticular membrane (Bar: 10 m). D – Detail of the vulvar aperture with a flap structure and papillae (thin arrow) nearby and cuticular transversal striations (star) (Bar: 10 m). E – Detail of the anus and cuticular transversal striations (star) (Bar: 10 m).
V.A. Chagas-Moutinho et al. / Acta Tropica 130 (2014) 162–166
165
Fig. 3. Scanning electron microscopy of male of Aspidodera lanfredi n.sp. A – General view of posterior region of male showing the sucker, pair of spiculae (S), spear-shaped digitiform projection (d), and caudal papillae (thin arrow) (Bar: 50 m). B – Lateral view of the cloaca showing the gubernaculum (g), ad-cloacal papillae (thin arrow), and cuticular transversal striations (star) (Bar: 10 m). C – Detail of the sucker showing caudal papillae (thin arrow), pedunculated papilla on the edge of the sucker (thin arrow), and cuticular transversal striations (star) (Bar: 10 m). D – Detail of the cloaca showing the cuticular membranous projections, ad-cloacal papillae (thin arrow), and cuticular transversal striations (star) (Bar: 10 m).
a gutter-shaped gubernaculum are present (Figs. 1D, E and 3B). The cloacal aperture has an edge with a pair of papillae and membranous cuticular projections (Fig. 3B and D). The posterior region ends in a digitiform projection (Figs. 1D, E and 3A). Holotype: Body length 4.93 mm. Body width 0.25 mm. Esophagus length with bulb 1.28 mm. Posterior bulb 0.21 mm long and 0.17 mm wide. Cephalic cap 0.11 mm long. Right spicule 0.72 mm long and left spicule 0.71 mm long. Gubernaculum 0.10 mm long. Nineteen pairs of papillae (observed by light microscopy). Allotype: Body length 5.54 mm. Body width 0.38 mm. Esophagus length with bulb 1.01 mm. Posterior bulb 0.23 mm long and 0.15 mm wide. Cephalic cap 0.14 mm long. Elliptical eggs 0.05 mm long and 0.04 mm wide. Male: Body length 4.9–6.32 mm. Body width 0.25–0.33 mm. Cephalic cap 0.10–0.13 mm long. Esophagus length with bulb 0.84–1.28 mm. Posterior bulb 0.20–0.21 mm long and 0.16–0.23 mm wide. Right spicule 0.76–0.87 mm long and left spicules 0.71–0.86 mm long. Gubernaculum 0.10–0.15 mm long. Twenty-five pairs of caudal papillae (observed by SEM). Females: Body length 4.91–6.66 mm. Body width 0.32–0.48 mm. Esophagus length with bulb 0.93–1.15 mm. Posterior bulb 0.22–0.31 mm long and 0.18–0.30 mm wide. Cephalic cap 0.12–0.16 mm long. Elliptical eggs 0.058 mm–0.069 mm long and 0.041–0.047 mm wide.
3.2. Taxonomic summary Type host: Didelphis aurita (Marsupialia: Didelphidae) WiedNeuwied, 1826. Site of infection: Large intestine. Type locality: Didelphis aurita – Cachoeiras de Macacu, Rio de Janeiro, Brazil (22◦ 17 52.8 S, 41◦ 41 53.5 W). Type specimens: - Holotype: XXXX - Allotype: XXXX - Paratypes: XXXXX Etymology: The species was named in honor of Dr. Reinalda Marisa Lanfredi, the head of the Laboratório de Biologia de Helmintos Otto Wucherer. 4. Discussion Most taxonomic studies involving Aspidoderidae nematodes are based on the structures found on the anterior end. Inglis (1957) highlighted the importance of the structures present on the head of these nematodes. Santos et al. (1990), in a revision of the genus Aspidodera, established a new key based on the cephalic cordons
166
V.A. Chagas-Moutinho et al. / Acta Tropica 130 (2014) 162–166
to identify these nematodes. The structures of the cephalic cap and cephalic cordons are easily visible by light microscopy. When the nematodes were visualized by Light Microscopy (LM), we observed that the outstanding difference between Aspidodera lanfredi and the other nematodes from the genus Aspidodera was the morphology of the cephalic cap. The cephalic caps of A. fasciata, A. binansata, A. vazi and A. sogandaresi are longer than the cephalic cap of A. lanfredi. A. subulata and A. scoleciformes have typical squareshaped cephalic caps. In A. subulata, the cephalic cap is very short. The cephalic cap of A. raillieti is most similar to that of A. lanfredi. Using scanning electron microscopy, Chagas-Moutinho et al. (2007) demonstrated in Aspidodera raillieti that the cephalic cordons are actually formed by a groove that surrounds the cuticular projections emitted by the lips. Jiménez-Ruiz et al. (2008) observed the same. Our observations agree with these previous studies. Thus, classification based only on the number and shape of the cephalic cordons could lead to an incorrect classification. In contrast to the similarity observed by light microscopy analyses, A. lanfredi differs from A. raillieti in the structures that compose the cephalic cap. A detailed SEM analysis made it possible to observe that the lips of A. lanfredi have cuticular projections that are not present on A. raillieti. Jimenez-Ruiz et al. (2006) described a digitiform projection on the left lateroventral lip of A. soganderesi. This structure was also observed by Chagas-Moutinho et al. (2007) in A. raillieti. Using scanning electron microscopy, we observed a membranous cuticular projection on the left lateroventral lip that fits on the right lateroventral lip. Another structure that compounds the cephalic cordons is the interlabial projections. These projections were observed in nematodes of the genus Aspidodera, and the morphology of these projections is different in each species of this genus. The interlabial projections of A. lanfredi are larger than the interlabial projections of A. raillieti observed by Chagas-Moutinho et al. (2007). Additionally, a pore-like structure was observed at the apex of the interlabial projections. This structure was described by Jiménez-Ruiz et al. (2008) as an inner papilla, and it was also observed in A. scoleciformes by Sant’Anna et al. (unpublished data). Santos et al. (1990) reported the presence of two papillae on each lip of A. raillieti. Jimenez-Ruiz et al. (2006) described the presence of one papilla and one amphid on the dorsal margin, one circular medial papillae on the lateroventral lips and one papilla on the dorsal lip on A. soganderesi. Chagas-Moutinho et al. (2007) described one pair of papillae and one amphid on the lateroventral lips of A. raillieti. In the present work, we observed a pair of papillae, one amphid and a papillae-like nodule on the ventrolateral lips. Jiménez-Ruiz et al. (2008) suggested a new combination for Nematomystes raillieti (Travassos, 1914) mainly based on the characteristics of the papillae and amphid on the ventrolateral lips and on the interlabial projection. However, we do not agree with this change. The criteria utilized were not sufficient to support this change. In the present work, we observed a pattern of distribution of the papillae that different from that shown by Jiménez-Ruiz et al. (2008) for Nematomystes scapteromi. In that work, the authors showed that the pair of papillae and the amphid formed a vertical line. In the present work, we observed that the pair of papillae formed a horizontal line parallel to amphid. They also described the presence of two papillae on the dorsal lip as a character of Aspidoderidae nematodes. In the present work, we did not observe papillae on the dorsal lips, a result also reported by ChagasMoutinho et al. (2007). The use of scanning electron microscopy allowed us to observe membranous cuticular projections coating the oral vestibule.
Chagas-Moutinho et al. (2007) noted that the oral vestibule was lined by a cuticular membrane with a serrated margin. These cuticular membranes seem to be used to close the triangular oral aperture. The size of the spicules is a criterion that permits separating the species of the genus Aspidodera. The spicules of A. scoleciformes are longer than the spicules of A. lanfredi. The spicules of A. subulata, A. fasciata, A. binansata, A. ansirupta and A. soganderesi are shorter. The spicules of A. vazi and A. raillieti are similar to the spicules of A. lanfredi. However, when we observed the cephalic cap, it was possible to separate these species. Chagas-Moutinho et al. (2007) demonstrated that the number of caudal papillae on males was not a good criterion for classification of these species because the number of caudal papillae observed by LM and SEM could be different. In the present work, we also observed this variation. By LM, 19 pairs of caudal papillae were observed, and by SEM, 25 pairs were observed. SEM analyses also allowed the visualization of three pairs of caudal papillae on females. These papillae were found near the vulva aperture. This is the first observation of these structures on nematodes of the genus Aspidodera. 5. Conclusion The use of scanning electron microscopy is a useful tool to analyze the structures that compound the cephalic cap of the nematodes of the genus Aspidodera and to observe other structures that cannot be observed by light microscopy. The combined use of light and scanning electron microscopy is important to obtain a more precise species description and separation. Acknowledgments We are grateful to Dr. David Straker for English revision and valuable suggestions. This work is in memoriam of Dr. Reinalda Marisa Lanfredi. We received financial support from CAPES, CNPq, CAPES-PROCAD and FAPERJ fellowships. References Chagas-Moutinho, V.A., Oliveira-Menezes, A., Cárdenas, M.Q., Lanfredi, R.M., 2007. Further description of Aspidodera raillieti (Nematoda: Aspidoderidae) from Didelphis marsupialis (Mammalia: Didelphidae) by light and scanning electron microscopy. Parasitol. Res. 101, 1331–1336. Inglis, W.G., 1957. The comparative anatomy and systematic significance of the head in the nematoda family Heterakidae. Proc. Zool. Soc. Lond. 128, 133–143. Jimenez-Ruiz, F.A., Gardner, S.L., Varela-Stokes, A.S., 2006. Aspidoderidae from North America, with the description of a new species of Aspidodera (Nematoda: Heterakoidea). J. Parasitol. 92, 847–854. Jiménez-Ruiz, F.A., Gardner, S.L., Noronha, D., Pinto, R.M., 2008. The systematic position of Lauroiinae Skrjabin and schikobalova, 1951 (Nemata: Heterakoidea: Aspidoderidae), as revealed by the analyses of traits used in its diagnosis. Cladistics 24, 459–476. Mafra, A.C.A., Lanfredi, R.M., 1998. Reevaluation of Physaloptera bispiculata (Nematoda: Spirurida) by light and scanning electron microscopy. J. Parasitol. 84, 582–588. Pinto, R.M., Gomes, D.C., 1980. Contribuic¸ão ao conhecimento da fauna helmintológica da região amazônica, Nematódeos. Atas Soc. Biol. Rio de Janeiro 21, 65–74. Railliet, A., Henry, A., 1912. Quelques nematodes parasites des Reptiles. Bull. Soc. Path. Exot. 5, 251–259. Railliet, A., Henry, A., 1913. Observations sur les nématodes du genre Aspidodera Railliet et Henry, 1912. Bull. Hist. Nat. Paris 19, 93–99. Santos, C.P., Lent, H., Gomes, D.C., 1990. The genus Aspidodera Railliet and Henry, 1912 (Nematoda: Heterakoidea): revision, new synonyms and key of species. Rev. Bras. Biol. 50, 1017–1031. Travassos, L., 1913. Sobre as espécies brasileiras da subfamília Heterakinae Railliet e Henry, 1912. Mem. Inst. Oswaldo Cruz 5, 271–318.
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
New Aspidoderidae species parasite of Didelphis aurita (Mammalia: Didelphidae): A light and scanning electron microscopy approach V.A. Chagas-Moutinho a,b,∗ , V. Sant’Anna a,b , A. Oliveira-Menezes a,c , W. De Souza b a
Laboratório de Biologia de Helmintos Otto Wucherer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Bloco G, Ilha do Fundão, 21941-902 Rio de Janeiro, RJ, Brazil Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Bloco G, Ilha do Fundão, 21941-902 Rio de Janeiro, RJ, Brazil c Pólo Avanc¸ado Universidade Federal do Rio de Janeiro, Macaé, Brazil b
a r t i c l e
i n f o
Article history: Received 22 November 2011 Received in revised form 25 September 2013 Accepted 4 October 2013 Available online 12 October 2013 Keywords: Taxonomy Morphology Scanning electron microscopy Aspidoderidae Aspidodera
a b s t r a c t Nematodes of the family Aspidoderidae (Nematoda: Heterakoidea) Skrjabin and Schikobalova, 1947, are widely distributed in the Americas. The family Aspidoderidae includes the subfamilies Aspidoderinae Skrjabin and Schikobalova, 1947, and Lauroiinae Skrjabin and Schikobalova, 1951. These two subfamilies are delineated by the presence or absence of cephalic cordons at the anterior region. The nematodes in the subfamily Aspidoderinae, which includes the genus Aspidodera Railliet and Henry, 1912, are represented by nematodes with anterior cephalic cordons at the anterior end. The nematodes of the genus Aspidodera Railliet and Henry, 1912, are found in the cecum and large intestine of mammals of the orders Edentata, Marsupialia and Rodentia. Species within this genus have many morphological similarities. The use of scanning electron microscopy allows the specific characterization of the species within this genus. In the present work, we describe a new species of Aspidodera parasite of the large intestine of Didelphis aurita (Mammalia: Didelphidae) Wied-Neuwied, 1826, collected from Cachoeiras de Macacu, Rio de Janeiro. The combination of light and scanning electron microscopy allowed us a detailed analysis of this nematode. © 2013 Published by Elsevier B.V.
1. Introduction Nematodes of the family Aspidoderidae (Nematoda: Heterakoidea) Skrjabin and Schikobalova, 1947, are widely distributed throughout the Americas, occurring in Brazil, Mexico, Panama, Paraguay, Trinidad, Argentina, the United States of America, Peru, Bolivia, Guatemala, Venezuela and Suriname (Pinto and Gomes, 1980; Santos et al., 1990). The family Aspidoderidae includes the subfamilies Aspidoderinae Skrjabin and Schikobalova, 1947, and Lauroiinae Skrjabin and Schikobalova, 1951 (Jiménez-Ruiz et al., 2008). Nematodes are separated into these subfamilies according to the presence or absence of cephalic cordons. The subfamily Aspidoderinae, which includes the genus Aspidodera Railliet and Henry, 1912, is composed of nematodes with anterior cephalic cordons. The nematodes of the genus Aspidodera Railliet and Henry, 1912, are parasites of mammals of the orders Edentata, Marsupialia and Rodentia (Santos et al., 1990). These nematodes parasitize the ceca
∗ Corresponding author at: Laboratório de Biologia de Helmintos Otto Wucherer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS, Av. Carlos Chagas Filho Bloco G, Ilha do Fundão, CEP: 21949-902 Rio de Janeiro, RJ, Brazil. Tel.: +55 21 22606963. E-mail address: [email protected] (V.A. Chagas-Moutinho). 0001-706X/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.actatropica.2013.10.005
and large intestines of their hosts (Jimenez-Ruiz et al., 2006). The main morphological characteristic used to identify species of the genus Aspidodera is an anterior end adorned with cephalic cordons with six longitudinal loops and three lips, each of which have a pair of papillae; an esophagus with a terminal bulb; a ventral sucker on males, a pair of spiculae and one gubernaculum; and a posterior region that ends in a digitiform projection (Santos et al., 1990; Chagas-Moutinho et al., 2007). Currently, the genus Aspidodera is composed of the species A. scoleciformes (Diesing, 1851) Railliet and Henry, 1912, A. subulata (Molin, 1860) Railliet and Henry, 1912, A. fasciata (Schneider, 1866) Railliet and Henry, 1913, A. binansata Railliet and Henry, 1913, A. raillieti Travassos, 1913, A. ansirupta Proenc¸a, 1937, A. vazi Proenc¸a, 1937, A. lacombae Vicente, 1964, and A. sogandaresi Jiménez-Ruiz, Gardner and Varela-Stokes, 2006. Species within this genus possess many morphological similarities, and light microscopy is sometimes insufficient to establish a correct classification of the nematodes of this genus. Scanning electron microscopy is a useful tool for identifying and characterizing species within this genus. The aim of this study was to record the Aspidoderidae parasites of D. aurita. A nematode was analyzed by light microscopy and scanning electron microscopy and proposed as a new species named Aspidodera lanfredi.
V.A. Chagas-Moutinho et al. / Acta Tropica 130 (2014) 162–166
2. Materials and methods Two D. aurita specimens were captured in Rio de Janeiro (22◦ 17 52.8 S, 41◦ 41 53.5 W), Brazil, by staff from the Laboratório de Vertebrados–Instituto de Biologia–Universidade Federal do Rio de Janeiro (capture license no. 099/06-RJ of the Instituto Brasileiro de Meio Ambiente e dos Recursos Naturais Renováveis). The marsupials were necropsied, and adult nematodes were collected from the large intestine. The nematodes were rinsed in 0.9% NaCl solution, fixed and stored in AFA solution at 60 ◦ C (Mafra and Lanfredi, 1998).
163
For morphometric studies, the nematodes were clarified in 50% phenol and visualized with the aid of a Zeiss Standard 20 microscope. Drawings were made with the aid of a Lucida camera. Measurements are in millimeters, except when indicated. For scanning electron microscopy (SEM), the nematodes were washed in 0.1 M cacodylate buffer pH 7.2, post-fixed in 1% OsO4 and 0.8 K3 Fe(CN)6 for 1 h, dehydrated in a graded series of ethanol solutions (30–100%) for 1 h for each step, critical point-dried in CO2 , sputter-coated with gold and examined using a Jeol JSM-5310 scanning electron microscope.
Fig. 1. Light microscopy of Aspidodera lanfredi n.sp. A – General view of anterior region showing the cephalic cap, the excretory pore, the esophagus with a terminal bulb, and the intestine with a pyriform dilatation of the anterior portion (Bar: 0.1 mm). B – Detail of the cephalic cap showing the lips (Bar: 0.1). C – Detail of the cephalic cap showing the cordons (Bar: 0.1 mm). D – Ventral view of the posterior region of the male showing the sucker, the cloaca, the spiculae, the gubernaculum, the caudal projection, and the caudal papillae (Bar: 0.1 mm). D – Lateral view of the posterior region of the male showing the sucker, the cloaca, the spiculae, the gubernaculum, the caudal projection, and the caudal papillae (Bar: 0.1 mm).
164
V.A. Chagas-Moutinho et al. / Acta Tropica 130 (2014) 162–166
3. Results 3.1. Description 3.1.1. Aspidodera lanfredi n. sp Diagnosis: Nematodes with slender bodies, tapered ends and white when alive. A cephalic cap with cephalic cordons (Fig. 1A–C) and oral aperture surrounded by three lips, one dorsal and two lateroventral, were present (Fig. 1A–C). The esophagus is long and has a terminal bulb (Fig. 1A). The excretory pore is ventrally located at the anterior third of the body (Fig. 1A). The intestine has a pyriform projection on the anterior portion (Fig. 1A). By SEM, we observed that each lip emits two slender cuticular projections toward the posterior region (Fig. 2A and C). A pair of papillae and one amphid are present on the apex of the lateroventral lips (Fig. 2A and C). A papillae-like nodule is located near the amphid on the lateroventral lips (Fig. 2C). The left lateroventral lip contains a membranous cuticular projection that fits into the right lateroventral lip (Fig. 2A and C). The right lateroventral lip contains two acute projections that fit into the left lateroventral lip and the dorsal lip, respectively (Fig. 2A). The dorsal lip possesses
two lateral projections (Fig. 2A). Membranous cuticular projections coating the oral vestibule are present on the internal margin of each lip. These projections seem to be able to close the oral aperture (Fig. 2A and C). The interlabial projections are robust and have a wide base that is tapered toward the apex (Fig. 2A and B). A pore-like structure is present on the apex of the interlabial projections (Fig. 2B). The oral aperture is triangular (Fig. 2A and C). The cuticle on the cephalic region is smooth and transversally striated along the body (Figs. 2A, C–E and 3A–D). Lateral alae are absent. The posterior region ends in a digitiform projection (Figs. 1D, E and 2A). In females, the posterior region becomes tapers gradually, ending in a digitiform projection. The vulval aperture contains a flap structure and three pairs of papillae nearby (Fig. 2D). Elliptical eggs were observed in the uterus. The anus is located in the caudal extremity and forms a transversal fissure (Fig. 2E). In males, the posterior region is ventrally bent (Figs. 1D, E and 3A). A ventral sucker with a chitinous ring (Figs. 1D, E and 3A and C) is present. There is a papilla on the internal margin of the sucker (Fig. 3C). A pair of spicules (Figs. 1D, E and 3A), with the apex spear-shaped (Fig. 3A), and
Fig. 2. Scanning electron microscopy of Aspidodera lanfredi n.sp. A – General view of the cephalic cap showing the lips with two slender projections (p), interlabial projections (i); a pair o papillae (thin arrow) and one amphid (empty head arrow) on latero-ventral lips; dorsal lip with two rounded projections (a) on lateral margin; right latero-ventral lip with two sharp projections (Pa); left latero-ventral lip with a membranous cuticular projection (large arrow); and oral vestibule covered by a cuticular membrane (asterisk) (Bar: 10 m). B – Detail of the pore-like structure (Pl) on the apex of interlabium (Bar: 5 m). C – Detail of the apical region of the lips showing the pair of papillae (thin arrow), the amphid (empty head arrow) and the papillae-like nodule (full head arrow) present on latero-ventral lips; membranous cuticular projection on left latero-ventral lip and the oral vestibule (asterisk) covered by a cuticular membrane (Bar: 10 m). D – Detail of the vulvar aperture with a flap structure and papillae (thin arrow) nearby and cuticular transversal striations (star) (Bar: 10 m). E – Detail of the anus and cuticular transversal striations (star) (Bar: 10 m).
V.A. Chagas-Moutinho et al. / Acta Tropica 130 (2014) 162–166
165
Fig. 3. Scanning electron microscopy of male of Aspidodera lanfredi n.sp. A – General view of posterior region of male showing the sucker, pair of spiculae (S), spear-shaped digitiform projection (d), and caudal papillae (thin arrow) (Bar: 50 m). B – Lateral view of the cloaca showing the gubernaculum (g), ad-cloacal papillae (thin arrow), and cuticular transversal striations (star) (Bar: 10 m). C – Detail of the sucker showing caudal papillae (thin arrow), pedunculated papilla on the edge of the sucker (thin arrow), and cuticular transversal striations (star) (Bar: 10 m). D – Detail of the cloaca showing the cuticular membranous projections, ad-cloacal papillae (thin arrow), and cuticular transversal striations (star) (Bar: 10 m).
a gutter-shaped gubernaculum are present (Figs. 1D, E and 3B). The cloacal aperture has an edge with a pair of papillae and membranous cuticular projections (Fig. 3B and D). The posterior region ends in a digitiform projection (Figs. 1D, E and 3A). Holotype: Body length 4.93 mm. Body width 0.25 mm. Esophagus length with bulb 1.28 mm. Posterior bulb 0.21 mm long and 0.17 mm wide. Cephalic cap 0.11 mm long. Right spicule 0.72 mm long and left spicule 0.71 mm long. Gubernaculum 0.10 mm long. Nineteen pairs of papillae (observed by light microscopy). Allotype: Body length 5.54 mm. Body width 0.38 mm. Esophagus length with bulb 1.01 mm. Posterior bulb 0.23 mm long and 0.15 mm wide. Cephalic cap 0.14 mm long. Elliptical eggs 0.05 mm long and 0.04 mm wide. Male: Body length 4.9–6.32 mm. Body width 0.25–0.33 mm. Cephalic cap 0.10–0.13 mm long. Esophagus length with bulb 0.84–1.28 mm. Posterior bulb 0.20–0.21 mm long and 0.16–0.23 mm wide. Right spicule 0.76–0.87 mm long and left spicules 0.71–0.86 mm long. Gubernaculum 0.10–0.15 mm long. Twenty-five pairs of caudal papillae (observed by SEM). Females: Body length 4.91–6.66 mm. Body width 0.32–0.48 mm. Esophagus length with bulb 0.93–1.15 mm. Posterior bulb 0.22–0.31 mm long and 0.18–0.30 mm wide. Cephalic cap 0.12–0.16 mm long. Elliptical eggs 0.058 mm–0.069 mm long and 0.041–0.047 mm wide.
3.2. Taxonomic summary Type host: Didelphis aurita (Marsupialia: Didelphidae) WiedNeuwied, 1826. Site of infection: Large intestine. Type locality: Didelphis aurita – Cachoeiras de Macacu, Rio de Janeiro, Brazil (22◦ 17 52.8 S, 41◦ 41 53.5 W). Type specimens: - Holotype: XXXX - Allotype: XXXX - Paratypes: XXXXX Etymology: The species was named in honor of Dr. Reinalda Marisa Lanfredi, the head of the Laboratório de Biologia de Helmintos Otto Wucherer. 4. Discussion Most taxonomic studies involving Aspidoderidae nematodes are based on the structures found on the anterior end. Inglis (1957) highlighted the importance of the structures present on the head of these nematodes. Santos et al. (1990), in a revision of the genus Aspidodera, established a new key based on the cephalic cordons
166
V.A. Chagas-Moutinho et al. / Acta Tropica 130 (2014) 162–166
to identify these nematodes. The structures of the cephalic cap and cephalic cordons are easily visible by light microscopy. When the nematodes were visualized by Light Microscopy (LM), we observed that the outstanding difference between Aspidodera lanfredi and the other nematodes from the genus Aspidodera was the morphology of the cephalic cap. The cephalic caps of A. fasciata, A. binansata, A. vazi and A. sogandaresi are longer than the cephalic cap of A. lanfredi. A. subulata and A. scoleciformes have typical squareshaped cephalic caps. In A. subulata, the cephalic cap is very short. The cephalic cap of A. raillieti is most similar to that of A. lanfredi. Using scanning electron microscopy, Chagas-Moutinho et al. (2007) demonstrated in Aspidodera raillieti that the cephalic cordons are actually formed by a groove that surrounds the cuticular projections emitted by the lips. Jiménez-Ruiz et al. (2008) observed the same. Our observations agree with these previous studies. Thus, classification based only on the number and shape of the cephalic cordons could lead to an incorrect classification. In contrast to the similarity observed by light microscopy analyses, A. lanfredi differs from A. raillieti in the structures that compose the cephalic cap. A detailed SEM analysis made it possible to observe that the lips of A. lanfredi have cuticular projections that are not present on A. raillieti. Jimenez-Ruiz et al. (2006) described a digitiform projection on the left lateroventral lip of A. soganderesi. This structure was also observed by Chagas-Moutinho et al. (2007) in A. raillieti. Using scanning electron microscopy, we observed a membranous cuticular projection on the left lateroventral lip that fits on the right lateroventral lip. Another structure that compounds the cephalic cordons is the interlabial projections. These projections were observed in nematodes of the genus Aspidodera, and the morphology of these projections is different in each species of this genus. The interlabial projections of A. lanfredi are larger than the interlabial projections of A. raillieti observed by Chagas-Moutinho et al. (2007). Additionally, a pore-like structure was observed at the apex of the interlabial projections. This structure was described by Jiménez-Ruiz et al. (2008) as an inner papilla, and it was also observed in A. scoleciformes by Sant’Anna et al. (unpublished data). Santos et al. (1990) reported the presence of two papillae on each lip of A. raillieti. Jimenez-Ruiz et al. (2006) described the presence of one papilla and one amphid on the dorsal margin, one circular medial papillae on the lateroventral lips and one papilla on the dorsal lip on A. soganderesi. Chagas-Moutinho et al. (2007) described one pair of papillae and one amphid on the lateroventral lips of A. raillieti. In the present work, we observed a pair of papillae, one amphid and a papillae-like nodule on the ventrolateral lips. Jiménez-Ruiz et al. (2008) suggested a new combination for Nematomystes raillieti (Travassos, 1914) mainly based on the characteristics of the papillae and amphid on the ventrolateral lips and on the interlabial projection. However, we do not agree with this change. The criteria utilized were not sufficient to support this change. In the present work, we observed a pattern of distribution of the papillae that different from that shown by Jiménez-Ruiz et al. (2008) for Nematomystes scapteromi. In that work, the authors showed that the pair of papillae and the amphid formed a vertical line. In the present work, we observed that the pair of papillae formed a horizontal line parallel to amphid. They also described the presence of two papillae on the dorsal lip as a character of Aspidoderidae nematodes. In the present work, we did not observe papillae on the dorsal lips, a result also reported by ChagasMoutinho et al. (2007). The use of scanning electron microscopy allowed us to observe membranous cuticular projections coating the oral vestibule.
Chagas-Moutinho et al. (2007) noted that the oral vestibule was lined by a cuticular membrane with a serrated margin. These cuticular membranes seem to be used to close the triangular oral aperture. The size of the spicules is a criterion that permits separating the species of the genus Aspidodera. The spicules of A. scoleciformes are longer than the spicules of A. lanfredi. The spicules of A. subulata, A. fasciata, A. binansata, A. ansirupta and A. soganderesi are shorter. The spicules of A. vazi and A. raillieti are similar to the spicules of A. lanfredi. However, when we observed the cephalic cap, it was possible to separate these species. Chagas-Moutinho et al. (2007) demonstrated that the number of caudal papillae on males was not a good criterion for classification of these species because the number of caudal papillae observed by LM and SEM could be different. In the present work, we also observed this variation. By LM, 19 pairs of caudal papillae were observed, and by SEM, 25 pairs were observed. SEM analyses also allowed the visualization of three pairs of caudal papillae on females. These papillae were found near the vulva aperture. This is the first observation of these structures on nematodes of the genus Aspidodera. 5. Conclusion The use of scanning electron microscopy is a useful tool to analyze the structures that compound the cephalic cap of the nematodes of the genus Aspidodera and to observe other structures that cannot be observed by light microscopy. The combined use of light and scanning electron microscopy is important to obtain a more precise species description and separation. Acknowledgments We are grateful to Dr. David Straker for English revision and valuable suggestions. This work is in memoriam of Dr. Reinalda Marisa Lanfredi. We received financial support from CAPES, CNPq, CAPES-PROCAD and FAPERJ fellowships. References Chagas-Moutinho, V.A., Oliveira-Menezes, A., Cárdenas, M.Q., Lanfredi, R.M., 2007. Further description of Aspidodera raillieti (Nematoda: Aspidoderidae) from Didelphis marsupialis (Mammalia: Didelphidae) by light and scanning electron microscopy. Parasitol. Res. 101, 1331–1336. Inglis, W.G., 1957. The comparative anatomy and systematic significance of the head in the nematoda family Heterakidae. Proc. Zool. Soc. Lond. 128, 133–143. Jimenez-Ruiz, F.A., Gardner, S.L., Varela-Stokes, A.S., 2006. Aspidoderidae from North America, with the description of a new species of Aspidodera (Nematoda: Heterakoidea). J. Parasitol. 92, 847–854. Jiménez-Ruiz, F.A., Gardner, S.L., Noronha, D., Pinto, R.M., 2008. The systematic position of Lauroiinae Skrjabin and schikobalova, 1951 (Nemata: Heterakoidea: Aspidoderidae), as revealed by the analyses of traits used in its diagnosis. Cladistics 24, 459–476. Mafra, A.C.A., Lanfredi, R.M., 1998. Reevaluation of Physaloptera bispiculata (Nematoda: Spirurida) by light and scanning electron microscopy. J. Parasitol. 84, 582–588. Pinto, R.M., Gomes, D.C., 1980. Contribuic¸ão ao conhecimento da fauna helmintológica da região amazônica, Nematódeos. Atas Soc. Biol. Rio de Janeiro 21, 65–74. Railliet, A., Henry, A., 1912. Quelques nematodes parasites des Reptiles. Bull. Soc. Path. Exot. 5, 251–259. Railliet, A., Henry, A., 1913. Observations sur les nématodes du genre Aspidodera Railliet et Henry, 1912. Bull. Hist. Nat. Paris 19, 93–99. Santos, C.P., Lent, H., Gomes, D.C., 1990. The genus Aspidodera Railliet and Henry, 1912 (Nematoda: Heterakoidea): revision, new synonyms and key of species. Rev. Bras. Biol. 50, 1017–1031. Travassos, L., 1913. Sobre as espécies brasileiras da subfamília Heterakinae Railliet e Henry, 1912. Mem. Inst. Oswaldo Cruz 5, 271–318.
