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Myxobolus cordeiroi n. sp., a parasite of Zungaro jahu (Siluriformes: Pimelodiade) from Brazilian Pantanal: Morphology, phylogeny and histopathology
Veterinary Parasitology 162 (2009) 221–229
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Myxobolus cordeiroi n. sp., a parasite of Zungaro jahu (Siluriformes: Pimelodiade) from Brazilian Pantanal: Morphology, phylogeny and histopathology§ E.A. Adriano a,*, S. Arana b, A.L. Alves c, M.R.M. Silva d, P.S. Ceccarelli e, F. Henrique-Silva f, A.A.M. Maia d a Departamento de Cieˆncias Biolo´gicas, Universidade Federal de Sa˜o Paulo (UNIFESP), Rua Professor Artur Riedel, 275, Jardim Eldorado, CEP 09972-270 Diadema, SP, Brazil b Departamento de Histologia e Embriologia da Universidade Estadual de Campinas (UNICAMP), Caixa Postal 6109, CEP 13083-970 Campinas, SP, Brazil c Departamento de Biologia, Universidade Estadual Paulista-UNESP, Avenida 24-A n8 1515, Caixa Postal 199, CEP 13506-900 Rio Claro, SP, Brazil d Departamento de Cieˆncias Ba´sicas, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de Sa˜o Paulo (USP), Rua Duque de Caxias Norte, 225, CEP 13635-900 Pirassununga, SP, Brazil e Centro Nacional de Pesquisa e Conservac¸a˜o de Peixes Continentais (CEPTA), Instituto Chico Mendes de Conservac¸a˜o da Biodiversidade (ICMBio), Rodovia SP 201, Km 6.5, Caixa Postal 64, CEP 13630-970 Pirassununga, SP, Brazil f Departamento de Gene´tica e Evoluc¸a˜o da Universidade Federal de Sa˜o Carlos-UFSCAR, Rodovia Washington Luis, Km 235, Caixa Postal 676, CEP 13.565-905 Sa˜o Carlos, SP, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 December 2008 Received in revised form 5 March 2009 Accepted 14 March 2009
This work is part of an ongoing investigation into the characteristics of Myxozoan parasites of freshwater fish in Brazil and was carried out using morphology, histopathology and molecular analysis. A new Myxosporea species (Myxobolus cordeiroi) is described infecting the jau´ catfish (Zungaro jahu). Fifty jau´ specimens were examined and 78% exhibited plasmodia of the parasite. The plasmodia were white and round, measuring 0.3–2.0 mm in diameter and the development occurred in the gill arch, skin, serosa of the body cavity, urinary bladder and eye. The spores had an oval body and the spore wall was smooth. Partial sequencing of the 18S rDNA gene resulted in a total of 505 bp and the alignment of the sequences obtained from samples in different organs revealed 100% identity. In the phylogenetic analysis, the Myxobolus species clustered into two clades—one primarily parasites of freshwater fish and the other primarily parasites of marine fish. M. cordeiroi n. sp. was clustered in a basal position in the freshwater fish species clade. The histological analysis revealed the parasite in the connective tissue of the different infected sites, thereby exhibiting affinity to this tissue. The plasmodium was surrounded by an outer collagen capsule of fibers with distinct orientation from the adjacent connective tissue and an inner layer composed of delicate collagen fibrils—more precisely reticular fibers. The development of the parasite in the cornea and urinary bladder caused considerable stretching of the epithelium. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Zungaro jahu Myxobolus cordeiroi n. sp. Myxozoa jau´ Pantanal Brazil
1. Introduction
§
Work supported by FAPESP (Proc. no. 06/59075-6). * Corresponding author. Tel.: +55 11 4049 3300; fax: +55 11 4043 6428. E-mail address: [email protected] (E.A. Adriano).
0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.03.030
The Brazilian Pantanal is the largest floodplain in the world and has approximately 150,000 km2. It comprises several important rivers, such as the Paraguay, Cuiaba´ and Miranda rivers and it is part of the upper Paraguay
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River Basin (Mateus et al., 2004; Ceccarelli et al., 2007). A great diversity of fish is found in this area, with 264 known species (Britski et al., 2006; Buckup et al., 2007). One of these species, Zungaro jahu (Ihering, 1898), is a carnivorous pimelodid, popularly known as jau´. The species is endemic to the rivers that form the Plata River Basin (Parana´, Paraguay and Uruguay). It occurs in Brazil, Argentina, Bolivia and Paraguay (Lundberg and Littmann, 2003) and is an important commercial and sport fish (Mateus et al., 2004). This catfish lives in deep waters in river channels, mainly near waterfalls (Britski et al., 2006; Ceccarelli et al., 2007; Buckup et al., 2007). It reaches sizes up to 150 kg and is the largest Neotropical catfish (Alves et al., 2007). In fish farms, individuals reach around 4 kg in the first year (Projeto Pacu, 2008). The jau´ population has undergone a drastic reduction due to overexploitation and the catch of this species has been prohibited in some regions (Resende, 2003). Changes in its habitat caused by anthropogenic action (especially the construction of hydroelectric plants) constitute another important threat to the species. Thus, some Brazilian research institutes have developed jau´ cultivation methods with the aim of reintroducing specimens into the environment (Cemig, 2007), whereas the species has been cultivated in fish farms as a sport and ornamental fish (Projeto Pacu, 2008). Myxosporea are among the most important fish parasites (Schmahl et al., 1989), with about 1500 currently known species (Azevedo et al., 2005). These parasites mainly infect fish, but some are parasites of reptiles and amphibians (Eiras, 2005). Among the Myxosporea, species of the genus Myxobolus are, so far, the most commonly found in fish, with more than 700 known species throughout the world (Eiras et al., 2005). In South America, 23 Myxobolus species have been described thus far and only 4 have been reported to infect pimelodids (Gio´ia and Cordeiro, 1996; Cellere et al., 2002). With the use of classical zoological methods, it is very difficult to determine the validity of morphologically similar Myxosporea with identical tissue affinity and that develop in taxonomically closely related host species (Molna´r et al., 2002). Thus, the taxonomic classification of Myxosporea based on morphology alone (Lom and Arthur, 1989) has been refined with the application of molecular biological methods (Molna´r et al., 2008, 2007, 2006; Andree et al., 1999). The present study is part of an ongoing investigation into the characteristics of Myxosporea parasites of freshwater fish in Brazil. Morphological, scanning electron microscopy, histopathological and molecular data were used to describe a new Myxobolus species found simultaneously infecting several organs in Z. jahu from the Brazilian Pantanal. 2. Materials and methods Fifty adult specimens of jau´ (Z. jahu) were collected on five expeditions to the Pantanal Mato-Grossense in central Brazil. Specimens were caught in the Aquidauna River and Miranda River in the southern region of the Pantanal in November 2001, October 2002, May 2003
and November 2003. Specimens were caught in rivers and lakes within the Pantanal National Park in the central region and in the Cuiaba´ River in the northern region of the Pantanal in June 2004, October 2004 and July 2005. Immediately after collection, the fish were transported alive to the field laboratory mounted nearby, where they were measured, weighed and necropsied. Cysts and/or infected organs were fixed in 10% buffered formalin for taxonomic and histopathological analyses and in 100% ethanol for molecular analysis. Morphological and morphometric studies of the spores were based on fixed mature spores (Lom and Arthur, 1989) obtained from the plasmodia in the different organs (40 spores from each organ). Measurements were performed on computer equipped with the Axivision 4.1 image capturing system coupled to an Axioplan 2 Zeiss Microscope. Spore dimensions (mm) were expressed as mean standard deviation (SD). Smears containing free spores were stained with Giemsa solution and mounted in a low-viscosity mounting medium (CytosealTM) as permanent slides. For the scanning electron microscopy, histological sections of plasmodia fixed and embedded using routine histological procedures were cut into sections of 10 mm, deposited on a coverslip coated with albumin, oven-dried for 48 h, deparaffinized with xylol, re-dehydrated, postfixed in 1% OsO4 in a cacodylate buffer for 30 min at 4 8C, washed in the same buffer, dehydrated in ethanol, critical point-dried in CO2, covered with metallic gold and examined under a Joel JMS 35 microscope operated at 15 kV (Adriano et al., 2002). For the molecular analysis, plasmodia obtained from different organs (skin, urinary bladder and eye) of Z. jahu caught in the Pantanal National Park and the Cuiaba´ River in July 2005 were removed from the host tissue, ruptured with the aid of a needle and the contents were collected in a 1.5 ml microcentrifuge tube. DNA was extracted using a Wizard1 Genomic DNA Purification kit (Promega, USA), following the manufacturer’s tissue protocol. DNA content was determined by biospectrophotometry at 260 nm. The following the oligonucleotide primers were used in the polymerase chain reaction (PCR) amplification: Forward MX5: 50 -CTGCGGACGGCTCAGTAAATCAGT-30 and Reverse MX3: 50 -CCAGGACATCTTAGGGCATCACAGA-30 , specific to the family Myxobolidae, as described by Andree et al. (1999). PCR was carried out in a final volume of 50 ml, which contained 10–50 ng of extracted DNA, 1 Taq DNA Polymerase buffer (Invitrogen), 0.2 mmol of dNTP (Invitrogen), 1.5 mmol of MgCl2, 10 pmol of each primer (Invitrogen), 2.5 U of Taq DNA polymerase (Invitrogen) and distilled water. A PTC-100 (MJ Research inc.) thermal cycler was used. Amplification was performed in the following manner: 35 cycles, preceded by a denaturation step at 95 8C for 5 min, denaturation (95 8C for 60 s), annealing (59 8C for 60 s) and extension (72 8C for 120 s), finished with an extended elongation step at 72 8C for 5 min. PCR products were submitted to electrophoresis on 1.0% agarose gel (BioAmerica) in a TBE buffer (0.045 M Tris–borate, 0.001 M EDTEA pH 8.0), stained with Vistra Green and analyzed in a
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FLA-3000 (Fugi) scanner. Size of the amplified fragments was estimated by comparisons with the 100 bp DNA Ladder (Invitrogen). The fragments were then purified using the Wizard SV Gel and PCR Clean-up System (Promega) and cloned into a pTZ57R/T, using the InsT/A cloning kit (Fermentas). Positive clones were selected using the blue-white screening and sequenced with the same oligonucleotide primers used to the amplification. At least five positive clones per sample were sequenced using the ET-Dye Terminator Kit (GE Healthcare) in a MegaBace 1000 automatic sequencer (GE Healthcare). A standard nucleotide-nucleotide BLAST (blastn) search was conducted (Altschul et al., 1997). Sequences of Myxobolus species obtained from three organs of Z. jahu were aligned for comparisons with one another and with sequences obtained from GenBank, using DAMBE (Xia and Xie, 2001). Nucleotide saturation was analyzed for the 18S rDNA gene by plotting uncorrected p distances against absolute distance values. Phylogenetic analyses were conduced using the MEGA 4.0 program (Tamura et al., 2007) for the neighbor-joining (NJ), minimal evolution (ME) and maximum parsimony phylogenetic methods. The Kimura two-parameter (K2P) evolution sequence model was used in the analysis. Bootstrap analysis (1000 replicates) was employed to assess the relative robustness of the branches of the NJ, ME and MP trees, using the MEGA 4.0 program. The species Ceratomyxa sparusaurati and C. shasta were used as the outgroup in the phylogenetic analysis. For the histological analysis, fragments of infected organs fixed in 10% buffered formalin were embedded
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in paraffin, cut into serial sections 4 mm thick and stained with hematoxylin/eosin, Sirius Red and reticulin stain. 3. Results Among the 50 specimens of jau´ examined in the present study, 39 (78%) had plasmodia of an unknown parasite from the genus Myxobolus. Among the infected fish, 76.9% were infected in the gill arch, 23.1% had the parasite in the skin, 20.5% were infected in serosa of the body cavity, 17.9% had the parasite in urinary bladder and 7.7% were infected in the eyes. Infection occurred in more than one organ in 25.6%. Plasmodia were always present in the connective tissue of the organs. 3.1. Description of Myxobolus cordeiroi sp. n. (Figs. 1–16) The polysporic plasmodia were white, round and measured 0.3–2.0 mm in diameter (Figs. 1 and 2). The development of the parasite was asynchronic, with different developmental stages along the periphery of the plasmodium and mature spores in the internal region (Figs. 10 and 13). Formalin-fixed mature spores had an oval body in the frontal view, with the anterior extremity more slender than the posterior extremity (Figs. 3–5). Light and scanning electron microscopy showed the presence of smooth valves, with suture folds and a sutural protrusion forming a rim around the spores (Figs. 4 and 5). In the lateral view, the spores were symmetric, convex and had a conspicuous
Figs. 1–5. Morphological aspects of plasmodia and spores of Myxobolus cordeiroi n. sp. Fig. 1: plasmodia in the gill arch of jau´. Scale bar = 1.5 mm. Fig. 2: plasmodium in the eye. Scale bar = 1.5 mm. Figs. 3 and 4: spores in fresh preparations. Note the sutural rim around the spore (arrow) (3 bar = 10 mm and 4 = 5 mm). Fig. 5: scanning electron microscopy. Note the suture folds and a rim around the spores (arrows) Scale bar = 10 mm.
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suture line (Figs. 3–5). The polar capsules were elongated and equal in size, with the anterior ends close to one another. The polar filaments were coiled in 5–6 turns perpendicular to the axis of the capsule (Fig. 16). Table 1 displays the dimensions of the spores in the different infection sites. Type host Site of infection Prevalence Locality
Type material
Etymology
Zungaro jahu Humboldt, 1821 (Pimelodidae). Gill arch, skin, serosa of the body cavity, urinary bladder and eye. 39/50 (78%) of the Z. jahu were infected. Pantanal Mato-Grossense (Aquidauana, Cuiaba´, Miranda and Paraguay rivers) in the Paraguay basin, Brazil. Slides with stained spores (syntype) have been deposited in the collection of the Museum of Natural History, Institute of Biology, State University of Campinas (Unicamp), State of Sa˜o Paulo, Brazil (accession number: ZUEC 24). The species name (M. cordeiroi) is in homage to Dr. Nelson da Silva Cordeiro, one of the pioneers in the study of Myxozoa in Brazil, who retired in 2006.
In the molecular analysis, the specific primer pair MX5MX3 successfully amplified an approximately 1600 bp fragment of the 18S rDNA gene in the spores obtained from plasmodia found infecting the urinary bladder, eye and skin of Z. jahu. The alignment of sequences of the samples from the urinary bladder, eye and skin revealed 100% identity. The BLAST search using the partial 18S rDNA sequence data (505 bp of the fragment 50 end) of the Myxobolus species parasite of Z. jahu did not match any of the Myxozoa available in the GenBank. The partial 18S rDNA sequences data of M. cordeiroi n. sp. have been deposited in GenBank under the accession number FJ827757. In the phylogenetic analysis (based on the alignment of 599 bp, with 210 informative characters and complete deletion), the Myxobolus species clustered into two monophyletic clades in the three phylogenetic methods (Fig. 17). Clade A was further divided into four monophyletic clades (Clades A1 to A4), among which, Clades A4 and A3 were composed only by Myxobolus parasite species of cyprinids fish. Clade A2 grouped four Myxobolus parasite species of salmonids (M. cerebralis, M. arcticus, M. insidiosus and M. neurobius), two parasite species of cyprinids (M. djragini and M. bramae AF085177) and M. sandrae, a parasite of Sander lucioperca (Percidae). Clade A1 was the basal unit for Clade A and was composed of M. cordeiroi n. sp., a parasite of Z. jahu and a unique South American freshwater fish parasite as well as a unique parasite of pimelodids. Monophyletic Clade B clustered five Myxobolus species: M. procerus, a parasite of the freshwater fish Percopsis omiscomaycus (Percopsidae), was the basal unit for Clade B and sister group of a clade composed of four parasite species of marine fish (M. bizerti, M. ichkeulensis, M. exiguus and M. muelleri AY129314, all parasites of mullet). Histological analysis revealed that plasmodia occurred in the connective tissue in all infection sites (Figs. 6–15). There was a collagen capsule surrounding the plasmodium, which had fibers with distinct orienta-
Figs. 6–8. Histological section of eye of the jau´ with two plasmodia of M. cordeiroi n. sp. in the connective tissue of corneal stroma. Note the stretching of the corneal epithelium in Fig. 6 (arrows). Scale bar = 300 mm. Fig 7: Inset of Fig. 6 showing amplified interface of plasmodium (P) and host tissue (H). Note a collagen capsule (CC) composed of interlaced fibers. Fig. 8: details of Fig. 7, showing fiber organization. Scale bars = 10 mm. Sirius red stain.
tion from the adjacent connective tissue and an inner layer composed of delicate collagen fibrils, which were reticular fibers in contact with the plasmodium (Fig. 11). In the eye, the plasmodia were located in the connective tissue of corneal stroma. Parasite development caused stretching of the corneal epithelium, which, at some points, was formed by only one cell layer (Figs. 6–8). In skin of the surface of the opercula, plasmodia occurred in the adipose tissue between the skin and the opercular bone tissue (Fig. 9). In the gill arch, plasmodia occurred in the base of the organ (Fig. 12). In the urinary bladder, plasmodia occurred in the tunica mucosa, producing stretching of the epithelium (Fig. 14). No inflammatory reaction was observed in any of the infection sites. 4. Discussion Morphological and morphometric studies revealed a constant pattern in the spores obtained from the plasmodia of Myxobolus found infecting the gill arch, skin, serosa of the body cavity, urinary bladder and eye of Z. jahu. This
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Table 1 Spore dimensions of Myxobolus cordeirus n. sp. from several organs of the jau´. Measurements are given in mm and expressed as the mean standard deviation. Location of plasmodia
Gill arch
Skin
Serosa
Urinary bladder
Eye
Spore length Spore width Spore thickness
11.1 0.2 7.3 0.1 5.5 0.1
10.9 0.4 7.5 0.3 5.6 0.1
11.3 0.3 7.1 0.2 5.2 03
11.2 0.2 7.3 0.3 5.4 0.2
10.8 0.5 7.2 0.2 5.5 0.2
5.4 0.3 1.4 0.1
5.3 0.2 1.4 0.2
5.3 0.1 1.4 0.2
5.4 0.2 1.5 0.3
5.2 0.3 1.5 0.2
Polar capsule Length Width
suggests a unique Myxobolus species that causes simultaneous infection in several organs. This hypothesis was corroborated by the partial 18S rDNA gene sequence of spores obtained from the skin, urinary bladder and eye, which demonstrated 100% identity in the alignment sequences. According to Molna´r et al. (2002), zoological methods alone make it is very difficult to determine the validity of morphologically similar myxosporea with identical tissue affinity and developing in taxonomically closely related host species. This same concept can be applied to morphologically similar myxosporea found infecting different organs in the same host species, since the site of infection cannot be used as a
taxonomic criterion and spore morphology may not be suitable for validating these myxosporea species. In such conditions, molecular methods are an important taxonomic tool. The morphology and morphometric parameters of the Myxobolus species parasite of the jau´ were compared with the 23 Myxobolus species described infecting South American freshwater fish (Eiras et al., 2005; Adriano et al., 2006; Martins and Onaka, 2006; Casal et al., 2006). The spores of the Myxobolus parasite of the jau´ resembled those of M. pygocentris Penido, 1927, which is a parasite of Pygocentrus piraya; M. associates Nemeczek, 1926, which is a parasite of Leporinus mormyrops; and the oval spores of M. colossomatis Molna´r and Be´ke´si, 1992,
Figs. 9–11. Histological section of the opercula of jau´ specimen infected by Myxobolus cordeiroi n. sp. Fig. 9: plasmodia (P) in the adipose tissue (AT) between the skin (S) and the opercular bone tissue (B). Scale bar = 200 mm. Sirius red stain. Fig. 10: Inset of Fig. 9, showing the periphery of the plasmodia (PP), with young sporogonic stages (YSS), an outer collagen capsule (CC) and an inner layer of reticular fibers (RF) in contact with the plasmodia. Scale bar = 10 mm. Fig. 11: section of plasmodium (P) stained with reticulin, showing the reticular fiber layer. Scale bar = 10 mm.
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Figs. 12–15. Histological section of organs from jau´ specimen infected by M. cordeiroi n. sp. Fig. 12: A plasmodium (P) developing in the connective tissue (CT) of gill arch (GA). (GF: gill filament). H&E stain. Scale bar = 200 mm. Fig. 13: Parasite–host interface, showing plasmodium with mature spores (msp) in the inner layer, young sporogonic stages (yss) in the peripheral layer and a collagen capsule (CC) surrounding it. (CT: connective tissue, E: epidermis). Sirius red stain. Scale bar = 20 mm. Fig. 14: Plasmodium (P) in the tunica mucosa of the urinary bladder. H&E stain. Scale bar = 200 mm. Fig. 15: Inset of Fig. 14. Note the stretching of the epithelium (EP) and the collagen capsule (CC) surrounding the plasmodium (P). Scale bar = 10 mm.
which is a parasite of Colossoma macropomum. However, M. pygocentris has longer, wider spores (15–16 mm 9–11 mm) as well as longer and wider polar capsules (9– 11 mm 3–4 mm) (Gio´ia and Cordeiro, 1996; Eiras et al., 2005). M. associatus has longer and wider spores (15 mm 10 mm), longer polar capsules (7 mm) and spores of two different shapes (round and oval) (Gio´ia and Cordeiro, 1996; Eiras et al., 2005). M. colossomatis has thinner spores (3.7 mm) and spores of two shapes (ellipsoidal and oval) (Molna´r and Be´ke´si, 1992). The Myxobolus parasite species of the jau´ further differs from these species with regard to host species and infection sites in some cases, as M. associatus infects the kidney and M. pygocentrus infects the intestine (Gio´ia and Cordeiro, 1996; Eiras et al., 2005). This is the first molecular study on a myxosporea parasite in a South American freshwater fish, which
renders the comparison of molecular results with Myxobolus spp. from this region impossible. However, the BLAST search using the partial 18S rDNA sequence of Myxobolus parasite species of Z. jahu did not match any of the Myxozoa available in the GenBank. Thus, the Myxobolus species studied here differs in morphological aspects from other South American Myxobolus spp. and differs on the molecular level from Myxobolus species reported in the GenBank. It could therefore be considered a new species, for which the name M. cordeiroi n. sp. is proposed. The presence of Myxobolus species clustered in two monophyletic clades in the phylogenetic analysis was supported by high bootstrap values, with Clade A containing primarily parasites of freshwater fish and Clade B containing primarily parasites of marine fish and this result is in agreement with that of Fiala (2006). This
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Fig. 16. Schematic representation of mature spores of M. cordeiroi n. sp.: (A) frontal view; (B) lateral view. Scale bar = 5 mm.
is an important phylogenetic observation and should drive future phylogeography studies on the origin and colonization of Myxobolus in South American freshwater fish. However, Clade A formed several smaller clades (A1–A4), in which there was a clear grouping of species according to the host family. Clades A4 and A3 were formed only by Myxobolus parasite species of cyprinids, whereas there was a predominance of parasite species of salmonids in Clade A2. Finally, Clade A1 was composed of the new Myxobolus parasite species of Z. jahu. A similar situation was observed for Clade B, which clustered five species: M. procerus, which is a parasite of the freshwater percopsid P. omiscomaycus, and further divided into another clade strongly supported by bootstrap values, originating a monophyletic clade composed of four parasite species of marine mugilids (Mugil cephalus and Liza ramada). The results presented here contribute to studies on Myxobolus taxonomy and systematics, as this was the first phylogenetic analysis of Myxobolus species involving a South American host fish. The fact that M. cordeiroi n. sp. parasite of Z. jahu appeared alone in a distinct clade (A1) may reflect two conditions: (a) the existence of a group of Myxobolus species that are phylogenetically closely related to parasite species of South American fish; and (b) the existence of a group of Myxobolus species that are phylogenetically closely related to parasite species of pimelodids. The clades found in the present study demonstrated a strong tendency toward Myxobolus spp. clusters according to host family rather than geographic region, as species from North America (M. martini, M. algonquinensis, M. siddalli, M. smithi) and Europe (M. macrocapsularis, M. parviformis, M. impressus, M. dispar) clustered together in Clade A4. This hypothesis may be confirmed with the future study of molecular phylogeny in other Myxobolus parasite species of different host families of South American freshwater fish. The phylogeny presented in this study is in agreement with that presented by Molna´r et al. (2006), who
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found M. bramae (AF507968) by Eszterbauer (2004) and M. bramae (AF085177) by Andree et al. (1999) – both parasite species of Abramis brama (Cyprinidae) – occupying distinct positions: Clade A4 and A2, respectively. In further agreement with Molna´r et al. (2006), our results also present M. muelleri (AY325284) by Eszterbauer (2004), which is a parasite of Leuciscus cephalus (Cyprinidae), and M. muelleri (AY129314) by Bahri et al. (2003), which is a parasite of L. ramada (Mugilidae), in very different positions: Clade A4 and Clade B, respectively. The double position of M. bramae and M. muelleri in this phylogenetic analysis demonstrates that each one of these taxa represents two Myxobolus species, and these results are according to Ferguson et al. (2008), the ones who suggested that these differences were resulted of erroneous identifications. The histopathological analysis revealed that the development of M. cordeiroi n. sp. occurred in the connective tissue in all organs, demonstrating the affinity of the parasite to this tissue. Similar tissue specificity has been reported in other studies. Adriano et al. (2006) found M. cuneus infecting the connective tissue of several organs in Piaractus mesopotamicus (wall of the arterioles of the gill filaments, serosa capsule of the gall bladder, middle layer and subepithelial connective tissue of the urinary bladder, connective tissue between the rays of the fins, subcutaneous tissue of the head surface and fibrous capsule spleen). Cone and Easy (2005) report Myxobolus diaphanous Fantham et al. (1940) infecting the loose connective tissue of a variety of organs in Fundulus diaphanus in Canada. However, it is difficult to compare the real tissue affinity of the vast majority of Myxobolus species reported in the literature as infecting multiple organs, since most studies do not offer a detailed reference to the tissue in which parasite development occurred. However, Myxobolus species reported simultaneously parasitizing different organs, in fact, could have a tissue affinity rather than non-specificity by organ of infection. No inflammatory responses were observed in the organs infected by M. cordeiroi n. sp. However, the pathogenic potential of M. cordeiroi n. sp. may be related with the capacity of the parasite to induce tissue compression during its development, as occurred in the cornea and urinary bladder, where the development of the parasite produced considerable stretching of the epithelium. As M. cordeiroi n. sp. exhibits connective tissue affinity, this characteristic may indicate a parasite with the potential for massive infection and considerable injury to the host. Furthermore, the presence of plasmodia in the cornea may largely affect the visual perception of the host, thereby altering the prey– predator relationship. Given the lack of knowledge on pathogens that affect Z. jahu, data from the present study on the taxonomy and histopathology of M. cordeiroi n. sp. found infecting wild specimens of the jau´ could be of considerable importance in the handling of the species in cultivation.
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Fig. 17. Phylogenetic tree of neighbor-joining, minimal evolution and maximum parsimony, showing relationship between M. cordeiroi n. sp and other Myxobolus spp. based on partial 18S rDNA sequences. GenBank accession numbers are given after species name and habitats of the species host are between parentheses (FW = freshwater, E = estuarine and M = marine), according to Fishbase (2008). Numbers above nodes indicate bootstrap confidence levels (gaining more than 50% support) for NJ, ME and MP, respectively. The dashed lines indicate the differences between the three trees, taking maximum parsimony as basic topology.
Acknowledgments The authors thank Ricardo Afonso Torres de Oliveira (CEPTA/ICMBio) for the aid in the fish necropsy; Dr. Jose´ Augusto Ferraz de Lima, manager of the Pantanal National Park and Dr. Laerte Batista de Oliveira Alves, manager of the National Center for Research and Conservation of Continental Fishes (CEPTA/ICMBio) for
support in the fieldwork; and Richard Boike for editing the English. References Adriano, E.A., Arana, S., Cordeiro, N.S., 2006. Histopathology and ultrastructure of Myxobolus cuneus n. sp. infecting the connective tissue of Piaractus mesopotamicus (Pisces: Characidae) cultivated in Brazil. Parasite 13, 137–142.
E.A. Adriano et al. / Veterinary Parasitology 162 (2009) 221–229 Adriano, E.A., Arana, S., Ceccarelli, P.C., Cordeiro, N.S., 2002. Light and scanning electron microscopy of Myxobolus porofilus sp. n. (Myxosporea: Myxobolidae) infecting the visceral cavity of Prochilodus lineatus (Pisces: Characiformes; Prochilodontidae) cultivated in Brazil. Fol. Parasitol. 49, 259–262. Alves, C.B.M., Silva, L.G.M., Godinho, A.L., 2007. Radiotelemetry of a female jau´, Zungaro jahu (Ihering, 1898) (Siluriformes: Pimelodidae), passed upstream of Funil Dam, rio Grande. Brazil. Neotrop. Ichthyol. 5, 229–232. Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402., http://www.ncbi.nlm.nih.gov/blast. Andree, K.B., Sze´kely, C., Molna´r, K., Gresoviac, S.J., Hedrick, R.P., 1999. Relationships among members of the genus Myxobolus (Myxozoa: Bivalvidae) based on small subunit ribosomal DNA sequences. J. Parasitol. 85, 68–74. Azevedo, C., Corral, L., Matos, E., 2005. Ultrastructure of Triangulamyxa amazoˆnica n. gen. and n. sp. (Myxozoa, Myxosporea), a parasite of the Amazonian freshwter fish, Sphoeroides tetudineus (Teleostei, Tetrodontidae). Eur. J. Protistol. 41, 57–63. Bahri, S., Andree, K.B., Hedrick, R.P., 2003. Morphological and phylogenetic studies of marine Myxobolus spp. from mullet in Ichkeul Lake, Tunisia. J. Euk. Microbiol. 50, 463–470. Britski, H.A., Silimon, K.Z.S., Lopes, B.S., 2006. Peixes do Pantanal: manual de identificac¸a˜o, 2nd ed. Embrapa/CPAP, Brası´lia, 2007. 227 pp. Buckup, P.A., Menezes, N.A., Ghazzi, M.S., 2007. Cata´logo das espe´cies de peixes de a´gua doce do Brasil. Museo Nacional, Rio de Janeiro, 195 p. Casal, G., Matos, E., Azevedo, C., 2006. A new myxozoan parasite from the Amazonian fish Metynnis argenteus (Teleostei, Characidae): light and electron microscope observations. J. Parasitol. 92, 817–821. Cellere, E.F., Cordeiro, N.S., Adriano, E.A., 2002. Myxobolus absonus sp. n. (Myxozoa: Myxosporea) parasitizing Pimelodus maculatus (Siluriformes: Pimelodidae), a South American freshwater fish. Mem. Inst. Oswaldo Cruz 97, 79–80. Ceccarelli, P.S., Adriano, E.A., Santos, S.M.C., Rego, R.F., Silva, L.O.L., 2007. Levantamento Quali-quantitativo da fauna parasitolo´gica de peixes do Pantanal Mato-Grossense. In: Centro Nacional de Pesquisa e Gesta˜o de Recursos Pesqueiros Continentais-Cepta. Pesquisas patolo´gicas e gene´ticas em recursos pesqueiros da Bacia do Alto Paraguai. Ibama, Pirassununga, pp. 16–116. Cemig., 2007. Projeto visa salvar peixe jau´ do perigo de extinc¸a˜o. http:// www.odebate.com.br/index.php?option=com_content&task=view&id=5645&Itemid=21. Accessed on 18/09/2008. Cone, D.K., Easy, R.H., 2005. Suplemental diagnosis and molecular taxonomy of Myxobolus diaphanous (Fanthan, Porter et Richardson, 1940) (Myxozoa) parasitizing Fundulus diaphanous (Cyprinodontiformes) in Nova Scotia, Canada. Folia Parasitol. 52, 217–222. Eiras, J.C., 2005. On overview on the myxosporean parasites in amphibians and reptiles. Acta Parasitol. 50, 267–275. Eiras, J.C., Molnar, K., Lu, Y.S., 2005. Synopsis of the species of Myxobolus Butschli, 1882 (Myxozoa: Myxosporea: Myxobolidae). Syst. Parasitol. 61, 1–46. Eszterbauer, E., 2004. Genetic relationship among gill-infecting Myxobolus species (Myxosporea) of cyprinids: molecular evidence of importance of tissue-specificity. Dis. Aquatic. Org. 58, 35–40.
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Ferguson, J.A., Atkinson, S.D., Whipps, C.M., Kent, M.L., 2008. Molecular and morphological analysis of Myxobolus spp. of salmonid fishes with the description of a new Myxobolus species. J. Parasitol. 94, 1322– 1334. Fiala, I., 2006. The phlogeny of Myxosporea (Myxozoa) based on small subunit ribosomal RNA gene analysis. Int. J. Parasitol. 36, 1521–1534. Fishbase - World Wide Web electronic publication. www.fishbase.org, version (04/2008). Accessed in 18/09/2008. Gio´ia, I., Cordeiro, N.S., 1996. Brazilian myxosporidian’s check-list (Myxozoa). Acta Protozool. 35, 137–149. Lom, J., Arthur, J.R., 1989. A guideline for the preparation of species description in Myxosporea. J. Fish Dis. 12, 151–156. Lundberg, J.G., Littmann, M.W., 2003. Family Pimelodidae (Long-whiskered catfishes). In: Reis, R.E., Kullander, S.O., Ferraris, Jr., C.J. (Eds.), Check list of the freshwater fishes of South and Central America. Edipucrs, Porto Alegre, pp. 432–446. Mateus, L.A.F., Penha, J.M.F., Petrere, M., 2004. Fishing resources in the rio Cuiaba´ basin, Pantanal do Mato Grosso, Brazil. Neotrop. Ichthyol. 2, 217–227. Martins, M.L., Onaka, E.M., 2006. Henneguya garavelli n. sp. and Myxobolus peculiaris n. sp. (Myxozoa: Myxobolidae) in the gills of Cyphocharax nagelli (Osteichthyes: Curimatidae) from Rio do Peixe Reservoir, Sa˜o Jose´ do Rio Pardo, Sa˜o Paulo, Brazil. Vet. Parasitol. 137, 253–261. Molna´r, K., Cech, G., Sze´kely, C., 2008. Infection of the heart of the common bream, Abramis brama (L.), with Myxobolus s.l. dogieli (Myxozoa, Myxobolidae). J. Fish Dis. 31, 613–620. Molna´r, K., Eszterbauer, E., Sze´kely, C., Da´n, A´., Harrach, B., 2002. Morphological and molecular biological studies on intramuscular Myxobolus spp. of cyprinid fish. J. fish Dis. 25, 643–652. Molna´r, K., Marton, S.Z., Eszterbauer, E., Sze´kely, C., 2007. Description of Myxobolus gayerae sp. n. and re-description of Myxobolus leuciscini infecting the European chub from the Hungarian stretch of the River Danube. Dis. Aquatic Org. 78, 147–153. Molna´r, K., Marton, S.Z., Eszterbauer, E., Sze´kely, C., 2006. Comparative morphological and molecular studies on Myxobolus spp. infecting chub from the River Danube, Hungary, and description of Myxobolus muellericus sp. n. Dis. Aquat. Org. 73, 49–61. Molna´r, K., Be´ke´si, L., 1992. Description of a new Myxobolus species, M. colossomatis n. sp. from the teleost Colossoma macropomum of the Amazon river basin. J. Appl. Ichthyol. 9, 57–63. Projeto Pacu, 2008. Jau´. http://www.projetopacu.com.br/portugues/ index.html?p=produtos. Assessed on 20/09/2008. Resende, E.K., 2003. Migratory fishes of the Paraguay-Parana´ Basin excluding the upper Parana´ Basin. In: Carolsfeld, J., Harvey, B., Ross, C., Baer, A. (Eds.), Migratory Fishes of South America: Biology, Fisheries and Conservation Status. National library of Canada Cataloguing in Publication Data, Canada, pp. 103-155. Schmahl, G., Mehlhorn, H., Taraschewski, H., 1989. Treatment of fishparasites. 7. Effects of Sym. Triazinone (Toltrazuril) on developmental stages of Myxobolus sp. Bu¨tschli, 1882 (Myxosporea, Myxozoa): a light and electron microscopic study. Eur. J. Protistol. 25, 26–32. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599. Xia, X., Xie, Z., 2001. DAMBE: data analysis in molecular biology and evolution. J. Hered. 92, 371–373.
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Myxobolus cordeiroi n. sp., a parasite of Zungaro jahu (Siluriformes: Pimelodiade) from Brazilian Pantanal: Morphology, phylogeny and histopathology§ E.A. Adriano a,*, S. Arana b, A.L. Alves c, M.R.M. Silva d, P.S. Ceccarelli e, F. Henrique-Silva f, A.A.M. Maia d a Departamento de Cieˆncias Biolo´gicas, Universidade Federal de Sa˜o Paulo (UNIFESP), Rua Professor Artur Riedel, 275, Jardim Eldorado, CEP 09972-270 Diadema, SP, Brazil b Departamento de Histologia e Embriologia da Universidade Estadual de Campinas (UNICAMP), Caixa Postal 6109, CEP 13083-970 Campinas, SP, Brazil c Departamento de Biologia, Universidade Estadual Paulista-UNESP, Avenida 24-A n8 1515, Caixa Postal 199, CEP 13506-900 Rio Claro, SP, Brazil d Departamento de Cieˆncias Ba´sicas, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de Sa˜o Paulo (USP), Rua Duque de Caxias Norte, 225, CEP 13635-900 Pirassununga, SP, Brazil e Centro Nacional de Pesquisa e Conservac¸a˜o de Peixes Continentais (CEPTA), Instituto Chico Mendes de Conservac¸a˜o da Biodiversidade (ICMBio), Rodovia SP 201, Km 6.5, Caixa Postal 64, CEP 13630-970 Pirassununga, SP, Brazil f Departamento de Gene´tica e Evoluc¸a˜o da Universidade Federal de Sa˜o Carlos-UFSCAR, Rodovia Washington Luis, Km 235, Caixa Postal 676, CEP 13.565-905 Sa˜o Carlos, SP, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 December 2008 Received in revised form 5 March 2009 Accepted 14 March 2009
This work is part of an ongoing investigation into the characteristics of Myxozoan parasites of freshwater fish in Brazil and was carried out using morphology, histopathology and molecular analysis. A new Myxosporea species (Myxobolus cordeiroi) is described infecting the jau´ catfish (Zungaro jahu). Fifty jau´ specimens were examined and 78% exhibited plasmodia of the parasite. The plasmodia were white and round, measuring 0.3–2.0 mm in diameter and the development occurred in the gill arch, skin, serosa of the body cavity, urinary bladder and eye. The spores had an oval body and the spore wall was smooth. Partial sequencing of the 18S rDNA gene resulted in a total of 505 bp and the alignment of the sequences obtained from samples in different organs revealed 100% identity. In the phylogenetic analysis, the Myxobolus species clustered into two clades—one primarily parasites of freshwater fish and the other primarily parasites of marine fish. M. cordeiroi n. sp. was clustered in a basal position in the freshwater fish species clade. The histological analysis revealed the parasite in the connective tissue of the different infected sites, thereby exhibiting affinity to this tissue. The plasmodium was surrounded by an outer collagen capsule of fibers with distinct orientation from the adjacent connective tissue and an inner layer composed of delicate collagen fibrils—more precisely reticular fibers. The development of the parasite in the cornea and urinary bladder caused considerable stretching of the epithelium. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Zungaro jahu Myxobolus cordeiroi n. sp. Myxozoa jau´ Pantanal Brazil
1. Introduction
§
Work supported by FAPESP (Proc. no. 06/59075-6). * Corresponding author. Tel.: +55 11 4049 3300; fax: +55 11 4043 6428. E-mail address: [email protected] (E.A. Adriano).
0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.03.030
The Brazilian Pantanal is the largest floodplain in the world and has approximately 150,000 km2. It comprises several important rivers, such as the Paraguay, Cuiaba´ and Miranda rivers and it is part of the upper Paraguay
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River Basin (Mateus et al., 2004; Ceccarelli et al., 2007). A great diversity of fish is found in this area, with 264 known species (Britski et al., 2006; Buckup et al., 2007). One of these species, Zungaro jahu (Ihering, 1898), is a carnivorous pimelodid, popularly known as jau´. The species is endemic to the rivers that form the Plata River Basin (Parana´, Paraguay and Uruguay). It occurs in Brazil, Argentina, Bolivia and Paraguay (Lundberg and Littmann, 2003) and is an important commercial and sport fish (Mateus et al., 2004). This catfish lives in deep waters in river channels, mainly near waterfalls (Britski et al., 2006; Ceccarelli et al., 2007; Buckup et al., 2007). It reaches sizes up to 150 kg and is the largest Neotropical catfish (Alves et al., 2007). In fish farms, individuals reach around 4 kg in the first year (Projeto Pacu, 2008). The jau´ population has undergone a drastic reduction due to overexploitation and the catch of this species has been prohibited in some regions (Resende, 2003). Changes in its habitat caused by anthropogenic action (especially the construction of hydroelectric plants) constitute another important threat to the species. Thus, some Brazilian research institutes have developed jau´ cultivation methods with the aim of reintroducing specimens into the environment (Cemig, 2007), whereas the species has been cultivated in fish farms as a sport and ornamental fish (Projeto Pacu, 2008). Myxosporea are among the most important fish parasites (Schmahl et al., 1989), with about 1500 currently known species (Azevedo et al., 2005). These parasites mainly infect fish, but some are parasites of reptiles and amphibians (Eiras, 2005). Among the Myxosporea, species of the genus Myxobolus are, so far, the most commonly found in fish, with more than 700 known species throughout the world (Eiras et al., 2005). In South America, 23 Myxobolus species have been described thus far and only 4 have been reported to infect pimelodids (Gio´ia and Cordeiro, 1996; Cellere et al., 2002). With the use of classical zoological methods, it is very difficult to determine the validity of morphologically similar Myxosporea with identical tissue affinity and that develop in taxonomically closely related host species (Molna´r et al., 2002). Thus, the taxonomic classification of Myxosporea based on morphology alone (Lom and Arthur, 1989) has been refined with the application of molecular biological methods (Molna´r et al., 2008, 2007, 2006; Andree et al., 1999). The present study is part of an ongoing investigation into the characteristics of Myxosporea parasites of freshwater fish in Brazil. Morphological, scanning electron microscopy, histopathological and molecular data were used to describe a new Myxobolus species found simultaneously infecting several organs in Z. jahu from the Brazilian Pantanal. 2. Materials and methods Fifty adult specimens of jau´ (Z. jahu) were collected on five expeditions to the Pantanal Mato-Grossense in central Brazil. Specimens were caught in the Aquidauna River and Miranda River in the southern region of the Pantanal in November 2001, October 2002, May 2003
and November 2003. Specimens were caught in rivers and lakes within the Pantanal National Park in the central region and in the Cuiaba´ River in the northern region of the Pantanal in June 2004, October 2004 and July 2005. Immediately after collection, the fish were transported alive to the field laboratory mounted nearby, where they were measured, weighed and necropsied. Cysts and/or infected organs were fixed in 10% buffered formalin for taxonomic and histopathological analyses and in 100% ethanol for molecular analysis. Morphological and morphometric studies of the spores were based on fixed mature spores (Lom and Arthur, 1989) obtained from the plasmodia in the different organs (40 spores from each organ). Measurements were performed on computer equipped with the Axivision 4.1 image capturing system coupled to an Axioplan 2 Zeiss Microscope. Spore dimensions (mm) were expressed as mean standard deviation (SD). Smears containing free spores were stained with Giemsa solution and mounted in a low-viscosity mounting medium (CytosealTM) as permanent slides. For the scanning electron microscopy, histological sections of plasmodia fixed and embedded using routine histological procedures were cut into sections of 10 mm, deposited on a coverslip coated with albumin, oven-dried for 48 h, deparaffinized with xylol, re-dehydrated, postfixed in 1% OsO4 in a cacodylate buffer for 30 min at 4 8C, washed in the same buffer, dehydrated in ethanol, critical point-dried in CO2, covered with metallic gold and examined under a Joel JMS 35 microscope operated at 15 kV (Adriano et al., 2002). For the molecular analysis, plasmodia obtained from different organs (skin, urinary bladder and eye) of Z. jahu caught in the Pantanal National Park and the Cuiaba´ River in July 2005 were removed from the host tissue, ruptured with the aid of a needle and the contents were collected in a 1.5 ml microcentrifuge tube. DNA was extracted using a Wizard1 Genomic DNA Purification kit (Promega, USA), following the manufacturer’s tissue protocol. DNA content was determined by biospectrophotometry at 260 nm. The following the oligonucleotide primers were used in the polymerase chain reaction (PCR) amplification: Forward MX5: 50 -CTGCGGACGGCTCAGTAAATCAGT-30 and Reverse MX3: 50 -CCAGGACATCTTAGGGCATCACAGA-30 , specific to the family Myxobolidae, as described by Andree et al. (1999). PCR was carried out in a final volume of 50 ml, which contained 10–50 ng of extracted DNA, 1 Taq DNA Polymerase buffer (Invitrogen), 0.2 mmol of dNTP (Invitrogen), 1.5 mmol of MgCl2, 10 pmol of each primer (Invitrogen), 2.5 U of Taq DNA polymerase (Invitrogen) and distilled water. A PTC-100 (MJ Research inc.) thermal cycler was used. Amplification was performed in the following manner: 35 cycles, preceded by a denaturation step at 95 8C for 5 min, denaturation (95 8C for 60 s), annealing (59 8C for 60 s) and extension (72 8C for 120 s), finished with an extended elongation step at 72 8C for 5 min. PCR products were submitted to electrophoresis on 1.0% agarose gel (BioAmerica) in a TBE buffer (0.045 M Tris–borate, 0.001 M EDTEA pH 8.0), stained with Vistra Green and analyzed in a
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FLA-3000 (Fugi) scanner. Size of the amplified fragments was estimated by comparisons with the 100 bp DNA Ladder (Invitrogen). The fragments were then purified using the Wizard SV Gel and PCR Clean-up System (Promega) and cloned into a pTZ57R/T, using the InsT/A cloning kit (Fermentas). Positive clones were selected using the blue-white screening and sequenced with the same oligonucleotide primers used to the amplification. At least five positive clones per sample were sequenced using the ET-Dye Terminator Kit (GE Healthcare) in a MegaBace 1000 automatic sequencer (GE Healthcare). A standard nucleotide-nucleotide BLAST (blastn) search was conducted (Altschul et al., 1997). Sequences of Myxobolus species obtained from three organs of Z. jahu were aligned for comparisons with one another and with sequences obtained from GenBank, using DAMBE (Xia and Xie, 2001). Nucleotide saturation was analyzed for the 18S rDNA gene by plotting uncorrected p distances against absolute distance values. Phylogenetic analyses were conduced using the MEGA 4.0 program (Tamura et al., 2007) for the neighbor-joining (NJ), minimal evolution (ME) and maximum parsimony phylogenetic methods. The Kimura two-parameter (K2P) evolution sequence model was used in the analysis. Bootstrap analysis (1000 replicates) was employed to assess the relative robustness of the branches of the NJ, ME and MP trees, using the MEGA 4.0 program. The species Ceratomyxa sparusaurati and C. shasta were used as the outgroup in the phylogenetic analysis. For the histological analysis, fragments of infected organs fixed in 10% buffered formalin were embedded
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in paraffin, cut into serial sections 4 mm thick and stained with hematoxylin/eosin, Sirius Red and reticulin stain. 3. Results Among the 50 specimens of jau´ examined in the present study, 39 (78%) had plasmodia of an unknown parasite from the genus Myxobolus. Among the infected fish, 76.9% were infected in the gill arch, 23.1% had the parasite in the skin, 20.5% were infected in serosa of the body cavity, 17.9% had the parasite in urinary bladder and 7.7% were infected in the eyes. Infection occurred in more than one organ in 25.6%. Plasmodia were always present in the connective tissue of the organs. 3.1. Description of Myxobolus cordeiroi sp. n. (Figs. 1–16) The polysporic plasmodia were white, round and measured 0.3–2.0 mm in diameter (Figs. 1 and 2). The development of the parasite was asynchronic, with different developmental stages along the periphery of the plasmodium and mature spores in the internal region (Figs. 10 and 13). Formalin-fixed mature spores had an oval body in the frontal view, with the anterior extremity more slender than the posterior extremity (Figs. 3–5). Light and scanning electron microscopy showed the presence of smooth valves, with suture folds and a sutural protrusion forming a rim around the spores (Figs. 4 and 5). In the lateral view, the spores were symmetric, convex and had a conspicuous
Figs. 1–5. Morphological aspects of plasmodia and spores of Myxobolus cordeiroi n. sp. Fig. 1: plasmodia in the gill arch of jau´. Scale bar = 1.5 mm. Fig. 2: plasmodium in the eye. Scale bar = 1.5 mm. Figs. 3 and 4: spores in fresh preparations. Note the sutural rim around the spore (arrow) (3 bar = 10 mm and 4 = 5 mm). Fig. 5: scanning electron microscopy. Note the suture folds and a rim around the spores (arrows) Scale bar = 10 mm.
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suture line (Figs. 3–5). The polar capsules were elongated and equal in size, with the anterior ends close to one another. The polar filaments were coiled in 5–6 turns perpendicular to the axis of the capsule (Fig. 16). Table 1 displays the dimensions of the spores in the different infection sites. Type host Site of infection Prevalence Locality
Type material
Etymology
Zungaro jahu Humboldt, 1821 (Pimelodidae). Gill arch, skin, serosa of the body cavity, urinary bladder and eye. 39/50 (78%) of the Z. jahu were infected. Pantanal Mato-Grossense (Aquidauana, Cuiaba´, Miranda and Paraguay rivers) in the Paraguay basin, Brazil. Slides with stained spores (syntype) have been deposited in the collection of the Museum of Natural History, Institute of Biology, State University of Campinas (Unicamp), State of Sa˜o Paulo, Brazil (accession number: ZUEC 24). The species name (M. cordeiroi) is in homage to Dr. Nelson da Silva Cordeiro, one of the pioneers in the study of Myxozoa in Brazil, who retired in 2006.
In the molecular analysis, the specific primer pair MX5MX3 successfully amplified an approximately 1600 bp fragment of the 18S rDNA gene in the spores obtained from plasmodia found infecting the urinary bladder, eye and skin of Z. jahu. The alignment of sequences of the samples from the urinary bladder, eye and skin revealed 100% identity. The BLAST search using the partial 18S rDNA sequence data (505 bp of the fragment 50 end) of the Myxobolus species parasite of Z. jahu did not match any of the Myxozoa available in the GenBank. The partial 18S rDNA sequences data of M. cordeiroi n. sp. have been deposited in GenBank under the accession number FJ827757. In the phylogenetic analysis (based on the alignment of 599 bp, with 210 informative characters and complete deletion), the Myxobolus species clustered into two monophyletic clades in the three phylogenetic methods (Fig. 17). Clade A was further divided into four monophyletic clades (Clades A1 to A4), among which, Clades A4 and A3 were composed only by Myxobolus parasite species of cyprinids fish. Clade A2 grouped four Myxobolus parasite species of salmonids (M. cerebralis, M. arcticus, M. insidiosus and M. neurobius), two parasite species of cyprinids (M. djragini and M. bramae AF085177) and M. sandrae, a parasite of Sander lucioperca (Percidae). Clade A1 was the basal unit for Clade A and was composed of M. cordeiroi n. sp., a parasite of Z. jahu and a unique South American freshwater fish parasite as well as a unique parasite of pimelodids. Monophyletic Clade B clustered five Myxobolus species: M. procerus, a parasite of the freshwater fish Percopsis omiscomaycus (Percopsidae), was the basal unit for Clade B and sister group of a clade composed of four parasite species of marine fish (M. bizerti, M. ichkeulensis, M. exiguus and M. muelleri AY129314, all parasites of mullet). Histological analysis revealed that plasmodia occurred in the connective tissue in all infection sites (Figs. 6–15). There was a collagen capsule surrounding the plasmodium, which had fibers with distinct orienta-
Figs. 6–8. Histological section of eye of the jau´ with two plasmodia of M. cordeiroi n. sp. in the connective tissue of corneal stroma. Note the stretching of the corneal epithelium in Fig. 6 (arrows). Scale bar = 300 mm. Fig 7: Inset of Fig. 6 showing amplified interface of plasmodium (P) and host tissue (H). Note a collagen capsule (CC) composed of interlaced fibers. Fig. 8: details of Fig. 7, showing fiber organization. Scale bars = 10 mm. Sirius red stain.
tion from the adjacent connective tissue and an inner layer composed of delicate collagen fibrils, which were reticular fibers in contact with the plasmodium (Fig. 11). In the eye, the plasmodia were located in the connective tissue of corneal stroma. Parasite development caused stretching of the corneal epithelium, which, at some points, was formed by only one cell layer (Figs. 6–8). In skin of the surface of the opercula, plasmodia occurred in the adipose tissue between the skin and the opercular bone tissue (Fig. 9). In the gill arch, plasmodia occurred in the base of the organ (Fig. 12). In the urinary bladder, plasmodia occurred in the tunica mucosa, producing stretching of the epithelium (Fig. 14). No inflammatory reaction was observed in any of the infection sites. 4. Discussion Morphological and morphometric studies revealed a constant pattern in the spores obtained from the plasmodia of Myxobolus found infecting the gill arch, skin, serosa of the body cavity, urinary bladder and eye of Z. jahu. This
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Table 1 Spore dimensions of Myxobolus cordeirus n. sp. from several organs of the jau´. Measurements are given in mm and expressed as the mean standard deviation. Location of plasmodia
Gill arch
Skin
Serosa
Urinary bladder
Eye
Spore length Spore width Spore thickness
11.1 0.2 7.3 0.1 5.5 0.1
10.9 0.4 7.5 0.3 5.6 0.1
11.3 0.3 7.1 0.2 5.2 03
11.2 0.2 7.3 0.3 5.4 0.2
10.8 0.5 7.2 0.2 5.5 0.2
5.4 0.3 1.4 0.1
5.3 0.2 1.4 0.2
5.3 0.1 1.4 0.2
5.4 0.2 1.5 0.3
5.2 0.3 1.5 0.2
Polar capsule Length Width
suggests a unique Myxobolus species that causes simultaneous infection in several organs. This hypothesis was corroborated by the partial 18S rDNA gene sequence of spores obtained from the skin, urinary bladder and eye, which demonstrated 100% identity in the alignment sequences. According to Molna´r et al. (2002), zoological methods alone make it is very difficult to determine the validity of morphologically similar myxosporea with identical tissue affinity and developing in taxonomically closely related host species. This same concept can be applied to morphologically similar myxosporea found infecting different organs in the same host species, since the site of infection cannot be used as a
taxonomic criterion and spore morphology may not be suitable for validating these myxosporea species. In such conditions, molecular methods are an important taxonomic tool. The morphology and morphometric parameters of the Myxobolus species parasite of the jau´ were compared with the 23 Myxobolus species described infecting South American freshwater fish (Eiras et al., 2005; Adriano et al., 2006; Martins and Onaka, 2006; Casal et al., 2006). The spores of the Myxobolus parasite of the jau´ resembled those of M. pygocentris Penido, 1927, which is a parasite of Pygocentrus piraya; M. associates Nemeczek, 1926, which is a parasite of Leporinus mormyrops; and the oval spores of M. colossomatis Molna´r and Be´ke´si, 1992,
Figs. 9–11. Histological section of the opercula of jau´ specimen infected by Myxobolus cordeiroi n. sp. Fig. 9: plasmodia (P) in the adipose tissue (AT) between the skin (S) and the opercular bone tissue (B). Scale bar = 200 mm. Sirius red stain. Fig. 10: Inset of Fig. 9, showing the periphery of the plasmodia (PP), with young sporogonic stages (YSS), an outer collagen capsule (CC) and an inner layer of reticular fibers (RF) in contact with the plasmodia. Scale bar = 10 mm. Fig. 11: section of plasmodium (P) stained with reticulin, showing the reticular fiber layer. Scale bar = 10 mm.
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Figs. 12–15. Histological section of organs from jau´ specimen infected by M. cordeiroi n. sp. Fig. 12: A plasmodium (P) developing in the connective tissue (CT) of gill arch (GA). (GF: gill filament). H&E stain. Scale bar = 200 mm. Fig. 13: Parasite–host interface, showing plasmodium with mature spores (msp) in the inner layer, young sporogonic stages (yss) in the peripheral layer and a collagen capsule (CC) surrounding it. (CT: connective tissue, E: epidermis). Sirius red stain. Scale bar = 20 mm. Fig. 14: Plasmodium (P) in the tunica mucosa of the urinary bladder. H&E stain. Scale bar = 200 mm. Fig. 15: Inset of Fig. 14. Note the stretching of the epithelium (EP) and the collagen capsule (CC) surrounding the plasmodium (P). Scale bar = 10 mm.
which is a parasite of Colossoma macropomum. However, M. pygocentris has longer, wider spores (15–16 mm 9–11 mm) as well as longer and wider polar capsules (9– 11 mm 3–4 mm) (Gio´ia and Cordeiro, 1996; Eiras et al., 2005). M. associatus has longer and wider spores (15 mm 10 mm), longer polar capsules (7 mm) and spores of two different shapes (round and oval) (Gio´ia and Cordeiro, 1996; Eiras et al., 2005). M. colossomatis has thinner spores (3.7 mm) and spores of two shapes (ellipsoidal and oval) (Molna´r and Be´ke´si, 1992). The Myxobolus parasite species of the jau´ further differs from these species with regard to host species and infection sites in some cases, as M. associatus infects the kidney and M. pygocentrus infects the intestine (Gio´ia and Cordeiro, 1996; Eiras et al., 2005). This is the first molecular study on a myxosporea parasite in a South American freshwater fish, which
renders the comparison of molecular results with Myxobolus spp. from this region impossible. However, the BLAST search using the partial 18S rDNA sequence of Myxobolus parasite species of Z. jahu did not match any of the Myxozoa available in the GenBank. Thus, the Myxobolus species studied here differs in morphological aspects from other South American Myxobolus spp. and differs on the molecular level from Myxobolus species reported in the GenBank. It could therefore be considered a new species, for which the name M. cordeiroi n. sp. is proposed. The presence of Myxobolus species clustered in two monophyletic clades in the phylogenetic analysis was supported by high bootstrap values, with Clade A containing primarily parasites of freshwater fish and Clade B containing primarily parasites of marine fish and this result is in agreement with that of Fiala (2006). This
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Fig. 16. Schematic representation of mature spores of M. cordeiroi n. sp.: (A) frontal view; (B) lateral view. Scale bar = 5 mm.
is an important phylogenetic observation and should drive future phylogeography studies on the origin and colonization of Myxobolus in South American freshwater fish. However, Clade A formed several smaller clades (A1–A4), in which there was a clear grouping of species according to the host family. Clades A4 and A3 were formed only by Myxobolus parasite species of cyprinids, whereas there was a predominance of parasite species of salmonids in Clade A2. Finally, Clade A1 was composed of the new Myxobolus parasite species of Z. jahu. A similar situation was observed for Clade B, which clustered five species: M. procerus, which is a parasite of the freshwater percopsid P. omiscomaycus, and further divided into another clade strongly supported by bootstrap values, originating a monophyletic clade composed of four parasite species of marine mugilids (Mugil cephalus and Liza ramada). The results presented here contribute to studies on Myxobolus taxonomy and systematics, as this was the first phylogenetic analysis of Myxobolus species involving a South American host fish. The fact that M. cordeiroi n. sp. parasite of Z. jahu appeared alone in a distinct clade (A1) may reflect two conditions: (a) the existence of a group of Myxobolus species that are phylogenetically closely related to parasite species of South American fish; and (b) the existence of a group of Myxobolus species that are phylogenetically closely related to parasite species of pimelodids. The clades found in the present study demonstrated a strong tendency toward Myxobolus spp. clusters according to host family rather than geographic region, as species from North America (M. martini, M. algonquinensis, M. siddalli, M. smithi) and Europe (M. macrocapsularis, M. parviformis, M. impressus, M. dispar) clustered together in Clade A4. This hypothesis may be confirmed with the future study of molecular phylogeny in other Myxobolus parasite species of different host families of South American freshwater fish. The phylogeny presented in this study is in agreement with that presented by Molna´r et al. (2006), who
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found M. bramae (AF507968) by Eszterbauer (2004) and M. bramae (AF085177) by Andree et al. (1999) – both parasite species of Abramis brama (Cyprinidae) – occupying distinct positions: Clade A4 and A2, respectively. In further agreement with Molna´r et al. (2006), our results also present M. muelleri (AY325284) by Eszterbauer (2004), which is a parasite of Leuciscus cephalus (Cyprinidae), and M. muelleri (AY129314) by Bahri et al. (2003), which is a parasite of L. ramada (Mugilidae), in very different positions: Clade A4 and Clade B, respectively. The double position of M. bramae and M. muelleri in this phylogenetic analysis demonstrates that each one of these taxa represents two Myxobolus species, and these results are according to Ferguson et al. (2008), the ones who suggested that these differences were resulted of erroneous identifications. The histopathological analysis revealed that the development of M. cordeiroi n. sp. occurred in the connective tissue in all organs, demonstrating the affinity of the parasite to this tissue. Similar tissue specificity has been reported in other studies. Adriano et al. (2006) found M. cuneus infecting the connective tissue of several organs in Piaractus mesopotamicus (wall of the arterioles of the gill filaments, serosa capsule of the gall bladder, middle layer and subepithelial connective tissue of the urinary bladder, connective tissue between the rays of the fins, subcutaneous tissue of the head surface and fibrous capsule spleen). Cone and Easy (2005) report Myxobolus diaphanous Fantham et al. (1940) infecting the loose connective tissue of a variety of organs in Fundulus diaphanus in Canada. However, it is difficult to compare the real tissue affinity of the vast majority of Myxobolus species reported in the literature as infecting multiple organs, since most studies do not offer a detailed reference to the tissue in which parasite development occurred. However, Myxobolus species reported simultaneously parasitizing different organs, in fact, could have a tissue affinity rather than non-specificity by organ of infection. No inflammatory responses were observed in the organs infected by M. cordeiroi n. sp. However, the pathogenic potential of M. cordeiroi n. sp. may be related with the capacity of the parasite to induce tissue compression during its development, as occurred in the cornea and urinary bladder, where the development of the parasite produced considerable stretching of the epithelium. As M. cordeiroi n. sp. exhibits connective tissue affinity, this characteristic may indicate a parasite with the potential for massive infection and considerable injury to the host. Furthermore, the presence of plasmodia in the cornea may largely affect the visual perception of the host, thereby altering the prey– predator relationship. Given the lack of knowledge on pathogens that affect Z. jahu, data from the present study on the taxonomy and histopathology of M. cordeiroi n. sp. found infecting wild specimens of the jau´ could be of considerable importance in the handling of the species in cultivation.
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Fig. 17. Phylogenetic tree of neighbor-joining, minimal evolution and maximum parsimony, showing relationship between M. cordeiroi n. sp and other Myxobolus spp. based on partial 18S rDNA sequences. GenBank accession numbers are given after species name and habitats of the species host are between parentheses (FW = freshwater, E = estuarine and M = marine), according to Fishbase (2008). Numbers above nodes indicate bootstrap confidence levels (gaining more than 50% support) for NJ, ME and MP, respectively. The dashed lines indicate the differences between the three trees, taking maximum parsimony as basic topology.
Acknowledgments The authors thank Ricardo Afonso Torres de Oliveira (CEPTA/ICMBio) for the aid in the fish necropsy; Dr. Jose´ Augusto Ferraz de Lima, manager of the Pantanal National Park and Dr. Laerte Batista de Oliveira Alves, manager of the National Center for Research and Conservation of Continental Fishes (CEPTA/ICMBio) for
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