INTRODUCTION
The Amazon basin contains the richest ichthyofauna in the world, with estimated 2500 of the 6000 species of Neotropical species (Goulding, Reference Goulding1980; Reis et al. Reference Reis, Kullander, Ferraris, Reis, Kullander and Ferraris2003). In the State of Acre in Western Amazonia, Brazil, c. 310 species of fish were catalogued, representing nearly 11% of all fish species recorded in Brazil (Acre, 2010).
According to the structural census report of fishing in the continental waters of North Brazil, the fishing practiced in the State of Acre is basically artisanal and for subsistence, representing the main protein source for local people (Vieira et al. Reference Vieira, Giarrizzo, Spach, Lana, Bianchini, Ribeiro, Niencheski, Filmann and Santos2006). In recent years, as a measure to reduce deforestation and improve the local economy, fish farming, mostly in already deforested and abandoned areas near or within small-dams, has been encouraged as a sustainable alternative to cattle-farming (Reis et al. Reference Reis, Honório, Barros, Kitron, Camara, Pereira, Keppeler, Silva-Nunes and Codeço2015). Thus, ichthyoparasitological studies in the Amazon should encompass, in addition to cultivated species, native species generally consumed by the local population.
In the State of Acre, Pimelodus blochii Valenciennes, 1840 is the fish eaten most often by the locals. The Pimelodidae are exclusively freshwater fishes, occurring in South America and Panama (Lundberg and Littmann, Reference Lundberg, Littmann, Reis, Kullander and Ferraris2003; Ribeiro and Lucena, Reference Ribeiro and Lucena2006). The genus Pimelodus Lacépède, 1803, is composed of c. 24 valid species, with 8, including P. blochii, from the Amazon Basin (Ribeiro and Lucena, Reference Ribeiro and Lucena2006).
This, the first helminthological survey on P. blochii from the Acre and Xapuri Rivers, Northwest Brazil, revealed 2 species of atractid nematodes in the intestine of this fish. The present paper includes the results of this survey, with the description of Orientatractis moraveci n. sp. and new morphological and ultrastructural data on Rondonia rondoni Travassos, Reference Travassos1920. In addition, new 18S rDNA and 28S rDNA sequences are presented with a phylogenetic analysis based on new and existing sequences of partial 18S rDNA.
MATERIALS AND METHODS
Specimens of the catfish P. blochii, collected between November, 2013 and April, 2014 by local fisherman operating in the Acre River (n = 120) (10°39′40″S, 68°30′19″W) and the Xapuri River (n = 120) (10°38′20″S, 68°32′08″W), were transferred fresh to the laboratory. Pimelodus blochii from Acre River were 8·8–25·7 (18·58 ± 2·12) cm long and from Xapuri River were 13·7–23·2 (14·27 ± 1·12) cm long. The organs were separated in Petri dishes and examined under a stereomicroscope. Prevalence and intensity data follow Bush et al. (Reference Bush, Lafferty, Lotz and Shostak1997). The nematodes recovered were washed in physiological saline and then fixed in 70% alcohol or hot 4% formaldehyde solution. For light microscopy, the nematodes were cleared with glycerine. Drawings were made with the aid of a Leica drawing attachment. After examination, the specimens were stored in vials of 70% ethanol. Measurements are presented in micrometres as the range followed by the mean in parentheses, unless otherwise stated. Specimens were deposited in the Helminthological Collection of the Instituto Oswaldo Cruz (CHIOC), Rio de Janeiro, Brazil.
For scanning electron microscopy (SEM), specimens were postfixed for 24 h at room temperature in a solution of 1% osmium tetroxide and 0·8% potassium ferrocyanide, dehydrated through a graded alcohol series, critical-point dried and sputter-coated with gold. The samples were examined using a JEOL JSM-6390 LV SEM, from the Plataforma de Microscopia Eletrônica do Instituto Oswaldo Cruz, at an accelerating voltage of 15 kV.
Genetic analysis
Genomic DNA was extracted using a QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer's instructions. The partial region spanning 18S rDNA was amplified by polymerase chain reaction (PCR) using a newly designed set of primers, Atractid_18SF (5′-CGT ATC GTT GCG TGA GAG GTG-3′) and Atractid_18SR (5′-AAA GTG TCG AAA CAG CAT TCC-3′), and PCR condition was performed under the following conditions: 94 °C for 3 min, followed by 40 cycles of 95 °C, 30 s, 53·7 °C, 30 s, 72 °C, 60 s and 72 °C for 5 min. PCR assays were carried out in a total volume of 15 µL containing 7·5 µL of 2 × GoTaq® Colorless Master Mix (Promega), 0·5 µL Mg2+ (50 mm concentration), 1·5 µL of each primers with final concentration at 0·5 µ m, 2·0 µL of cDNA sample and ultrapure water to complete. The partial region spanning 28S rRNA was amplified by PCR using the primers C1 (5′-ACCCGCTGAATTTAAGCAT-3′) and D2 (5′-TGGTCCGTGTTTCAAGAC-3′) (Hassouna et al. Reference Hassouna, Michot and Bachellerie1984, after Chisholm et al. Reference Chisholm, Morgan, Adlard and Whittington2001) under the following conditions: 95 °C for 5 min, 40 cycles at 95 °C, 60 s, 56·2 °C, 45 s, 72 °C, 60 s and 72 °C for 5 min.
PCR products were visualized after electrophoresis on a 1·5% agarose gel stained with SYBR green (Invitrogen). Amplified PCR products were purified with ExoSAP-IT (Affymetrix) following the manufacturer instructions. DNA cycle sequencing reactions were performed using BigDye v.3.1 chemistry (Applied Biosystems). The sequencing was performed in an ABI Prism 3100 sequence analyser using the same primer set. Sequences of both strands were generated, edited and aligned using the CLUSTAL W algorithm of the MEGA 6.0 package (Thompson et al. Reference Thompson, Higgins and Gibson1994; Tamura et al. Reference Tamura, Peterson, Peterson, Stecher, Nei and Kumar2011). The comparison for similarities with sequences from GenBank was performed using BLAST 2.0 (Altschul et al. Reference Altschul, Gish, Miller, Myers and Lipman1990). The best-fit substitution model of partial 18S dataset was GTR + I + G model of nucleotide substitution selected under the Akaike information criterion (AIC) using MrModelTest 2 with aid of PAUP4.0a147 (Nylander, Reference Nylander2004). Tree reconstructions were carried out using Bayesian Inference (BI) via the software MrBayes 3.2.6, where the Markov chain Monte Carlo (MCMC) was set to 2 × 106 generations, every 100th tree was sampled and the first 105 generations were omitted from phylogeny reconstruction; gamma shape parameter and proportion of invariants (PINVAR) were estimated from the dataset (Ronquist and Huelsenbeck, Reference Ronquist and Huelsenbeck2003). The remaining trees were used to generate a consensus tree and to calculate the Bayesian posterior probabilities (Bpp) of all branches using a majority-rule consensus approach. Maximum-likelihood (ML) analysis was constructed with PhyML 3·1 and nodal support was estimated by performing 1000 bootstrap replicates (Swofford, Reference Swofford2002). Tree topologies were visualized in FigTree 1.4.2 (2014). Taxa, for which sequences from GenBank were used for phylogenetic analysis, were KR139827 Spectatus spectatus, U94371 Cruzia americana, DQ503461 Raillietnema sp., EF375487 Labeonema sp., DQ118537 Nemhelix bakeri, LC018444 Cosmocercoides pulcher, DQ442679 R. rondoni, EF375487 Labeonema synodontisi and AF036607 Teratocephalus lirellus (as Outgroup), in addition to the new sequences of KX524511 R. rondoni and KX524513 O. moraveci n. sp.
RESULTS
A total of 240 P. blochii were examined, 120 from the Xapuri River and 120 from the Acre River. The two atractid nematode species found are described below.
Orientatractis moraveci n. sp. (Figs 1–3)
Fig. 1. Orientatractis moraveci n. sp. (A) Whole body showing oesophagus and sharply pointed tail; (B) cephalic end with four submedian lips with bicornuate (asterisks) and simple horns (a); two lateral lips with inverted U-shaped structure (b); amphids (Am) and small papillae surrounding the oral opening; (C) tail of male with spicules, gubernaculum and papillae; (D) detail of spicules and gubernaculum; (E) female tail with vulva and anal aperture.
Fig. 2. Scanning electron microscopy of Orientatractis moraveci n. sp. (A) Anterior region of the body with oral aperture; (B) Cephalic end showing submedian lips with bicornuate (arrowhead) and single horns (a); lateral lips showing inverted U-shaped structure with free tips (b), amphids (Am) and two rows of papillae (arrows); (C) lateral lip (l) with inverted U-shaped tips between two submedian lips (s) with single horn structure and amphid (Am); (D) detail of submedian lip with bicornuate tips (arrowhead) and single horn (a) and tips of lateral structure (b); (E) knob-like deirid (arrow) and excretory pore (*) in anterior third of the body; (F)–(G) detail of knob-like deirid.
Fig. 3. Scanning electron microscopy of Orientatractis moraveci n. sp. (A) general view of posterior region of male showing spicule and papillae; (B) detail of ad-cloacal and post-cloacal papillae and lateral longitudinal cuticular striation; (C) detail of tip tail of male; (D) male caudal papillae in lateral view; (E) lateral view of posterior region of female showing protuberant vulva and pointed tail; (F) detail of protuberant vulva and anus.
Cosmocercoidea Railliet, 1916
Atractidae Railliet, 1917
Orientatractis Petter, 1966
Type host: P. blochii Valenciennes 1840.
Site: Intestine.
Prevalence: Acre River: 13% (16 infected fishes of 120 examined). Xapuri River: 23% (28 infected fishes from 120 examined).
Intensity of infection: Acre River: 1–138 (26); Xapuri River: 1–273 (29).
Mean abundance: Acre River: 4; Xapuri River: 7.
Type locality: Xapuri River, Acre State, Brazil.
Other locality: Acre River, Acre State, Brazil.
Material deposited: CHIOC numbers 38326a (holotype) and 38326b (alotype) and 38326c (paratypes).
Morphological and ultrastructural data
Small sized whitish nematodes. Cuticle with transverse striations. Cephalic end of body blunt; posterior end of body slender, with very long, sharply pointed tail (Figs 1A, 2A and E). Oral opening surrounded by 2 lateral amphids and 8 papillae arranged in 2 circles (1B, 2B–D). Cephalic end with 6 lips: 4 submedian lips with bicornuate structures (2 subdorsal and 2 subventral) and single horned structure immediately posterior each, and 2 lateral lips with sclerotised, inverted U-shaped structure with free tips (Figs 1B, 2B–D). Large lateral amphids are located anterior to inverted U-shaped structure (Figs 2B and C). Dorsal and ventral grooves separate the right and left sides of cephalic extremity (Fig. 2D).
Male (measurements based on 10 adult specimens)
Body 2·09–3·13 (2·55) mm long; maximum width 95–135 (118). Length of entire oesophagus 271–386 (362); anterior part of oesophagus (corpus) 120–138 (133) long and 20–30 (26) wide; posterior part, including bulb, 138–253 (230) long and 50–75 (62) wide. Nerve ring and excretory pore 103–200 (155) and 126–208 (162), respectively, from anterior extremity (Figs 2E, G). Deirids located 238–333 (279) from anterior extremity (Figs 2E and F). Tail slender, 225–270 (252) long, sharply pointed (Figs 1C, 3A and C). Longitudinal cuticular striations observed laterally on posterior extremity (Figs 3A and B). There are 10 pairs of caudal papillae: 3 subventral pairs pre-cloacal, 2 pairs ad-cloacal (sometimes only 1 pair is visible) and 5 pairs post-cloacal. The post-cloacal are disposed as: 1 pair subventral followed by 1 pair lateral, and 1 pair subventral; 2 last pairs (1 subventral and 1 lateral) are close to each other, at the end of the second third of length of tail (Figs 1C, D, 3A–D). Spicules unequal, well sclerotized; larger (left) spicule 161–198 (181) long; smaller (right) 69–100 (87) long (Figs 3A, B and D). Gubernaculum 38–58 (50) long, with anterior lateral process (Figs 1C and D).
Female (measurements based on 10 specimens)
Body 1·98–2·71 (2·39) mm long; maximum width 142–200 (172) (Fig. 1A). Length of entire oesophagus 322–400 (360); anterior part of oesophagus (corpus) 101–147 (132); posterior part of oesophagus, including bulb, 202–253 (231) (Fig. 1A). Nerve ring and excretory pore 115–184 (135) and 138–232 (181), respectively, from anterior extremity. Deirids 230–333 (275) from anterior end. Vulva with extended lips, situated 57–76 (67) anterior to anus (Figs 1E, 3E and F). Vagina short, oriented anteriorly. Monodelphic uterus contains developing eggs, 175–266 (218) long and 92–142 (117) wide, and free larvae (Fig. 1A). Tail 237–294 (272) long.
Genetic data
Two new sequences of O. moraveci n. sp. were obtained in this study and were deposited in the GenBank under the accession numbers KX524513 (18S rDNA region with 731 base pairs) and KX524514 (28S rDNA with 734 base pairs). The sequence most similar to O. moraveci n. sp. with respect to the partial 18S rDNA region was Dirofilaria repens (AB973229) with a 97% similarity, 100% query cover and a maximum score of 1229. For the new sequence of the partial 28S rDNA region, the most similar sequence was C. pulcher (LCO18444) with an 84% similarity, 79% query cover and a maximum score of 571.
Remarks
Orientatractis moraveci n. sp. is the first species of the genus parasitizing fish in South America. A comparison of the data between O. moraveci n. sp. and the other species of the genus shows that the new species is mainly distinguished from its congeners by having: 10 pairs of caudal papillae (3 pairs pre-cloacal, 2 pairs ad-cloacal and 5 pairs post-cloacal); unequal spicules 161–198 and 69–100 long; and a gubernaculum 38–58 long with an anterior lateral process.
Etymology
The new species is named for Prof. Frantisek Moravec, from the Czech Academy of Sciences, Czech Republic, in recognition of his valuable contributions to our knowledge of the nematode parasites of fishes and his willingness to work with and teach researchers from all over the world.
Rondonia rondoni Travassos, Reference Travassos1920 (Figs 4–6)
Fig. 4. Rondonia rondoni. (A) Three rudimentary bi-lobed lips surrounded by inner and outer circles of four papillae and two amphids; (B) tail of male with spicules, gubernaculum and papillae; (C) detail of spicules and gubernaculum; (D) entire body of female showing oesophagus, eggs and larvae; (E) detail of the oesophagus; (F) tail of female with common aperture.
Fig. 5. Scanning electron and light microscopy of Rondonia rondoni. (A and B) Anterior region showing three bi-lobed lips, oral aperture, inner (black arrowhead) and outer (white arrowhead) circles of four papillae and amphids (Am); (C) general view of posterior region of male with sharply pointed tail and two parallel sub-lateral bands of small spines (arrows); inset: detail of small spines; (D) male tail with papillae; (E) ventro-lateral view of papillae; (F) lateral view of posterior region showing double papilla (arrow).
Fig. 6. Scanning electron microscopy of females of Rondonia rondoni. (A) posterior region of female showing cloacal aperture; (B) detail of cloacal aperture; (C) female tail tip detail showing different patterns of cuticular striations.
Site: Intestine.
Host: P. blochii Valenciennes 1840.
Prevalence: Acre River: 8% (9 infected fishes from 120 examined); Xapuri River: 19% (23 infected fishes from 120 examined).
Intensity of infection: Acre River: 1–20 (6); Xapuri River: 1–226 (29).
Mean abundance: Acre River: 0·5; Xapuri River: 6.
Locality: Acre and Xapuri Rivers, Acre State, Brazil.
Material deposited: CHIC n. 38324 (vouchers)
Morphological and ultrastructural data
Medium sized nematodes. Cuticle smooth, transversally striated. Head end rounded. Mouth with 3 rudimentary bi-lobed lips (1 dorsal and 2 ventral) surrounded by inner and outer circles each of 4 papillae and 2 amphids (Figs 4A, 5A and B).
Male (measurements based on 10 specimens)
Body 3·9–4·9 (4·4) mm long and 165–230 (204) wide. Oesophagus 555–610 (589) in total length; anterior end forms pharynx 13–20 (17) long; oesophageal corpus 310–340 (326) long and 43–68 (54) wide; posterior part of oesophagus, including bulb, 240–275 (263) long and 50–105 (63) wide at anterior narrow part; bulb 88–130 (104) long and 88–103 (93) wide. Nerve ring and excretory pore 358–457 (406) and 715–1040 (902), respectively, from anterior extremity. Tail conical, slender, 230–325 (278) long (Figs 4B, 5C and D). Caudal papillae: 4 pairs pre-cloacal (3 simple and 1 double pair), 2 pairs ad-cloacal and 6 pairs post-cloacal (4 subventral and 2 lateral) (Figs 4B, 5D–F). Spicules unequal and dissimilar; large spicule 150–183 (165) long; small spicule 78–100 (87) long; gubernaculum simple, 33–40 (36) long (Figs 4B and C). SEM observations demonstrated 2 parallel ventral rows of minute spines on posterior third of body, ending close to cloaca (Fig. 5C).
Female (measurements based on 10 specimens)
Body of gravid females 4·65–7·02 (5·57) mm long and 330–460 (407) wide (Fig. 4D). Oesophagus 615–695 (658) in total length; anterior pharynx 13–18 (16) long; oesophageal corpus 345–380 (367) long and 53–75 (66) wide; posterior part, including bulb, 270–315 (291) long and 60–95 (74) wide at narrowest region; bulb 108–123 (115) long and 108–120 (116) wide (Figs 4D and E). Nerve ring and excretory pore 430–470 (450) and 830–1240 (991), respectively, from anterior extremity. Ovary in mid-body; uterus contains developing eggs, 305–435 (364) long and 185–220 (203) wide, and free larvae; vagina short; vulva opens into rectum (Figs 4D–F, 6A and B). Tail conical, slender, 230–450 (322) long, with different patterns of cuticular striations close to tip (Figs 4F and 6C).
Genetic data
A new sequence of 28S rDNA of R. rondoni obtained in this study was deposited in the GenBank under the accession number KX524512 (722 base pairs). An additional sequence of the 18S rDNA region with 745 base pairs was also deposited under the accession number KX524511. The BLAST results for the new 28S rDNA sequence showed that the closest similarity was with C. americana (U94757) with a 86% similarity, a 55% query cover and a maximum score of 422. The result for the partial 18S rDNA sequence of R. rondoni indicated a 99% similarity, 100% query cover and a maximum score of 1360 with R. rondoni (DQ442679).
Phylogenetic tree of the Cosmocercoidea
Our sequences of the partial 18S rDNA gene of R. rondoni and O. moraveci n. sp. were used in a phylogenetic analysis with other sequences from the Cosmocercoidea previously deposited in the GenBank, with the free-living nematode T. lirellus used as the outgroup (Fig. 7). The topology of the tree included representatives of three families: Atractidae, Cosmocercidae and Kathlaniidae. Atractids, represented by R. rondoni and O. moraveci n. sp. sequences, compose a clade with a statistical support of 0·88 (Bpp) and 82% (ML). The cosmocercid clade with N. bakeri, C. pulcher and Railletnema sp., had a statistical support of 0.0.93 (Bpp) and 68% (ML). Cruzia americana, a kathlaniid, was in the same clade with the cosmocercids (Bpp = 1 and ML = 97%), whereas another kathlaniid, S. spectatus, appeared in a well separated clade (Bpp = 1; value not showed in ML). Labeonema synodontisi, an atractid, appears close to Raillietnema sp., nesting with the Cosmocercidae (Bpp = 1 and ML = 100%). Consequently, the Atractidae appears closer to the Cosmocercidae than to the Kathlaniidae (Bpp = 1 and ML = 100%).
Fig. 7. Bayesian and ML trees from phylogenetic analyses of the 18S rDNA sequences from cosmocercoid nematodes (families Atractidae, Cosmocercidae and Kathlaniidae). First number represents branch support of Bayesian posterior probability (for 2 × 106 generations; burn-in 105); the second number shows the ML bootstrap value (from 1000 replications). ML, maximum-likelihood.
DISCUSSION
The Orientatractis Petter, Reference Petter1966 and Klossinemella Costa, Reference Costa1961 are close related genera differing in the number and shape of sclerotized structures surrounding the mouth. After González-Solís and Moravec (Reference González-Solís and Moravec2004), members of Klossinemella have 8 Y-shaped sclerotized structures and 4 single horns whereas in Orientatractis there are 4 bicornuate pieces and 4 single horns. The O. moraveci n. sp. is included in this genus by the presence of 4 bicornuate pieces and 4 single horns.
Species of the genus Orientatractis have been reported parasitizing fish, reptiles and anurans from Mexico, Thailand, Vietnam, Costa Rica and New Guinea (Petter, Reference Petter1966; Buckley, Reference Buckley1969; González-Solís and Moravec, Reference González-Solís and Moravec2004; Gibbons and Platt, Reference Gibbons and Platt2006; Bursey et al. Reference Bursey, Goldberg and Kraus2014; Moravec et al. Reference Moravec, Kamchoo and Pachanawan2015). Currently, this genus comprises the species Orientatractis levanhoai Petter, Reference Petter1966, Orientatractis leiperi Buckley, Reference Buckley1969, Orientatractis campechensis González-Solís and Moravec, Reference González-Solís and Moravec2004, Orientatractis chiapasensis González-Solís and Moravec, Reference González-Solís and Moravec2004, Orientatractis asymmetrica Gibbons and Platt, Reference Gibbons and Platt2006, Orientatractis hamabatrachos Bursey et al. Reference Bursey, Goldberg and Kraus2014 and Orientatractis mekongensis Moravec et al. Reference Moravec, Kamchoo and Pachanawan2015. Only three of these species parasitize fishes: O. chiapasensis from Vieja intermedia and Tomocichla tuba, and O. campechensis from Vieja bifasciata, all cichlid fishes from México, and O. mekongensis from Helicophagus leptorhynchus, a pangasiid fish from Thailand. Orientatractis moraveci n. sp. represents the first report of a species of the genus parasitizing a pimelodid fish in South America.
Orientatractis moraveci n.sp. differs from the O. chiapasensis mainly by the size of the large spicule (161–198 vs 204–238 µ m), distance vulva–anus (57–76 vs 92–129 µ m) and the size of tail of both males (225–270 vs 460–505 µ m) and females (237–294 vs 380–722 µ m). It is also differentiated from O. campechensis in the size of large spicule (161–198 vs 430–506) µ m, the vulva-anus distance (57–76 vs 76–106 µ m) and the size of tail of the males (225–270 vs 608–722 µ m) and females (237–294 vs 866–1246 µ m). Compared with these species, the number of post-cloacal papillae is also different (5 pairs vs 4 pairs). In the case of O. mekongensis, in addition to the host and considerable geographical separation, differences included the body length of the male (2099–3135 vs 5350–6660 µ m) and female (1985–2710 vs 7750–8950 µ m), the size of the large spicule (161–198 vs 306–384 µ m), the vulva-anus distance (57–76 vs 108–150 µ m) and the size of tail of the males (225–270 vs 435–680 µ m) and females (237–294 vs 952–1074 µ m).
Orientatractis moraveci n. sp. differs from O. hamabatrachos, an amphibian parasite from New Guinea, mostly by the vulva-anus distance (57–76 vs 102–153 µ m) and the size of the tail of the males (225–270 vs 306–408 µ m) and females (237–294 vs 510–612 µ m). The species parasitizing reptiles include O. asymmetrica from Costa Rica, which differs in the body length of the males (2099–3135 vs 4050–4290 µ m) and females (1985–2710 vs 3740–5500 µ m), the vulva-anus distance (57–76 vs 100–112 µ m) and the size of the tail of the males (225–270 vs 836–952 µ m) and females (237–294 vs 820–1080 µ m). The other reptile parasite, O. levanhoai, can be differentiated mainly by the body length of the males (2099–3135 vs 3300 µ m) and females (1985–2710 vs 3400 µ m) and the size of the tail of the males (225–270 vs 800 µ m) and females (237–294 vs 1000 µ m). The number of male caudal papillae reported in the species from amphibians and reptiles was also different.
Rondonia rondonia was briefly described by Travassos (Reference Travassos1920), with more detailed data being given a few years later (Travassos et al. Reference Travassos, Artigas and Pereira1928). Subsequently, only two additional nominal species have been assigned to the genus, namely Rondonia lophii Gállego-Berenger, Reference Gállego-Berenger1947, a fish parasite from Spain based only on female specimens, and Rondonia batrachogena Bursey et al. Reference Bursey, Goldberg and Kraus2014, from amphibians in New Guinea. Rondonia rondoni has been reported from varies fishes, such as members of the Characidae, Mulidae, Douradidae and Pimelodidae, in different river basins, such as the Paraguay, Paraná, Amazonas and São Francisco (Gállego-Berenger, Reference Gállego-Berenger1947; Costa, Reference Costa1963; Moravec et al. Reference Moravec, Kohn and Fernandes1992). Despite a number of previous studies, our SEM analyses enabled us to detect a pair of double papillae in the pre-cloacal region. Travassos et al. (Reference Travassos, Artigas and Pereira1928), when described R. rondoni, reported the presence of two parallel, chitinous projections in the pre-cloacal region of the male homologous to those observed in species of the genus Cosmocerca Diesing, 1861. These structures, seen for the first time using the SEM, are composed of rows of minute spines directed posteriorly.
The two nematodes from P. blochii studied here from the Acre and Xapuri Rivers presented differences in terms of their ecological indices (prevalence, intensity and abundance). Orientatractis moraveci n. sp. from the Xapuri River showed a higher prevalence (13·33 vs 23·33%), mean intensity (26·31 vs 29·18) and mean abundance (3·51 vs 6·08). The same result was obtained for R. rondoni, with especially large differences in prevalence (7·5 vs 19·17%), mean intensity (6 vs 29·22) and mean abundance (0·45 vs 5·6). These data showed that O. moraveci n. sp. and R. rondoni share the same host, site and geographical distribution, with increased ecological indexes in the Xapuri River, which is a smaller tributary of the Acre River.
In general, our phylogenetic reconstruction of the 18S rDNA region exhibited consistent data, with a high statistical support, for the studied representative members of the Cosmocercoidea. Species of this superfamily are able to parasitize a variety of hosts groups, such as freshwater fish (S. spectatus, O. moraveci n. sp. and R. rondoni), mammals (C. americana), amphibian (Raillietnema sp. and C. pulcher) and land snails (N. bakeri). Our sequences of R. rondoni and O. moraveci n. sp., together with a sequence of R. rondoni previously deposited in GenBank, grouped with the Atractidae clade with moderate statistical support. Additional analysis using a greater number of species of atractids might offer stronger phylogenetic support.
The new sequence of O. moraveci n. sp. confirms the position of this species within the Atractidae, with the Cosmocercidae as a sister group and the Kathlaniidae as a basal group. However, Pereira et al. (Reference Pereira, Tavares, Paiva, Scholz and Luque2015) found that the kathlaniid C. americana nested within the Cosmocercidae, and the inconsistent position of this species was also pointed out by Nadler et al. (Reference Nadler, Carreno, Mejía-Madrid, Ullberg, Pagan, Houston and Hugot2007) when analyzing the same 18S rDNA region. Raillietnema sp., C. pulcher and N. bakeri grouped within the Cosmocercidae with high Bpp support (0.0.93), but much less so for the bootstrap analysis (68%).
The position of Labeonema synodotisi (Vassiliadès, 1973) (=EF375487 Labeonema sp. in GenBank) collected from the African fish Synodontis ocellifer is also inconsistent. Koubková et al. (Reference Koubková, Baruš, Hodová and Šimková2008) proposed this new combination for this species, previously described as Raillietnema synodontisi Vassiliadès, 1973. However, Moravec and Van As (Reference Moravec and Van As2004) pointed out that R. synodontisi differs from Labeonema spp. in possessing two ovaries instead of one, and its larvae do not hatch in utero. Pereira et al. (Reference Pereira, Tavares, Paiva, Scholz and Luque2015) also used this Labeonema sequence in a phylogenetic analysis of the Cosmocercoidea, considering it very similar to the cosmocercoid genus Raillietnema Travassos, 1927. Labeonema synodontisi also nested with Raillietnema sp. in our analysis, with strong support, using new and existing atractid sequences. Therefore, both molecules (present study and Pereira et al. Reference Pereira, Tavares, Paiva, Scholz and Luque2015) and morphology (Moravec and Van As, Reference Moravec and Van As2004) suggest that L. synodontisi should be returned to its original genus as R. synodontisi.
The description of O. moraveci n. sp., a parasite of P. blochii, represents the first occurrence of a member of the genus in fishes of South America, and R. rondoni is reported for the first time parasitizing this same host from the Acre and Xapuri Rivers, Western Amazon, Brazil. New ultrastructural and molecular data derived from these species will assist the future phylogenetic analyses of groups within the Cosmocercoidea. The molecular data and previous morphological studies of L. synodontisi indicate that it should be returned to the genus Raillietnema.
ACKNOWLEDGEMENTS
The authors are grateful to Dr David Gibson from the Natural History Museum, London, for his valuable suggestion and to Ana Carolina Camargo, Ana Cristina Ferreira and Pedro Moraes for technical support.
FINANCIAL SUPPORT
The study was financially supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq- Universal – CPS), Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES-Parasitologia- CPS), Fundação Oswaldo Cruz (FIOCRUZ/IOC/PAEF – CPS), Instituto Federal do Acre (IFAC- PHOC/MTS) and Fundação de Amparo à Pesquisa do Acre (FAPAC-PHOC/MTS). M. T. Silva, V. A. C. Moutinho and C. P. Santos were sponsored by CNPq fellowships.
ETHICAL STANDARDS
This study was authorized by the Brazilian Institute Chico Mendes for Conservation of Biodiversity (ICMBio No 43450-1) in accordance with the guidelines of the Brazilian College for Animal Experiments (COBEA).
