Metazoan endoparasites of Pygocentrus nattereri ( Characiformes : Serrasalminae ) in the Negro River , Pantanal , Brazil Endoparasitas metazoários de Pygocentrus nattereri ( Caraciformes : Serrasalminae ) no rio Negro , Pantanal , Brasil

In the period of October 2007 to August 2008, 152 specimens of Pygocentrus nattereri were caught in the Negro River in the Nhecolândia region, central Pantanal wetland, State of Mato Grosso do Sul, Brazil. The specimens were necropsied and a total of 4,212 metazoan endoparasites were recovered, belonging to 10 taxons: Procamallanus (Spirocamallanus) inopinatus, Philometridae gen. sp., Eustrongylides sp., Brevimulticaecum sp., Contracaecum sp. (Nematoda), Echinorhynchus paranensis (Acanthocephala), Leiperia gracile, Sebekia oxycephala, Subtriquetra sp. 1 and Subtriquetra sp. 2 (Pentastomida). This is the first record of two parasite species from P. nattereri: E. paranensis and L. gracile.


Introduction
Pygocentrus nattereri Kner, 1858 is a gregarious and piscivorous species (SAZIMA; MACHADO, 1990).Its wide geographical distribution includes the tropical and neotropical regions of South America (Argentina, Bolivia, Brazil, Colombia, Ecuador, Guyana, Paraguay, Peru and Uruguay), mainly in the Amazon and Paraguay-Paraná river basins, coastal rivers in northeastern Brazil, and the Essequibo River basin (JÉGU, 2003).
Because of the lack of knowledge regarding the endoparasite fauna in this host, the aim of this study was to report and describe the species of metazoan endoparasites found in P. nattereri in the Negro River, Pantanal wetland, State of Mato Grosso do Sul, Brazil.

Materials and Methods
From October 2007 to August 2008, 152 specimens of P. nattereri were collected, pithed immediately after capture and preserved under refrigeration until the moment of necropsy.The necropsy, specimen collection, preparation and conservation of the endoparasites were performed according to Eiras et al. (2006).
Hosts measured 19.89 ± 3.11 (11.4-24.8)cm in standard length and weighed 376.36 ± 179.35 (49-853) g.The fish were caught with hooks and cast nets of different mesh sizes, in the main channel of the middle Negro River (19° 34' 29.2" S and 56° 14' 37.1" W), a tributary of the Paraguay River in the Nhecolândia subregion of the central Pantanal wetland, state of Mato Grosso do Sul, Brazil.Some parasite specimens were prepared for observation by means of scanning electron microscopy (SEM) according to Chiarini-Garcia (1997).Measurements of specimens in light microscopy (LM) were made with Leica (LAS Leica TM ) software.All measurements are in millimeters; means are followed by the range in parentheses.
These parasites were compared with specimens deposited in the Helminthological Collection of the Oswaldo Cruz Institute (CHIOC).Representative specimens were deposited in the Zoological Reference Collection at the Universidade Federal do Mato Grosso do Sul, together with a host voucher specimen (ZUFMS-PIS No. 3087).Parasitological descriptors were calculated according to Bush et al. (1997).The mean values of descriptors are followed by the standard deviation (±).
Remarks: The number of spiral thickenings in the buccal capsule of the specimens examined is close to that reported by Moravec (1998).However, the number of spiral thickenings differs among several reports, and these differences are considered as intraspecific variability (MORAVEC et al., 1997).The specimens described by Petter and Dlouhy (1985) showed 14-25 spiral thickenings.Rodrigues et al. (1991) and Moravec et al. (1993, 1997) reported 15-19, 8-17 and 13-22 spiral thickenings, respectively. Moravec et al. (1997) reported the presence of eight cephalic papillae arranged in two circles around the oral opening, differing from the present specimens which have only four cephalic papillae.Some specimens were compared to voucher specimen CHIOC 31.324,parasitic in Leporinus sp., from the Machado River, state of Rondônia, Brazil (GIESE et al., 2009).Similarities in the number of the spiral thickenings were observed, and although the voucher specimen was not measured, it was visibly longer and wider than our specimens.This voucher specimen is permanently mounted, and due to the impossibility of en face observation and the poor conservation state of the specimen, it was not possible to observe the number of cephalic papillae.
Remarks: Genus and species determination was not possible owing to the poor condition of this specimen.The family identification is according to the features of an adult female philometrid as described by Moravec (1998): anterior end rounded, peribuccal ring absent; mouth simple, without buccal capsule; oral opening surrounded by 6-8 cephalic papillae.The vulva and vagina were not observed in this specimen; Moravec (1998) mentioned that these structures are atrophied in gravid females.Recently, Philometra nattereri was described parasitizing P. nattereri in Amazonia (CÁRDENAS et al., 2012).Although the specimen recorded in the present study has not been identified, it differs in some aspects from the one described by Cárdenas et al. (2012), who reported a narrower range of widths (0.47-0.65) and 14 cephalic papillae.The infection site, also different, was reported as in the oculo-orbits and nasal cavity.
Remarks: The body length and width measurements are close to those reported for larval Eustrongylides ignotus Jäegerskiold, 1909, while the oesophagus length is close to those of larvae of E. tubifex (Nitzsch, 1819) (MORAVEC, 1998) (we found no report of the oesophagus length in E. ignotus).The buccal cavity is longer than that of E. tubifex (0.012) and closer to that reported for E. ignotus (0.060-0.097).Moravec et al. (1997), studying nematodes of freshwater fishes in Venezuela, described one larva of Eustrongylides sp., and reported a buccal cavity length (0.285) five times longer than in the present specimens.
Some of the present specimens were compared with the voucher specimen CHIOC 36.977, from Potamotrygon falkneri Castex & Maciel, 1963, collected in the Paraná River, State of Paraná, Brazil (LACERDA et al., 2008).In this specimen, the ventriculus could not be observed due to the overlapping of the body parts in the permanent mount.
Remarks: The measurements of Contracaecum sp.larvae in this study are close to those of Contracaecum sp.type 2 of Moravec (1998), with differences in the length and width of the ventriculus and in the length of the ventricular appendix (greater in the present specimens).Some specimens were compared with the voucher specimen CHIOC 35.521, from Geophagus brasiliensis (Quoy & Gaimard, 1824), collected in the Guandu River, State of Rio de Janeiro, Brazil (AZEVEDO et al., 2006), which is larger than the specimens found in the present study.Contracaecum sp.larvae have been reported in several fish species (MORAVEC et al., 1997;MORAVEC, 1998;MARTINS et al., 2005) Remarks: The dimensions of the trunk, proboscis and proboscis receptacle, number of rows and hooks, as well as the dimensions of the lemnisci and ovijector, are very similar to those reported for E. paranensis by Machado Filho (1959).According to Machado Filho (1959), the main difference observed between E. paranensis and other congeneric species is the presence of 14 longitudinal rows with 11 hooks per row on the proboscis.This specimen was considered adult, because it had well-developed reproductive structures, but non-gravid because it did not contain eggs.This is the first report of this species in P. nattereri.
Remarks: The measurements of these specimens are in agreement with those reported for larval stages of L. gracile in Salminus brevidens (syn.= S. brasiliensis, Cuvier, 1819) and are slightly larger than those for larvae found in H. malabaricus and Brachyplatystoma sp.Bleeker, 1862 described by Rego and Eiras (1989).The principal hooks of these specimens are larger than the accessory hooks, differing from specimens found in S. brevidens and H. malabaricus, where the accessory hooks were larger than the principal hooks (REGO; EIRAS, 1989).Some individuals were compared to the voucher specimens CHIOC 32.446, parasitic in Brachyplatystoma sp., from the Salobra River, State of Mato Grosso, Brazil; CHIOC 30.353; parasitic in H. malabaricus, from the State of Espírito Santo, Brazil; and CHIOC 29.889a-b, parasitic in Salminus brevidens from the Salobra River (REGO; EIRAS, 1989).
Although some of these specimens were darkened and in permanent mounts, it was possible to observe similarities among them, including the body length, number of annuli (more than 100) and the characteristics of the oral cadre, although the accessory hooks visually appeared to be smaller than the principal hooks in the voucher specimens.The report of this parasite in P. nattereri by Eiras et al. (2010) was based on the dissertation that reported the first record in this host and formed the basis of the present study, but Eiras et al. (2010) did not report any description of specimens, collection locality, or parasitological descriptors.
Remarks: The measurements and some morphological features of S. oxycephala are close to those reported by Rego and Eiras (1989), mainly in relation to body length, hooks and number of annuli.In this study, specimens of S. oxycephala had the principal hooks larger than the accessory hooks, differing from the description by Rego and Eiras (1989) for this same species in P. nattereri.Some of the specimens were compared to voucher specimens CHIOC 32.445, parasitic in P. nattereri, from the Cuiabá River, State of Mato Grosso, Brazil and CHIOC 32.447, parasitic in P. corruscans, also from the Cuiabá River (REGO; EIRAS, 1989).These specimens were similar in the shape of hooks, oral cadre, body dimensions and number of annuli.
Despite the observed similarity between the larvae of S. oxycephala and L. gracile, these larvae can be distinguished mainly by the body size, approximately three times longer; by the number of body annuli, approximately twice as numerous in L. gracile; and by the shape of the oral cadre, oval to elongate in Sebekia spp.and U-shaped in Leiperia spp.(OVERSTREET et al., 1985;REGO;EIRAS, 1989;JUNKER et al., 2000).
Remarks: According to Junker and Boomker (2006), the features observed in these two morphospecies are in agreement with those of the genus Subtriquetra Sambon, 1922 including the elliptical and dorsally flattened body, and the rounded oral cadre located between two pairs of simple, slender and sharply pointed hooks.
Records of larval stages of species of Subtriquetra parasitizing fish are scarce, and are mainly from South America, which prevented the specific identification of this morphospecies.These two morphotypes were considered distinct because of the differences in the shape of the oral cadre, hooks and spines, structures that were larger in Subtriquetra sp. 2. Some collected specimens were compared to some of the voucher specimens of the genus Subtriquetra, CHIOC 17.797, parasitic in H. malabaricus, from the Juparanã Lagoon, State of Espírito Santo, Brazil (TRAVASSOS; FREITAS, 1940) and CHIOC 11.424, parasitic in P. nattereri, from the Salobra River, State of Mato Grosso do Sul, Brazil (unknown publication).These parasites were similar in shape and dimensions of the body and oral cadre, and in the presence of ventral papillae on the anterior end of the body.

Figure 1 .
Figure 1.Micrographs of Procamallanus (Spirocamallanus) inopinatus: A -SEM micrograph of anterior end, apical view showing four cephalic papillae (black arrow).B -LM micrograph of lateral anterior end showing the spiral thickenings in the buccal capsule.

Figure 2 .
Figure 2. Micrographs of Leiperia gracile: A -SEM micrograph of anterior end, ventral view.B -LM micrograph showing the main and accessory hooks; (ab) = length of hook; (ac) = hook base; (bc) = length from base to extremity of hook blade.

Figure 3 .
Figure 3. SEM micrographs of Sebekia oxycephala: A -detail of annulus with spines on its edges.B -anterior end, ventral view.

Figure 4 .
Figure 4. Micrographs of Subtriquetra spp.: A -SEM micrograph showing rows of spines and chloride cells (black arrow).B -SEM micrograph of anterior end, ventral view of Subtriquetra sp. 1. C -LM micrograph showing measurements of simple hook (ab) = length of hook blade; (ac) = hook base; (bc) = length from base to extremity of hook blade.D -SEM micrograph of anterior end, ventral view of Subtriquetra sp. 2.
Based on one immature adult female specimen: Trunk elongate, slender, 12.19 long, 2.31 largest width; anterior end wider than posterior