A new species of Procamallanus Baylis, 1923 (Nematoda, Camallanidae) from Astronotusocellatus (Agassiz, 1831) (Perciformes, Cichlidae) in Brazil

Abstract A new species of Procamallanus Baylis, 1923 was found as a parasite of the fish Astronotusocellatus (Agassiz, 1831) from a lake in the Jardim Botânico Bosque Rodrigues Alves, Belém, Brazil. Procamallanusspiculastriatussp. n. has a smooth buccal capsule and a well-developed basal ring that is armed with four sclerotized tooth-like structures. The male of the new species is similar to the two species that are known from Brazilian fish, P.peraccuratus Pinto, Fábio, Noronha & Rolas, 1976, and P.annipetterae Kohn & Fernandes, 1988, by the absence of the gubernaculum. It differs from these two by the morphology of the buccal capsule, the number are arrangement of the caudal papillae in males, the size and morphology of the spicules and the shape of the tail of both sexes. Procamallanusspiculastriatussp. n. is the third species discovered in fish from Brazil. This finding extends the geographical distribution of the genus into the Brazilian Amazon.


Introduction
The genus Astronotus is comprised of two species, A. crassipinnis (Heckel, 1840) and A. ocellatus (Agassiz, 1831) (Perciformes: Cichlidae) (Kullander 2003;Froese and Pauly 2017). Astronotus ocellatus (known as Acaraú-açu; Apaiari; Oscar) is popular among aquarists, but it is not as popular for aquaculture because of its slow growth rate. Early attempts at cultivation were encouraged by the government in the early 1970s, but they were not successful they only succeeded in the introduction of the species into almost all of Brazil (Fontenele and Nepomuceno 1983). Because it is an introduced species, there has been little interest on studies of its parasites; to date, only three studies (Azevedo et al. 2007;Malta 1984;Neves et al. 2013) have included this species of fish.
Nematodes of the genus Procamallanus Baylis, 1923 (Camallanida, Procamallaninae) are predominately parasites of freshwater fish that are distributed over several zoogeographical regions (Moravec 1998;Hoffman 1999). Members of the genus are easily recognized by the presence of a buccal capsule that is strongly sclerotized, but without ridges (Baylis 1923;Moravec and Scholz 1991;Moravec and Thatcher 1997). Despite the importance of introduced species of fish as mechanisms of co-introduction of parasites into native populations (Bautista-Hernández et al. 2014), helminths of most of introduced species have not been studied. As part of an ongoing study of the helminths of vertebrates of eastern Brazil, samples of A. ocellatus were collected and necropsied. Procamallanin nematodes were found as parasites of these fish, but they could not be assigned to a known species; therefore, the new species is described herein.

Materials and methods
Forty specimens of A. ocellatus were collected from Iara Lake, at the Jardim Botânico Bosque Rodrigues Alves (1°25'49"S, 48°27'22"W), located in an urban area of the city of Belém, state of Pará, eastern Brazilian Amazon. Fish were collected during March to July 2015 with the aid of a casting net. Fish were transported alive to the Laboratório de Biologia Celular e Helmintologia "Profa. Dra. Reinalda Marisa Lanfredi", Instituto de Ciências Biológicas, Universidade Federal do Pará-UFPA, for necropsy. Nematodes were collected, washed in phosphate-buffered saline, fixed in AFA solution (93 parts 70% ethanol, 5 parts formalin, and 2 parts glacial acetic acid) (Pritchard and Kruse 1982), and stored in 70% ethanol. For light microscopy, helminths were cleared in Amman's lactophenol solution (Giese et al. 2009) and examined under an Olympus BX41 microscope with a drawing tube. Measurements are given in micrometers unless otherwise noted and are presented as the range (minimum and maximum values) followed by the mean in parentheses. For scanning electron microscopy (SEM), helminths were washed in phosphate-buffered saline with a pH of 7.0 (Sodium Phosphate Monobasic 3.12 g, Sodium Phosphate Dibasic 2.83 g, and 17 g Sodium Chloride in 200 ml of distilled water), post-fixed in 1% osmium tetroxide, dehydrated to the critical point using CO 2 , coated with gold+palladium, and studied using a scanning Material. Type specimens. Holotype male (MPEG 195), allotype female (MPEG 196), and four paratypes (MPEG 197;MPEG 198;MPEG 199;MPEG 200) were deposited in the Coleção de Invertebrados of the Museu Paraense Emílio Goeldi (MPEG), Belém, Pará, Brazil.
Etymology. The species name refers to the unique morphology of the spicules, which membranous alae that are supported by rays, giving them a striated appearance. Description.

Discussion
The family Camallanidae was established for species with a prominent, sclerotized buccal capsule (Railliet and Henry 1915). Yeh (1960) divided the family into two subfamilies, Camallaninae Railliet & Henry, 1915, for species with the buccal capsule divided into two halves, and Procamallaninae Yeh, 1960, for those with a single, cup-like buccal capsule. The new species with its cup-like buccal capsule composed of two lateral halves is identified as a member of Procamallaninae. Six genera have been assigned to Procamallaninae: Procamallanus, Spirocamallanus Olsen, 1952, Malayocamallanus Jothy & Fernando, 1970, Punctocamallanus Moravec & Scholz, 1991, Spirocamallanoides Moravec & Sey, 1988and Denticamallanus Moravec & Thatcher, 1997(Baylis 1923Olsen 1952;Jothy and Fernando 1970;Moravec and Scholz 1991;Moravec and Thatcher 1997;Rigby and Rigby 2014). The genus Procamallanus consists of approximately 40 known species distributed worldwide, but only two species have been reported from Brazil, P. peraccuratus and P. annipetterae , both from southern Brazil (Pinto et al. 1976;Petter and Dlouhy 1985;. The new species is assigned to Procamallanus because its cuplike buccal capsule with smooth walls (without striations); according to Moravec and Thatcher (1997) the main characteristic of this genus is the presence of a smooth buccal capsule in both sexes.
The new species can be distinguished from all known members of the genus outside of Brazil in having tooth-like structures on the basal ring of the buccal capsule. In addition to the above characteristic, it differs from P. annulatus Yamaguti, 1955 (Indonesia), P. elatensis Fusco & Overstreet, 1979 (Israel), P. laeviconchus Wedl, 1861 (Egypt), P. planoratus Kulkarni, 1935 (India) and P. pseudolaeviconchus Moravec & Van As, 2015 (Botswana) by the absence of a sclerotized gubernaculum, present in the other five species (Wedl 1861;Kulkarni 1935;Yamaguti 1955;Fusco and Overstreet 1979;Moravec and Van As 2015). P. spiculastriatus can be distinguished from P. pacificus Moravec, Justine, Würtz, Taraschewski & Sasal, 2006 (New Caledonia) also not found in Brazil, by the absence of the small processes (mucrons) (sensu Moravec et al. 2006) on the tip of the tail.
Two species of Procamallanus have been found in Brazil: P. peraccuratus Pinto, Fábio, Noronha & Rolas, 1976, from Geophagus brasiliensis (Quoy & Gaimard) and Australoheros facetus (Jenyns) (both Cichlidae) in the State of Espirito Santo (Southern Region of Brazil) and P. annipetterae (= P. petterae Kohn & Fernandes, 1988, from Corydoras paleatus (Jenyns) in the Iguaçu River, State of Paraná (south of Brazil) (Pinto et al. 1976;. Pinto et al. (1976) suggested that the characteristics of the buccal capsule and the morphological and morphometric data of each taxon should be considered for the differentiation of species, a view shared by Moravec (1998).
Procamallanus spiculastriatus sp. n. has tooth-like structures on the basal ring of the buccal capsule similar to these in P. annipetterae although the new species has four distinct tooth-like structures, whereas these are six in P. annipetterae as described by Petter (1990; see also her fig. 5A) in addition the letter species is distinct.  provide no info on the number of cephalic papillae in females; only in the holotype male P. spiculastriatus sp. n. can be distinguished from P. annipetterae by the tail shape (conical in female of the new species vs. pointed in both sexes with a marked long and narrow posterior part); number of caudal papillae (three precloacal, two adcloacal, and five postcloacal vs. four precloacal and four postcloacal), shape of oral opening (circular in P. spiculastriatus vs. oval in P. annipetterae) and morphometric parameters such as spicule length (smaller spicule 229-284 µm, larger spicule 312-355 µm vs. 150-160 µm and 180-210 µm, respectively) and length of the tail (184 µm in males and 208 µm in females vs. 336 µm in males and 281 µm in females), comparisons made based on the description of .
Procamallanus spiculastriatus sp. n. resembles P. peraccuratus in the morphology of the buccal capsule, oral opening circular and presence of caudal alae of males, but differs by the presence of four internal sclerotized tooth-like structures on the basal ring, the presence of two postcloacal dorsal papillae, and the presence of spicules with alate distal end supported by sclerotized rays of P. spiculastriatus and those characters are absent in P. peraccuratus (Moravec et al. 1993;Moravec 1998). Despite sharing hosts from the same family (Cichlidae), the species differ with respect to species of host and the biomes where they are found: P. spiculastriatus sp. n. is a parasite of A. ocellatus (biome Amazonia), whereas P. peraccuratus is a parasite of G. brasiliensis and Au. facetus (biome Brazilian Atlantic Forest). This finding extends the geographical distribution of the genus into the Brazilian Amazon. Additional morphometric comparisons between P. spiculastriatus sp. n. and the two species found in Brazil are presented in Table 1.
The six genera currently assigned to Procamallaninae were reduced to subgenera of Procamallanus by Moravec (1998). However, Moravec (1998) did not provide any arguments why the former genera should be demoted to a lower level. Rigby and Rigby (2014) noted the overlap in the diagnostic characteristics of the buccal capsules of these taxa. Two molecular phylogenetic studies (Černotíková et al. 2011;Choud-   a Abbreviations: L = length; W = width, e = esophagus, DL = dorsolateral papillae; Ratio L/Oc and esophagus = Length of entire oesophagus and buccal capsule representing of body length. b Derived or calculated from the original publication of the species description. hury and Nadler 2016), further revealed that Procamallanus and Spirocamallanus are paraphyletic. We find that phylogenetic relationships within Procamallaninae have not been evaluated in an objective analysis using both morphological and molecular data. There is no evidence that such different taxa should be assigned to the same genus-level group (i.e., that they should be recognized as sub-genera rather than distinct genera, which is an arbitrary decision). Therefore, based on the current knowledge of the studied group we follow the concept accepting the full generic status of all recognized genus-group names within Procamallaninae. Even in the absence of a rigorous test of the monophyly of the genera, all but Procamallanus have contain species with consistent features; all known species of Procamallanus have smooth-walled buccal capsules without tooth-like structures except for P. spiculastriatus sp. n. and P. annipetterae, both of which have tooth-like structures on the basal ring. This suggests that these two species might represent a distinct genus. Currently, we do not feel confident in describing a new genus without the support of a formal phylogenetic analysis. Among the camallanid species parasitizing A. ocellatus, only Spirocamallanus inopinatus from northern Brazil (Moravec 1998;Thatcher 2006) and Camallanus sp. from Midwestern Brazil have been reported (Kohn et al. 1985;Vicente et al. 1985).