parasitizing

: Three piranha species, Serrasalmus maculatus , S. marginatus and Pygocentrus nattereri, living sympatrically in the lower Paraná River (Argentina) were examined searching for nasal monogeneans to know its diversity and distribution. Four species of monogeneans belonging to Rhinoxenus were found parasitizing the nasal cavities. Two new species are described, and new morphological data of 2 previously described species is provided. Rhinoxenus argentinensis n. sp. is characterized by having the male copulatory organ (MCO) as a coiled tube with a reel shaped-base; an elongated accessory piece articulated to base of MCO; a sinistral vagina with a sclerotized vestibule, and a sclerotized cap of the ventral anchor laterally modified forming a triangular expansion. Rhinoxenus paranaensis n. sp. is characterized by having a MCO as a coiled tube with a reel-shaped base; an accessory piece with an elongate proximal portion, a dilated distal portion with digitiform projections articulated to base of MCO; a sinistral vagina with 5-6 cuticular spine-shaped processes, and a sclerotized cap of the ventral anchor modified laterally forming a bilobate expansion. Additionally, multivariate discriminant analyses showed significant morphometric differences in the ventral anchors among Rhinoxenus species parasitizing ‘piranhas ’.


INTRODUCTION
Dactylogyrids of the genus Rhinoxenus Kritsky, Thatcher and Boeger, 1988 are parasites of the nasal cavities in characiform fish from the Neotropical region (Domingues and Boeger 2005).The genus is considered to be monophyletic, and its members have been suggested as suitable markers of the coevolutionary history of their hosts (Boeger et al. 1995, Domingues andBoeger 2005).
clade was reviewed by Domingues and Boeger (2005), who suggested that several processes, such as cospeciation, duplication, dispersion and extinction are required to explain the extant hostparasite associations.These processes apparently have differentially occurred on some Rhinoxenus clades, and the authors identified those species from piranhas, R. piranhus Kritsky, Boeger and Thatcher, 1988 and R. euryxenus Domingues and Boeger, 2005, as examples of cospeciation.
In this work, representatives of the three piranha species, living sympatrically in the lower Paraná River and its tributaries of the Provinces of Santa Fe, entre Ríos and Buenos Aires, Argentina, were examined in search of nasal monogeneans.
The purpose was to know the diversity of the Rhinoxenus genus in the region and provide further evidences on its distribution.As a result, 2 new species are proposed, and supplementary information is provided for 2 previously described species.
Methods of parasites collection, preparation and measurement were carried out as described by Kritsky et al. (1986Kritsky et al. ( , 1988) ) and Rossin et al. (2017).Note that the authors of the new taxa are different from the authors in this paper; see article 50.1 and Recommendation 50A of the International Code of Zoological Nomenclature.
For the three-dimensional reconstructions of sclerotized structures, examinations, as well as image pre and post processing steps of Z-series, were conducted using the open source Fiji software (Schindelin et al. 2012).Stacks that showed a significant attenuation associated with increasing imaging depth were corrected with a linearly or exponentially increasing factor along the z-axis.Stacks were then deconvolved using the CMle algorithm with the Huygens software (SVI, Hilversum, Netherlands), which provided a major improvement in the signal-to-noise ratio of the confocal stack, as well as a modest increase in resolution.Afterward, the brightness and contrast of the stacks were adjusted in Fiji, and threedimensional isosurface reconstructions of the pieces were obtained with the three-dimensional plug-in viewer.A resampling factor of two was always used, and the threshold value was adjusted to obtain the desired level of detail.Finally, these three-dimensional models were processed in the open source software Blender (www.blender.org).The surfaces corresponding to objects of interest were isolated from surrounding debris, and evident artefacts due to fusion with adjacent debris were corrected by editing the model mesh.The location of these edited patches is clearly marked in purple in the supplementary videos.For the final presentation, a smoothing filter (factor: 0.5, repeats: 10) was applied to the meshes, and threedimensional renders were produced.Full 360° rotation videos of the models were also produced and exported in AVI format.
Measurements are given in micrometers, as range followed by the mean and number of specimens measured in parentheses.Prevalence and mean abundance were calculated according Bush et al. (1997).
Morphometric differences among the ventral anchors of Rhinoxenus species found in piranhas were explored using canonical analysis of principal coordinates (CAP; Anderson andWillis 2003, Anderson et al. 2008).Potential over-parameterization was prevented by choosing the number of PCO axes (m) that maximized a leave-one-out allocation success to groups (Anderson and Robinson 2003).CAP analyses were based on euclidean distances on 9 morphometric variables of ventral anchors (Table I); these measurements were taken from 6 individuals of each of the four host species (Table I ;.To test for significant morphometric differences of the ventral anchors among Rhinoxenus species, a permutation trace test (sum of squared canonical eigenvalues) was applied; the P-value was obtained after 9999 permutations.CAP analyses were performed by PeRMANOVA + for PRIMeR package (Anderson et al. 2008).
Type specimens are deposited in the Helminthological Collection of the Museo de la Plata (HCMlP), la Plata, Argentina.Host nomenclature follows Froese and Pauly (2018).

Rhinoxenus piranhus
The selected orthonormal PCO axes (n = 3), described 100% of the variation in the data cloud, with a 100% of correct allocations (Fig. 31).
When vectors corresponding to the Spearman correlations of morphometric measurements were superimposed the CAP biplot, greater values for most measurements characterized R. euryxenus, sharing a longer shaft with R. argentinensis n. sp.On the other hand, R. piranhus was characterized by larger values of total length and base length, while R. paranaensis n. sp.Characterized by the lower values for most measurements.

DISCUSSION
The genus Rhinoxenus comprises eight species of parasites from nasal cavities of Neotropical characiform fish, which have been recorded in rivers of Brazil and French guiana (Kritsky et al. 1988, Boeger et al. 1995, Domingues and Boeger 2005).This is the first report of the Rhinoxenus species in Argentina, thus, adding two new species and increasing to ten the species richness of Rhinoxenus.
Regarding the phylogeny of Rhinoxenus, Domingues and Boeger (2005) proposed the monophyly of this genus.These authors suggests that R. piranhus and R. euryxenus cluster together supported by the saucer-like point of the ventral anchor as a synapormophy with a consistency of 100%.The presence of this feature in both new species from piranhas supports that conclusion.Furthermore, the presence of a triangular hole in the base of the ventral anchor is a characteristic present on the 4 species that parasitize the nasal cavities of piranhas.
According to Domingues and Boeger (2005), R. euryxenus and R. piranhus differ from each other by the morphology of ventral anchors (with shaft strongly recurved near midlength in R. euryxenus vs. relatively straight in R. piranhus), among other features.The morphology of the ventral anchors is highly variable across the members of this genus (see Domingues and Boeger 2005).However, in the light of the new evidence, these structures are corroborated to be quite homogeneous in shape in those species parasitizing piranhas.The four species exhibit enough differences (in the shape of the sclerotized cap, the size and shape of both the lateral expansion and the medial concavity, and in the curvature and diameter of the shaft) to readily differentiate them from each other.
The presence of R. piranhus in P. nattereri is reported for the first time in the Paraná basin (Kritsky et al. 1988), but this species was not found in S. marginatus or in S. maculatus as previously reported along the upper Paraná (S. maculatus identified as S. spiropleura) (Domingues and Boeger 2005).Similarly, R. euryxenus was found mainly in S. marginatus, but also in S. maculatus (only 1 specimen in 37 examined fish) in the low Paraná River.Except for the finding of one specimen  I. of R. euryxenus in S. maculatus, each Rhinoxenus species parasitized a single species of piranha in Argentina, despite the sympatry of these hosts.This indicates a high host specificity contrasting to the low specificity observed in piranhas from Brazilian rivers (Table II).Unfortunately, the existing literature only reports the presence of different species, and no quantitative data on prevalence or abundance are available to test the host specificity among these parasites.For instance, the presence of R. euryxenus in Leporinus agassizii (Anostomidae) was considered accidental by Domingues and Boeger (2005), and other cases of accidental infestations could occur.
According to Jégu (2003), 25 species of piranhas are distributed in the Amazon basin, 16 in the Orinoco, 9 in rivers of the guyanas, 3 in the Paraguay-Paraná Rivers, and 2 in the São Francisco River, and their phylogeny and phylogeography have been intensely studied (Hubert et al. 2007, Freeman et al. 2007, Ortí et al. 2008).Indeed, this latitudinal gradient in species richness of serrasalmids across South American rivers, along with their known phylogenetic relationships, provide an excellent opportunity for further studies on host-parasite coevolutionary processes, as well as on drivers of parasite specificity.

TABLE I Comparative measurements (in µm) of ventral anchors of 4 Rhinoxenus species parasitizing piranhas. R. argentinensis n. sp.
(Domingues and Boeger 2005)om most of the eight previously described congeners based on the morphology of the ventral anchors.Among the members of this genus, only 2 species, R. piranhus and R. euryxenus, share a saucer-like distal point in ventral anchors with the new species(Domingues and Boeger 2005).However, R. argentinensis n. sp.differs from R. piranhus by having a sclerotized cap laterally modified into a triangular expansion, whereas, in R.
MARÍA A. ROSSIN et al.ReMARKSRhinoxenus paranaensis n. sp. also has a saucerlike distal point of the ventral anchor, sharing this characteristic with R. piranhus, R. euryxenus and R