Diversity of myxozoans parasitizing the catfish Rhamdia quelen (Siluriformes: Heptapteridae), in southeastern Brazil, based on morphological and molecular evidence

Rhamdia quelen is a commercially important fish in South America. During the survey of myxozoan infections in fishes from Pardo River, Paranapanema River basin, São Paulo State, Brazil, we describe three new species of Henneguya found parasitizing gills of R. quelen: Henneguya bagre n. sp., Henneguya breviscauda n. sp and Henneguya novaerae n. sp. The descriptions were based on myxospores morphology and small subunit ribosomal DNA partial sequences. Phylogenetic analysis showed a clade formed by species that parasitize Siluriformes, with Henneguya jundiai as a sister species of Henneguya bagre n. sp., Henneguya breviscauda n. sp and Henneguya novaerae n. sp. Our study indicates that the parasites infecting R. quelen belong to a lineage of myxozoans infecting Heptapteridae fishes. Using molecular and morphological characterization, the species were identified as new species for the genus Henneguya. Based on our analysis we recommend monitoring the presence of these parasites in farmed fishes, to analyze possible pathologies caused by them.


Results
In total, 20 specimens of R. quelen were collected (7 females, 13 males, 15.4-29.5 cm TL, 60.1-480.0 g). Although equal efforts were made in all localities, the number of captured hosts was different between the locations. Eleven specimens were collected in locality 1, two specimens in locality 2, and seven specimens in locality 3. Plasmodia of Henneguya spp. were observed in gill filaments of 11 specimens (55% prevalence). Specific prevalence can be seen in Table 1  Henneguya breviscauda n. sp.  Site of infection: histozoic, gill filaments (interfilamental type). Type-material: A glass slide with myxospores (hapantotype) was deposited in the collection of the Instituto Nacional de Pesquisa da Amazônia (INPA), Brazil (Num. INPA91). The ssrDNA partial sequences were deposited in GenBank with accession numbers OP070158-OP070160.
Etymology: The specific name is derived from the name of the locality (Nova Era Resort) where the host was collected.
Myxospores: The morphological data are based on the observation of myxospores isolated from a single plasmodium. The myxospores found were elongated and ellipsoidal in frontal view. Two elongate, elliptical shell valves with two medium tapering caudal appendages. Average measurements: total length 20.9 ± 0.9 (19.2-21.8) µm, body length 11.2 ± 0.5 (10.5-12.4) µm, body width 5.1 ± 0.3 (4.6-6.1) µm, caudal appendages length 9.8 ± 0.9 (8.2-11.1) µm. Myxospores were biconvex in the lateral view with a thickness of 3.8 ± 0.2 (3.6-4.1) µm. The polar capsules were equal in size and measured 5.9 ± 0.6 (4.9-6.7) µm in length and 1.6 ± 0.1 (1.4-1.8) µm in width, occupying about half of the sporoplasm. Seven coils in the polar tubules present inside the polar capsule perpendicular to the longitudinal axis of the polar capsule were observed. Binucleated sporoplasm and symmetrical valves. Etymology: The specific name is derived from the Latin words: brevis (small) and cauda (tail) and referred to the small size of their caudal appendages.
Myxospores: The morphological data are based on the observation of myxospores isolated from a single plasmodium. The myxospores found were elongated and ellipsoidal in frontal view. Two elongated, elliptical shell valves with two small tapering caudal appendages. Average measurements: total length 17.1 ± 0.9 (15.9-18.7) µm, body length 10.7 ± 0.4 (9.6-10.8) µm, body width 5.3 ± 0.5 (4.4-6.1) µm, caudal appendages length 7.2 ± 0.8   Remark. Henneguya novaerae n. sp., Henneguya bagre n. sp., and Henneguya breviscauda n. sp. were distinguished from the other Henneguya spp. by the morphological or molecular way. The three new species were compared with each other and with species previously described parasitizing Siluriformes fishes from the Americas (Table 2), and all Henneguya species already described, based on the morphometric characteristics of the myxospores.
The three new species described in this study are visually similar and the myxospore body is almost identical in morphology and morphometry. However, there is a clear difference concerning the size of the caudal appendage of the species (14.7 ± 1.4 µm vs. 9.8 ± 0.9 µm vs. 7.2 ± 0.8 µm), which also causes a distinction in the size of the total length of the myxospore (24.9 ± 0.9 µm vs. 20.9 ± 0.9 µm vs. 17.1 ± 0.9 µm). Henneguya breviscauda n. sp. also has polar capsules of different sizes, which is not observed in the other two species. Henneguya novaerae n. sp. presents the width of the body of the myxospore smaller than Henneguya bagre n. sp. (3.8 ± 0.3 µm vs. 5.1 ± 0.3 µm), also differentiating the two species. Furthermore, the molecular differences reported above support the discrimination of the species.
Henneguya novaerae sp. was compared with other Henneguya spp. that parasitizes R. quelen. Henneguya jundiai is the species that most resembles Henneguya novaerae sp. However, there are morphometric differences regarding the length and width of the myxospore body. For H. jundiai the maximum reported spore body length was 10.0 µm, with an average of 9.5 µm, while for Henneguya novaerae n. sp. the measurements found were 10.7 ± 0.5 (10.1-11.6) µm. Concerning the width of the myxospore body, the opposite occurs, with H. jundiai having larger measurements than those found for Henneguya novaerae n. sp. (4.6 ± 0.4 (4.1-5.5) µm vs. 3.8 ± 0.3 (3.2-4.2) µm, respectively). Furthermore, H. jundiai parasitizes the gill arch of R. quelen, while Henneguya novaerae n. sp. parasitizes the gill filaments. The molecular comparison with 106 different nucleotides (equivalent to 6.6% of the total) in the partial sequences available for the species reinforces the distinction between these two species. About the other two Henneguya spp. described parasitizing R. quelen, Henneguya novaerae n. sp. presented smaller measures in almost all characteristics, besides the molecular differences that can be observed in the phylogenetic analysis.
In comparison with species that parasitize Siluriformes fishes from the Americas, the species that most resembled Henneguya novaerae n. sp. was Henneguya pseudoplatystoma Naldoni, Arana, Maia, Ceccarelli, Tavares,   (13.7 µm) to that observed in the two new species, but the myxospores body length is greater (14.3 µm), differentiating them.
All other species used for comparison did not show morphological or morphometric characteristics resembling the new species. At least one morphometric feature, the number of turns of the polar tubules (varying at least 3 turns), or partial genetic sequences, differed in comparison with the new species.

Discussion
In the present study, new species of myxozoans were described based on morphometric characteristics, as well as with phylogenetic support, and the sum of the evidence indicated conclusively the existence of three new species, denominated Henneguya novaerae n. sp, Henneguya bagre n. sp. and Henneguya breviscauda n. sp.
The sequence similarities and variables sites are important evidence for species identification and discrimination of closely related myxozoans species 15,26,27 . Although H. jundiai has been described in the same main watershed where this study was carried out, geographically close, and morphologically similar, our molecular analyzes showed differences between the referred species and the new species. Furthermore, the preferred location for parasitizing the gills was also different, H. jundiai parasitizing the gill arches and the new species found in the filaments.
The phylogenetic analysis indicated an affinity of Henneguya spp. with the hosts, with a strong evolutionary signal, as reported by Carriero et al. 28 , Vieira et al. 29,30 , and Úngari et al. 31 . In the family Myxobolidae, the hosts are the primary evolutionary factors, which contribute to the few monophyletic clades observed in the phylogenies 32,33 . In this study, the arrangement of the species was determined primarily by the order of the host, followed by the family of the host. The new species are inserted in a polyphyletic clade composed of species that infect Siluriformes fishes, along with other Henneguya/Myxobolus species that parasitize R. quelen. The phylogenetic tree clearly distinguishes the new species from the other Henneguya spp. deposited in GenBank. This arrangement further corroborates the effectiveness of the ssrDNA gene for analyses of the phylogenetic relationships among myxozoans, given that closely related species can be extremely variable, with their characteristics being determined primarily by the process of coevolution with their respective hosts 34 . www.nature.com/scientificreports/ Abrunhosa et al. 25 and Negrelli et al. 24 reported the presence of species that parasitize Esociformes fishes in their phylogenetic analyses, as observed in this study. These data corroborate the close phylogenetic relationships between myxozoans species that parasitize fishes from the families Esocidae and Heptapteridae, as only genetically similar species were used for this analysis in this study.
Unfortunately, there is no ssrDNA data available for H. rhamdia in the GenBank, making it impossible to genetically compare them to the new species or to evaluate the phylogenetic relationships of these species. Future molecular and phylogenetic analysis of H. rhamdia is highly recommended to enable a definitive comparison and study of the relationship between species.
Differences were observed concerning the prevalence of each myxozoan described in this study about the collection localities of the hosts. Henneguya novaerae n. sp. was the only species found at two different localities, being close to the source and mouth of the Pardo River. Henneguya bagre n. sp. and Henneguya breviscauda n. sp. were only found at a single locality, and the fish parasitized with Henneguya breviscauda n. sp. were not parasitized by another myxozoan species. There was co-infection of Henneguya novaerae n. sp. and Henneguya bagre n. sp. at locality 3. Although the different number of hosts collected per point may have interfered with the prevalence found, it is possible that different populations of Henneguya spp. inhabit different populations of R. quelen along the Pardo River. The various natural and artificial geographic barriers can contribute to this, but future studies are necessary to clarify this statement.
The place of formation of plasmodia is an important taxonomic character for the differentiation of histozoic myxozoan species 35 . Unfortunately, because of the small number of infected hosts, priority was given to molecular identifications rather than histological preparations. Despite this, visual observations and macroscopic photos helped us to determine the position and shape of plasmodia in the gills. Future studies should determine which tissue of the gill is the specific site of infection for the new species.
The present study describes Henneguya novaerae n. sp., Henneguya bagre n. sp., and Henneguya breviscauda n. sp., gill parasites of R. quelen from Brazil. This is an important contribution to the understanding of the microparasite fauna of the siluriform species of the Neotropical region. The study indicates too that molecular characteristics and microhabitat preferences in the hosts are important for distinguishing morphologically similar species. Based on our analysis we recommend monitoring the presence of these parasites in farmed fish, to analyze possible pathologies caused by them. Table 2. Comparative data for the three new species described and Henneguya spp. from siluriform fishes from South America. All measurements are in µm. TL = total length; ML = myxospore body length; MW = myxospore body width; CA = caudal appendages length; PL = polar capsule length; PW = polar capsule width; TH = thickness; T = number of turns of the polar filament; SI = site of infection. a Larger polar capsules; b Smaller polar capsules.  36 . The morphological measurements of the fresh myxospores followed the recommendations of Lom and Arthur 37 . The measurements of 30 myxospores were performed using a computerized image analysis system with differential interference contrast (DIC) (Leica Application Suite, V3; Leica Microsystems, Wetzlar, Germany). Morphometric data were obtained from myxospores from one plasmodium of the type-host. All measurements are presented in micrometers (μm) and are expressed as means, followed by the standard deviation (SD), and range in parentheses. Illustrations of the myxospores were produced with the aid of a camera lucida mounted on a Leica DMLS microscope with phasecontrast optics. The final images were edited using CorelDRAW X8 software (Corel Corporation, Canada).
DNA extraction, amplification, sequencing, and alignments. Samples containing plasmodia were removed from the gills and fixed in absolute ethanol (100%). Genomic DNA was extracted from the species using the DNeasy® Blood & Tissue Kit (Qiagen, Valencia, CA, USA), following the manufacturer's protocol. At least one of the samples used to obtain the partial sequences came from the type-host species. Access to the genetic data was authorized by the Brazilian Ministry of Environment (Sisgen A8A1D2B). Fragments of ssrDNA were amplified using the primers and cycling conditions presented in Table 3. Conventional polymerase chain reaction (PCR) amplifications were performed on a final volume of 25 µL using ready-to-go PCR beads (GE Healthcare) with extracted DNA (3.0 µL) and 1.0 µL of each PCR primer. PCR products (2.0 µL) were run on an agarose gel (1%) using GelRed™ fluorescent nucleic acid dye and loading buffer to confirm amplicon size and yield. The PCR reaction products for the ssrDNA gene were purified with magnetic beads from the Ampure XP kit (Beckman Coulter) following the manufacturer's protocol and sequenced using the same set of PCR amplification primers. Automated sequencing was performed directly on purified PCR products using a BigDye v.3.1 Terminator Cycle Sequencing Ready Reaction kit on an ABI 3500 DNA genetic sequencer (Applied Biosystems).
The new sequences were assembled and edited using Sequencher v. 5.2.4 (Gene Codes, Ann Arbor, MI, USA). The similarity of the partial sequences obtained with other sequences available in the GenBank database was obtained through searches in the basic local alignment search tool (BLAST). The species most genetically similar (> 80%) to the new species were included in the phylogenetic analyses. The alignments were performed using the MUSCLE algorithm implemented on Geneious 7.1.3 38 with default settings.
Phylogenetic analyses. The best-fitting models for nucleotide substitution selected by jModelTest software 43 using the Akaike information criterion was GTR + I + G. Phylogenies were reconstructed for each alignment under Bayesian inference (BI) using MrBayes v. 3.2 44 . and Maximum Likelihood (ML) using PhyML 45 with 1000 replicates. The BI was run using four Markov chain Monte Carlo searches with 10,000,000 generations and sampling tree topologies every 100 generations. The burning was set to the first 30% of generations; the consensus trees were estimated using the remaining topologies. The nodes with posterior probabilities and bootstrap values greater than 0.95/65 were considered valid. The final trees were edited using CorelDRAW X8.