Phylogeny and ultrastructure of Myxobolus rangeli n. sp. (Myxozoa, Bivalvulida), a histozoic parasite in Siluriformes fish from the Amazon region

Abstract A new species of Myxobolus parasitizing the arterial bulb and cardiac musculature of the freshwater fish Pimelodus ornatus Kner, 1858, from the Arari river in the municipality of Cachoeira do Arari, island of Marajó, Pará, Brazil, was described. In the present study, the observed prevalence of myxozoan parasites in the heart tissue of the hosts was 20% (6/30). The myxozoans observed had mature biconvex spores, slightly rounded, an anterior end with two pyriform polar capsules and a posterior end with very evident sporoplasm, measuring 8 ± 0.2 μmin length. The spore width was 5.8 ± 0.4 μm, with a thickness of 3.4 ± 0.2μm. The length of the polar capsules was 3.6 ± 0.3 μm and the width was 1.2 ± 0.2μm, with 6 to 7 turns of the polar filament. The divergences observed, regarding the morphometric and genetic structure of SSU rDNA, in relation to other Myxobolidae already described in the literature, confirm the description of the new species Myxobolus rangeli n. sp.


Introduction
Myxozoans make up a group of extremely diverse metazoan parasites that develop endoparasitic interactions with aquatic organisms, especially fish (Lom & Dyková, 2006). Despite all the accumulated knowledge about the diversity of this group (Eiras et al., 2014;Abdel-Ghaffar et al., 2017;Okamura et al., 2018), the interactions and responses of the hosts to these organisms have only been partially revealed (Okamura et al., 2015;Naldoni et al., 2020).
Representatives of the genus Myxobolus Bütschli, 1882, are among the most frequently reported myxozoans that establish infectious relationships in freshwater fish. They are considered to be an important group of pathogens that can cause environmental and economic damage (Eiras et al., 2014;Molnár & Eszterbauer, 2015;Sindeaux-Neto et al., 2021). In the Amazon region, recent studies have described occurrences of Myxobolus sp. and Myxobolus marajoensis in the intestinal muscle of Siluriformes fish on the island of Marajó, Pará, Brazil (Abrunhosa et al., 2016(Abrunhosa et al., , 2017. Also in the state of Pará, Capodifoglio et al. (2019Capodifoglio et al. ( , 2020a described Myxobolus matosi, Myxobolus longissimus, Myxobolus colossomatis, Myxobolus arapiuns and Myxobolus pirapitingae, parasitizing Characiformes hosts in the Tapajós river basin; and Sindeaux-Neto et al. (2021) published the description of the species Myxobolus freitasi, a parasite of the central nervous system of Gymnotiformes fish in the Peixe-Boi river basin, in the eastern portion of the Brazilian Amazon region.
The genus Pimelodus La Cépède, 1803, is the most species-rich group of fish in the family Pimelodidae in the Neotropical region (Garavello & Shibatta, 2007). Theseare small feral catfish commonly known as "mandi". Myxozoan infections of the genus Myxobolus in hosts of this genus of Siluriformes were described by Cellere et al. (2002), in a study carried out in the Paraná river basin, Brazil. The present study deals with redescription andit used aspects of the ultrastructure and molecular biology of new species of Myxobolus that were described by Matos et al. (2014).

Materials and Methods
Thirty fish of the species Pimelodus ornatus Kner, 1858, from the Arari river, were examined. They were caught in an area adjacent to the municipality of Cachoeira do Arari (01° 00'S; 48° 57' W) on the island of Marajó, in the state of Pará (Brazil). They were bought alive from artisanal fishermen, transported and kept in aquariums for up to 72 hours at the Carlos Azevedo Research Laboratory at the Federal Rural University of the Amazon (UFRA). They were then euthanized by means of anesthesia using tricaine methanesulfonate (MS222 Sandoz) at a concentration of 50 mg/L, inaccordance with the procedures approved by the ethics committee for animal experimentation. Following this, necropsy was performed to search for myxosporean infections, through dissection.
During the necropsy, the organs were examined under a stereomicroscope and tissue cysts were collected and analyzed by means of optical microscopy. The presence of parasites was verified.
Tissue fragments were subjected to the transmission electron microscopy (TEM) technique. Specimens were prepared by fixing them in 5% glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.2) for 12 h at 4 °C, followed by washing in the same buffer for 12 h at 4 °C and post-fixation in 2% osmium tetroxide buffered with 0.2 M sodium cacodylate for 3 h. The material was dehydrated in an ascending series of ethanol concentrations, followed by propylene oxide, before incorporation into Epon resin. Semi-thin sections were stained with methylene blue, and ultra-thin sections with double contrast with uranyl acetate and lead citrate. These were observed under a JEOL 100CXII transmission electron microscope, which was operated at 60 kV in the Laboratory of Structural and FunctionalBiology of the Institute of Biological Sciences, at UFPA (LBEF/ICB/UFPA).
For scanning electron microscopy (SEM), fragments of parasitized tissues and free spores were fixed in 5% glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.2) for 12 h at 4 °C, followed by washing in the same buffer for 12 h at 4 °C and post-fixation in 2% OsO 4 buffered with 0.2 M sodium cacodylate for 3 h. The material was then dehydrated in an ascending series of ethanol concentrations, freeze-dried to a critical point, coated with gold and examined under aTESCANVega 3 LMU tabletop electron microscope at the Ultrastructure Laboratory of the UFRA Institute of Animal Health and Production (LU/ISPA/UFRA).
For molecular and phylogenetic analyses, myxosporid cysts were removed and fixed in 80% ethanol. DNA was extracted using the PureLink® Genomic DNA mini-kit (Invitrogen, USA), following the protocol provided by the manufacturer. The DNA samples were quantified in a Biodrop Duo spectrophotometer (Biodrop) and subjected to the polymerase chain reaction (PCR) technique in order to obtain the partial sequence of the small subunit ribosomal DNA (SSU rDNA), using primers that have been recommended in the literature. The ERIB1/ERIBI10 primer sets (Barta et al., 1997) were used in the first round of amplification, followed by the MC3/MC5 primers (Molnár et al., 2002), nested from the first round, and the ACT3f/ACT3r primers (Hallett & Diamant, 2001), semi-nested in the first pair with ERIB1 and ERIB10.
The amplifications were performed in a final reaction volume of 25 µl, containing 1 x ReddyMix PCR Master mixture (Thermo Scientific, USA), 75 mM Tris-HCl (pH 8.8), 20 mM KCl, 1.5 mM MgCl 2 , 0.2 mM of each nucleotide triphosphate (Thermo Scientific, USA), 10 pmol of each primer, 1.25 U of Taq DNA polymerase (Thermo Scientific, USA) and the DNA model (10-50 ng/µl). The reaction protocol for the ERIB1/ERIB10primers consisted of an initial extension at 95°C for 5 minutes, followed by 35 cycles of 95°C for 60 seconds, 56°C for 60 seconds and 72°C for 120 seconds, with a final extension step of 72°C for 10 min. For the other reactions, the reaction protocol was 95°C for 5 minutes, followed by 35 cycles of 95°C for 30 seconds, annealing temperatures of 56°C (nested PCR) or 58°C (semi-nested PCR) for 30 seconds and 72°C for 60 seconds, with a final extension step of 72°C for 10 min.
Subsequently, 3 µl of the PCR mixture was electrophoresed on 1% agarose gel with 1X Tris-borate-EDTA (TBE), stained with SYBR® Safe (Invitrogen, USA) and viewed under blue light. The PCR products were purified by means of DNA GFX ™ PCR and use of a gel strip purification kit (GE Healthcare, UK), in accordance with the manufacturer's instructions. The sequencing reactions were conducted using the Big Dye Terminator v3.1 cycle sequencing kit (Applied Biosystems, USA), following the manufacturer's instructions, in an ABI 3100 genetic analyzer (Applied Biosystems, USA).
The sequences obtained through this procedure were aligned in the BioEdit software (Hall, 2007) and any ambiguous bases were clarified using the respective chromatograms. Sequences of the SSU rDNA gene of myxozoan species that had previously been deposited in NCBI GenBank (Sayers et al., 2022) were aligned in Clustal X 1.8 (Thompson et al., 1997). Similarity scores greater than 80% in the Basic Local Alignment Search Tool (BLAST) were used as a criterion for selecting GenBank strings for inclusion in the analysis. The jModelTest software, version 0.1.1 (Guindon & Gascuel, 2003;Posada, 2008) was used to identify the best nucleotide replacement model for the data set.
Bayesian inferences were implemented in MrBayes, version 3.1.2 (Ronquist & Huelsenbeck, 2003).The Markov chain Monte Carlo method was used to search for two simultaneous executions of four chains of 10,000,000 generations, in which each 500 th tree was sampled. A consensus tree was generated by means of the TreeAnnotator v1.8.4 tool, with a burn-in of 10%, and this was edited and plotted in FigTree v.1.4.3 (Rambaut et al., 2018). The reliability of the phylogenetic findings was verified by means of likelihood mapping analysis, in Tree-Puzzle 5.2 (Schmidt et al., 2002). Genetic distances were calculated in PAUP* 4.0b1 (Swofford, 2002), using the standard p parameter for the SSU rDNA gene.
Illustrative drawings were made from photomicrographs that had been obtained with the aid of a camera coupled to a computer.

Results
The macroscopic analysis showed the presence of whitish cysts, located in the cardiac musculature and in the region of the bulbus arteriosus of six specimens of P.ornatus, containing pyriform spores. The spore valves were symmetrical, with two equal polar capsules and a binucleated sporoblast (Figure 1).
Electron microscopy was performed onmature spores present in the bulbus arteriosus and in the heart muscle ( Figure 2), and the presence of two identical conical valves was shown ( Figures. 3A and 3C). The two polar capsules (PCs) were equal in size, pyriform and elongated, and they converged at the apex of the spore, with a polar filament (PF) presenting 6-7 turns ( Figure 3B).    Description of cysts: The cysts were found to have an oval shape, showing a whitish color and averaging 286 µm (213-408) in length and 175 µm (122-245) in width.
Histopathology: The infection was characterized macroscopically by hypertrophy of the bulbus arteriosus and cardiac muscle that exhibited normal staining. Microscopic analysis of the bulbus arteriosus and cardiac muscle revealed infection by Myxobolus rangeli n sp., which was organized in the form of cysts on the pericardium, and with free spores in the myocardium. In association with areas of myocardial infection by free spores, there was degeneration and multifocal necrosis of muscle fibers, and discrete accumulation of mixed inflammatory infiltrate. It was concluded that the fish presented myocarditis secondary to infection by Myxobolus rangeli n sp.
Representative sequence: the SSU rDNA sequence of M. rangeli n. sp. has been deposited at GenBank under the accession number MT990755.
A partial sequence of 1,805 bp corresponding to the SSU rDNA was applied through sequencing spores of Myxobolus rangeli n. sp. found in the heart of P. ornatus.
In the phylogenetic tree generated by means of Bayesian inferences, two main groups were formed (Figure 4), which both presented high nodal support (posterior probability). The clade formed by parasites of freshwater fish was divided into two subclades: the first comprised hosts of the orders Perciformes and Characiformes; while the second was formed by parasites of the genera Henneguya and Myxobolus, which are parasites of hosts of the order Siluriformes. Myxobolus rangeli n. sp. was present in the subclade of parasites of siluriform fish in South America, composed of a subclade of hosts of the family Pimelodidae, which presented high nodal support (posterior probability). Likelihood analysis, which generated 10,000 combinations of quartets, further confirmed the topology of the phylogenetic tree ( Figure 4B). The alignment of the sequence contained 92.9% of the phylogeny within the tree. Only 5% of the quartets were partially resolved, while 2.1% were unresolved. For pairwise comparisons, a new alignment was obtained, including only the species of Myxobolus that were parasites of Pimelodidae hosts, grouped with M. rangeli n. sp. The minimum genetic distance (p) was 4.2%, between Myxobolus rangeli n. sp. and M. flavus (KF296346), and the distance was 6.4% in relation to M. tapajosi (MF193890) ( Table 2). Table 2. Genetic distances (p-distances) between all Myxobolides species in the Pimelodidae fish subclate (Figure 3).

Discussion
The morphology of the myxospores observed in the cardiac tissue of Pimelodus ornatus is consistent with the general characteristics defined for the genus Myxobolus Bütschli, 1882 (family Myxobolidae) (Lom & Dyková, 2006). Myxobolus infections in the cardiac musculature of different species of fish are rarely recorded in the scientific literature. Examples are the occurrence of M. bulbocordis in the heart of Barbus sharpeyi in Iran (Masoumian et al., 1996) and M. muscularis in the fibers of the heart and skeletal muscle of Chelon ramada in Portugal (Rocha et al., 2019).
Polyphyletism is a striking feature in most myxozoan genera (Kent et al., 2001;Azevedo et al., 2021). In addition to the combination of morphological and molecular analyses, factors related to the environment and tropism in relation to the host have been shown to be important in establishing the evolutionary relationships of this group (Fiala, 2006;Rocha et al., 2019). It was seenin the results described here that there were clear indications of grouping in relation to the type of environment, thus forming a well-defined clade of myxozoa that infect freshwater fish.
These results reinforce the hypothesis that host affinity is a strong evolutionary signal for myxobolids (Moreira et al., 2014;Vieira et al., 2018) and that the origin and irradiation of these parasites reflect the evolution of their hosts (Azevedo et al., 2021). The phylogeny presented here reinforces this finding, since the sequences analyzed were grouped according to the order and family of the hosts.
The morphological and morphometric characteristics of the spores and the partial sequence of the SSU rDNA gene obtained in the present study enabled description of Myxobolus parasites of the cardiac musculature of specimens of Pimelodus ornatusthat were native to the island of Marajó, Brazil (Matos et al., 2014). The description of the new species, Myxobolus rangeli n. sp. contributes to knowledge of the biodiversity and phylogenetic relationships of myxozoan parasites of freshwater fish in the eastern portion of the Amazon region.