New morphological data and molecular diagnostic of Henneguya friderici ( Myxozoa : Myxobolidae ) , a parasite of Leporinus friderici ( Osteichthyes : Anostomidae ) from southeastern Brazil

The myxozoan Henneguya friderici is a parasite of the gills, intestine, kidney and liver of Leporinus friderici, a characiform fish belonging to the family Anostomidae. Forty-two specimens of L. friderici that had been caught in the Mogi Guaçú River, state of São Paulo, were studied. Elongated white plasmodia were found in the gill filaments of 10 host specimens (24%). The mature spores had an ellipsoidal body with polar capsules of equal size and caudal length greater than body length. This study also described 18S rDNA sequencing of H. friderici infecting the gill filaments. This produced a sequence of 1050 bp that demonstrated significant genetic differences with previously described species of Henneguya. Similarity analysis using sequences from species that clustered closest to those produced by this study showed that the species with greatest genetic similarity to H. friderici was H. leporinicola, with 94% similarity.


Introduction
The diversity of known myxozoans has grown greatly since the early work of Kudo (1919).Around 2.200 species have now been described (LOM & DYKOVÁ, 2006) and these represent around 18% of cnidarian species diversity, as far as is currently known (OKAMURA et al., 2015).Henneguya Thélohan, 1892, is one of the most diverse genera of Myxosporea and currently includes more than 200 known and described species (LOM & DYKOVÁ, 2006).This widespread genus includes typical coelozoic and histozoic species and predominantly infects marine and freshwater fish (EIRAS & ADRIANO, 2012).
Of these, around 28 have been found to infect fish species of the order Characiformes.
Identification of the species in this genus, like those in other genera of myxozoans, is based almost exclusively on spore morphology.In the class Myxosporea, morphology has been the main criterion for classification of species (KUDO, 1933;MOLNÁR, 1994).In fact, this method has always failed to identify highly similar species that are found in the same infection site and host and which only have subtle differences in spore structures (YE et al., 2012).Fortunately, this problem has been solved through molecular approaches (SMOTHERS et al., 1994;ANDREE et al., 1999;HOLZER et al., 2004).18S rDNA is the molecular marker that has most commonly been used for detection, identification and phylogenetic analysis on myxozoans (HOLZER et al., 2006).The difficulties of relying on spore morphology for species identification have led authors to recommend that SSU rDNA sequencing should be included when new species are described (ANDREE et al., 1999;KENT et al., 2001;LOM & DYKOVÁ, 2006).
Leporinus friderici (Bloch, 1794) is a characiform fish belonging to the family Anostomidae that is, popularly known in Brazil as "piau".It is widely distributed in the Amazon and Paraguay river basins (FROESE & PAULY, 2016).Among the species of Henneguya, only Henneguya friderici (CASAL et al., 2003) has been reported from L. friderici.
Henneguya friderici was found infecting the gills, intestine, kidney and liver of "piau" from an estuarine region of the Amazon River, in the state of Pará, Brazil.Relative organelle preservation occurred in the liver tissue and, in some cases, development of the parasite caused gradual and generalized degeneration in the intestine, gills and kidney (CASAL et al., 2003) The present paper supplements the original description of H. friderici with new data on morphology and 18S rDNA sequencing on samples from gill filaments of L. friderici from the Mogi Guaçú River, state of São Paulo, Brazil.The new data support the original diagnosis by Casal et al. (2003).

Materials and Methods
Forty-two specimens of L. friderici were caught by local fishermen with nets and hooks in the Mogi Guaçú River near Pirassununga, state of São Paulo, Brazil (21°55'36" S; 47°22'6" W), between January 2014 and January 2016.Gills extracted from the fish were placed in Petri dishes with tap water and were examined for myxozoans using a dissecting microscope.Infected gill filaments were preserved using two different methods: frozen (for spore measurements) and in 95% ethanol (for DNA analysis).
Parasitological examinations were conducted using standard methods with the aid of an optical microscope (Olympus BX51) with differential interference contrast (DIC).Images were captured using a 3.2 mp UC30 digital camera and were analyzed by means of photomicrography software (Cell D 3.4, Olympus Soft Imaging Solutions GmbH, Germany).At least 30 measurements were made for each relevant spore dimension, following the guidelines of Lom & Arthur (1989).
Gill filaments from three hosts were used for DNA extraction by means of the DNeasy Blood & Tissue Kit, following the manufacturer's instructions (QIAGEN Inc., California, USA).The polymerase chain reaction (PCR) was performed as described by Whipps et al. (2015) in 50 µl reaction volumes of the Quick-Load Taq 23 Master Mix (New England Biolabs, Ipswich, Massachusetts, USA), with 0.5 µM of each primer and 3 µl of template DNA.A first round of amplification targeting the small subunit (SUU) rDNA was performed using the primers 18E and 18R (WHIPPS et al., 2003), followed by a second round of PCR with 18E and Myxgen2R (KENT et al., 2000) or with 18R and Myxgen3F (KENT et al., 2000).The amplifications were performed in a C1000 TM thermal cycler (BioRad Laboratories, Hercules, California, USA) with initial denaturation at 95 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, 56 °C for 45 s and 68 °C for 90 s, and a final extension at 72 °C for 7 min.Product amplification was evaluated by observation on 1% agarose gel, and the remainder of the sample was purified using the E.Z.N.A. Cycle Pure Kit (Omega Bio-Tek, Norcross, Georgia, USA).DNA was quantified using a DNA spectrophotometer (NanoDrop Technologies, Wilmington, Delaware, USA).Sequencing reactions were carried out by means of the ABI BigDye Terminator Cycle Sequencing Ready Reaction Kit version 3.1, using the ABI3730xl Genetic Analyzer (Applied Biosystems, Foster City, California, USA).Contiguous sequences were assembled in Geneious (Geneious version 9, created by Biomatters, available from (http://www.geneious.com/)and were deposited in GenBank (Table 1).

Results
Henneguya friderici cysts were found in gill filaments from ten piau specimens, i.e. 24% of the total examined.The elongated white plasmodia measured approximately 2 mm in length.The mature spores were ellipsoid in frontal view and the valves were symmetrical New morphological data and molecular diagnostic of H. friderici and convex in lateral view.The polar capsules were elongated and equal in size and occupied a little less than half of the spore body (Figure 1).Spores (N = 30) were 12.8 ± 2.1 (7.4-14.8)µm in length, 4.4 ± 0.4 (3.4-5.2) µm in width and 32.8 ± 2.6 (2.49-40) µm in total length.The bifurcated caudal processes were cylindrical, equal in size, 19.6 ± 2.2 (16.1-24.4)µm in length, and extended behind the spore.Two equal capsules were pyriform, tapering toward their anterior end and occupying nearly half of the spore, and they measured 5.1 ± 0.5 (3.7-5.9)µm in length and 1.5 ± 0.1 (1.2-1.8)µm in width (Figure 1).Table 2 provides a comparison between the data on the spore dimensions, infection sites and host of H. friderici obtained in this study and the data from the original descriptions.
The 18S rDNA sequencing on H. friderici spores resulted in a sequence containing 1050 bp, which was deposited in the GenBank database under accession number KY315824.This sequence was used for phylogenetic analysis.A BLAST comparison between the sequence obtained and other myxosporean sequences available in GenBank revealed that the 18S rDNA sequence of H. friderici had 92% similarity to that of Henneguya leporinicola Martins, Similarity analysis using sequences from species that clustered closest to those produced by the present study showed that the species with greatest genetic similarity to H. friderici was H. leporinicola, with 94% similarity.The ML and BI phylogenetic tree (Figure 2) showed that H. friderici appears as a sister species of H. leporinicola in a subclade composed mainly of myxosporean parasites of Characiformes and Esociformes.

Discussion
Henneguya friderici was described by Casal et al. (2003) infecting the gills, intestine, kidney and liver of L. friderici in the Amazon River, near Belém, state of Pará, Brazil.Its description was based on morphological and ultrastructural data.This was, in the past, the main method for characterization and identification of myxosporeans (MOLNÁR, 2002).However, Kent et al. (2001) and Lom & Dyková (2006) suggested that amplification of 18S rDNA is fundamental for describing new species of myxosporeans, because of the difficulties of characterizing the spores morphologically.
The present study provided 18S rDNA sequencing on H. friderici that was found infecting the gill filaments of host caught in the Mogi Guaçú River in the state of São Paulo.This enabled phylogenetic   5.1 ± 0.5 (3.7-5.9)1.5 ± 0.1 (1.2-1.8)

Gill filament
Present study LS: length of the spore; WS: width of the spore; AL: length of the tail; TL: total length of the spore; PCL: polar capsules length; PCW: polar capsules width.
analysis on this parasite.The 18S rDNA gene is used in molecular systematics for determining relationships among myxozoans because it is highly variable between very closely related species (KENT et al., 2001).The morphometric and morphological data obtained in the present study clearly confirmed the identification of the species as H. friderici, which was originally described by Casal et al. (2003) (Table 2).Molnár (2002) divided the formation of gill-located myxosporean plasmodia into three types: (1) lamellar; (2) filamental; and (3) gill arch.Among these, the filamental type is subdivided into four types: (1) vascular; (2) epithelial; (3) intrachondral; and (4) basifilamental.In the present study, the H. friderici plasmodia developed on the filamental epithelium of the gills and deformed the gill filaments (Figure 3).
The prevalence of H. friderici in piau was 24%.This was close to the 30% reported by Casal et al. (2003), considering all the infected organs of L. friderici.However, in fish from the Mogi Guaçú River, infection was only observed in the gill filaments.Furthermore, these results corroborated data from other studies conducted in South America in which species of Henneguya were found at the same infection site (NALDONI et al., 2009(NALDONI et al., , 2014)).
These supplementary data on the morphology, 18S rDNA sequencing and phylogeny of H. friderici may facilitate accurate diagnoses and better understanding of the phylogenetic relationships of this parasite.Fiala (2006) indicated that host preference is very important and that myxosporean species could group together according to fish host species.Although host geographical origin is particularly important, tissue tropism in myxosporean evolution has also been revealed in numerous phylogenetic studies (ANDREE et al., 1999;KENT et al., 2001;ESZTERBAUER, 2004;FIALA, 2006).

Figure 2 .
Figure 2. Maximum Likelihood from phylogenetic analysis of the sequences of 18S rDNA gene of Henneguya friderici associated with the closest species indicated by the analysis of Max Score by BLAST of the NCBI platform.First number of nodal support is from maximum likelihood bootstrap (1000 replications), the second number shows Bayesian posterior probability (for 4 × 10 6 generations; burn-in = 4 × 10 3 ).Sample from the present study is in bold.

Table 1 .
List of myxozoan whose sequences were used for analyses and the obtained in the present study.

Table 2 .
Comparison of the characteristics of Henneguya fridericiwith similar species.