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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).
Currently, more than 44 species of Henneguya are known to infect South America fish (EIRAS, 2002; EIRAS & ADRIANO, 2012; CARRIERO et al., 2013; NALDONI et al., 2014). 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 (CellD 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 C1000TM 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).
Table 1 List of myxozoan whose sequences were used for analyses and the obtained in the present study.
| Parasite | GenBank accession No. | Host | Country | Reference |
|---|---|---|---|---|
| Ceratomyxa shasta | AF001579 | Oncorhynchus mykiss | USA | Bartholomew et al. (1997) |
| Henneguya adiposa | EU492929 | Ictalurus punctatus | USA | Griffin et al. (2009) |
| Henneguya bulbosus | KM000055 | Ictalurus punctatus | USA | Rosser et al. (2014) |
| Henneguya. cerebralis | JX131380 | Thymallus nigrescens | Mongolia | Batueva et al. (2013) |
| Henneguya. corruscans | JQ654971 | Pseudoplatystoma corruscans | Brazil | Adriano et al. (2012) |
| Henneguya creplini | EU732597 | Zingel zingel | Hungary | Eszterbauer et al. (2006) |
| Henneguya cuniculator | KF732840 | Pseudoplatystoma corruscans | Brazil | Naldoni et al. (2014) |
| Henneguya cutanea | AY676460 | Abramis brama | Hungary | Kallert et al. (2005) |
| Henneguya dogieli | KJ725078 | Siniperca chuatsi | China | Unpublished |
| Henneguya doneci | LC011456 | Carassius gibelio | China | Li et al. (2015) |
| Henneguya doneci | EU344898 | Carassius auratus | China | Unpublished |
| Henneguya doneci | HM146129 | Carassius gibelio | China | Ye et al. (2012) |
| Henneguya doori | HDU37549 | Perca fluviatilis | Canada | Siddall et al. (1995) |
| Henneguya exilis | AF021881 | Ictalurus punctatus | USA | Lin et al. (1999) |
| Henneguya friderici | KY315824 | Leporinus friderici | Brazil | Present study |
| Henneguya gurlei | DQ673465 | Ameiurus nebulosus | USA | Iwanowicz et al. (2008) |
| Henneguya ictaluri | AF195510 | Ictalurus punctatus | USA | Pote et al. (2000) |
| Henneguya jocu | KF264964 | Lutjanus jocu | Portugal | Azevedo et al. (2014) |
| Henneguya leporinicola | KP980550 | Leporinus macrocephalus | Brazil | Capodifoglio et al. (2015) |
| Henneguya lobosa | EU732600 | Esox lucius | Germany | Eszterbauer et al. (2006) |
| Henneguya maculosus | KF296344 | Pseudoplatystoma corruscans | Brazil | Carriero et al. (2013) |
| Henneguya mississippiensis | KP404438 | Ictalurus punctatus | USA | Rosser et al. (2015) |
| Henneguya multiplasmodialis | JQ654969 | Pseudoplatystoma corruscans | Brazil | Adriano et al. (2012) |
| Henneguya pellis | FJ468488 | Ictalurus punctatus | USA | Griffin et al. (2009) |
| Henneguya pellucida | KF296352 | Piaractus mesopotamicus | Brazil | Carriero et al. (2013) |
| Henneguya piaractus | KF597016 | Piaractus mesopotamicus | Brazil | Müller et al. (2013) |
| Henneguya pseudoplatystoma | KP981638 | Pseudoplatystoma corruscans | Brazil | Milanin et al. (2015) |
| Henneguya pseudorhinogobii | AB447994 | Rhinogobius sp. | Japan | Kageyama et al. (2009) |
| Henneguya psorospermica | EU732602 | Esox lucius | Germany | Eszterbauer et al. (2006) |
| Henneguya rhinogobii | AB447992 | Rhinogobius sp. | Japan | Kageyama et al. (2009) |
| Henneguya rotunda | KJ416130 | Salminus brasiliensis | Brazil | Moreira et al. (2014a) |
| Henneguya salminicola | AF031411 | Oncorhynchus kisutch | Canada | Hervio et al. (1997) |
| Henneguya sp. | JQ411297 | Oncorhynchus masou masou | Japan | Yokoyama et al. (2012) |
| Henneguya sp. | KR704889 | Cirrhinus mrigala | India | Unpublished |
| Henneguya sp. | EU732601 | Esox lucius | Hungary | Eszterbauer et al. (2006) |
| Henneguya sp. | EU732599 | Perca fluviatilis | Hungary | Eszterbauer et al. (2006) |
| Henneguya sp. | JQ690355 | Carassius auratus | China | Unpublished |
| Henneguya sutherlandi | EF191200 | Ictalurus punctatus | USA | Griffin et al. (2008) |
| Henneguya visibilis | KC771143 | Leporinus obtusidens | Brazil | Moreira et al. (2014b) |
| Henneguya zikaweiensis | KR020026 | Carassius auratus | China | Zhang et al. (2015) |
| Henneguya zschokkei | HZU13827 | Prosopium williamsonii | USA | Smothers et al. (1994) |
| Henneguya zschokkei | AF378344 | Prosopium williamsonii | Canada | Kent et al. (2001) |
Alignments were subjected to maximum likelihood (ML) and Bayesian inference (BI) (rates = invgamma) analyses; additionally, Tamura & Nei (TRN) distance values were performed using Geneious. ML and BI trees were calculated under the TRN + I + G model for the sequences of the rDNA 18S, using PHYML (GUINDON & GASCUEL, 2003) and MrBayes (HUELSENBECK & RONQUIST, 2001) Geneious plug-ins for ML and BI, respectively. These models were selected using jModelTest2 (DARRIBA et al., 2012). Nucleotide frequencies were estimated from the data (A = 0.2824, C = 0.1634, G = 0.2826, T = 0.2715). Six rates of nucleotide substitution were (AC) = 1.0000, (AG) = 3.2212, (AT) = 1.0000, (CG) = 1.0000, (CT) = 6.0419, (GT) = 1.0000; proportion of invariable sites = 0.1460; gamma distribution = 0.3960 estimated with 4 rate categories. ML nodal support was estimated by 1000 nonparametric bootstrap replications. Bayesian posterior probability were determined running the Markov chains (two runs and four chains) for 4 × 106 generations, discarding the initial 1/4 of sampled trees (trees sample every 4 × 103 generations) as burn in fraction. Phylogenetic trees were rooted using Ceratonova shasta (Noble, 1950) as outgroup based upon previous Myxobolidae phylogenies (ADRIANO et al., 2009; CAPODIFOGLIO et al., 2015; NALDONI et al., 2015).
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 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.
Figure 1 (a) Mature spore of Henneguya friderici parasite of gill filaments of Leporinus friderici in frontal view with Nomarski interference contrast; (b) schematic of Henneguya friderici myxospore demonstrating the polar capsule, spore capsule, and caudal processes. Scale bar 10 μm.
Table 2 Comparison of the characteristics of Henneguya friderici with similar species.
| Species | LS | WS | AL | TL | PCL | PCW | Host | Site | Reference |
|---|---|---|---|---|---|---|---|---|---|
| Henneguya leporinicola | 7.6(5.5-8.7) | 4.2 (3.6-4.9) |
21.8 (12.9-32.2) |
- | 3.0 (2.0-3.6) | 1.6 (1.2-2.0) | Leporinus macrocephalus | Gills | Martins et al. (1999) |
| Henneguya azevedoi | 12.0(11-13) | 3.2 (3-4) | 39.4 (37-40) | 56.4 (52-58) | 6.3 (6-7) | 2.1 (2-3) | Leporinus obtusidens | Gill lamellae | Barassa et al. (2012) |
| Henneguya caudicula | 11.3 (11-12) |
5.4 (5-6) | 3.4 (3-4) | 14.7 (14-16) | 3.7 (3-4) | 1.5 | Leporinus lacustris | Gill filament | Eiras et al. (2008) |
| Henneguya friderici | 10.4 (9.6-11.8) |
5.7 (4.8-6.6) |
23.3 (19.1-28.7) |
33.8 (28.7-39.3) |
4.9 (4.2-5.9) | 2.1 (1.5-2.6) |
Leporinus friderici | Gills | Casal et al. (2003) |
| Henneguya schizodon | 13.1 (12-14) |
3.3 (3.4) | 16.3 (15-17) | 28.9 (27-30) | 5.4 (5-6) | 1.3 (1-1.5) | Schizodon fasciatus | Kidney | Eiras et al. (2004) |
| Henneguya visibilis | 10.8 ± 0.6 | 3.9 ± 0.2 | 18 ± 1.2 | 26.8 ± 1.1 | 4.9 ± 0.3 | 1.4 ± 0.1 | Leporinus obtusidens | Connective tissue | Moreira et al. (2014b) |
| Henneguya friderici | 12.8 ± 2.1 (7.4-14.8) | 4.4 ± 0.4 (3.4-5.2) | 19.6 ± 2.2 (16.1-24.4) | 32.8 ± 2.6 (2.49-40) | 5.1 ± 0.5 (3.7-5.9) |
1.5 ± 0.1 (1.2-1.8) | Leporinus friderici | 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.
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, Souza, Moraes & Moraes, 1999 (KP980550) and 89% similarity to that of H. bulbosus Rosser, Griffin, Quiniu, Khoo & Pote, 2014 (KM000055).
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.
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 × 106 generations; burn-in = 4 × 103). Sample from the present study is in bold.
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 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).
Figure 3 Plasmodia of Henneguya friderici infecting the gill filaments of Leporinus friderici. Scale bar = 10 mm.
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, 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).
