Vol. 107: 129–139, 2013 DISEASES OF AQUATIC ORGANISMS Published December 12 doi: 10.3354/dao02668 Dis Aquat Org

Prevalence, intensity, and phylogenetic analysis of Henneguya piaractus and Myxobolus cf. colossomatis from farmed in

Maria Isabel Müller1,*, Edson Aparecido Adriano1,2, Paulo Sérgio Ceccarelli3, Marcia Ramos Monteiro da Silva4, Antonio Augusto Mendes Maia4, Marlene Tiduko Ueta1

1Department of Biology, Institute of Biology, Universidade Estadual de , Rua Monteiro Lobato, 255, CEP: 13083-862, Campinas, SP, Brazil 2Department of Biological Sciences, Universidade Federal de , CEP: 09972-270, Diadema, SP, Brazil 3National Research and Conservation Center for Continental Fish, Instituto Chico Mendes de Conservação da Biodiversidade, CEP: 13630-970, Pirassununga, SP, Brazil 4Department of Basic Sciences, Faculty of Animal Science and Food Engineering, Universidade de São Paulo, CEP: 13635-900, Pirassununga, SP, Brazil

ABSTRACT: Henneguya piaractus and Myxobolus colossomatis (Myxosporea: Myxobolidae) are commonly found in the characid Piaractus mesopotamicus, an important fish farm species in Brazil. This paper describes the prevalence, mean intensity, molecular phylogeny, ultrastructure, and histology of H. piaractus and M. cf. colossomatis found infecting specimens of P. mesopotam- icus collected from fish farms in the state of São Paulo, Brazil. A total of 278 fish were collected from 3 fish farms between February 2008 and July 2010. Parasite prevalence and mean intensity varied throughout the study period, and according to location and year. A phylogenetic tree, plac- ing South American species in a global context, showed a clear tendency among myxosporean species to cluster according to host families. Ultrastructural analysis for M. cf. colossomatis showed the plasmodial wall with numerous projections toward host cells and phagocytic activity. Histopathological data showed hyperplasia caused by H. piaractus in highly infected fish. Histo- logical and ultrastructural analysis of H. piaractus showed results similar to those that have previ- ously been reported.

KEY WORDS: Piaractus mesopotamicus · Henneguya piaractus · Myxobolus cf. colossomatis · Ultrastructure · Phylogeny · São Paulo

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INTRODUCTION structure (Fiala & Bartosová 2010) that mainly infect aquatic hosts and have a wide host range (Feist & Due to the expansion of the aquaculture industry, Longshaw 2006). Interest in the group has increased which has been developing worldwide since the simultaneously with the development of aquaculture, 1990s (FAO 2012), fish disease has become an impor- as a number of species of the group can cause serious tant research area (Barassa et al. 2003). Myxozoa outbreaks of disease among farmed fish species comprise an important group of fish pathogens. This (Feist & Longshaw 2006). Species of the genera phylum is composed of highly specialized metazoan Myxo bolus and Henneguya are among the most parasites with an extremely reduced body size and common fish parasites (Eiras et al. 2005, Eiras &

*Email: [email protected] © Inter-Research 2013 · www.int-res.com 130 Dis Aquat Org 107: 129–139, 2013

Adriano 2012). Here we evaluated myxosporeans with 2.5% glutaraldehyde in 0.1 M sodium cacody- found in farmed Piaractus mesopotamicus (Holm- late buffer (pH 7.2) for scanning electron microscopy. berg 1887), a large characid commonly known in After washing in the same buffer, the preparations Brazil as . It is naturally distributed in the were dehydrated in ethanol, critical-point dried with

Paraná, Paraguay, and Uruguay basins (Godoy 1975) CO2, coated with metallic gold, and examined using and is one of the most important fish species in a JEOL JSM 35 microscope operating at 15 kV (Adri- Brazilian fish farming (Adriano et al. 2005). Pacu is ano et al. 2002a). the second most produced species native to South Plasmodia were fixed in 2.5% glutaraldehyde in America in Brazilian fish farming, with 21 245 t pro- 0.1 M cacodylate buffer (pH 7.4) for 12 h, washed in duced in 2010, together with another 21 621 t of a glucose-saline solution for 2 h and post-fixed in

tambacu (a hybrid resulting from the crossing of OsO4, performed at 4°C, for transmission electron pacu and Colossoma macropomum) (MPA microscopy observation. After dehydration in an ace- 2012). tone series, the material was embedded in EMbed The aim of this study was to evaluate the preva- 812 resin. Ultrathin sections double stained with lence and mean intensity of Henneguya piaractus uranyl acetate and lead citrate were examined using Martins et Souza, 1997 and Myxobolus cf. colossoma- an LEO 906 electron microscope operating at 60 kV tis Molnár et Békési, 1993. Morphological, histologi- (Adriano et al. 2005). cal, and ultrastructural analysis and 18S rRNA gene The contents of the plasmodia were collected for sequencing, together with phylogenetic analysis, molecular analysis in a 1.5 ml microcentrifuge tube were also performed. and fixed in 99% ethanol. DNA was extracted using the Qiagen Dneasy® Blood and Tissue kit, following the manufacturer’s instructions, with a final volume MATERIALS AND METHODS of 100 µl. DNA content was determined using a NanoDrop 2000 spectrophotometer (Thermo Scien- In total, 278 specimens of Piaractus mesopotamicus tific) at 260 nm. Polymerase chain reaction (PCR) (6.8 to 53.0 cm in length) were collected from 3 fish was performed in a final volume of 25 µl, containing farms located in the state of São Paulo from February 10 to 50 ng of extracted DNA, 1× Taq DNA Poly- 2008 to July 2010: 218 fish from the Pirassununga merase buffer (Invitrogen), 0.2 mmol of dNTP (Invit- farm (21° 55’ 52’’ S, 47° 22’ 29’’ W) between February rogen), 1.5 mmol of MgCl2, 0.2 pmol of each primer 2008 and July 2010, 50 fish from the farm (Invitrogen), 0.25 µl (1.25 U) of Taq DNA poly- (22° 28’ 17’’ S, 47° 00’ 40’’ W) be tween February 2009 merase (Invitrogen), and ultrapure (MilliQ) water. and July 2010, and 10 fish from the farm Fragments of the small subunit rDNA gene were (22° 26’ 11’’ S, 46° 49’ 20’’ W), in October 2010 only. amplified using the primer set ERIB1 (forward) and Fish were collected using nets and fishing rods and ACT1R (re verse), MYXGEN4f (forward), and ERIB10 transported alive to the laboratory, where they were (reverse; Table 1) (Barta et al. 1997, Andree et al. killed by transection of the spinal cord, before being 1999, Hallett & Diamant 2001, Diamant et al. 2004). measured and necropsied. The gills were immedi- An initial denaturation step was carried out at 95°C ately screened for myxozoans using a stereomicro- for 5 min, followed by 35 cycles of denaturation scope, and measurement of the spores was per- (95°C for 60 s), annealing (62°C for 60 s), and exten- formed in accordance with studies by Lom & Arthur sion (72°C for 120 s), and a final extended elonga- (1989). Measuring was performed using a computer tion step at 72°C for 5 min. PCR products were elec- equipped with Axivision 4.1 software, linked to an trophoresed in 1.0% agarose gel (Bio America) in a Axioplan 2 Zeiss microscope. TBE buffer (0.045 M Tris-borate, 0.001 M EDTA, pH Free spores were fixed in methanol, stained with 8.0), stained with ethidium bromide and analyzed in Giemsa solution (pH 7.2). A low viscosity mounting an FLA-3000 (Fugi) scanner. The size of the ampli- medium, CytosealTM, was used to create permanent fied fragments was estimated by comparison with slides. Fragments of gills containing cysts were fixed the 1 kb DNA Ladder (Invitrogen). Amplicons were in buffered 10% formalin for 24 h and placed in purified using the QIAquick® PCR Purification Kit paraffin for histopathological analysis. Sections (4 µm (Qiagen). The sequencing was prepared using the thick) were then stained with hematoxylin-eosin same primer pairs used in the PCR and also the (Adriano et al. 2002a). MC5-MC3 primer pair (Table 1) (Molnár et al. 2002) Spores were deposited on a cover slip coated with and was performed with the BigDye ® Terminator poly-L-lysine and fixed for 2 h at room temperature v3.1 cycle sequencing kit (Applied Biosystems) in Müller et al.: Myxosporeans in Piaractus mesopotamicus 131

Table 1. Primers used to amplify and sequence 18S fragments of Henneguya piaractus and Myxobolus cf. colossomatis. F: foward; R: reverse

Primer Sequence (5’ to 3’) Source

ERIB1 F ACC TGG TTG ATC CTG CCA G Barta et al. (1997) ACT1R R AAT TTC ACC TCT CGC TGC CA Hallett & Diamant (2001) MYXGEN4f F GTG CCT TGA ATA AAT CAG AG Diamant et al. (2004) ERIB10 R CTT CCG CAG GTT CAC CTA CGG Barta et al. (1997) MC5 F CCT GAG AAA CGG CTA CCA CAT CCA Molnár et al. (2002) MC3 R GAT TAG CCT GAC AGA TCA CTC CAC GA Molnár et al. (2002)

an ABI 3730 DNA se quencing analyzer (Applied software (SAS Institute), and Duncan’s multiple Biosystems). range test was applied to compare the averages of For each sequence, a standard nucleotide−nucleo- prevalence and mean intensity of infection for loca- tide BLAST (blastn) search (Altschul et al. 1997) was tion, year, and species (p ≤ 0.05 significance level). conducted to verify similarity with other myxo - sporean sequences in GenBank. The partial se quen - ces of the 18S rDNA obtained from the species con- RESULTS sidered in the present study were visualized, edited, and aligned with BioEdit 7.1.3.0 (Hall 1999) software Henneguya piaractus (Figs. 1, 3, & 4) and Myxobo- using the ClustalW algorithm (Thompson et al. 1994). lus cf. colossomatis (Figs. 2, 3, & 5) were found to The phylogenetic positions of the partial 18S rDNA infect Piaractus mesopotamicus in all 3 of the evalu- gene of Henneguya piaractus and Myxobolus cf. ated fish farms. H. piaractus plasmodia were poly- colossomatis was compared with all the sequences of sporic, white, and round to ellipsoidal (majority), and Myxobolus/Henneguya parasites of freshwater and were found in the gill lamellae (Figs. 1 & 3). M. cf. brackish water fishes stored in GenBank. To ensure colossomatis plasmodia were polysporic, round, and the accuracy of the analysis, GenBank sequences white and were found in the branchial arches, gill smaller than that of M. cf. colossomatis (the smaller ra kers, and in a basifilamental position (Fig. 2). Mea - sequence produced in the sequencing of the present surements of fresh spores (n = 30) of both parasite study) were not used in analysis. species and data of previous records of H. piaractus Using Akaike’s information criterion (AIC), the and M. colossomatis are provided in Table 2. best evolutionary model of nucleotide substitution Prevalence for both species varied from high was determined with the jModeltest (Posada 2008) (>50%) to moderate (up to 50%) for fish gathered program, which identified GTR+I+G as the best from the Pirassununga, Mogi Mirim, and Itapira fit. From the data, the nucleotide frequencies (A = farms (Table 3). Taking into account the collection 0.2408, C = 0.1867, G = 0.2818, T = 0.2907) and the period, prevalence also varied from high to moderate 6 rates of nucleotide substitution (AC = 1.2421, AG = (Table 3), with the exception of fish gathered from 3.6268, AT = 1.6702, CG = 0.6062, CT = 5.2143, GT = the Mogi Mirim farm in 2010 which had lower preva- 1.0000) were estimated. Gamma distribution was lence (p < 0.05). 0.3380. These parameters were used for maximum The highest prevalence of Henneguya piaractus at likelihood analysis, which was performed in PhyML the Pirassununga fish farm (71.2%) was recorded in 3.0 (Guindon et al. 2010) with bootstrap confidence 2008, with prevalence decreasing during subsequent values calculated using 100 replicates. The in-group years (Table 3). The highest prevalence of Myxobo- was composed of 46 species, and Ceratomyxa shasta lus cf. colossomatis at Pirassununga was recorded in and C. seriolae were used as the outgroup. FigTree 2010 (73.5%), and the lowest percentage was re cor- v1.3.1 (Rambaut 2009) software was used to visualize ded in 2008 (53.1%). M. cf. colossomatis was more the resulting tree. prevalent in Pirassununga than in the other fish Prevalence and mean intensity of infection data farms, with significant results (p < 0.05) and Duncan were calculated annually according to Bush et al. values of 63.3, compared to 21.3 (Mogi) and 16.6 (1997). Intensity was evaluated by counting the total (Itapira). plasmodia in each infected organ (gills). One-way The ANOVA found significant results for preva- ANOVA was conducted using PROC GLM SAS 9.1 lence versus location and year and for mean intensity 132 Dis Aquat Org 107: 129–139, 2013

versus year (p < 0.05). The Duncan test found that Pirassununga had a higher prevalence and mean intensity than the other fish farms. For Duncan analy- sis, overall data for prevalence (76.2 for 2009, 30.6 for 2010, and 23.2 for 2008) and mean intensity (56.1 for 2009, 16.4 for 2010, and 8.7 for 2008) was significant in 2009 in comparison with 2010 and 2008. Annual mean intensity data presented for Henneguya piar- actus (Pirassununga) was 39.8 cysts fish−1 in 2008, although this de creased considerably in subsequent years (Table 3). The mean intensity for the Mogi Mirim and Itapira fish farms was high throughout the study period (Table 3). In 2009, mean intensity was low for M. cf. colossomatis at the Pirassununga and Itapira fish farms but was high at Mogi Mirim. Histological studies showed that plasmodia of Hen- neguya piaractus were intralamellar, located be tween the gill lamellar epithelium and the capillary, causing accentuated dilatation and deformation of infected gill lamellae (Fig. 1). Plasmodia of Myxobolus cf. colossomatis were distributed throughout the tissue of the branchial arches, gill rakers, and basifilamental Fig. 1. Henneguya piaractus. Light photomicrographs showing portion. The parasite formed plasmodia among the (A) spores stained with Giemsa solution. Scale bar = 10 µm. arch of the gill and gill rakers and in a basifilamental (B) Histological sections of Piaractus mesopotamicus showing 2 plasmodia producing compression in the neighboring position, between the osseous and cartilaginous tissue lamellae (arrows). Stained with HE. Scale bar = 100 µm (Fig. 2). No tissue damage was observed.

Fig. 2. Myxobolus cf. colossomatis infecting Piaractus mesopotamicus. Light photomicrographs of parasites in the gills. (A) Plasmodium in the base of the filament. (B) Plasmodia in the gill rakers (arrows). Scale bar = 200 µm. (C) Mature fresh spores. Scale bar = 10 µm. (D) Histological section showing the plasmodium full of spores in the gill arch (arrow), stained with HE. Scale bar = 100 µm Müller et al.: Myxosporeans in Piaractus mesopotamicus 133

Scanning electron microscopy for Henneguya piaractus showed rup- tured intralamellar plasmodia, with some external spores (Fig. 3A,B). Ultrastructural analyses showed that the single plasmodial wall was con- nected to the ectoplasm zone through numerous and extensive pinocytotic canals, and the earliest stages of sporogenesis were observed at the periphery of the endoplasm, with mature spores occurring in the central region of the plasmodia (Fig. 4). Scanning electron microscopy for Myxobolus cf. colossomatis showed mature pear-shaped to sub-spherical spores (Fig. 3C). Ultrastructural ana - lysis revealed the plasmodia in direct contact with the host tissue. The plasmodial wall consisted of a single membrane, which showed numerous projections toward host cells and phagocytic activity. There was no pino cytotic canal observed in the ecto- plasm. Numerous mitochondria were found in the periphery of the endo- plasm, and in the innermost layer the earliest stages of sporogenesis and Fig. 3. Scanning electron microscopy. (A) Normal gill lamellae (white arrows) mature spores were ob served (Fig. 5). of Piaractus mesopotamicus and a lamella infected by a plasmodium of Hen- Regarding molecular analysis, the neguya piaractus (large black arrow). Note the presence of numerous spores primer pairs ERIB1−ACT1R and (small black arrows). Scale bar = 50 µm. (B) Spores of H. piaractus (black arrows). Scale bar = 20 µm. (C) Spores of Myxobolus cf. colossomatis. Scale MYXGEN4f−ERIB10 successfully am - bar = 10 µm plified 2 fragments, of approximately

Table 2. Henneguya piaractus and Myxobolus (cf.) colossomatis. Comparative data of measurements (in µm) made in this study com- pared to data presented in previous studies. LPC: length of polar capsules; WPC: width of polar capsules; NCF: number of coils of polar filaments; nd: no data

Total Body Spore LPC WPC Tail NCF Site of infection Source length length width length and host

H. piaractus 52.5 12.7 3.6 6.7 1.2 41.2 nd Gills − P. mesopotamicus Martins & Souza (1997) 59.6±2.3 12.8±0.7 4.1±0.2 6.5±0.4 1.2±0.2 46.4±2.1 8−9 Gills − P. mesopotamicus Adriano et al. (2005) 61.5±0.91 21.1±0.62 6.7±0.40 9.8±0.28 1.9±0.37 40.5±1.02 10−11 Gills − P. mesopotamicus Azevedo et al. (2010) 63.5±7.4 14.2±1.0 4.1±0.3 6.8±0.75 1.6±0.2 49.4±7.4 8−9 Gills − P. mesopotamicus Present study M. colossomatis 11.8 nd 6.9 6.0 2.1 nd 7−8 Several organs − Molnár & Békési (1993) (11.4−12.2) (6.6−7.2) (5.8−6.6) (1.8−2.5) Colossoma macropomum 9.55 nd 4.98 5.52 1.57 nd nd Blood − C. macropomum Maciel et al. (2011) (9.08−10.28) (4.64−5.11) (4.90–6.30) (1.31−1.70) M. cf. colossomatis 10.3±0.7 nd 6.4±0.8 4.4±0.42 1.8±0.22 nd 7−8 Gills − P. mesopotamicus Present study 134 Dis Aquat Org 107: 129–139, 2013

in this study were deposited in Gen- Bank under accession numbers KF - 597016 for H. piaractus and KF597017 for M. cf. colossomatis. Phylogenetic analysis resulted in a topology that showed myxosporean species forming 2 distinct clades (Cla des A and B; Fig. 6). Clade A di - vided further, forming clades A1 and A2, where A1 then divided further, forming 2 subclades, the smallest com- posed of Myxo bolus oliveirai Milannin et al. 2010 and Henneguya piaractus, and the largest formed by Henneguya spp. parasites of siluriforms of the families Ictaluridae and Pimelodidae. Clade A2 consisted only of Henne - guya spp., parasites of perciforms of several families. The large clade B divided further to form B1 and B2. Clade B1 also further divided forming 2 subclades, the larger of which was composed of parasite species of cy- prinid hosts, and the smaller was formed by M. cf. colossomatis and par- asites of siluriforms, H. mystusia Sar - kar, 1985 and H. basifilamentalis Mol- nár et al. 2006. Clade B2 clustered 3 Henneguya spp. parasites of salmo - nids (Fig. 6).

DISCUSSION

The morphometric values of Hen- neguya piaractus obtained in this Fig. 4. Henneguya piaractus infecting Piaractus mesopotamicus. Transmission study were very close to those of Mar- electron microscopy of plasmodium in the gills. (A) Section showing aspects of tins & Souza (1997) and Adriano et al. the host−parasite interaction. Note numerous long pinocytic canals (black arrows) connecting the thin plasmodial wall (white arrows) to the conspicuous (2005). In both of these studies, as in ectoplasm (ec) of the plasmodium (P), mitochondria (m), and generative cells the present study, the H. piaractus (gc). H: host cells. Scale bar = 1 µm. (B) Detail of periphery of plasmodia show- samples were obtained from Piaractus ing the thin layer of granular material (white arrows) between the plasmodial mesopotamicus from fish farms in the wall and the host cells (H), long pinocytic canals distributed throughout the ectoplasm (ec), and some mitochondria (M) inside of the plasmodia (P). Scale state of São Paulo, Brazil. However, bar = 0.5 µm. (C) Detail of young spore section in the sporoplasm (spl). Note distinct morphometric values were the 2 nuclei (n), some sporoplasmosomes (black arrow), and the points of obtained by Azevedo et al. (2010), junctions of the valves (white arrows). Scale bar = 0.5 µm who examined P. meso potamicus from the Pantanal Brazilian wetland region 1000 and 1200 bp, respectively, of the 18S rDNA (see Table 2). Based on these differences, Azevedo et gene for Henneguya piaractus and Myxobolus cf. al. (2010) proposed the redescription of H. piaractus. colossomatis found in the gills of Piaractus meso- However, in the redes cription proposal, the authors potamicus. Sequencing of the 18S rDNA gene re - did not consider data reported by Adriano et al. sulted in sequences of 1914 bp for H. piaractus and (2005), comparing their results only with the data of 1341 bp for M. cf. colossomatis. Se quences ob tained Martins & Souza (1997). The morphometric values Müller et al.: Myxosporeans in Piaractus mesopotamicus 135

ob tained in the present study corroborate those pre- is possible that the species found by Azevedo et al. viously reported by Martins & Souza (1997) and Adri- (2010) in P. mesopotamicus from the Pantanal Bra - ano et al. (2005) but differ in several aspects from zilian wetland region is not H. piaractus, but this those obtained by Azevedo et al. (2010). Therefore, it requires confirmation by molecular study. Myxobolus colossomatis was des- cribed by Molnár & Békési (1993) as infecting the connective tissue of sev- eral organs of Colossoma macropo- mum taken from a fish farm in the Amazon. Martins et al. (1999) reported M. colossomatis infecting the kidney, liver, spleen, muscle, and gall bladder of Piaractus mesopotamicus and kid- ney of C. macropomum from a fish farm in the state of São Paulo, Brazil, but these authors did not provide mor- phometric data. Since then, other authors have considered this parasite of P. mesopotamicus to be M. colosso- matis (Adriano et al. 2002b, Campos et al. 2008). However, this parasite has not had its 18S rDNA gene sequenced in samples obtained from C. macropo- mum or from P. meso po tamicus. In the present study, morphological, ultra- strutural, histological, and 18S rDNA sequencing data of samples of this parasite obtained from P. me so po ta - micus are presented. There was little difference between the morphometric data presented in Table 2 and data reported by Molnár & Békési (1993) and Maciel et al. (2011) from samples from C. ma cropomum, and the data ob tained in the present study from samples from P. piaractus. These differences were not enough to sup- port discrimination as different taxa. There fore, as the 18S rDNA sequen- cing data presented here are from a different host to that of the original descriptions, to avoid future confusion Fig. 5. Myxobolus cf. colossomatis infecting Piaractus mesopotamicus. Trans- we prefer to identify the parasite as mission electron microscopy of plasmodium in the gills. (A) Section showing Myxobolus cf. colossomatis, and await aspects of the host−parasite interaction. Note the presence of expansions of future molecular confirmation that it the plasmodial wall toward the host (black arrows), numerous mitochondria really is M. colossomatis. (white arrow), and some young developmental stages (yds) in ectoplasm (ec). Little is known about myxozoan Inside the sporoblasts (spb), young spores (ys), and immature spores (im) are present. Scale bar = 2 µm. (B) Detail of the periphery of the plasmodium show- prevalence in freshwater fish in Brazil, ing young spores (ys), numerous mitochondria (black arrows) in ectoplasm either in aquaculture or in nature (ec), and expansions of the plasmodial wall toward the host (white arrows) and (Martins & Onaka 2006). Itapira fish phagocytosis activity (large arrow). Scale bar = 1 µm. (C) Enlarged part of (A) farm had a high prevalence of Henne - showing detail of the sporoblast with 2 developing spores. Note 3 polar cap- sules (pc), nuclei of capsulogenic cells (✱), polar filaments (black arrows), and guya piaractus (100%). However, the lipid inclusions (white arrows). Scale bar = 2 µm data of this fish farm are the results of 136 Dis Aquat Org 107: 129–139, 2013

Table 3. Henneguya piaractus and Myxobolus cf. colossomatis. Mean values of parasites from the gills of Piaractus mesopotamicus collected at 3 fish farms during February 2008 to July 2010. Intensity was measured as the number of plas- modia in the gills. Different superscripts (a and b) indicate significant differences among samples between years, for the Piras- sununga and Mogi Mirim fish farms (p < 0.05)

Parameter Pirassununga Mogi Mirim Itapira 2008 2009 2010 Total 2009 2010 Total 2009

Total no. of hosts 94 90 34 218 20 30 50 10 Prevalence (%) H. piaractus 71.2a 54.5b 50b 56.8 65a 16.6b 36 100 M. cf. colossomatis 53.1b 63.3b 73.5a 60.5 55a 9b 40 50 Mean intensity H. piaractus 39.8a 5.7b 5.4b 24.5 64.3a 51.6a 83.7 47.1 Range 1−1183 1−33 1−20 2−389 1−606 5−115 M. cf. colossomatis 5.4b 7.3b 6.6b 6.0 57.5a 19.8b 40.6 7.0 Range 1−37 1−30 1−27 5−340 2−85 1−16

Fig. 6. Maximum likelihood phylo genetic tree showing the position of Henneguya piaractus and Myxobolus cf. colossomatis among other myxo sporeans of these 2 genera based on par- tial 18S rDNA sequences. GenBank accession num bers and host taxa are given in front of species names. C: Cypriniformes; Sil: Siluri- formes; P: Perciformes; Sal: Salmoniformes (Froese & Pauly 2013). Numbers above nodes indicate bootstrap confidence levels Müller et al.: Myxosporeans in Piaractus mesopotamicus 137

only one sampling done in 2009, and this high preva- pends on the locality of the infection, and which lence appears to be related to an outbreak during the organ or tissue is damaged. Regarding gill infection, spring of 2009, when fish also showed moderate the formation of cysts at sites other than the gill fila- prevalence of Myxobolus cf. colossomatis and were ments and/or lamellae is not of major pathogenic also heavily infected by other parasite taxa, espe- importance (Feist & Longshaw 2006), as evidenced cially monogeneans (Anacanthorus penilabiatus) and by M. cf. colossomatis. protozoans (Ichthyophthirius multifilis), and a large The ultrastructural aspects of Henneguya piaractus number of fish died. Unfortunately, information is not were meticulously explored in a prior study by Adri- available to allow the causes of this parasite outbreak ano et al. (2005). The results of the present study to be identified. However in the region of the present resemble those obtained by these authors, except study, spring is a season with several climatic in the absence of phagocytic activity, which was not changes, and according to Coutant (1998), outbreaks ob served in the present study. In respect to the re - of disease during the changing of the seasons are description proposed by Azevedo et al. (2010), be - often reported at fish farms, especially if environ- sides the morphologic differences shown in Table 2, mental conditions, such as changes in aquatic param- these authors did not report the presence of pinocytic eters, climate stress, and introduction of pathogens, canals linking the plasmodial wall to the ectoplasm, are not favorable, as these factors can increase the which was shown by Adriano et al. (2005), and also in susceptibility of the hosts to parasites, and provoke an our study. imbalance in the host/parasite/environment system. This is the first ultrastructural study of Myxobolus Pirassununga had higher prevalence and mean cf. colossomatis. The results revealed that the plas- intensity values than Mogi Mirim fish farm. This fact modial wall was composed of a single membrane, in may be associated with the characteristics of the direct contact with the host cells, and the existence of Pirassununga fish farm, which belongs to a Brazilian phagocytic activity. The presence of phagocytic government research institution (CEPTA-ICMBio). activity has been observed in Kudoa quadratum This is an old farm, founded in 1939, and has a long (Uspenskaya 1982), Myxobolus cerebralis, Sphaero - history of developing handling techniques for neo - spora sp. (Lom & Dyková 1995), and in Henneguya tropical fishes, mainly Piaractus mesopotamicus , the piaractus (Adriano et al. 2005). The sporogenesis of focus of our study. P. mesopotamicus were intro- M. cf. colossomatis follows the pattern of other Myxo - duced to the Pirassununga fish farm earlier than to bolus species (Current et al. 1979, Casal et al. 1996, the Mogi Mirim fish farm, which is younger than the 2002, Adriano et al. 2006), with early stages of forma- facility at Pirassununga. This fact may have influ- tion of spores in the pe ri phery of the plasmodium, enced the development of myxosporean species, as and with more mature spores located centrally. well as the establishment of adequate conditions to Phylogenetic analysis showed clade A1 was promote development and transmission. On the other formed by 2 subclades, with the smallest, albeit with hand, the main goal of the Mogi Mirim fish farm is low bootstrap support, grouping Mycobolus oliveirai, reproduction, and at each production cycle, the parasite of Brycon spp., and Henneguya piaractus, ponds are drained. Meanwhile, the aim of the Piras- parasite of characids, both of which are characiforms. sununga fish farm is not to optimize productivity, so The largest subclade clustered Henneguya spp., the this handling method is not routinely used. Based on parasite species of North American ictalurids, form- transmission of myxozoans by oligochaetes, the influ- ing a grouping with strong bootstrap support, and ence of this handling technique appears to be clear in South American pimelodids, forming a grouping the prevalence and intensity of H. piaractus and with acceptable bootstrap support. Clade B1 had a M. cf. colossomatis between Pirassununga and Mogi sister clade, albeit with low bootstrap support, with Mirim fish farms. the grouping formed by H. mystusia and H. basifila- Henneguya piaractus is an important pathogen mentalis, parasites of an Asian siluriform of the fam- and causes pathological alterations in the gills of cul- ily Bagridae, and M. cf. colossomatis, parasite of the tivated pacu (Martins & Souza 1997, Adriano et al. characid Piaractus mesopotamicus. The grouping of 2005). The results of the present study corroborate these 3 species was strongly supported. these findings. Inner topologies within marine and freshwater Myxobolus cf. colossomatis was found infecting the branches suggest that myxosporeans cluster accord- gill arches, and there was some evidence of basifila- ing to various characteristics (Fiala 2006). Some mental infection, but no major pathology was found. grouped by spore morphology (Salim & Dresser For myxozoans, the importance of pathology de - 2000), and some revealed relationships which de - 138 Dis Aquat Org 107: 129–139, 2013

pended on the site of infection (Andree et al. 1999, LITERATURE CITED Holzer et al. 2004, Whipps et al. 2004). The results of Adriano EA, Arana S, Ceccarelli PS, Cordeiro NS (2002a) the present study show a clear tendency of myxo - Light and scanning microscopy of Myxobolus porofilus sporean species to cluster according to host family, sp. n. (Myxosporea: Myxobolidae) infecting the visceral corroborating the results obtained by Fiala (2006), cavity of Prochilodus lineatus (Pisces: : Ferguson et al. (2008), Naldoni et al. (2011), and Prochilodontidae) cultivated in Brazil. Folia Parasitol (Ceske Budejovice) 49: 259−262 Adriano et al. (2012), except for the presence of Adriano EA, Arana S, Ceccarelli PS, Cordeiro NS (2002b) Myxobolus cf. colossomatis, parasite of the characid Prevalência de parasitos do filo Myxozoa em pacu (Piar- Piaractus mesopotamicus. This species appears dis- actus mesopotamicus) (Osteichthyes: Characidae) em tant from the other myxo sporean parasites of characi- rios do pantanal Mato-grossense, Brasil. Bol Téc CEPTA forms, clustering as a basal species of the clade com- 15: 31−38 Adriano EA, Arana S, Cordeiro NS (2005) Histology, ultra- posed of the parasites of bagrid hosts. However, this structure and prevalence of Henneguya piaractus clustering with parasites of siluriforms coincides with (Myxosporea) infecting the gills of Piaractus mesopotam- the grouping of Henneguya piaractus and M. olivei - icus (Characidae) cultivated in Brazil. Dis Aquat Org 64: rai, which are parasites of characiforms but clustered 229−235 Adriano EA, Arana S, Cordeiro NS (2006) Myxobolus cuneus as sister taxa in a clade composed of parasites of silu- n. sp. (Myxosporea) infecting the connective tissue of riforms. Such information suggests a close relation- Piaractus mesopotamicus (Pisces: Characidae) in Brazil: ship between the ancestors of myxosporean parasites histopathology and ultrastructure. Parasite 13: 137−142 of siluriforms and characiforms, fish orders that are Adriano EA, Arana S, Alves AL, Silva MRM, Ceccarelli PS, widely distributed across the South American conti- Henrique-Silva F, Maia AAM (2009) Myxobolus cor dei - roi n. sp., a parasite of Zungaro jahu (Siluriformes: nent (Nelson 2006). Pimelodidae) from Brazilian Pantanal: morphology, phy- However, only a few species of myxosporean para- logeny and histopathology. Vet Parasitol 162:221−229 sites of South American freshwater fish identified Adriano EA, Carriero MM, Maia AAM, Silva MRM, Naldoni using the 18S rDNA gene have been sequenced and J, Ceccarelli PS, Arana S (2012) Phylogenetic and host- parasite relationship analysis of Henneguya multiplas- deposited in GenBank. To date, sequences of 3 myxo- modialis n. sp. infecting Pseudoplatystoma spp. in Brazil- sporean parasites of siluriforms (Henneguya eirasi, ian Pantanal wetland. Vet Parasitol 185:110−120 H. multiplasmodialis, and H. corruscans), and 3 par- Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, asites of characiforms, viz. the 2 species se quenced in Miller W, Lipman DJ (1997) Gapped BLAST and PSI- BLAST: a new generation of protein database search this study, together with Myxobolus oliveirai, are programs. Nucleic Acids Res 25:3389−3402 available. M. cordeiroi (Adriano et al. 2009) was Andree KB, Sékely C, Molnár K, Gresoviac SJ, Hedrick RP described infecting the pimelodid Zungaro jahu and (1999) Relationships among members of the was the first myxosporean species from South Amer- Myxobolus (Myxozoa: Bivalvidae) based on small sub- ica to have its 18S rDNA gene sequenced and unit ribosomal DNA sequences. J Parasitol 85:68−74 Azevedo C, Marques DKS, Casal G, Amaral CMC, Silva EV, deposited. However, as it consisted of only 505 bp, it Matos P, Matos E (2010) Ultrastructural re-description of was not used in this study. Henneguya piaractus (Myxozoa), a parasite of the fresh- Given the lack of data concerning the sequencing water fish Piaractus mesopotamicus (Teleostei, Characi- of myxosporean parasites of South American fish dae) from the Paraguai River, Brazil. Acta Protozool 49: 115−120 available in GenBank, the evolutionary history of Barassa B, Cordeiro NS, Arana S (2003) A new species of myxosporean parasites of characiforms will only be Henneguya, a gill parasite of Astianax altiparanae better understood after the sequencing of a signifi- (Pisces: Characidae) from Brazil, with comments on cant number of species. histopathology and seasonality. Mem Inst Oswaldo Cruz 98: 761−765 Barta JR, Martin DS, Liberator PA, Dashkevicz M and others Acknowledgements. We thank R. A. Torres de Oliveira (1997) Phylogenetic relationships among eight Eimeria (CEPTA/ ICMBio) for help in collecting fish, A. X. Linhares species infecting domestic fowl inferred using complete for help with statistical analysis, M. M. Carriero for help in small subunit ribosomal DNA sequences. J Parasitol 83: phylogenetic analysis, and the anonymous reviewers for 262−271 their helpful suggestions that improved this article. This Bush AO, Lafferty KD, Lotz JM, Shostak AW (1997) Para- study is part of the PhD dissertation of M.I.M. supported by sitology meets ecology on its own terms: Margolis et al. CAPES (doctorate fellowship 2008/2012 with PDEE fellow- revisited. J Parasitol 83: 575−583 ship 2010/2011, proc. no. BEX 2034/10-7) through the Grad- Campos CM, Moraes JRE, Moraes FR (2008) Histopatologia uate Program in Parasitology of the Department of Parasi - de fígado, rim e baço de Piaractus mesopotamicus, Pro - tology (IB-UNICAMP). PROCAD NF 69/2010 provided chilodus lineatus e Pseudoplatystoma fasciatum parasita- additional funding for the supply of materials. E.A.A. re - dos por myxosporídeos, capturados no rio Aquidauana, ceived a research productivity grant from the Brazilian Sci- Mato Grosso so Sul, Brasil. Braz J Vet Parasitol 17: entific Development Agency CNPq. 200−205 Müller et al.: Myxosporeans in Piaractus mesopotamicus 139

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Editorial responsibility: Dieter Steinhagen, Submitted: December 4, 2013; Accepted: September 10, 2013 Hannover, Germany Proofs received from author(s): November 18, 2013