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 Piaractus mesopotamicus in Brazil Maria Isabel Müller1,*, Edson Aparecido Adriano1,2, Paulo Sérgio Ceccarelli3, Marcia Ramos Monteiro da Silva4, Antonio Augusto Mendes Maia4, Marlene Tiduko Ueta1 1Department of Animal Biology, Institute of Biology, Universidade Estadual de Campinas, Rua Monteiro Lobato, 255, CEP: 13083-862, Campinas, SP, Brazil 2Department of Biological Sciences, Universidade Federal de São Paulo, 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 Resale or republication not permitted without written consent of the publisher 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 meso potamicus (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 pacu. 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 tambaqui 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 Nano Drop 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 Mogi Mirim farm (In vitrogen), 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 Itapira 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.