Parasitology International 63 (2014) 303–307

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Phylogeny, ultrastructure and histopathology ofMyxobolus lomi sp. nov.,a parasite of Prochilodus lineatus (Valenciennes, 1836) (Characiformes:Prochilodontidae) from the Peixes River, São Paulo State, Brazil

Rodney Kozlowiski Azevedo a,⁎, Diego Henrique Mirandola Dias Vieira b, Gustavo Henrique Vieira c,Reinaldo José Silva b, Edilson Matos d, Vanessa Doro Abdallah a

a USC — Universidade Sagrado Coração, Bauru, São Paulo, 17011-160, Brazilb UNESP — Univ Estadual Paulista, Campus de Botucatu, Instituto de Biociências, Departamento de Parasitologia, Botucatu, São Paulo, 18618-970, Brazilc UNESP — Univ Estadual Paulista, Campus de Botucatu, Instituto de Biociências, Departamento de Física e Biofísica, Botucatu, São Paulo, 18618-970, Brazild UFRA— Universidade Federal Rural da Amazônia, Laboratório de Pesquisa Carlos Azevedo, Belém, Pará 66077-901, Brazil

⁎ Corresponding author. Tel.: +55 1121077297.E-mail address: azevedork@hotmail.com (R.K. Azeved

1383-5769/$ – see front matter © 2013 Elsevier Ireland Lhttp://dx.doi.org/10.1016/j.parint.2013.11.008

a b s t r a c t

a r t i c l e i n f o

Article history:Received 29 April 2013Received in revised form 9 October 2013Accepted 14 November 2013Available online 27 November 2013

Keywords:PhylogenyUltrastructureHistopathologyMyxobolus lomi sp. nov.Prochilodus lineatusBrazil

This paper presents the morphological, histological, molecular and ultrastructural data on Myxobolus lomi sp.nov., a parasite of the gill filaments of Prochilodus lineatus from the Peixes River (48º06′38″W; 22º 49′53.1″S),São Paulo State, Brazil. From 20 P. lineatus specimens examined, 90.0% (n = 18) were infected. The plasmodiawere white and round, measuring 250 to 300 μm in diameter and the development occurred in the base of thegill filament. The spores showed symmetrical and smooth valves, with the polar filament having 8 to 11 coils.A thorough comparison with all the Myxobolus species described so far is provided. A partial sequencing of the18S rDNA gene revealed approximately 1600-bp. The Myxobolus species parasite of P. lineatus did not matchany of the Myxozoa available in GenBank. In the phylogenetic analysis, M. lomi sp. nov. is clustered with tenother species and only four of these parasites were from gills. Histological analysis of P. lineatus gills infectedbyM. lomi sp. nov. revealed numerous well-delimited cysts at the base of the primary lamella, between connec-tive tissue and bone, next to the gill arteries. However no pronounced inflammatory response was found at theinfection site.

© 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

The South American Continent contains one of the biggest hydro-graphic networks in the world, in which a great variety of fish speciesreside [1]. Brazil shows a highly diversified freshwater fish fauna withapproximately 8000 species, corresponding to 24% of all freshwaterfish species in the world [2]. Myxozoans infecting freshwater and ma-rine fishes represent an important pathogenic group with a worldwidedistribution [3]. The genus Myxobolus Bütschli, 1882 (Myxobolidae) isone of the largest myxosporean groups and its members are importantpathogens of freshwater and marine fish in several geographical areas[4,5].

A synopsis of theMyxobolus species with 744 records was preparedby [4]. Another synopsis recording 792 species, seven of them in am-phibians, was prepared by [5]. Several new species have been detailedsince then. There are currently 31 described Myxobolus species

o).

td. All rights reserved.

parasitizing fish in Brazil. The number of these described species is sur-prisingly low when matched with the fish species registered in Brazil.According to [6] various authors have been discussing the importanceof the species as a pathogen, because some infect economically impor-tant types of fish and others infect cultivable fish, thus possibly causinglarge commercial damage with high mortality rates.

It has been demonstrated that the identification of such histozoicparasites by usingmorphologic characteristics alone is often insufficientto account for a precise diagnosis. The employment of molecularmethods by means of SSU rDNA together with morphologic markers,have been enabling trustful discrimination among species, regardlessof their life stage [7].

This paper describes a newMyxobolus species parasitizing the gills ofProchilodus lineatus (Valenciennes, 1836), based on light microscopy,molecular biology, ultrastructure and histopathology remarks.P. lineatus is a Central American and South American species of ray-finned fish that inhabits the basin of the Paraná River and theParaguay River, the Paraiba do Sul River in Brazil and the San JuanRiver in Nicaragua, is a migratory species of great economic importanceboth in fisheries and aquaculture [8].

304 R.K. Azevedo et al. / Parasitology International 63 (2014) 303–307

2. Materials and methods

In 2011, twenty specimens of P. lineatus, which is of great commer-cial importance in some rivers in São Paulo State, were collected fromPeixes River in the municipality of Anhembi (48º06′38″W; 22º 49′53.1″S) in São Paulo State, Brazil. Immediately after each collection,the fish were killed by transection of the spinal cord and thennecropsied. The fish were dissected and the cysts were removed fromthe gill filaments of different specimens and examined under a lightmi-croscope equipped with differential interference contrast microscopy(Leica DMLB 5000, Leica Microsystems, Wetzlar, Germany). Measure-ments were obtained using a computerized image analysis system(LAS, Leica Microsystems). Spore dimensions (μm) were expressed asmean ± standard deviation, followed by the range and the number ofspecimens measured in parentheses. The illustration was made withthe aid of a camera lucida mounted on a Leica DMLSmicroscope. For ul-trastructural studies, small fragments of the gill lamellae containingcyst-like plasmodia were excised and fixed in 3% glutaraldehyde in0.2 M sodium cacodylate buffer (pH 7.2) at 4 °C for 10 h. After beingrinsed overnight in the same buffer at 4 ºC and post-fixed in 2% osmiumtetroxide in the same buffer for 3 h at 4 °C, the fragments weredehydrated through an ascending ethanol series followed by propyleneoxide and embedded in Epon. Semi-thin sections were stained withmethylene blue-Azure II and observed by DIC optics. Ultra-thin sectionswere double-stained with aqueous uranyl acetate and lead citrate andobserved under a JEOL 100CXII transmission electron microscope(TEM) operated at 60 kV.

Collected gill fragmentswere put into a Karnovsky's solution and thedehydrationwas initiated afterwards in increasing concentrations of al-cohol (3 washings into 70°GL replaced at every two hours; followingthat the material has remained in alcohol 95°GL for 4 h). The materialunderwent a resin-alcohol mixture, having remained there for 12 h.The material was finally transferred to an infiltration resin and laterthe inclusion of the material with the resin was performed. Afterthis procedure, 3 μm cuts were made. The sections were stained withtoluidine blue and hematoxylin-eosin, then visualized through lightmicroscope.

Formolecular analysis, the plasmodiawere taken from the host's tis-sue and collected into a 1.5 mL microcentrifuge tube. Myxobolus DNAwas extracted by means of the QIAmp DNA Mini Kit (Qiagen,Germany), according to the manufacturer's instructions. The DNA wasthen quantified and qualified through a Nano Drop spectrophotometerreadingusing 1.0 μL from the sample and 0.7% agarose gel electrophore-sis (1080 ηg/μl). The samples were diluted with TE and the work con-centration was 100 ηg/μl. The 18S rDNA was amplified with the ERIB1and ERBIB10 universal primers [9]. Nested PCR reactions were per-formed by using the MX5-MX3 primer set [10]. The PCR reactionswere made in microtubes of 0.2 mL out of total volumes of 25 μL, with10 pmol of each primer (25 μM), 2.5 μL of TaKaRa Taq® Hot Start ver-sion 10× PCR buffer, 5 mmol of dNTPs, 0.625U of TaKaRa Taq® HotStart Taq Polymerase, 13.75 μL H2O and 2 μL from the sample. The incu-bation was performed in an Eppendorf Mastercycler Personal thermalcycler, with initial denaturing at 95 °C for 10 min, followed by 30 cycleswith denaturing at 95 °C for 1 min, annealing at 48 °C for 1 min and ex-tension at 72 °C for 2 min, in addition to a final extension at 72 °C for10 min. The amplification efficiency was monitored by the electropho-resis from the reaction in 1.5% agarose gel prepared in a 1X TBE buffer(0.09 M Tris-Borate; 0.002 EDTA) and stained with ethidium bromide(0.1 μg/mL) in 100 V for 40 min. The size of the amplified productswas compared with the 250 bp standard and later photographedunder UV transillumination. The bands obtained from the reactionproducts were cut out from the agarose gel and purified by means ofillustra GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare).After the purification, the sample DNA was quantified by NanoDropreading (Eppendorf) in 260 nm absorbance (A260). The sequencing re-actions were performed bymeans of the ABI PRISM Big Dye Terminator

Cycle Sequencing Ready Reaction Kit (Applied Biosystems), which con-tains initiators that flank in the forward (5′–3′) and reverse (3′–5′) di-rections. The reactions were made in 0.2 mL microtubes with totalvolumes of 10 μL, containing 2.0 μL of ABI PRISM Big Dye TerminatorCycle Sequencing Ready Reaction Kit (Applied Biosystems), 0.5 μL ofthe forward primer (10 μM) and 2.0 μL of the PCR obtained product.The same reactions were repeated by using reverse primer (10 μM).The incubation was made in a thermal cycler (PTC 200, MJ Research),with an initial denaturing at 96 °C for 5 min, followed by 35 cycleswith denaturing at 96 °C for 30 s, annealing at 58 °C for 60 s and exten-sion at 60 °C for 4 min. The products obtained by sequencingwere sub-mitted to precipitation. Eighty μg of isopropanol 80% were initiallyadded to the microtubes containing the reaction products. The sampleswere then centrifuged at 16.100 g for 15 min and the supernatant wasdiscarded. After adding 150 μg of ethanol 70%, another centrifugationwas performed, and the supernatant was discarded. The elimination ofthe exceeding alcohol was made in the concentrator (Concentrator5301, Eppendorf). The samples were diluted in 2.2 μL of Blue Dextranwith an addition of 5° Dye (Applied Biosystems) in a 1:8 concentrationfor further application to denaturing gel of 5% urea-polyacrylamide(Long Ranger Singel Packs/BMA). The sequencingwasmade in an auto-matic DNA Sequencer ABI Prism 377 (Applied Biosystems), with fixedvoltage at 300 V and time interval for collection of fluorescent signs at2400 scans/h. The electrophoretic race was performed in a TBE 1X buff-er (0.09 M Tris-Borate; 0.002 EDTA) for 3 h and 30 min at 200 W, at anapproximate temperature of 51 °C. During the electrophoresis, the fluo-rescence detected at the laser scanner areawas collected and stocked bythe Data Collection 2.6 software (Applied Biosystems). The sampleswere analyzed in triplicate and the data were later analyzed by the Se-quencing Analysys 3.4 software (Applied Biosystems) through thetransformation of the fluorescence intensity into peaks correspondingto the nucleotides (chromatogram).

A standard nucleotide–nucleotide Basic Local Alignment Search Tool(BLAST) (blastn) search was conducted [11]. The sequence of theMyxobolus species obtained from P. lineatuswas alignedwith sequencesobtained in the GenBank using CLC Sequence Viewer (Aarhus, Denmark).To evaluate the position of the Myxobolus species obtained fromP. lineatus in relation to other Myxobolus spp., phylogenetic analyseswere conducted using the phylogenetic methods maximum likelihood(ML) using MEGA 5.0 Software [12] (Tempe, USA). An initial tree forthe heuristic search was obtained by applying the Neighbor-Joiningmethod to a matrix of pairwise distances estimated using the MaximumComposite Likelihood (MCL) approach. Bootstrap analysis (1000 repli-cates) was employed to assess the relative robustness of the branchesof trees. The species Ceratomyxa sparusaurati Sitjà-Bobadilla et al. 2007were used as outgroups in the phylogenetic analyses.

3. Results

Taxonomic summaryPhylumMyxozoa Grassé, 1970Class Myxosporea Bütschli, 1881Order Bivalvulida Shulman, 1959Family Myxobolidae Thélohan, 1892Genus Myxobolus Bütschli, 1882Myxoboluslomi sp. nov.Diagnosis.(Figs. 1–7)Rounded plasmodia with synchronous development and measure-

ments from 250 to 300 μm were found at the base of the gill filament.The spores had a valvular wall 0.9–1.2 μm thick all around their body,and the valves were symmetrical and smooth. The spores were a littlelonger than wide, rounded, tapering anteriorly. They were 14.2 ± 1.4(11.8–15.8, n = 30) μm long by 11.1 ± 1.5 (8.7–12.5, n = 30) μmwide. No mucus envelope was observed at the surface of the spore.The two polar capsules (PCs) were unequal in size, pyriform, pointedapically, circular in cross-section and convergent towards the apex of

Figs. 1–4. 1. Light photomicrographs of mature spore ofMyxobolus lomi sp. nov. in a freshpreparation; 2. Schematic representation ofmature spores ofM. lomi sp. nov.; 3. Histolog-ical sections of gill filaments from Prochilodus lineatus infected by plasmodia ofM. lomi sp.nov. (arrow); 4 Plasmodia ofM. lomi sp. nov. showing synchronous development.

Figs. 5–6. Transmission electronmicrographs of themyxosporeanMyxobolus lomi sp. nov.infecting the freshwater fish Prochilodus lineatus. 5. Ultrastructural details of a longitudinalsection of a polar capsule (PC), showing different sections of the polar filament (PF), polarcapsuleswall consisted of a singlemembrane (arrowwhite) and detail of the apical regionof the polar capsule (arrowhead); 6. Transverse section of the polar capsules showing twonucleus (*) and two polar capsules with polar filament (PF) and suture line of the sporewall (arrowhead).

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the spore. The PCs, which extend up to about 1/2 of the total length ofthe spores, the larger ones were 6.4 ± 0.9 (5.2–7.9, n = 30) μm longand 3.1 ± 0.7 (2.3–4.0, n = 30) μm wide and the smaller ones were6.0 ± 0.8 (4.7–7.4, n = 30) μm long and 2.9 ± 0.5 (2.2–4.2, n = 30)μmwide, with the polar filament (PF) having 8 to 11 coils and orientat-ed obliquely to the longitudinal axis of the PC. Sporoplasm binucleateand with a great iodinophilous vacuole.

In the molecular analysis, the specific primer pair MX5-MX3 suc-cessfully amplified an approximately 1600-bp fragment of the 18SrDNA gene in the spores obtained from plasmodia found infecting thegill filaments of P. lineatus. The triplicated samples were presented100% of similarity when sequenced and aligned. The BLAST searchusing the partial 18S rDNA sequence data (1,527 bp) of the Myxobolusspecies parasite of P. lineatus did not match any of the Myxozoa avail-able in the GenBank. The sequence data showed 73% similarity withMyxobolus oliveirai, the most closely related specie [11].

In the phylogenetic analyses, the Myxobolus species clustered intosix distinct lineages. These remaining species clustered in a monophy-letic group composed of numerous species.Myxobolus lomi sp. nov. clus-tered with ten other Myxobolus species, being only four of theseparasites from gills.

Type host. P. lineatus (Valenciennes, 1836), (Characiformes,Prochilodontidae).

Type locality. Peixes River in the municipality of Anhembi (48º06′38″W; 22º 49′53.1″S) in the State of São Paulo, Brazil.

Site of infection. Spores located in the gill filaments.Prevalence. 90.0% (18/20).Typematerial. One glass slidewith semi thin sections of the cyst con-

taining spores (Hapantotype) and one slide with fresh spores were de-posited in the Myxozoa Type Slide Collection at the “Instituto Nacionalde Pesquisa da Amazônia — INPA”, Manaus, Brazil under the numbers

INPA 017 and INPA 018 respectively. The 18S rDNA sequence was de-posited in GenBank under the accession KF677014.

Etymology. The specific name (M. lomi) is in homage to Dr. Jiři Lom,professor at the Academy of Sciences of the Czech Republic, who signif-icantly contributed for improving our knowledge on Myxozoa.

4. Discussion

The morphologic and morphometric characteristics of M. lomi sp.nov. were compared to the characteristics of all Myxobolus species de-scribed in South America [3,13] and in other geographic regions [4,5].Among the 31Myxobolus species described in Brazil to date,Myxobolusporofilus Adriano et al. 2002 was described parasitizing the visceral cav-ity of P. lineatus. When the results of the present paper are compared tothe ones obtained from the different Myxobolus species previously de-scribed in Brazilian fish, a number of morphologic differences is noted,especially regarding the spore and PC dimensions and shapes, as wellas PFC number, position and organization. From the 31 species detailedparasitizing the Brazilian fish, Myxobolus heckelii Azevedo et al. 2009;Myxobolus insignis Eiras et al. 2005; Myxobolus metynnis Casal et al.2006 and Myxobolus noguchii Pinto, 1928 have the total length similarto the M. lomi sp. nov. When the M. lomi sp. nov. characteristics arecompared to the ones found in the species from other continents [4],those having the total length similar to theM. lomi sp. nov. are selected.This way, theMyxobolus amieti Fomena et al., 1985 is included infectingCtenopoma nanum's spleen and eyes in Cameroon; Myxobolus andhrae(Landsberg and Lom, 1991) parasitizing the outer wall of theOphiocephalus punctatus's intestine in India;Myxobolus eucalii (Guilford,1965) detailed in the Eucalia inconstans's skull and pectoral fins in theU.S.; Myxobolus mylopharyngodoni Nie and Yin, 1973 parasitizingMylopharyngodon piceus's liver, gills and fins in China and finallyM. varicorhini Dzhalilov and Daniyarov, 1975 parasitizing its host tegu-ment, liver and spleen in Central Asia (Table 1). Despite the similarity

Fig. 7. Condensed phylogenetic tree showing relationship between Myxobolus lomi sp. nov. and other 18 nucleotide sequences ofMyxobolus spp. based on partial 18S rDNA. The evolu-tionary history was inferred using the Maximum likelihood (ML) method. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history ofthe taxa analyzed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The ML treewas obtained using the Neighbor-Joining method. Genbank accessions are given after species name. Numbers above nodes indicate bootstrap confidence levels.

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with the spore length, the other characteristics of these species are quitedifferent regarding theM. lomi sp. nov. spores. Also, their hosts are verydifferent phylogenetically, and their geographic locations are also quitedifferent from one another.

TheMyxobolus species studied here differs in morphological aspectsfrom other Myxobolus spp. and differs on the molecular level fromMyxobolus species reported in the GenBank. It could therefore be con-sidered a new species, for which the nameM. lomi sp. nov. is proposed.

This is the second report of a myxosporean species infecting fishfrom the genus Prochilodus fromBrazil; however theM. porofilus specieswas not sequenced by [14] and therefore the molecular comparisonwith this species is not possible, but the BLAST search using the partial18S rDNA sequence ofM. lomi sp. nov. did notmatch any of theMyxozoaavailable in GenBank.

The molecular phylogenetic analysis was based on the comparativeanalyses of the 18S rDNAgene in 18 species ofMyxobolus (approximate-ly 1500 bp) obtained from GenBank. To enhance the accuracy of theanalysis, smaller species were not used. The results, with high bootstrap

Table 1Comparison of measurements from Muxobolus lomi sp. nov. with species from Myxobolus more

Species LS WS LPC

M. lomi sp. nov. 14.2 (11.8–15.8) 11.1 (8.7–12.5) 6.4 (5.2–.9);6.0 (4.7–7.4)

M. heckelii 12.7 (12.2–13.1) 6.6 (6.3–6.9) 2.9 (2.7–3.3)M. insignis 14.5 (14–15) 11.3 (11–12) 7.6 (7–8)M. metynnis 13.1 (12.9–13.5) 7.8 (7.5–8.3) 5.2 (5.0–5.5)M. noguchii 13.6 8.5 6.8M. porofilus 5.7 4.8 1.6M. amieti 14.0 (11.3–15.8) 7.4 (5.4–8.7) 8.4 (6.0–10.0)M. andhrae 13.5 (12.1–15.7) 6.4 (5.7–8.6) 9.0 (8.6–10.0)M. eucalii 14.4 (12.0–15.6) 9.9 (8.4–10.8) 11.1 (9.6–12.0)M. mylopharyngodoni 14.2 (12–15.6) 11.1 (10.8–12) 9.2 (8.4–10.8)M. varicorhini 11.8–16 10.6–11.8 5.9–7.1

Abbreviations: LS, length of the spore; WS, width of the spore; LPC, length of the polar capsulesments are in micrometers, except for the cyst size (mm).

values, showed theMyxobolus species clustering as amonophyletic unit.Myxobolus cordeiroi Adriano et al. 2009 was the most divergent speciesof the genus in this analysis andM. oliveiraiMilanin et al. 2010 appearsto be the sister group of the large monophyletic clade composed by theremainingMyxobolus species.

Myxobolus lomi sp. nov. is grouped into a monophyletic clade A withthe speciesM. oliveirai and ten species by themethod of ML.MyxobolusM. lomi sp. nov. was clustered in a monophyletic subclade B that is sup-ported by high bootstrap values (98) with other fish parasites of fresh-water. Myxobolus M. lomi sp. nov. was clearly presented in a separatebranch of clade and other species phylogenetically close toMyxobolus.

This is thefirst phylogenetic study on aMyxobolus species parasite ofa South American prochilodontid host. From Brazil, only the 18S rDNAgene sequences of M. cordeiroi, with around 500 bp and M. oliveirai,with 1527 bp have been deposited in GenBank. Additionally ultrastruc-tural andhistopathological results reported in this study support thehy-pothesis of species description ofM. lomi sp. nov. as a new species to beincluded in the phylogeny ofMyxobolus. This result strongly contributes

near morphologic.

WPC NC Cyst (mm) Site of infection

3.1 (2.3–4.0);2.9 (2.2–4.2)

8–11 0.25–0.3 Gill filaments

1.7 (1.4–2.0) 4–5 X Gills4.2 (3–5) 6 X Gills3.2 (3.0–3.6) 8–9 X Orbicular region2.2 X X Gills1.1 3 3–5 Body-cavity1.9 (1.4–2.5) X 0.13–0.265 x 0.125–0.25 Spleen, eye1.7 (1.4–2.1) X 1 Outer wall of intestine3.7 (3.0–4.8) 9–11 0.2 Cranium, pectoral fins7.8 (7.2–8.4) 6–7 0.172 × 0.132 Gills, kidneys, fins2.5–4.1 X X Skin, kidneys, spleen

;WPC, width of the polar capsules; NC, number of coils of the polar filament; All measure-

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to the taxonomic study of species of Myxobolus and comprehension ofkinship among the other species of parasites of freshwater fish.

The histological analysis on P. linetaus gills infected byM. lomi sp. nov.revealed numerouswell-delimited cysts at the base of the primary lamel-la, between connective tissue andbone.However, no pronounced inflam-matory response was found at the infection site, which is similar to theresults found by other authors in Myxozoa species described in Brazil[6,10,14–17]. As cysts were found close to the gill arteries, a possibleblood compression may occur with the increasing number of cysts, thusimpairing the gill function of the species infected by theM. lomi sp. nov.

The ultrastructural analysis revealed that the plasmodium and polarcapsules wall consisted of a single membrane, two equal symmetricaland smooth valves, each forming the spore and one binucleatesporoplasm contained some sporoplasmosomes.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

The authors would like to thank Carlos Cesar Ramos, the technicianfrom the Electron Microscopy Laboratory (Department of Pathology)of the Medical School of Botucatu University-UNESP who assisted withthe transmission electron microscopy. Authors would like to thankElisa Pinto de Oliveira for editing the English. Sponsorships: FAPESP(2010/06564-5, 2011/00010-0), CNPq (312590/2009-1), PROCADCAPES (Auxílio 032-2006).

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