DISEASES OF AQUATIC ORGANISMSDis Aquat Org

Vol. 112: 139–148, 2014doi: 10.3354/dao02794

Published December 2

INTRODUCTION

The Florida pompano Trachinotus carolinus is atro pical/subtropical fish species of the family Caran -gidae, common on the western coast of the AtlanticOcean. This species is appreciated both as a gameand food fish, presenting high commercial value forthe aquaculture industry (Riley et al. 2009). Never-theless, few parasitological surveys have been con-ducted on the Florida pompano inhabiting the Brazil-

ian coast, and to date we know of only 1 report ofmicrosporidian infection (Casal et al. 2012).

The genus Henneguya Thélohan, 1892 is the sec-ond largest within the class Myxosporea Bütschli,1881, comprising nearly 200 species (Eiras 2002,Eiras & Adriano 2012), the majority of which infectfreshwater fish (Lom & Dyková 2006). Worldwide, 34species are known to infect marine fish hosts (Eiras2002, Eiras & Adriano 2012, Khlifa et al. 2012, Li et al.2012, Azevedo et al. 2014), of which only H. jocu was

© Inter-Research 2014 · www.int-res.com*Corresponding author: azevedoc@icbas.up.pt

Ultrastructure and phylogeny of the parasiteHenneguya carolina sp. nov. (Myxozoa), from the marine fish Trachinotus carolinus in Brazil

S. Rocha1,2, G. Casal1,3, P. Garcia4, E. Matos5, S. Al-Quraishy6, C. Azevedo1,2,6,*

1Laboratory of Animal Pathology, Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), University of Porto, 4050-123 Porto, Portugal

2Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, 4050-313 Porto, Portugal3Department of Sciences, High Institute of Health Sciences-North (CESPU), 4585-116 Gandra, Portugal

4AQUOS-Aquatic Organism Health Laboratory, Aquaculture Department, Federal University of Santa Catarina (UFSC), 88040-900 Florianópolis, SC, Brazil

5Carlos Azevedo Research Laboratory, Federal Rural University of Amazonia (UFRA), 66077-901 Belém, PA, Brazil6Zoology Department, College of Sciences, King Saud University, 11451 Riyadh, Saudi Arabia

ABSTRACT: Microscopic and molecular procedures are used to describe a new myxosporean spe-cies, Henneguya carolina sp. nov., found infecting the intestine of the marine teleost fish Trachino-tus carolinus on the southern Atlantic coast of Brazil. Spherical to ellipsoid cysts, measuring up to~750 µm, display synchronous development. Mature myxospores are ellipsoidal with a bifurcatedcaudal process. Myxospore body length, width, and thickness are 12.7 ± 0.8 (12.0−13.4) µm, 8.8 ±0.6 (7.5−9.6) µm, and 5.8 ± 0.4 (5.0−6.4) µm, respectively; 2 equal caudal processes are 16.8 ± 1.1(15.9−18.0) µm long, and the total myxospore length is 29.4 ± 0.8 (28.4−30.4) µm. Two pyriformpolar capsules measure 5.0 ± 0.5 (4.6−5.6) × 2.4 ± 0.4 (1.9−2.9) µm, and each contains a polar fila-ment forming 3 to 4 coils. Sporoplasm is binucleated and presents a spherical vacuole surroundedby numerous globular sporoplasmosomes. Molecular analysis of the small subunit rRNA gene bymaximum parsimony, neighbor joining, and maximum likelihood reveals the parasite clusteringtogether with other myxobolids that are histozoic in marine fish of the order Perciformes, therebystrengthening the contention that the host phylogenetic relationships and aquatic environmentare the strongest evolutionary signal for myxosporeans of the family Myxobolidae.

KEY WORDS: Myxozoa · Florida pompano · Fish-infecting · Marine · Perciformes · Intestine ·Ultrastructure · SSU rRNA · Host affinity

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Dis Aquat Org 112: 139–148, 2014

described from Brazil (Azevedo et al. 2014). This geo-graphical region, however, is home to approximately50 freshwater Henneguya species (Eiras 2002, Eiras& Adriano 2012, Kozlowiski de Azevedo et al. 2013,Carriero et al. 2013, Müller et al. 2013, Moreira et al.2014), most of which have been described using onlylight microscopy observations and diagrammaticdrawings for morphologic differentiation (Eiras 2002,Eiras & Adriano 2012), with some studies also usingtransmission electron microscopy (TEM) for ultra-structural characterization (Adriano et al. 2005a,b,Azevedo et al. 2009, 2011, Barassa et al. 2012). Morerecently, the use of combined microscopic and mole -cular procedures for the description of myxosporeanshas resulted in the description of 9 Brazilian Henne -guya species, whose small subunit rRNA (SSU rRNA)sequences are available in GenBank (Carriero et al.2013, Müller et al. 2013, Azevedo et al. 2014, Moreiraet al. 2014). Overall, GenBank provides informationon the SSU rRNA gene for 48 Henneguya species: 37from freshwater fish hosts, and 11 from marine fishhosts.

For this polyphyletic genus, the reduced number ofmolecularly characterized species hinders the discern-ment of taxonomic and phylogenetic relationships,given the species-rich state of the Henne guya−Myxobolus myxobolid complex (Fiala 2006, Liu et al.2010, Li et al. 2012). Several studies have sought toidentify the factors influencing the phylogenetic rela-tionships within different myxosporean genera, reveal-ing myxospore morphology and tissue tropism, aswell as host phylogeny, aquatic environment, andgeographical distribution, as possible evolutionarysignals for species clustering (Kent et al. 2001, Eszter-bauer 2004, Fiala 2006, Gleeson & Adlard 2012). Thepresent study provides a comprehensive combina-tion of morphological and molecular data for thedescription of H. carolina sp. nov., a myxosporeanparasite infecting the intestine of the marine perci-form Trachinotus carolinus on the Bra zilian southernAtlantic coast.

MATERIALS AND METHODS

Seventeen specimens of the marine perciform fishTrachinotus carolinus Linnaeus, 1766 (Teleostei, Ca -ran gi dae) (Brazilian common name ‘Pampo’), werecollected from the Brazilian southern Atlantic coast(27° 34’ S, 48° 25’ W), near Barra da Lagoa in Flori-anópolis, State of Santa Catarina, Brazil. Dissectionwas followed by a parasitological survey of severalorgans and tissues. Infected tissues were examined

and photographed using light microscopy (LeitzDialux 20), equipped with differential interferencecontrast optics. Myxospore morphometry was deter-mined from fresh material.

Transmission electron microscopy

For TEM, fragments of parasitized tissue werefixed in 3% glutaraldehyde buffered in 0.2 M sodiumcacodylate (pH 7.4) at 4°C for 24 h, washed overnightin the same buffer at the same temperature, andpost-fixed in 2% osmium tetroxide with the samebuffer for 3 h at 4°C. The samples were then dehy-drated in an ascending graded series of ethanol andoxide propylene, followed by embedding in EPON.Semi-thin sections were stained with methyleneblue-Azur II. Ultrathin sections were double-con-trasted with uranyl acetate and lead citrate, exam-ined, and photographed using a JEOL 100 CXII TEM(JEOL Optical), operating at 60 kV.

DNA extraction, amplification, and sequencing

Fragments of parasitized tissue were fixed and pre-served in 80% ethanol at 4°C before genomic DNAextraction, which was performed using a GenEluteTM

Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich), following the manufacturer’s instructions.The DNA was stored in 50 µl of TE buffer at −20°Cuntil further use.

Amplification of the SSU rRNA gene was achievedusing both universal and specific myxosporean pri -mers: the 5’-end with the primers 18e (5’-CTG GTTGAT CCT GCC AGT-3’) (Hillis & Dixon 1991) andACT3r (5’-ATT GTT CGT TCC ATG-3’); the 3’-endwith the primers ACT3f (5’-CAT GGA ACG AACAAT-3’) (Hallett & Diamant 2001) and 18r (5’-CTACGG AAA CCT TGT TAC G-3’) (Whipps et al. 2003);and the overlapping sequence by pairing the primerMyxospec F (5’-TTC TGC CCT ATC AAC TTG TTG-3’) (Fiala 2006) with the 18r primer. PCRs were per-formed in 50 µl reactions using 10 pmol of eachprimer, 10 nmol of each dNTP, 2.5 mM MgCl2, 5 µl10× Taq polymerase buffer, 1.5 units Taq DNA poly-merase (Nzytech), and 50 to 100 ng of genomic DNA.The reactions were run on a Hybaid PxE Thermo -cycler (Thermo Electron), with initial denaturation at95°C for 3 min, followed by 35 cycles of 94°C for 45 s,53°C for 45 s, and 72°C for 90 s. The final elongationstep was performed at 72°C for 7 min. Aliquots (5 µl)of the PCR products were electrophoresed through a

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Rocha et al.: Ultrastructure and phylogeny of Henneguya carolina sp. nov.

1% agarose 1× Tris-acetate-EDTA buffer (TAE) gelstained with ethidium bromide. PCR products werepurified using a single-step enzymatic cleanup thateliminates unincorporated primers and dNTPs by theExoFast method.

The PCR products from different regions of theSSU rRNA gene were sequenced directly. The se -quencing reactions were performed using a BigDyeTerminator v1.1 from the Applied Biosystems Kit andwere run on an ABI3700 DNA analyzer (Perkin-Elmer, Applied Biosystems).

Distance and phylogenetic analysis

To evaluate the phylogenetic position of the newHenneguya species amongst its closest relatives se-quenced to date, namely other myxobolids, 41 SSUrRNA sequences from GenBank were obtained andanalyzed according to the highest similarity score. Te -tracapsuloides bryosalmonae (U70623) and Budden -brockia plumatellae (AY074915) were selec ted asthe outgroup. The alignment was performed withClustalW in MEGA 5.05 software (Tamura et al. 2011),with an opening gap penalty of 10 and a gap ex ten -sion of 4 for both paired and multiple alignments.Subsequent phylogenetic and molecular evolutionaryanalyses were conducted using MEGA 5.05.

The phylogenetic analysis was performed usingmaximum parsimony (MP), neighbor joining (NJ),and maximum likelihood (ML) methodologies. ForMP, the close neighbor interchange heuristic optionwith a search factor of 1 and random initial tree addi-tion of 500 replicates was performed. For NJ, weused Kimura 2-parameter as the substitution modelwith a gamma distribution (shape parameter = 1.4).For ML, the general time reversible substitutionmodel with 4 gamma-distributed rate variationamong sites was performed. All positions with lessthan 95% site coverage were eliminated from alltrees, resulting in a total of 1001 positions in the finaldataset. The bootstrap consensus tree was inferredfrom 500 replicates for MP, NJ, and ML.

A second alignment was performed for the SSUrRNA sequence of the new Henneguya species andthe SSU rRNA sequences presenting the highest sim-ilarity score in BLAST (clustering in the same clade),resulting in a total of 2174 bp positions in the finaldataset. Distance estimation was carried out inMEGA 5.05, using the Kimura 2-parameter modeldistance matrix for transitions and transversions. Allambiguous positions were removed for each se -quence pair.

RESULTS

Collected and analyzed fish did not present externalsymptoms of infection or disease. Microscopic analysis(Figs. 1 & 2) revealed the presence of cysts adjacent tothe intestinal mucosa of several specimens. Morpho-logical and ultrastructural features (Figs. 3 to 8) of themature spores identified the parasite as a member ofthe genus Henneguya, according to the classificationprovided by Lom & Dyková (2006):Phylum: Myxozoa Grassé, 1970Class: Myxosporea Bütschli, 1881Order: Bivalvulida Shulman, 1959Family: Myxobolidae Thélohan, 1892Genus: Henneguya Thélohan, 1892Species: Henneguya carolina sp. nov.

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Figs. 1 & 2. Light micrographs of Henneguya carolina sp. nov.infecting the intestine of the teleost fish Trachinotus caroli-nus. Fig. 1. Semi-thin section of a cyst, spherical to ellipsoidal,and containing numerous myxospores. Fig. 2. Isolated freefresh mature ellipsoidal myxospores with bifurcated caudalprocesses (arrowheads) extending from the posterior pole.Notice the vacuole (arrow) in the basal portion of the

myxospore’s body

Dis Aquat Org 112: 139–148, 2014

Description

Cysts: Spherical to ellipsoidal, measuring up to ~750 µm,adjacent to the intestinal mucosa of infected speci-mens. Cyst walls in close contact with the intestinalepithelium; formed by numerous collagen-like fibrils,

externally surrounded by some fibroblasts (Figs. 1 & 3).Free mature myxospores also in the intestinal lumen(Fig. 2). Development synchronous, with cysts con-taining both immature and mature myxospores. Im-mature myxospores recognized by developing val -vogenic, capsulogenic, and sporoplasmic cells (Fig. 4).

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Figs. 3−8. Transmission electron micrographs of Henneguya carolina sp. nov. infecting the intestine of the teleost fish Tra-chinotus carolinus. Fig. 3. Cyst containing several myxospores (S). The cyst wall (W), formed by numerous collagen-like fibrils,is externally surrounded by fibroblasts (F). Fig. 4. Immature myxospore displaying the 2 valvogenic cells (VC) uniting alongthe suture line (arrowheads), as well as 1 of the 2 capsulogenic cells (CC), inside of which the polar filament (PF) coils. Notethe bilayered wall of the polar capsule (arrow), consisting of an outer electron-dense layer and an inner electron-lucent layer.Fig. 5. Longitudinal section of a myxospore showing its wall comprised of 2 valves, from which 2 caudal processes extend(arrows). In the anterior pole, 1 of the 2 polar capsules (PC) displaying its polar filament (PF) coiled along the inner wall; andin the posterior pole, the sporoplasm (Sp). Fig. 6. Posterior pole of a myxospore in longitudinal section, showing the sphericalvacuole (Va) and sporoplasmosomes (Sps) that constitute the sporoplasm. Notice the 2 caudal processes (arrows). Fig. 7. Trans-verse section of a myxospore allowing the visualization of the 2 equal-sized polar capsules at the same level, showing the coilsof the polar filament (PF). Fig. 8. Apical portion of a polar capsule (PC) in longitudinal section, displaying the cap-like structure(arrow) through which the polar filament is released, located near the myxospore’s suture line (arrowheads). Notice the lipidic

globules (*)

Rocha et al.: Ultrastructure and phylogeny of Henneguya carolina sp. nov.

Mature myxospores: Ellipsoidal with a bifurcatedcaudal process (Figs. 2 & 5). Myxospore body length:12.7 ± 0.8 (12.0−13.4) µm; myxospore body width:8.8 ± 0.6 (7.5−9.6) µm; myxospore body thickness:5.8 ± 0.4 (5.0−6.4) µm; 2 equal caudal processes:16.8 ± 1.1 (15.9−18.0) µm; total myxospore length:29.4 ± 0.8 (28.4−30.4) µm. Morphometry based on themeasurement of 25 mature myxospores. Myxosporewall thin and smooth, comprised of 2 symmetricalvalves, each extending to form a caudal process atthe posterior end of the myxospore body (Figs. 5 & 6).Caudal processes composed of the same material con-stituting the valves; the latter united along a straightsuture line (Fig. 8). Two pyriform, equal-sized polarcapsules located side by side at the myxo spore’santerior pole, 5.0 ± 0.5 (4.6−5.6) µm long and 2.4 ± 0.4(1.9−2.9) µm wide, each containing an isofilar polarfilament forming 3 to 4 coils (Figs. 4, 5, 7, & 8). Walldouble-layered, about 0.23 µm thick, formed by anouter electron-dense layer and an inner electron-lucent layer (Figs. 4 & 8), and delimiting the electron-dense substance composing the polar capsule matrix.Extrusion pore of the polar capsules located close tothe apical portion of the valves, near the suture line(Fig. 8). A well-defined and homogeneous binucleatesporoplasm, located at the myxo spore’s posteriorpole, presented a spherical vacuole, which measuredabout 3.0 µm in diameter and appeared surroundedby several globular sporoplasmosomes (Figs. 5 & 6).The ultrastructural features described here are re -presented in a schematic drawing (Fig. 9), providingbetter perception of the myxo spore’s morphology.Type host: The marine teleost Trachinotus carolinusLinnaeus, 1766 (Teleostei, Carangidae).Type locality: Brazilian southern Atlantic coast(27° 34’ S, 48° 25’ W), in the Barra da Lagoa nearthe city of Florianópolis, State of Santa Catarina,Brazil.Site of infection: Cysts adjacent to the intestinal mucosa.Prevalence: 9 infected of 17 fish analyzed (53%).Pathology: other than ultrastructural evidence of con-

nective tissue/fibroblast capsule, no other gross ormicroscopic evidence of other host response.Etymology: The specific epithet ‘carolina’ derivesfrom the specific epithet of the host species.Type specimens: Slides containing semi-thin sectionsof the hapantotype were deposited in the Interna-tional Protozoan Type Slides Collection at the Insti-tuto Nacional de Pesquisa da Amazônia (INPA), Ma -naus, State of Amazonas, Brazil, reference 021/ 14,and in the Type Slide Collection of the Laboratory ofAnimal Pathology at the Interdisciplinary Centre ofMarine and Environmental Research, Porto, Portu-gal, reference CIIMAR 2014.5.

Molecular analysis

The consensus DNA sequence of the SSU rRNAgene, composed of 2090 bp, was deposited in Gen-Bank (accession number KJ509923). In total, 41 SSUrRNA sequences from the genera Henneguya andMyxobolus were aligned with the SSU rRNA se -quence of H. carolina sp. nov. MP, NJ, and ML phylo-genetic trees constructed for the selected SSU rRNAsequences revealed H. carolina sp. nov. clusteringtogether with Henneguya sp. IF-2006 and H. jocu,which infect marine fish of the families Carangidaeand Lutjanidae, respectively, both of which belong tothe order Perciformes. This topo logy is supported bybootstrap values of 70% for MP, 81% for NJ, and69% for ML (Fig. 10). To gether, these 3 species form1 of the 2 subclades constituting the clade comprisingall sequenced myxobolids histozoic in marine fish ofthe order Perciformes. The other subclade comprisesH. lateolabracis, H. akule, H. cy nos cioni, H. ogawai,H. yo koyamai, Myxobolus sp. AASAB-2013, H. pagri,H. mauritaniensis, and H. tunisiensis.

Pairwise comparisons among the SSU rRNA sequen -ces available to date for the marine perciform-infect-ing Henneguya species showed that H. carolina sp.nov. exhibited the highest identity to Henneguya sp.IF-2006 (90.3%) and H. jocu (82.3%) (Table 1).

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Fig. 9. Schematic draw-ing of a myxospore ofHenneguya carolina sp.nov. in longitudinal sec-tion, depicting the inter-nal and external organi-zation described herein

Dis Aquat Org 112: 139–148, 2014144

Fig. 10. Maximum parsimony (MP) tree of the SSU rRNA sequence of Henneguya carolina sp. nov. and other se lected myxo-zoan species. The numbers on the branches are bootstrap confidence levels on 500 replicates for MP, neighbor-joining (NJ),and maximum likelihood (ML) trees. There were a total of 1001 positions in the final dataset. GenBank accession numbers are

given in parentheses after the species name

Rocha et al.: Ultrastructure and phylogeny of Henneguya carolina sp. nov.

DISCUSSION

The morphological aspects of the myxospores de-scribed here are consistent with the characteristicsdefined for the genus Henneguya by Lom & Dyková(2006). Considering that myxosporean phylogenybased on molecular data of the SSU rRNA gene hasrevealed the aquatic environment and organ of infec-tion to be stronger evolutionary signals than myxo -spore morphology (Andree et al. 1999, Kent et al.2001, Eszterbauer 2004, Holzer et al. 2004, Fiala2006), both of these aspects are considered for com-parative purposes. About 34 Henneguya species havebeen recorded from marine fish hosts, most from thebulbus arteriosus, gills, and kidney, with only 5 spe-cies recorded from the gastrointestinal tract (Eiras2002, Eiras & Adriano 2012). Morphological compari-son of H. carolina sp. nov. to the latter allowed therecognition and differentiation of several specific as-pects (Table 2): H. pre-intestinalis and H. zahoorihave smaller myxospores (Ozaki & Isizaki 1941, Bhatt& Siddiqui 1964); H. ocellata presents smaller totalmyxospore length (Iversen & Yokel 1963); H. shackle-toni displays longer caudal processes (Brickle et al.2006); and the caudal processes of H. ogawai areshorter and its unequal polar capsules smaller (Li etal. 2012). The remaining marine Henneguya speciesare morphologically differentiated from H. carolinasp. nov. by their longer caudal processes, with the ex-ception of H. akule and H. yokoyamai (Work et al.2008, Li et al. 2012). H. akule is differentiated by itshigher total myxo spore length (Work et al. 2008), andH. yokoyamai by possessing unequal and smaller po-lar capsules (Li et al. 2012).

The nearly 170 freshwater and anadromous Hen-neguya species recorded to date have mainly beendescribed from the gills, kidney, and skin (Eiras 2002,

Eiras & Adriano 2012), accounting for only about 12species that infect the gastrointestinal tract, whichdiffer from H. carolina sp. nov. in morphometry andother specific aspects: H. branchialis, H. ghaffari,H. gigas, H. pellucida, H. rhinogobii, H. sinensis,H. tangschensis, and H. zikawiensis have longer cau-dal processes (Sikama 1938, Chen & Hsieh 1960,Ashmawy et al. 1989, Ali 1999, Eiras 2002 and refer-ences therein, Adriano et al. 2005a); H. ovaliformisdisplays smaller caudal processes (Eiras 2002 andreferences therein); H. hainanensis, H. tenuis, andHenneguya sp. from Acanthogobius flavimanus pre -sent smaller myxospores and polar capsules (Vaney& Conte 1901, Chen & Ma 1998, Baxa et al. 2013).Overall, no other freshwater Henneguya speciesresembles the parasite described here (Eiras 2002,Eiras & Adriano 2012). Molecular analysis of the SSUrRNA gene further supports the parasite describedhere as a new species of the genus Henneguya,herein termed H. carolina sp. nov.

Recognizing the evolutionary signals influencingmyxosporean phylogeny is mandatory for the unveil-ing of these parasites’ evolution and taxonomic posi-tioning. Most myxosporean genera are polyphyletic(Andree et al. 1999, Kent et al. 2001, Carriero et al.2013), demanding the implementation of compre-hensive morphologic and molecular analyses to discern their phylogenetic relationships. The genusHenneguya is no exception, especially consideringthe species-rich state of the Henneguya−Myxobolusmyxobolid complex (Fiala 2006, Liu et al. 2010, Li etal. 2012). H. carolina sp. nov. establishes phyloge-netic relationships for MP, NJ, and ML in concor-dance with previously published cladograms (Khlifaet al. 2012, Li et al. 2012, Carriero et al. 2013, Moreiraet al. 2014). For myxobolids, molecular data on theSSU rRNA gene have revealed myxospore morphol-

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ID Species GenBank 1 2 3 4 5 6 7 8 9 10 11

(1) Henneguya carolina sp. nov. KJ509923 –(2) Henneguya sp. IF-2006 DQ377706 0.097 –(3) Henneguya jocu KF264964 0.177 0.159 –(4) Henneguya yokoyamai AB693053 0.185 0.171 0.181 –(5) Henneguya tunisiensis GQ340975 0.185 0.177 0.179 0.115 –(6) Henneguya ogawai AB693051 0.187 0.173 0.174 0.038 0.113 –(7) Henneguya pagri AB183748 0.191 0.187 0.188 0.095 0.076 0.093 –(8) Henneguya cynoscioni JN017203 0.200 0.177 0.186 0.112 0.118 0.112 0.107 –(9) Henneguya lateolabracis AB183747 0.200 0.192 0.207 0.140 0.138 0.143 0.120 0.134 –(10) Henneguya akule EU016076 0.206 0.189 0.208 0.143 0.133 0.139 0.127 0.141 0.108 –(11) Henneguya mauritaniensis JQ687060 0.206 0.200 0.190 0.105 0.071 0.107 0.072 0.108 0.128 0.139 –(12) Myxobolus sp. AASAB-2013 KC711053 0.226 0.215 0.204 0.094 0.113 0.102 0.094 0.133 0.156 0.158 0.100

Table 1. Pairwise differences obtained by Kimura-2 parameter analysis for the small subunit rRNA sequences of Henneguyaspecies infecting marine fish of the order Perciformes

Dis Aquat Org 112: 139–148, 2014

ogy to be an unreliable evolutionary signal for thediscernment of phylogenetic relationships. The clas-sic taxonomic distinction between the Henneguyaand Myxobolus genera is based on the presence andabsence, respectively, of a caudal process (Lom &Dyková 2006). Nevertheless, molecular based studiesshow the inadequacy of using this morphologicaspect as the key character for the differentiation ofthese genera (Kent et al. 2001, Liu et al. 2010, Li et al.2012), thus suggesting that new myxobolid systemat-ics await proposal.

Recent studies have suggested host affinity as thestrongest evolutionary signal for myxobolids, namelyHenneguya and Myxobolus (Carriero et al. 2013,Moreira et al. 2014). The tree topology presentedhere strengthens this contention, since the SSU rRNAsequences analyzed cluster according to the familyand order of the fish host, forming well-defined cladesof myxobolids infecting fish of the orders Siluri-formes, Mugiliformes, Perciformes, Cypriniformes, andSalmoniformes. H. carolina sp. nov. is no exception,as it clusters within the clade comprising histozoicmyxobolids parasitizing marine Perciformes.

Phylogenetic studies of Myxozoa have also shownthat the aquatic environment inhabited by the host isan important evolutionary signal (Kent et al. 2001,Fiala 2006). In concordance, the tree topology pre-sented here shows the Henneguya species infectingfreshwater Perciformes clustering together within aseparate clade. Other than the clade of marine Perci-formes, the tree topology also displays a clade of mar -ine Mugiliformes; however, these are not sister clades.Instead, the marine clade of Perciformes appearspositioned at the basis of the marine clade of Mugili-formes, which is sister to a freshwater clade of Siluri-formes. Some species do not form a main mar ine clade,as several studies have shown for myxo sporean gen-era (Kent et al. 2001, Fiala 2006); this is probably dueto the fact that the analysis performed consideredonly myxobolid sequences, as it was not aim of thisstudy to broadly analyze myxozoan phylogeny.

Tissue tropism constitutes an important evolution-ary signal for several myxosporean genera (Andreeet al. 1999, Eszterbauer 2004, Holzer et al. 2004, Fiala2006); however, evaluating its importance for thetaxo nomy of myxobolids represents a difficult task.Although Henneguya species do not appear to clus-ter according to the organ of infection, this could bedue to the fact that molecular data available for thisgenus correspond to a small portion of its totality,with most sequences corresponding to freshwaterspecies infecting the gills and tegument, and a few tomarine species infecting the heart. Some studies also

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al.

mac

lovi

nu

sIs

lan

ds

(34.

5−65

.5)

(9.5

−14

.5)

(7.0

−11

.0)

(5.4

−8.

6)(2

5.0−

51.0

)(3

.0−

5.0)

(2.5

−3.

5)(2

006)

H. o

gaw

aiA

can

thop

agru

s Ja

pan

21.1

11

.0

6.9

5.9

10.0

4.

3 1.

9 −

Li

et a

l.

sch

leg

elii

(19.

2−23

.4)

(8.9

−12

.2)

(6.3

−7.

5)(5

.2−

6.6)

(8.4

−12

.7)

(3.8

−5.

2)(1

.4−

2.3)

(201

2)

Tab

le 2

. Hen

neg

uya

spec

ies

infe

ctin

g th

e g

astr

oin

test

inal

trac

t of m

arin

e fi

sh h

osts

. TS

L: t

otal

sp

ore

len

gth

; SB

L: s

por

e b

ody

len

gth

; SB

W: s

por

e b

ody

wid

th; S

BT

: sp

ore

bod

y th

ick

nes

s; C

PL

: cau

dal

pro

cess

len

gth

; PC

L: p

olar

cap

sule

len

gth

; PC

W: p

olar

cap

sule

wid

th; F

C: n

o. o

f fil

amen

t coi

ls. D

ata

are

mea

n ±

SD

an

d/o

r ra

ng

e; d

imen

sion

s g

iven

in

µm

; (–)

not

pro

vid

ed o

r n

ot a

pp

lica

ble

Rocha et al.: Ultrastructure and phylogeny of Henneguya carolina sp. nov.

consider that myxosporean reports should be morespecific in identifying the exact site of infection,especially in the case of Henneguya and Myxobolusdescriptions (Molnár 2002, Liu et al. 2013).

The molecular analysis presented here supportsprevious findings (Fiala 2006, Ferguson et al. 2008,Carriero et al. 2013), revealing a strong tendency ofHenneguya and Myxobolus species to cluster evolu-tionarily according to the phylogenetic relationshipsand aquatic environment of their fish hosts, with mor-phology and geography playing a less important role.Given that fish are polyphyletic (Benton 1998), theorigins and radiations of myxo sporean parasitesprobably reflect the evolution of their fish hosts, aspreviously noted by Carriero et al. (2013). However,the limited molecular data concerning myxozoan par-asites, as well as the unclear evolution of fish groups,makes the existent information insufficient to ascer-tain this possibility with any coherence.

Acknowledgements. This study was partially supported bythe Eng. António de Almeida Foundation (Portugal); theFoundation for Science and Technology (FCT) (Portugal),within the scope of the PhD fellowship grant SFRH/ BD/92661/2013 to S.R., and the project PTDC/ MAR/ 116838/2010; the project EUCVOA (NORTE-07-0162-FEDER-000116)(Portugal); the Foundation ‘Conselho Nacional de Desen-volvimento Científico e Tecnológico’ (CNPq) and the Foun-dation ‘Coordenação de Aperfeiçoamento de Pessoal deNível Superior’ (CAPES; Brazil); and King Saud University(Saudi Arabia), within the scope of Project no. RGP-002). Wethank Prof. Maurício Laterça Martins (UFSC) for providingthe laboratory facilities in which the fish necropsy and pre-liminary microscopic observations were performed. Thiswork complies with the current laws of the countries inwhich it was performed.

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Editorial responsibility: Sven Klimpel,Frankfurt, Germany

Submitted: March 28, 2014; Accepted: July 23, 2014Proofs received from author(s): October 30, 2014