Phylogeny, ultrastructure and histopathology of Myxobolus lomi sp. nov.

,
a parasite of Prochilodus lineatus (Valenciennes, 1836) (Characiformes:
Prochilodontidae) from the Peixes River, So Paulo State, Brazil
Rodney Kozlowiski Azevedo
a,
, Diego Henrique Mirandola Dias Vieira
b
, Gustavo Henrique Vieira
c
,
Reinaldo Jos Silva
b
, Edilson Matos
d
, Vanessa Doro Abdallah
a
a
USC Universidade Sagrado Corao, Bauru, So Paulo, 17011-160, Brazil
b
UNESP Univ Estadual Paulista, Campus de Botucatu, Instituto de Biocincias, Departamento de Parasitologia, Botucatu, So Paulo, 18618-970, Brazil
c
UNESP Univ Estadual Paulista, Campus de Botucatu, Instituto de Biocincias, Departamento de Fsica e Biofsica, Botucatu, So Paulo, 18618-970, Brazil
d
UFRA Universidade Federal Rural da Amaznia, Laboratrio de Pesquisa Carlos Azevedo, Belm, Par 66077-901, Brazil
a b s t r a c t a r t i c l e i n f o
Article history:
Received 29 April 2013
Received in revised form 9 October 2013
Accepted 14 November 2013
Available online 27 November 2013
Keywords:
Phylogeny
Ultrastructure
Histopathology
Myxobolus lomi sp. nov.
Prochilodus lineatus
Brazil
This paper presents the morphological, histological, molecular and ultrastructural data on Myxobolus lomi sp.
nov., a parasite of the gill laments of Prochilodus lineatus from the Peixes River (480638W; 22 4953.1S),
So Paulo State, Brazil. From 20 P. lineatus specimens examined, 90.0% (n = 18) were infected. The plasmodia
were white and round, measuring 250 to 300 m in diameter and the development occurred in the base of the
gill lament. The spores showed symmetrical and smooth valves, with the polar lament having 8 to 11 coils.
A thorough comparison with all the Myxobolus species described so far is provided. A partial sequencing of the
18S rDNA gene revealed approximately 1600-bp. The Myxobolus species parasite of P. lineatus did not match
any of the Myxozoa available in GenBank. In the phylogenetic analysis, M. lomi sp. nov. is clustered with ten
other species and only four of these parasites were from gills. Histological analysis of P. lineatus gills infected
by M. 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 inammatory response was found at the
infection 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 sh species
reside [1]. Brazil shows a highly diversied freshwater sh fauna with
approximately 8000 species, corresponding to 24% of all freshwater
sh species in the world [2]. Myxozoans infecting freshwater and ma-
rine shes represent an important pathogenic group with a worldwide
distribution [3]. The genus Myxobolus Btschli, 1882 (Myxobolidae) is
one of the largest myxosporean groups and its members are important
pathogens of freshwater and marine sh in several geographical areas
[4,5].
A synopsis of the Myxobolus species with 744 records was prepared
by [4]. Another synopsis recording 792 species, seven of them in am-
phibians, was prepared by [5]. Several new species have been detailed
since then. There are currently 31 described Myxobolus species
parasitizing sh in Brazil. The number of these described species is sur-
prisingly low when matched with the sh species registered in Brazil.
According to [6] various authors have been discussing the importance
of the species as a pathogen, because some infect economically impor-
tant types of sh and others infect cultivable sh, thus possibly causing
large commercial damage with high mortality rates.
It has been demonstrated that the identication of such histozoic
parasites by using morphologic characteristics alone is often insufcient
to account for a precise diagnosis. The employment of molecular
methods by means of SSU rDNA together with morphologic markers,
have been enabling trustful discrimination among species, regardless
of their life stage [7].
This paper describes a newMyxobolus species parasitizing the gills of
Prochilodus 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-
nned sh that inhabits the basin of the Paran River and the
Paraguay River, the Paraiba do Sul River in Brazil and the San Juan
River in Nicaragua, is a migratory species of great economic importance
both in sheries and aquaculture [8].
Parasitology International 63 (2014) 303307
Corresponding author. Tel.: +55 1121077297.
E-mail address: azevedork@hotmail.com (R.K. Azevedo).
1383-5769/$ see front matter 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.parint.2013.11.008
Contents lists available at ScienceDirect
Parasitology International
j our nal homepage: www. el sevi er . com/ l ocat e/ par i nt
2. Materials and methods
In 2011, twenty specimens of P. lineatus, which is of great commer-
cial importance in some rivers in So Paulo State, were collected from
Peixes River in the municipality of Anhembi (480638W; 22 49
53.1S) in So Paulo State, Brazil. Immediately after each collection,
the sh were killed by transection of the spinal cord and then
necropsied. The sh were dissected and the cysts were removed from
the gill laments of different specimens and examined under a light mi-
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 (m) were expressed as
mean standard deviation, followed by the range and the number of
specimens measured in parentheses. The illustration was made with
the aid of a camera lucida mounted on a Leica DMLS microscope. For ul-
trastructural studies, small fragments of the gill lamellae containing
cyst-like plasmodia were excised and xed in 3% glutaraldehyde in
0.2 M sodium cacodylate buffer (pH 7.2) at 4 C for 10 h. After being
rinsed overnight in the same buffer at 4 C and post-xed in 2% osmium
tetroxide in the same buffer for 3 h at 4 C, the fragments were
dehydrated through an ascending ethanol series followed by propylene
oxide and embedded in Epon. Semi-thin sections were stained with
methylene blue-Azure II and observed by DIC optics. Ultra-thin sections
were double-stained with aqueous uranyl acetate and lead citrate and
observed under a JEOL 100CXII transmission electron microscope
(TEM) operated at 60 kV.
Collectedgill fragments were put into a Karnovsky's solution andthe
dehydration was initiated afterwards in increasing concentrations of al-
cohol (3 washings into 70GL replaced at every two hours; following
that the material has remained in alcohol 95GL for 4 h). The material
underwent a resin-alcohol mixture, having remained there for 12 h.
The material was nally transferred to an inltration resin and later
the inclusion of the material with the resin was performed. After
this procedure, 3 m cuts were made. The sections were stained with
toluidine blue and hematoxylin-eosin, then visualized through light
microscope.
For molecular analysis, the plasmodia were taken fromthe host's tis-
sue and collected into a 1.5 mL microcentrifuge tube. Myxobolus DNA
was extracted by means of the QIAmp DNA Mini Kit (Qiagen,
Germany), according to the manufacturer's instructions. The DNA was
then quantied and qualied through a Nano Drop spectrophotometer
reading using 1.0 L fromthe sample and 0.7%agarose gel electrophore-
sis (1080 g/l). The samples were diluted with TE and the work con-
centration was 100 g/l. The 18S rDNA was amplied with the ERIB1
and ERBIB10 universal primers [9]. Nested PCR reactions were per-
formed by using the MX5-MX3 primer set [10]. The PCR reactions
were made in microtubes of 0.2 mL out of total volumes of 25 L, with
10 pmol of each primer (25 M), 2.5 L of TaKaRa Taq

Hot Start ver-

sion 10 PCR buffer, 5 mmol of dNTPs, 0.625U of TaKaRa Taq

Hot
Start Taq Polymerase, 13.75 L H
2
Oand 2 L fromthe sample. The incu-
bation was performed in an Eppendorf Mastercycler Personal thermal
cycler, with initial denaturing at 95 C for 10 min, followed by 30 cycles
with 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 nal extension at 72 C for
10 min. The amplication efciency 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 g/mL) in 100 V for 40 min. The size of the amplied products
was compared with the 250 bp standard and later photographed
under UV transillumination. The bands obtained from the reaction
products were cut out from the agarose gel and puried by means of
illustra GFX

PCR DNA and Gel Band Purication Kit (GE Healthcare).

After the purication, the sample DNA was quantied by NanoDrop
reading (Eppendorf) in 260 nm absorbance (A
260
). The sequencing re-
actions were performed by means of the ABI PRISMBig Dye Terminator
Cycle Sequencing Ready Reaction Kit (Applied Biosystems), which con-
tains initiators that ank in the forward (53) and reverse (35) di-
rections. The reactions were made in 0.2 mL microtubes with total
volumes of 10 L, containing 2.0 L of ABI PRISM Big Dye Terminator
Cycle Sequencing Ready Reaction Kit (Applied Biosystems), 0.5 L of
the forward primer (10 M) and 2.0 L of the PCR obtained product.
The same reactions were repeated by using reverse primer (10 M).
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 cycles
with 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 sequencing were sub-
mitted to precipitation. Eighty g of isopropanol 80% were initially
added to the microtubes containing the reaction products. The samples
were then centrifuged at 16.100 g for 15 min and the supernatant was
discarded. After adding 150 g of ethanol 70%, another centrifugation
was performed, and the supernatant was discarded. The elimination of
the exceeding alcohol was made in the concentrator (Concentrator
5301, Eppendorf). The samples were diluted in 2.2 L of Blue Dextran
with an addition of 5 Dye (Applied Biosystems) in a 1:8 concentration
for further application to denaturing gel of 5% urea-polyacrylamide
(Long Ranger Singel Packs/BMA). The sequencing was made in an auto-
matic DNA Sequencer ABI Prism 377 (Applied Biosystems), with xed
voltage at 300 V and time interval for collection of uorescent signs at
2400 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 an
approximate temperature of 51 C. During the electrophoresis, the uo-
rescence detected at the laser scanner area was collected and stocked by
the Data Collection 2.6 software (Applied Biosystems). The samples
were analyzed in triplicate and the data were later analyzed by the Se-
quencing Analysys 3.4 software (Applied Biosystems) through the
transformation of the uorescence intensity into peaks corresponding
to the nucleotides (chromatogram).
A standard nucleotidenucleotide Basic Local Alignment Search Tool
(BLAST) (blastn) search was conducted [11]. The sequence of the
Myxobolus species obtained fromP. lineatus was aligned with sequences
obtained inthe GenBank using CLC Sequence Viewer (Aarhus, Denmark).
To evaluate the position of the Myxobolus species obtained from
P. lineatus in relation to other Myxobolus spp., phylogenetic analyses
were conducted using the phylogenetic methods maximum likelihood
(ML) using MEGA 5.0 Software [12] (Tempe, USA). An initial tree for
the heuristic search was obtained by applying the Neighbor-Joining
method to a matrix of pairwise distances estimated using the Maximum
Composite Likelihood (MCL) approach. Bootstrap analysis (1000 repli-
cates) was employed to assess the relative robustness of the branches
of trees. The species Ceratomyxa sparusaurati Sitj-Bobadilla et al. 2007
were used as outgroups in the phylogenetic analyses.
3. Results
Taxonomic summary
Phylum Myxozoa Grass, 1970
Class Myxosporea Btschli, 1881
Order Bivalvulida Shulman, 1959
Family Myxobolidae Thlohan, 1892
Genus Myxobolus Btschli, 1882
Myxoboluslomi sp. nov.
Diagnosis.(Figs. 17)
Rounded plasmodia with synchronous development and measure-
ments from 250 to 300 m were found at the base of the gill lament.
The spores had a valvular wall 0.91.2 m thick all around their body,
and the valves were symmetrical and smooth. The spores were a little
longer than wide, rounded, tapering anteriorly. They were 14.2 1.4
(11.815.8, n = 30) m long by 11.1 1.5 (8.712.5, n = 30) m
wide. No mucus envelope was observed at the surface of the spore.
The two polar capsules (PCs) were unequal in size, pyriform, pointed
apically, circular in cross-section and convergent towards the apex of
304 R.K. Azevedo et al. / Parasitology International 63 (2014) 303307
the spore. The PCs, which extend up to about 1/2 of the total length of
the spores, the larger ones were 6.4 0.9 (5.27.9, n = 30) m long
and 3.1 0.7 (2.34.0, n = 30) m wide and the smaller ones were
6.0 0.8 (4.77.4, n = 30) m long and 2.9 0.5 (2.24.2, n = 30)
m wide, with the polar lament (PF) having 8 to 11 coils and orientat-
ed obliquely to the longitudinal axis of the PC. Sporoplasm binucleate
and with a great iodinophilous vacuole.
In the molecular analysis, the specic primer pair MX5-MX3 suc-
cessfully amplied an approximately 1600-bp fragment of the 18S
rDNA gene in the spores obtained from plasmodia found infecting the
gill laments of P. lineatus. The triplicated samples were presented
100% of similarity when sequenced and aligned. The BLAST search
using the partial 18S rDNA sequence data (1,527 bp) of the Myxobolus
species parasite of P. lineatus did not match any of the Myxozoa avail-
able in the GenBank. The sequence data showed 73% similarity with
Myxobolus oliveirai, the most closely related specie [11].
In the phylogenetic analyses, the Myxobolus species clustered into
six 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 these
parasites from gills.
Type host. P. lineatus (Valenciennes, 1836), (Characiformes,
Prochilodontidae).
Type locality. Peixes River in the municipality of Anhembi (4806
38W; 22 4953.1S) in the State of So Paulo, Brazil.
Site of infection. Spores located in the gill laments.
Prevalence. 90.0% (18/20).
Type material. One glass slide withsemi thinsections 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 Nacional
de Pesquisa da Amaznia 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 specic name (M. lomi) is in homage to Dr. Jii 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 31 Myxobolus species described in Brazil to date, Myxobolus
porolus Adriano et al. 2002 was described parasitizing the visceral cav-
ity of P. lineatus. When the results of the present paper are compared to
the ones obtained from the different Myxobolus species previously de-
scribed in Brazilian sh, a number of morphologic differences is noted,
especially regarding the spore and PC dimensions and shapes, as well
as PFC number, position and organization. From the 31 species detailed
parasitizing the Brazilian sh, 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 similar
to the M. lomi sp. nov. When the M. lomi sp. nov. characteristics are
compared to the ones found in the species from other continents [4],
those having the total length similar to the M. lomi sp. nov. are selected.
This way, the Myxobolus amieti Fomena et al., 1985 is included infecting
Ctenopoma nanum's spleen and eyes in Cameroon; Myxobolus andhrae
(Landsberg and Lom, 1991) parasitizing the outer wall of the
Ophiocephalus punctatus's intestine in India; Myxobolus eucalii (Guilford,
1965) detailed in the Eucalia inconstans's skull and pectoral ns in the
U.S.; Myxobolus mylopharyngodoni Nie and Yin, 1973 parasitizing
Mylopharyngodon piceus's liver, gills and ns in China and nally
M. varicorhini Dzhalilov and Daniyarov, 1975 parasitizing its host tegu-
ment, liver and spleen in Central Asia (Table 1). Despite the similarity
Figs. 14. 1. Light photomicrographs of mature spore of Myxobolus lomi sp. nov. in a fresh
preparation; 2. Schematic representation of mature spores of M. lomi sp. nov.; 3. Histolog-
ical sections of gill laments fromProchilodus lineatus infected by plasmodia of M. lomi sp.
nov. (arrow); 4 Plasmodia of M. lomi sp. nov. showing synchronous development.
Figs. 56. Transmission electron micrographs of the myxosporean Myxobolus lomi sp. nov.
infecting the freshwater sh Prochilodus lineatus. 5. Ultrastructural details of a longitudinal
section of a polar capsule (PC), showing different sections of the polar lament (PF), polar
capsules wall consistedof a single membrane (arrowwhite) and detail of the apical region
of the polar capsule (arrowhead); 6. Transverse section of the polar capsules showing two
nucleus (*) and two polar capsules with polar lament (PF) and suture line of the spore
wall (arrowhead).
305 R.K. Azevedo et al. / Parasitology International 63 (2014) 303307
with the spore length, the other characteristics of these species are quite
different regarding the M. lomi sp. nov. spores. Also, their hosts are very
different phylogenetically, and their geographic locations are also quite
different from one another.
The Myxobolus species studied here differs in morphological aspects
from other Myxobolus spp. and differs on the molecular level from
Myxobolus species reported in the GenBank. It could therefore be con-
sidered a new species, for which the name M. lomi sp. nov. is proposed.
This is the second report of a myxosporean species infecting sh
fromthe genus Prochilodus fromBrazil; however the M. porolus species
was not sequenced by [14] and therefore the molecular comparison
with this species is not possible, but the BLAST search using the partial
18S rDNAsequence of M. lomi sp. nov. didnot matchany of the Myxozoa
available in GenBank.
The molecular phylogenetic analysis was based on the comparative
analyses of the 18S rDNAgene in18 species of Myxobolus (approximate-
ly 1500 bp) obtained from GenBank. To enhance the accuracy of the
analysis, smaller species were not used. The results, with high bootstrap
values, showed the Myxobolus species clustering as a monophyletic unit.
Myxobolus cordeiroi Adriano et al. 2009 was the most divergent species
of the genus in this analysis and M. oliveirai Milanin et al. 2010 appears
to be the sister group of the large monophyletic clade composed by the
remaining Myxobolus species.
Myxobolus lomi sp. nov. is grouped into a monophyletic clade A with
the species M. oliveirai and ten species by the method of ML. Myxobolus
M. lomi sp. nov. was clustered in a monophyletic subclade B that is sup-
ported by high bootstrap values (98) with other sh parasites of fresh-
water. Myxobolus M. lomi sp. nov. was clearly presented in a separate
branch of clade and other species phylogenetically close to Myxobolus.
This is the rst phylogenetic study on a Myxobolus species parasite of
a South American prochilodontid host. From Brazil, only the 18S rDNA
gene sequences of M. cordeiroi, with around 500 bp and M. oliveirai,
with 1527 bp have been deposited in GenBank. Additionally ultrastruc-
tural andhistopathological results reportedinthis study support the hy-
pothesis of species description of M. lomi sp. nov. as a newspecies to be
included in the phylogeny of Myxobolus. This result strongly contributes
Fig. 7. Condensed phylogenetic tree showing relationship between Myxobolus lomi sp. nov. and other 18 nucleotide sequences of Myxobolus 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 of
the 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 tree
was obtained using the Neighbor-Joining method. Genbank accessions are given after species name. Numbers above nodes indicate bootstrap condence levels.
Table 1
Comparison of measurements from Muxobolus lomi sp. nov. with species from Myxobolus more near morphologic.
Species LS WS LPC WPC NC Cyst (mm) Site of infection
M. lomi sp. nov. 14.2 (11.815.8) 11.1 (8.712.5) 6.4 (5.2.9);
6.0 (4.77.4)
3.1 (2.34.0);
2.9 (2.24.2)
811 0.250.3 Gill laments
M. heckelii 12.7 (12.213.1) 6.6 (6.36.9) 2.9 (2.73.3) 1.7 (1.42.0) 45 X Gills
M. insignis 14.5 (1415) 11.3 (1112) 7.6 (78) 4.2 (35) 6 X Gills
M. metynnis 13.1 (12.913.5) 7.8 (7.58.3) 5.2 (5.05.5) 3.2 (3.03.6) 89 X Orbicular region
M. noguchii 13.6 8.5 6.8 2.2 X X Gills
M. porolus 5.7 4.8 1.6 1.1 3 35 Body-cavity
M. amieti 14.0 (11.315.8) 7.4 (5.48.7) 8.4 (6.010.0) 1.9 (1.42.5) X 0.130.265 x 0.1250.25 Spleen, eye
M. andhrae 13.5 (12.115.7) 6.4 (5.78.6) 9.0 (8.610.0) 1.7 (1.42.1) X 1 Outer wall of intestine
M. eucalii 14.4 (12.015.6) 9.9 (8.410.8) 11.1 (9.612.0) 3.7 (3.04.8) 911 0.2 Cranium, pectoral ns
M. mylopharyngodoni 14.2 (1215.6) 11.1 (10.812) 9.2 (8.410.8) 7.8 (7.28.4) 67 0.172 0.132 Gills, kidneys, ns
M. varicorhini 11.816 10.611.8 5.97.1 2.54.1 X X Skin, kidneys, spleen
Abbreviations: LS, length of the spore; WS, width of the spore; LPC, length of the polar capsules; WPC, width of the polar capsules; NC, number of coils of the polar lament; All measure-
ments are in micrometers, except for the cyst size (mm).
306 R.K. Azevedo et al. / Parasitology International 63 (2014) 303307
to the taxonomic study of species of Myxobolus and comprehension of
kinship among the other species of parasites of freshwater sh.
The histological analysis on P. linetaus gills infected by M. lomi sp. nov.
revealed numerous well-delimitedcysts at the base of the primary lamel-
la, betweenconnective tissue andbone. However, no pronouncedinam-
matory response was found at the infection site, which is similar to the
results found by other authors in Myxozoa species described in Brazil
[6,10,1417]. As cysts were found close to the gill arteries, a possible
blood compression may occur with the increasing number of cysts, thus
impairing the gill function of the species infected by the M. lomi sp. nov.
The ultrastructural analysis revealed that the plasmodiumand polar
capsules wall consisted of a single membrane, two equal symmetrical
and smooth valves, each forming the spore and one binucleate
sporoplasm contained some sporoplasmosomes.
Conict of interest
The authors declare no conict of interest.
Acknowledgments
The authors would like to thank Carlos Cesar Ramos, the technician
from the Electron Microscopy Laboratory (Department of Pathology)
of the Medical School of Botucatu University-UNESP who assisted with
the transmission electron microscopy. Authors would like to thank
Elisa Pinto de Oliveira for editing the English. Sponsorships: FAPESP
(2010/06564-5, 2011/00010-0), CNPq (312590/2009-1), PROCAD
CAPES (Auxlio 032-2006).
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