Molecular characterization of Sphaerospora molnari (Myxozoa), the agent of gill sphaerosporosis in common carp Cyprinus carpio carpio


Eszterbauer, E.; Sipos, D.; Forró, B.; Ová, P.Barto.; Holzer, A.S.

Diseases of Aquatic Organisms 104(1): 59-67

2013


Sphaerospora molnari Lom, Dyková, Pavlásková and Grupcheva, 1983 often causes severe infections in the gills and skin of common carp fingerlings Cyprinus carpio carpio in Central Europe. Although most Sphaerospora spp. are coelozoic and affect the excretory system of fish, S. molnari develops mature spores in the epithelia of gill filaments, making it a rare representative of histozoic freshwater species within the genus. On the basis of a partial 18S rDNA sequence assigned as belonging to S. molnari, previous phylogenetic studies located the species within the Myxobolus clade. In the present study, S. molnari isolates from Hungary and the Czech Republic were characterized based on morphology, DNA sequence analysis and phylogenetic comparison. The obtained 3714 bp final consensus 18S rDNA sequence of the parasite showed several, sometimes extremely long inserts in the variable regions of the gene and differed considerably from the one published in GenBank in 2002. In situ hybridization confirmed the validity of the obtained DNA sequence and detected pre-sporogonic blood stages in the interstitium and blood vessels of the kidney. Phylogenetic analysis showed that S. molnari clusters within the Sphaerospora sensu stricto clade with a high support, revealing it as the first known histozoic member of the Sphaerospora subclade comprising parasites of freshwater fish.

Vol.
104:
59-67,2013
doi:
10.3354/dao02584
DISEASES
OF
AQUATIC
ORGANISMS
Dis
Aquat
Org
Published
April
29
Molecular
characterization
of
Sphaerospora
molnari
(Myxozoa),
the
agent
of
gill
sphaerosporosis
in
common
carp
Cyprinus
carpio
carpio
E.
Eszterbauer",
D.
Sipos
l
,
B.
Ferro
l
,
P.
Bartogove,
A.
S.
Holzer
2
'Institute
for
Veterinary
Medical
Research,
Centre
for
Agricultural
Research,
Hungarian
Academy
of
Sciences,
Budapest,
Hungary
2
Institute
of
Parasitology,
Biology
Centre
of
the
Academy
of
Sciences
of
the
Czech
Republic,
Ceske
Budejovice,
Czech
Republic
ABSTRACT:
Sphaerospora
molnari
Lom,
Dykova,
Pavlaskova
and
Grupcheva,
1983
often
causes
severe
infections
in
the
gills
and
skin
of
common
carp
fingerlings
Cyprinus
carpio
carpio
in
Cen-
tral
Europe.
Although
most
Sphaerospora
spp.
are
coelozoic
and
affect
the
excretory
system
of
fish,
S.
molnari
develops
mature
spores
in
the
epithelia
of
gill
filaments,
making
it
a
rare
represen-
tative
of
histozoic
freshwater
species
within
the
genus.
On
the
basis
of
a
partial
18S
rDNA
sequence
assigned
as
belonging
to
S.
molnari,
previous
phylogenetic
studies
located
the
species
within
the
Myxobolus
Glade.
In
the
present
study,
S.
molnari
isolates
from
Hungary
and
the
Czech
Republic
were
characterized
based
on
morphology,
DNA
sequence
analysis
and
phylogenetic
comparison.
The
obtained
3714
bp
final
consensus
18S
rDNA
sequence
of
the
parasite
showed
several,
sometimes
extremely
long
inserts
in
the
variable
regions
of
the
gene
and
differed
consid-
erably
from
the
one
published
in
GenBank
in
2002.
In
situ
hybridization
confirmed
the
validity
of
the
obtained
DNA
sequence
and
detected
pre-sporogonic
blood
stages
in
the
interstitium
and
blood
vessels
of
the
kidney.
Phylogenetic
analysis
showed
that
S.
molnari
clusters
within
the
Sphaerospora
sensu
stricto
Glade
with
a
high
support,
revealing
it
as
the
first
known
histozoic
member
of
the
Sphaerospora
subclade
comprising
parasites
of
freshwater
fish.
KEY
WORDS:
Sphaerosporosis
Histozoic
myxozoans
Blood
stages
Cyprinids
18S
rDNA
Phylogeny
In
situ
hybridization
Resale
or
republication
not
permitted
without
written
consent
of
the
publisher
INTRODUCTION
Sphaerospora
spp.
(Myxozoa)
are
common
endo-
parasites
of
fish,
with
the
highest
diversity
described
in
cyprinids
(Baska
&
Molnar
1988,
Sitja-Bobadilla
&
Alvarez-Pellitero
1994).
Most
members
of
the
genus
are
coelozoic
(i.e.
plasmodial
stages
of
parasite
locate
in
cavities
of
body
organs)
and
infect
the
excretory
system
of
fish.
Because
of
their
simple
spherical
or
sub-spherical
myxospore
shape
involving
2
valves,
2
polar
capsules
and
usually
2
uninucleate
sporo-
plasms,
species
identification
based
solely
on
mor-
phological
features
is
rather
difficult.
Thus,
it
is
not
surprising
that
particular
emphasis
is
put
on
using
molecular
tools
for
the
taxonomy
of
Sphaerospora
spp.
Following
the
description
of
the
genus
by
Theo-
han
in
1892
(Thelohan
1895),
it
took
more
than
a
cen-
tury
until
the
first
rDNA
sequences
of
Sphaerospora
spp.
were
published
(Kent
et
al.
1998,
2001).
Phylo-
genetic
studies
in
combination
with
the
examination
of
phenotypic
and
biological
features
(i.e.
spore
morphology,
tissue
tropism,
host
range,
etc.)
have
become
'mainstream'
in
myxozoan
research
(e.g.
Whipps
et
al.
2003,
Eszterbauer
2004,
Holzer
et
al.
2004,
Fiala
2006,
Molnar
et
al.
2010,
Fiala
&
Bar-
togova
2010).
Due
to
the
unknown
host
specificity
*Email:
eedit@vmri.hu
0
Inter-Research
2013
www.int-res.com
60
Dis
Aquat
Org
104:
59-67,
2013
and
geographical
distribution
of
many
myxozoan
taxa,
it
is
important
to
provide
a
combination
of
mor-
phological
and
molecular
features
for
a
given
myxo-
zoan
species.
An
upswing
in
Sphaerospora
taxonomy
was
experienced
when
the
number
of
known
Sphaerospora
18S
rDNA
sequences
increased
signif-
icantly,
and
larger
DNA
fragments
or
complete
sequences
were
used
for
phylogeny.
Having
studied
the
phylogeny
of
the
genus,
which
is
known
to
be
highly
polyphyletic,
Jirku
et
al.
(2007)
introduced
the
term
Sphaerospora
sensu
stricto
for
those
species
that
are
coelozoic
and
cluster
together
in
a
basal
phy-
logenetic
myxosporean
Glade.
Recently,
the
Glade
expanded
with
2
new
members,
S.
angulata
from
Prussian
carp
and
goldfish
and
S.
dykovae
from
com-
mon
carp,
both
of
which
develop
spores
in
the
kidney
tubules
(Holzer
et
al.
2013).
However,
a
few
18S
rDNA
sequences
of
Sphaerospora
spp.
still
cluster
outside
the
Sphaerospora
sensu
stricto
Glade,
i.e.
S.
testicularis
Sitja-Bobadilla
and
Alvarez-Pellitero,
1990,
S.
dicentrarchi
Sitja-Bobadilla
and
Alvarez-
Pellitero,
1992,
S.
elwhaiensis
Jones,
Fiala,
Prosperi-
Porta,
House
and
Mumford,
2011,
and
S.
molnari
Lom,
Dykova,
Pavlaskova
and
Grupcheva,
1983;
these
species
are
mostly
histozoic
parasites,
the
plas-
modia
of
which
develop
intra-
or
intercelullarly
in
tis-
sues
of
fish.
As
mixed
myxozoan
infections
are
common,
espe-
cially
in
cyprinid
fish,
with
>20
species
reported
in
common
carp
(Lom
&
Dykova
2006),
we
recently
started
to
confirm
the
identity
of
published
18S
rDNA
sequences
of
sphaerosporids
clustering
outside
the
Sphaerospora
sensu
stricto
Glade.
The
need
to
re-
analyze
these
Sphaerospora
spp.
has
already
been
highlighted
in
studies
dealing
with
myxozoan
phy-
logeny
and
the
relations
between
their
development
and
phylogenetic
data
(Fiala
2006,
Morris
&
Adams
2008,
Bartogova
et
al.
2011).
By
hybridization
of
spe-
cific
oligonucleotide
probes
to
target
DNA
in
histo-
logical
sections
(in
situ
hybridization
[ISH])
we
were
able
to
identify
erroneously
ascribed
Sphaerospora
sp.
entries
in
GenBank
(e.g.
Eszterbauer
2011,
Holzer
et
al.
2013).
Contrary
to
the
majority
of
Sphaerospora
spp.
and
most
of
the
sequenced
mem-
bers
of
Sphaerospora
sensu
stricto,
Sphaerospora
molnari
is
a
histozoic
species
that
develops
myx-
ospores
in
the
epithelia
of
gills
and
skin,
causing
gill
sphaerosporosis
in
common
carp
Cyprinus
carpio
carpio
fingerlings
in
Europe
(Iskov
1969,
Hamory
&
Molnar
1972,
Molnar
1979, 1980,
Waluga
1983).
Transelectromicroscopic
studies
by
Kaup
et
al.
(1995)
confirmed
the
histological
observations
that
the
monosporic
sporoblasts
of
S.
molnari
surrounded
by
an
envelope
cell
lay
isolated
in
the
gill
epithelium.
Before
1983,
S.
molnari
was
identified
as
S.
carassii;
thereafter,
it
was
distinguished
from
S.
carassii
Kudo,
1919
and
S.
chinensis
Lom,
Dykova,
Pavlaskova
and
Grupcheva,
1983
based
on
spore
morphology,
host
species
and
geographic
locality
(Lom
et
al.
1983).
S.
molnari
invades
the
epithelia
of
gill
filaments
and
lamellae
and
secondarily
also
those
of
the
skin
sur-
rounding
the
gill
cavity
and
the
nasal
pits.
Affected
epithelia
show
marked
dystrophic
changes
and
necrosis,
causing
secondary
bacterial
infections
and
leading
to
discharge
of
spores
into
the
environment
Molnar
(1979).
This
is
why
S.
molnari
is
regarded
as
a
serious
pathogen
of
common
carp
fingerlings
in
Central
Europe.
In
the
present
study,
we
provide
18S
rDNA
se-
quences
of
Sphaerospora
molnari
isolates
collected
from
Hungary
and
the
Czech
Republic.
Using
in
situ
hybridization,
we
confirm
the
validity
of
the
obtained
sequences
and
provide
insights
into
the
location
of
pre-sporogonic
stages
of
S.
molnari.
Furthermore,
we
clarify
the
phylogenetic
position
of
the
first
histozoic
member
of
the
freshwater
subclade
of
Sphaerospora
sensu
stricto
sequenced
to
date.
MATERIALS
AND
METHODS
Source
of
samples
Common
carp
Cyprinus
carpio carpio
fingerlings
were
captured
by
hand
with
a
net
in
an
earthen
pond
of
a
carp
hatchery
in
Hortobagy,
Hungary
(47°
35'
11.1"
N,
21°
02'
58.4"
E).
Fifty
individuals
were
subjected
to
parasitological
examination.
As
a
second
sampling
source,
0+
carp
fingerlings
were
collected
between
March
and
October
2011
and
2012
in
Chfegt'ovice
(n
=
23)
and
Jindfichfiv
Hradec
(n
=
46),
Czech
Republic
(49°
19'
25"
N,
14°
17'
12"
E
and
49°
09'
35"
N,
14°
10'
07"
E).
In
the
Hungarian
sam-
ples,
gill
arches,
gill
scrapings
and
kidney
smears
were
examined
by
light
microscopy.
Photomicro-
graphs
were
taken
of
the
observed
myxospores
using
a
Moticam
2000
(Motic,
VWR)
digital
microscope
camera
mounted
on
a
Zeiss
Axiostar
Plus
phase
con-
trast
microscope
(Carl
Zeiss).
Sphaerospora
molnari
myxospores
(n
=
50)
from
Hungarian
carp
were
measured
on
digital
images
using
the
software
Motic
Images
Plus
2.0
calibrated
against
a
digital
image
of
a
graticule.
Measurements
were
obtained
following
the
guidelines
of
Arthur
&
Lom
(1989)
and
Sitja-
Bobadilla
&
Alvarez-Pellitero
(1994).
The
tissue
sam-
ples
observed
by
microscopy
on
slides
were
collected
Eszterbauer
et
al.:
Molecular
characterization
of
Sphaerospora
molnari
61
for
subsequent
molecular
analysis
and
stored
at
-20°C.
Gill
arches
affected
by
myxosporean
infection
were
fixed
in
10
%
neutral
buffered
formalin
for
ISH.
Kidney
and
blood
samples
from
carp
fingerlings
orig-
inating
from
the
Czech
Republic
were
fixed
in
TNES-
urea
(Asahida
et
al.
1996)
for
DNA
analysis,
and
sev-
eral
kidneys
were
also
fixed
for
ISH
as
described
above.
DNA
extraction,
PCR
and
DNA
sequencing
Two
samples
from
each
sampling
site
were
thawed
and
then
homogenized
in
1.5
ml
microtubes
with
a
sterile
plastic
pestle
(Eppendorf).
Then,
tubes
con-
taining
the
homogenates
were
filled
with
dH
2
O,
mixed
by
vortexing
and
centrifuged
at
7000
x
g
for
5
min
on
a
tabletop
centrifuge.
Pellets
were
dissolved
in
500
pl
lysis
buffer
(100
mM
NaCl,
10
mM
Tris,
10
mM
EDTA,
0.2
%
sodium
dodecyl
sulphate,
and
0.4
mg
m1
-1
Proteinase
K)
and
incubated
at
55°C
for
5
h.
DNA
was
then
purified
using
the
MiniPrep
Express
Matrix
(BI0101,
Qbiogene)
as
per
Eszter-
bauer
(2004).
TNES-urea
fixed
samples
were
lysed
with
0.1
mg
m1
-1
Proteinase
K
(Sigma-Aldrich)
overnight
and
extracted
using
a
general
phenol-
chloroform
extraction
protocol.
Genomic
DNA
was
amplified
for
the
first
round
with
the
primer
pair
ERIB1-ERIB10
(Table
1).
The
total
volume
of
the
PCR
reactions
was
50
pl,
which
contained
-50
ng
DNA,
lx
Taq
PCR
reaction
buffer
(Fermentas,
Thermo
Fisher
Scientific),
1.5
mM
MgCl
2
,
0.2
mM
dNTP
mix
(Sigma-Aldrich),
25
pM
of
each
primer
and
2
units
of
Taq
DNA
Polymerase
(Fermentas,
Thermo
Fisher
Scientific).
Amplification
conditions
were
95°C
for
50
s,
59°C
for
50
s
and
72°C
for
180
s
for
35
cycles,
with
an
initial
denaturation
at
95°C
for
5
min
and
ter-
minal
extension
at
72°C
for
7
min.
As
a
second
round,
PCR
assays
with
the
same
cycling
conditions
but
with
a
shorter
(80
s)
elongation
step
and,
in
some
cases,
at
a
different
annealing
temperature
(given
in
paren-
thesis)
were
performed
with
the
following
primer
combinations:
MB5-ERIB10,
ERIB1-Act1R
(52°C),
ERIB1-Smo1800R,
Smo13130E-ERIB10,
Act1F-ERIB10
(52°C)
and
Act1F-Myx4R
(Table
1).
PCR
products
were
purified
with
a
MEGAquick-Spin
PCR
and
Agarose
Gel
DNA
Extraction
System
(INtRON
Biotechnology).
The
purified
PCR
products
were
used
for
direct
sequencing
with
an
ABI
BigDye
Terminator
v.
3.1
Cycle
Sequencing
kit
with
an
ABI
3100
Genetic
Ana-
lyzer
automated
DNA
sequencer
(Applied
Biosys-
tems,
Life
Technologies
and
Macrogen
Europe).
Besides
the
amplification
primers,
the
primers
listed
in
Table
1
were
used.
For
2
samples,
PCR
products
amplified
with
ERIB1-ERIB10
were
cloned
using
CloneJet
PCR
Cloning
Kit
as
per
manual
(Fermentas,
Thermo
Fisher
Scientific).
Alkaline
miniprep
was
used
for
plasmid
purification.
Five
clones
of
each
sample
were
subjected
to
partial
DNA
sequencing
using
the
universal
plasmid
primers
supplied
with
the
cloning
kit.
One
clone
was
sequenced
entirely
with
overlaps
in
both
directions
using
the
primers
listed
in
Table
1.
A
specific
PCR
assay
for
S.
molnari
Table
1.
Oligonudeotides
used
for
PCR
(P),
DNA
sequencing
(S)
and
in
situ
hybridisation
(I)
Name
Sequence
(5'—
3')
Use
Reference
ERIB1
ACC
TGG
TTG
ATC
CTG
CCA
G
P,
S
Barta
et
al.
(1997)
ACT1F
GGC
AGC
AGG
CGC
GCA
AAT
TAC
CCA
A
P,
S
Hallett
&
Diamant
(2001)
ACT1R
AAT
TTC
ACC
TCT
CGC
TGC
CA
P,
S
Hallett
&
Diamant
(2001)
MYX4R
CTG
ACA
GAT
CAC
TCC
ACG
AAC
P,
S
Hallett
&
Diamant
(2001)
SPHR
GTT
ACC
ATT
GTA
GCG
CGC
GT
S
Eszterbauer
&
Szekely
(2004)
ERIB10
CTT
CCG
CAG
GTT
CAC
CTA
CGG
P,
S
Barta
et
al.
(1997)
MB5
GGT
GAT
GAT
TAA
CAG
GAG
CGG
T
P,
S
Eszterbauer
(2004)
Smo1700F
GGA
GAT
GGT
GAC
GAG
ACA
TA
S
Present
study
Smo180OR
TGG
TAT
TAC
CGC
GGC
TGC
TG
P,
S
Present
study
Smo11200F
GCC
AAG
CGA
GCG
TCC
AAT
CA
S
Present
study
Smo1120OR
TTG
GAC
GCT
CGC
TTG
GCT
GT
S
Present
study
Smo11400F
CTC
CTT
CAC
TGT
GAG
CAA
CG
S
Present
study
Smo1190OR
GTA
CCG
AAG
AAC
GCA
CAC
GA
S
Present
study
Smo11800F
GTC
CAA
TTG
CTT
GAA
CCA
CC
S
Present
study
Smo1250OR
GCA
TGT
GTG
AGC
GTG
ATT
GT
S
Present
study
Smo13130F
TGT
ACT
GTT
GCC
GGT
GGA
TT
P,
S
Present
study
115FOA
CGT
GAA
CGA
GCG
CAA
CCA
CA
S
Present
study
SmSSU487F
GCC
TCT
CCA
CCT
GTG
TAT
G
P,
I
Present
study
SmSSU1307R
ACC
GTG
AGC
CAC
GCG
TAA
TG
P,
I
Present
study
62
Dis
Aquat
Org
104:
59-67,
2013
was
designed,
using
primers
SmSSU487F
and
SmSSU1307R
(Table
1)
with
all
PCR
parameters
as
described
above
apart
from
the
annealing
tempera-
ture,
which
was
set
as
66°C.
For
sequence
assembly,
the
STADEN
Sequence
Analysis
Package
v.
2001.0
(Staden
1996)
was
used.
In
situ
hybridization
Samples
for
ISH
were
fixed
in
10
%
neutral-
buffered
formalin
for
24
to
48
h,
dehydrated
in
an
ethanol
series
and
embedded
in
paraffin.
Six
pm
sec-
tions
were
cut
and
adhered
to
salinized
slides.
The
ISH
method
followed
established
protocols
(Holzer
et
al.
2003,
2010)
applying
a
DIG-alkaline
phosphatase
detection
system.
Briefly,
sections
were
deparaf
-
finized
and
rehydrated
in
a
graded
ethanol
series
and
then
equilibrated
in
Tris-buffered
saline
(TBS)
(pH
8.0).
Thereafter,
sections
were
permeabilized
with
100
pg
m1
-1
of
Proteinase
K
in
TBS
for
25
min
at
37°C.
Sections
were
then
washed
in
phosphate-
buffered
saline
(pH
7.4)
and
post-fixed
for
15
min
with
0.4
%
paraformaldehyde
in
phosphate-buffered
saline.
Fixative
was
rinsed
off
with
distilled
water,
and
the
sections
were
dried
at
45°C.
The
primers
SmSSU487F
and
SmSSU1307R
(Table
1)
were
com-
mercially
DIG-labeled
at
the
3'-end
(Sigma-Aldrich)
and
used
as
specific
probes.
The
specificity
of
the
probes
was
confirmed
by
DNA
sequence
comparison
via
BLAST
search
because
no
match
was
found
with
any
other
DNA
sequences
in
GenBank.
Gene
Frames
(Thermo
Fisher
Scientific)
were
adhered
to
the
slides,
and
hybridization
buffer
(4x
SSC
in
TBS
con-
taining
0.5
%
Ficoll,
0.5
%
polyvinylopyrrolidone,
0.5
%
bovine
serum
albumin
and
100
pg
m1
-1
calf
thy-
mus
DNA)
containing
1.5
ng
p1
-1
of
each
oligonucle-
otide
probe
was
added.
After
covering
the
Gene
Frames,
the
sections
were
denatured
for
4
min
at
90°C
and
hybridized
with
the
labeled
probes
for
2
h
at
45°C.
The
incubation
was
followed
by
a
non-strin-
gent
washing
in
2x
SSC
and
highly
stringent
wash-
ing
in
0.1x
SSC
containing
0.1
%
Tween
20
at
45°C.
Sections
were
then
transferred
into
TBS
(pH
7.5)
and
incubated
with
1:5000
anti-DIG-alkaline
phos-
phatase
Fab
fragments
(Roche
Diagnostics)
in
TBS
for
2
h
at
room
temperature.
Following
a
wash
in
TBS
(pH
8.0),
Vector
Blue
substrate
(Vectorlabs)
was
used
to
visualize
the
alkaline
phosphatase
linked
DIG-
labeled
probe
according
to
the
manufacturer's
recommendations.
Counterstaining
was
conducted
using
0.1
%
nuclear
fast
red.
Finally,
sections
were
dehydrated
through
an
alcohol
series
and
transferred
into
Xylene
Substitute
(Sigma-Aldrich)
and
perma-
nently
mounted
with
VectaMount
(Vectorlabs).
Phylogenetic
analysis
The
SSU
rDNA
sequences
of
all
available
Sphaerospora
s.
str.
spp.
and
selected
members
of
freshwater
and
marine
myxosporean
lineages
were
aligned
in
Clustal
X
v.
1.83
(Thompson
et
al.
1997)
using
default
parameters.
The
alignment
was
manu-
ally
edited,
and
the
ambiguous
regions
were
excluded
in
BioEdit
(v.
7.0.5.2;
Hall
1999).
Chloro-
myxum
leydigi
was
used
as
outgroup
in
all
analyses.
We
performed
maximum
parsimony
analysis
in
PAUP'
(v4.b10;
Swofford
2001),
using
a
heuristic
search
with
random
taxa
addition,
the
ACCTRAN
option,
TBR
swapping
algorithm,
all
characters
treated
as
unordered
and
gaps
treated
as
missing
data.
Clade
support
values
were
calculated
from
1000
bootstrap
replicates
with
random
sequence
additions.
Maximum
likelihood
analysis
was
calcu-
lated
in
RAxML
(Stamatakis
2006)
using
the
GTR
+
F
model.
Bootstraps
were
based
on
1000
replicates.
The
Bayesian
inference
(BI)
analysis
was
performed
in
MrBayes
v.
3.0
(Ronquist
&
Huelsenbeck
2003)
using
the
GTR
+
F
+
I
model
of
evolution.
Posterior
probabilities
were
estimated
from
1
000
000
via
2
independent
runs
of
4
simultaneous
Markov
chain-
Monte
Carlo
chains
with
every
100th
tree
saved.
The
burn-in
period
(100
000
generations)
was
determined
in
Tracer
v.
1.4.1
(Rambaut
&
Drummond
2007).
RESULTS
Characteristics
of
parasite
infection
Of
the
50
examined
fish
originating
from
Hungary,
gills
of
6
specimens
(12%
prevalence)
were
infected
with
subspherical
myxospores
developing
in
mono-
sporous
pseudoplasmodia
in
the
epithelium
of
gill
filaments.
The
morphological
examination
of
myxo-
spores
in
gill
scrapings
confirmed
that
the
observed
spores
belonged
to
Sphaerospora
molnari
(Fig.
1),
and
their
measurements
resembled
those
described
in
the
original
description
by
Shulman
(1966)
(Table
2).
In
2
fish
specimens,
a
massive
infection
was
observed
in
most
gill
filaments,
where
the
entire
filament
was
affected
by
the
parasite.
In
the
2
fish
whose
gills
were
used
for
ISH
and
DNA
sequence
analysis,
no
other
myxozoans
were
detected
in
the
course
of
dissection.
Srs
Hng.
e
4
01b.
Air
;lc
a
r
rs
ire
v7-
a
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4
Eszterbauer
et
al.:
Molecular
characterization
of
Sphaerospora
molnari
63
The
gill
smears
of
carp
from
the
Czech
Republic
were
not
studied
microscopically;
however,
the
18S
rDNA
of
Sphaerospora
molnari
was
sequenced
from
2
blood
samples
of
carp.
The
specific
S.
molnari
PCR
assay
showed
that
34.8%
(8
of
23)
of
common
carp
in
Chfegt'ovice
and
85%
(39
of
46)
of
common
carp
in
Jindfichfiv
Hradec
were
positive
for
S.
molnari
DNA
in
the
blood.
Positive
samples
were
found
all
through
the
sampling
period.
Figs.
1
to
4.
Sphaerospora
molnari
and
Cyprinus
carpio
carpio.
Fig.
1.
S.
molnari
myxospores
from
gill
of
common
carp
fin-
gerling
C.
carpio
carpio.
Fresh
preparation.
Bar
=
10
pm.
Inset:
S.
molnari
myxospore
with
mucous
envelope.
Fig.
2.
Gill
of
com-
mon
carp
fingerlings
heavily
infected
with
S.
molnari.
The
monosporous
pseudoplas-
modia
develop
in
high
numbers
in
the
epi-
thelium
of
the
gill
filaments.
Parasites
show
dark
blue
coloration.
In
situ
hy-
bridization.
Bar
=
200
pm.
Fig.
3.
Kidney
parenchyma
of
common
carp
fingerling
with
S.
molnari
developmental
stages.
Par-
asite
stages
are
dark
blue.
In
situ
hybridiza-
tion.
Bar
=
10
pm.
Fig.
4.
S.
molnari
blood
stages
(stained
dark
blue)
detected
in
a
kid-
ney
capillary
of
common
carp
fingerling.
In
situ
hybridization.
Bar
=
10
pm
Table
2.
Measurements
of
the
myxospores
of
Sphaerospora
molnari
and
other
relevant,
closely
related
Sphaerospora
spp.
developing
in
cyprinids.
Mean
values
±
standard
deviations
are
given
in
pm.
Minimum
and
maximum
values
are
in
parentheses.
SL:
spore
length;
SW:
spore
width,
PCL:
length
of
polar
capsule;
PCW:
width
of
polar
capsule;
PF:
polar
filament;
nd:
no
data
Species
Authors
Locality
Host
Tissue
SL
SW
PCL
PCW
PF
turns
Sphaerospora
Present
Hungary
Cyprinus
Gill
9.9
±
0.59
10.0
±
0.51
4.68
±
0.39
3.9
±
0.28
4-5
molnari
(n
=
50)
study
carpio
carpio
epithelium
(8.7-11.1) (9.0-10.9) (3.9-5.8)
(3.3-5.0)
S.
molnari
Lom
et
al.
Czech
C.
carpio
Gill
and
skin
10.3
10.5
4-5
4-4.5
4
(1983)
Republic
carpio
epithelium
(9.5-11)
(10-11)
(3-5)
S.
chinensis
Lom
et
al.
(1983)
Czech
Republic
C.
carpio
carpio
Gill
epithelium
7.4
7
nd
nd
5
S.
carassiia
Shulman
(1966)
Former
USSR
&
Japan
Carassius
carassius,
Carassius
gibelio,
C.
carpio
Gill
epithelium
8-13 8-13
4-5
nd
nd
Rut
lus
rutilus
S.
dykovae
b
Dykova.
&
Czech
C.
carpio
Kidney
7.3
7.2
1.7-2.3
1.3-1.6
4-5
Lom
(1982)
Republic
carpio
tubules
(6.0-8.0) (6.4-8.3)
aOriginally
described
by
Kudo
(1919)
b
Originally
described
as
S.
renicola
64
Dis
Aquat
Org
104:
59-67,
2013
DNA
sequence
analysis
Almost
complete
18S
rDNA
sequences
were
obtained
from
2
Hungarian
and
2
Czech
isolates
of
Sphaerospora
molnari.
The
3693
and
3714
bp
final
consensus
DNA
sequences
both
possessed
several,
sometimes
extremely
long
expansions
in
the
variable
regions
V2,
V4,
V5
and
V7
of
the
18S
rDNA
gene
region.
The
18S
rDNA
of
the
2
Hungarian
isolates
(GenBank
accession
number
JX431510)
were
identi-
cal,
while
the
Czech
isolates
(GenBank
accession
no.
JX431511),
which
were
also
identical
with
each
other,
differed
from
the
Hungarian
isolates
at
7
nucleotide
positions
(99.7
%
similarity).
The
1794
bp
partial
18S
rDNA
sequence
of
Myxozoa
sp.
ASH-
2012
isolate
bloodcarpCZ
that
has
recently
become
available
in
GenBank
(JQ801548)
was
identical
to
the
Czech
isolate
of
S.
molnari.
The
presently
exam-
ined
S.
molnari
isolates
and
the
one
(AF378345)
pre-
viously
submitted
to
GenBank
by
Kent
et
al.
(2001)
differed
considerably
in
primary
sequence
character-
istics
and
in
the
absence
of
relevant
expansion
seg-
ments,
except
in
region
V7.
The
two
18S
rDNA
sequences
possess
only
48.5
to
48.6
%
similarity
in
a
much
shorter,
2080
bp
long
alignment
of
the
gene.
ISH
ISH
using
a
Sphaerospora
molnari-specific
DNA
probe
confirmed
the
great
parasite
load
in
the
epithelium
of
gill
filaments
and
clearly
linked
the
S.
molnari
spore
morphotype
to
the
amplified
18S
rDNA
sequence.
Especially
in
the
apical
part
of
the
gill
filaments,
epithelial
hyperplasia
and
necrosis
of
gill
tissue
was
also
observable
(Fig.
2).
Interestingly,
large
numbers
of
pre-sporogonic
stages
of
the
para-
site,
measuring
between
3.5
and
5
pm
were
detected
in
the
blood
vessels
(Fig.
4).
Pre-sporogonic
stages
were
also
commonly
found
in
the
interstitial
tissue
of
the
kidney
(Figs.
3
&
4).
Phylogeny
Phylogenetic
analysis
showed
that
the
histozoic,
gill-dwelling
Sphaerospora
molnari
clusters
within
the
Sphaerospora
sensu
stricto
Glade
and
groups
with
a
high
support
with
the
coelozoic
kidney
parasites
S.
hankai
Lom,
Desser
and
Dykova,
1989,
S.
dykovae
(Dykova
&
Lom
1982)
and
S.
angulata
Fujita,
1912,
from
freshwater
fish.
The
exact
relationships
among
these
4
species
could
not
be
clearly
resolved.
Con-
firming
the
findings
of
prior
DNA
sequence
analysis,
the
18S
rDNA
of
the
examined
S.
molnari
isolates
and
the
one
submitted
by
Kent
et
al.
(2001)
were
located
distantly
on
the
phylogenetic
tree,
as
the
latter
one
grouped
outside
the
Sphaerospora
sensu
stricto
Glade
with
the
histozoic
Myxobolus
dade
(Fig.
5).
DISCUSSION
The
molecular
study
of
Sphaerospora
molnari,
the
agent
of
gill
and
skin
sphaerosporosis
in
common
carp
in
Central
Europe,
provided
several
novel
findings.
We
proved
that
S.
molnari
is
in
fact
a
member
of
the
Sphaerospora
sensu
stricto
Glade
and
most
likely
has
evolved
from
coelozoic
kidney
parasites
of
freshwater
fishes,
with
a
close
relationship
to
all
species
hitherto
sequenced
from
cyprinids.
For
a
long
time,
the
phylo-
genetic
position
of
S.
molnari
was
thought
to
be
dif-
ferent:
a
partial
18S
rDNA
assigned
as
S.
molnari
(AF378345)
but
sequenced
from
goldfish
Carassius
auratus
auratus
by
Kent
et
al.
(2001)
was
one
of
the
se-
quences
that
rendered
the
genus
Sphaerospora
as
polyphyletic,
as
it
clusters
with
histozoic
Myxobolus
species
from
freshwater
environments
(e.g.
Kent
et
al.
2001,
Eszterbauer
&
Szekely
2004,
Holzer
et
al.
2004,
Fiala
2006,
Bartagova
et
al.
2011).
Lom
et
al.
(1983)
stated
that
S.
molnari
is
a
gill
parasite
exclusively
oc-
curring
in
common
carp
Cyprinus
carpio carpio
and
differs
morphologically
from
S.
chinensis
from
the
gills
of
C.
carpio carpio
in
the
Far
East.
Furthermore,
they
distinguished
S.
molnari
from
S.
carassii,
which
was
originally
described
from
the
gills
of
Carassius
carassius
but
also
reported
from
C.
gibelio
and
Rutilus
rutilus
and
which,
thereby,
may
represent
a
species
complex.
While
the
relation
of
S.
molnari
to
these
spe-
cies
is
still
unclear
as
no
DNA
sequences
are
available,
we
suggest
that
they
may
possess
a
similar
evolutionary
history,
originating
from
sphaerosporids
in
the
excretory
system
of
cyprinids.
The
isolate
of
Kent
et
al.
(2001)
was
collected
from
the
gills
of
gold-
fish.
Usually,
shorter
18S
rDNA
sequences,
like
the
ones
belonging
to
the
freshwater
dade
as
defined
by
Fiala
(2006),
amplify
preferentially
over
the
extremely
long
18S
rDNA
sequences
of
Sphaerospora
sensu
stricto
members.
Due
to
commonly
occurring
mixed
infections,
we
suggest
that
the
sequence
of
Kent
et
al.
(2001)
requires
further
validation
before
its
potential
adscription
to
S.
carassii.
Holzer
et
al.
(2013)
recently
reported
that
Sphaero-
spora
angulata,
which
forms
spores
in
the
kidney
of
goldfish
and
Prussian
carp,
possesses
the
longest
18S
rDNA
(3469
bp)
known
so
far.
Our
findings
con-
Eszterbauer
et
al.:
Molecular
characterization
of
Sphaerospora
molnari
65
100/100/1.00
100/100/1.00
100/100/1.00
97/54/1.00
Sphaerospora
elegans
USA
(JX286618)
Sphaerospora
elegans
Scotland
(AJ609590)
Sphaerospora
sp.
ex
Pomoxis
nigromaculatus
(JX286621)
98/100/1s
Polysporoplasma
sp.
ex
Liza
ramada
(JX286626)
ff
00/100/1.00
Polysporoplasma
sp.
ex
Chelon
labrosus
(JX286625)
Polysporoplasma
spans
(JX286624)
Sphaerospora
truttae
(AM410773)
Myxozoa
sp.
ASH-2012
isolate
bloodcarpCZ
(JQ801548)
Sphaerospora
molnari
isolate
CZ
(1X431511)
Sphaerospora
molnari
isolate
HU/E115
(1X431510)
Sphaerospora
hankai
(JX286623)
Sphaerospora
dykovae
CZ
(4801533)
99/99/1.0-Sphaerospora
angulata
(JQ801527)
Sphaerospora
ranae
(EF211975)
00t6/0.80
Sphaerospora
cf.
ohlmacheri
(JX286619)
100/100/1.00
100/97/0.99
99/961.00
-/-/0.73
-/-/0.71
59/-/-
100/100/1.00
-HO
62
100/100/1.00
97/96/1
Sphaerospora
sp.
ex
Ptychadena
anchietae
(JX286622)
lootionaliSphaerospora
epinepheli
(HQ871152)
80/55/073
I
Sphaerospora
epinepheli
(HQ871153)
.1013
/
100
/
114
Sphaerospora
sparidarum
(1X286620)
Bipteria
formosa
(FJ790308)
Sphaerospora
fugu
(AB195805)
Sp
hae
rosp
ora
se
nsu
s
tr
ic
to
G
la
de
100/98/1.00
Kudoa
trifolia
(AM183300)
Parvicapsula
minibicornis
(DQ231038)
Ceratomyxa
sp.
ex
Notacanthus
bonaparte
(DQ377699)
Marine
lineage
96/54/1.00
58/40.87[
Myxobolus
hearti
(GU574808)
[
._
100
/
100
/
1
.
00
Sphaerospora
molnari
(AF378345)
Henneguya
doneci
(HM
146
1
29)
Myxobolus
lentisuturalis
(AY119688)
Myxobilatus
gasterostei
(AY495703)
100/100/1.00
1
Sphaeromyxa
hellandi
(DQ377693)
I
82/97/0.90
Zschokkella
nova
(DQ377688)
Chloromyxum
fluviatile
(GU471265)
Chloromyxum
leydigi
(DQ377710)
0.1
99/97/1.00
100/100/1.00
100/98/1.00
Freshwater
lineage
Fig.
5.
18S
rDNA-based
maximum
likelihood
(GTR
+
F
model)
tree
of
33
myxosporean
sequences
showing
Sphaerospora
mol-
nari
isolates
clustering
within
the
Sphaerospora
sensu
stricto
lade.
Newly
sequenced
taxa
in
bold.
Chloromyxum
leydigi
was
used
as
outgroup.
Maximum
likelihood/maximum
parsimony
bootstraps/Bayesian
posterior
probabilities
shown
at
nodes.
Dashes
indicate
bootstrap
values
<50
or
not
present
in
the
maximum
parsimony
or
Bayesian
tree.
NCBI
accession
numbers
shown
in
brackets.
18S
rDNA
sequence
of
S.
molnari
(AF378345)
previously
submitted
by
Kent
el
al.
(2001)
is
shaded
grey
firmed
that
the
18S
rDNA
of
S.
molnari
is
even
longer
(3714
bp
were
obtained
from
the
Czech
isolates),
which
is
highly
unusual,
even
among
Sphaerospora
spp.,
which
are
known
to
contain
sometimes
ex-
tremely
long
nucleotide
inserts
compared
to
other
myxozoan
genera
(Fiala
2006,
Holzer
et
al.
2007,
2013).
In
S.
molnari,
these
inserts
were
50
to
100
bp
longer
than
in
S.
angulata,
and
some
of
them
even
affected
unique
regions
where
other
sphaerosporids
do
not
exhibit
inserts.
Using
ISH,
we
were
able
to
specifically
detect
pro-
liferative
stages
of
Sphaerospora
molnari
in
kidney
capillaries
and
also
in
the
parenchyma.
The
species-
specific
assay
had
a
great
advantage
over
histology
as
the
misidentification
of
blood
stages
of
e.g.
S.
dykovae,
a
frequently
occurring
kidney
parasite
of
common
carp,
undoubtedly
could
be
avoided.
The
surprisingly
high
number
of
blood
stages
of
S.
mol-
nari
and
the
intercellular,
histozoic
occurrence
of
S.
molnari
in
the
kidney
interstitial
tissue,
detected
by
ISH,
presents
clear
similarities
with
coelozoic
sphaeroporids
forming
spores
in
the
renal
tubules
of
freshwater
fish,
as
they
are
known
to
proliferate
in
the
blood
(Baska
&
Molnar
1988)
and
invade
the
interstitial
tissue
of
the
kidney
in
order
to
penetrate
the
tubular
epithelia
(Holzer
et
al.
2003).
However,
we
never
observed
S.
molnari
in
the
intratubular
space.
S.
molnari
may
have
evolved
from
a
coelozoic
kidney
parasite
and
may
have
become
histozoic
in
the
gills
by
parasite
competition
in
the
kidney
tubules
of
carp
(i.e.
S.
dykovae).
It
is
likely
that
com-
petitive
exclusion
and
separation
forced
S.
molnari
to
explore
another
niche
in
the
host,
and
thereby
the
parasite
found
an
easy
option
for
spore
release.
66
Dis
Aquat
Org
104:
59-67,2013
Acknowledgements.
We
thank
H.
Peckova
for
her
assis-
tance
in
performing
PCR
on
the
Czech
parasite
isolates.
The
study
was
supported
by
the
Hungarian
Scientific
Research
Fund
(OTKA
K75873),
the
Czech
Science
Foundation
(505/12/G112
and
P506/11/P724)
and
the
Academy
of
Sci-
ences
of
the
Czech
Republic
(M200961205).
LITERATURE
CITED
Asahida
T,
Kobayashi
T,
Saitoh
K,
Nakayama
I
(1996)
Tissue
preservation
and
total
DNA
extraction
from
fish
stored
at
ambient
temperature
using
buffers
containing
high
con-
centration
of
urea.
Fish
Sci
62:727-730
Barta
JR,
Martin
DS,
Prof
ous-Juchelka
H,
Liberator
PA
and
others
(1997)
Phylogenetic
relationships
among
eight
Eimeria
species
infecting
domestic
fowl
inferred
using
complete
small
subunit
ribosomal
DNA
sequences.
J
Par-
asitol
83:262-271
Bartogova
P,
Freeman
M,
Yokoyama
H,
Caffara
M,
Fiala
I
(2011)
Phylogenetic
position
of
Sphaerospora
testicularis
and
Latyspora
scomberomori
n.
gen.
n.
sp.
(Myxozoa)
within
the
marine
urinary
lade.
Parasitology
138:
381-393
Baska
F,
Molnar
K
(1988)
Blood
stages
of
Sphaerospora
spp.
(Myxosporea)
in
cyprinid
fishes.
Dis
Aquat
Org
5:23-28
Dykova
I,
Lom
J
(1982)
Sphaerospora
renicola
n.
sp.,
a
myxosporean
from
carp
kidney,
and
its
pathogenicity.
Z
Parasitenkd
68:259-268
Eszterbauer
E
(2004)
Genetic
relationship
among
gill-infect-
ing
Myxobolus
species
(Myxosporea)
of
cyprinids:
molecular
evidence
of
importance
of
tissue-specificity.
Dis
Aquat
Org
58:35-40
Eszterbauer
E
(2011)
Erratum
to
'Molecular
phylogeny
of
the
kidney-parasitic
Sphaerospora
renicola
from
com-
mon
carp
(Cyprinus
carpio)
and
Sphaerospora
sp.
from
goldfish
(Carassius
auratus
auratus)'
.
Acta
Vet
Hung
59:
409-409
Eszterbauer
E,
Szekely
C
(2004)
Molecular
phylogeny
of
the
kidney-parasitic
Sphaerospora
renicola
from
common
carp
(Cyprinus
carpio)
and
Sphaerospora
sp.
from
gold-
fish
(Carassius
auratus
auratus).
Acta
Vet
Hung
52:
469-478
Fiala
I
(2006)
The
phylogeny
of
myxosporea
(Myxozoa)
based
on
small
subunit
ribosomal
RNA
gene
analysis.
Int
J
Parasitol
36:1521-1534
Fiala
I,
Bartogova
P
(2010)
History
of
myxozoan
character
evolution
on
the
basis
of
rDNA
and
EF-2
data.
BMC
Evol
Biol
10:228
Hall
TA
(1999)
BioEdit:
a
user-friendly
biological
sequence
alignment
editor
and
analysis
program
for
Windows
95/98/NT.
Nucl
Acids
Symp
Ser
41:95-98
Hallett
SL,
Diamant
A
(2001)
Ultrastructure
and
small-sub-
unit
ribosomal
DNA
sequence
of
Henneguya
lested
n.sp.
(Myxosporea),
a
parasite
of
sand
whiting
Sillago
analis
(Sillaginidae)
from
the
coast
of
Queensland,
Australia.
Dis
Aquat
Org
46:197-212
Hamory
G,
Molnar
K
(1972)
Diseases
of
fish
fingerlings
in
fish
farms,
caused
by
protozoans.
Magyar
Allatory
Lapja
27:358-360
(in
Hungarian)
Holzer
AS,
Sommerville
C,
Wootten
R
(2003)
Tracing
the
route
of
Sphaerospora
truttae
from
the
entry
locus
to
the
target
organ
of
the
host,
Salmo
salar
L.,
using
an
opti-
mized
and
specific
in
situ
hybridization
technique.
J
Fish
Dis
26:647-655
Holzer
AS,
Sommerville
C,
Wootten
R
(2004)
Molecular
rela-
tionships
and
phylogeny
in
a
community
of
myxospore-
ans
and
actinosporeans
based
on
their
18S
rDNA
sequences.
Int
J
Parasitol
34:1099-1111
Holzer
AS,
Wootten
R,
Sommerville
C
(2007)
The
secondary
structure
of
the
unusually
long
18S
ribosomal
RNA
of
the
myxozoan
Sphaerospora
truttae
and
structural
evolu-
tionary
trends
in
the
Myxozoa.
Int
J
Parasitol
37:
1281-1295
Holzer
AS,
Stewart
S,
Tildesley
A,
Wootten
R,
Sommerville
C
(2010)
Infection
dynamics
of
two
renal
myxozoans
in
hatchery
reared
fry
and
juvenile
Atlantic
cod
Gadus
morhua
L.
Parasitology
137:1501-1513
Holzer
AS,
Bartogova
P,
Peckova
H,
Tyml
T
and
others
(2013)
'Who's
who'
in
renal
sphaerosporids
(Bivalvulida:
Myxozoa)
from
common
carp,
Prussian
carp
and
gold-
fish—molecular
identification
of
cryptic
species,
blood
stages
and
new
members
of
Sphaerospora
s.
str.
Para-
sitology
140:46-60
Iskov
MP
(1969)
Sphaerosporosis
as
a
new
disease
of
carp.
Problemy parazitologii,
Proc
VIth
Sci
Conf
Parasitol,
Ukrainian
SSR
II,
Naukova
Dumka,
Kiev,
p
228-232
(in
Russian)
Jirkii
M,
Fiala
I,
Mod.rr
D
(2007)
Tracing
the
genus
Sphaero-
spora:
rediscovery,
redescription
and
phylogeny
of
the
Sphaerospora
ranae
(Morelle,
1929)
n.
comb.
(Myxo-
sporea,
Sphaerosporidae),
with
emendation
of
the
genus
Sphaerospora.
Parasitology
134:1727-1739
Kaup
FJ,
Kuhn
EM,
Korting
W
(1995)
Light
and
electron
microscopic
studies
on
the
sporogenesis
of
Sphaerospora
molnari
in
the
gill
lamellas
of
carp
(Cyprinus
carpio).
Berl
Munch
Tierarztl
Wochenschr
108:206-214
(in
German)
Kent
ML,
Khattra
J,
Hervio
DML,
Devlin
RH
(1998)
Riboso-
mal
DNA
sequence
analysis
of
isolates
of
the
PKX
myx-
osporean
and
their
relationship
to
members
of
the
genus
Sphaerospora.
J
Aquat
Anim
Health
10:12-21
Kent
ML,
Andree
KB,
El-Matbouli
M,
Bartholomew
JL
and
others
(2001)
Recent
advances
in
our
knowledge
on
Myxozoa.
J
Eukaryot
Microbiol
48:395-413
Kudo
RR
(1919)
Studies
on
Myxosporidia.
III.
Biol
Monogr
5:
241-503
Lom
J,
Arthur
JR
(1989)
A
guideline
for
the
preparation
of
species
descriptions
in
Myxosporea.
J
Fish
Dis
12:
151-156
Lom
J,
Dykova
I
(2006)
Myxozoan
genera:
definition
and
notes
on
taxonomy,
life-cycle
terminology
and
patho-
genic
species.
Folia
Parasitol
(Praha)
53:1-36
Lom
J,
Dykova
I,
Pavlaskova
M,
Grupcheva
G
(1983)
Sphaerospora
molnari
sp.
nov.
(Myxozoa:
Myxosporea),
an
agent
of
gill,
skin
and
blood
sphaerosporosis
of
com-
mon
carp
in
Europe.
Parasitology
86:529-535
Molnar
K
(1979)
Gill
sphaerosporosis
in
the
common
carp
and
grasscarp.
Acta
Vet
Acad
Sci
Hung
27:99-113
Molnar
K
(1980)
Cutaneous
sphaerosporosis
of
the
common
carp
fry.
Acta
Vet
Acad
Sci
Hung
28:371-374
Molnar
K,
Marton
S,
Szekely
C,
Eszterbauer
E
(2010)
Differ-
entiation
of
Myxobolus
spp.
(Myxozoa:
Myxobolidae)
infecting
roach
(Rudlus
rudlus)
in
Hungary.
Parasitol
Res
107:1137-1150
Morris
DJ,
Adams
S
(2008)
Sporogony
of
Tetracapsuloides
bryosalmonae
in
the
brown
trout
Salmo
trutta
and
the
role
of
the
tertiary
cell
during
the
vertebrate
phase
of
myxozoan
life
cycles.
Parasitology
135:1075-1092
Rambaut
A,
Drummond
AJ
(2007)
Tracer
v1.4.
Available
at
http://beast.bio.ed.ac.uk/Tracer
Eszterbauer
et
al.:
Molecular
characterization
of
Sphaerospora
molnari
67
)0.
Ronquist
F,
Huelsenbeck
JP
(2003)
MrBayes
3:
Bayesian
phylogenetic
inference
under
mixed
models.
Bioinfor-
matics
19:1572-1574
Shulman
SS
(1966)
The
myxosporidium
fauna
of
the
USSR.
Nauka,
Moscow-Leningrad
(in
Russian)
Sitja.-Bobadilla
A,
Alvarez-Pellitero
P
(1994)
Revised
classifi-
cation
and
key
species
of
the
genus
Sphaerospora
Davies,
1917
(Protozoa:
Myxosporea).
Res
Rev
Parasitol
54:67-80
)0.
Staden
R
(1996)
The
Staden
sequence
analysis
package.
Mol
Biotechnol
5:233-241
)0.
Stamatakis
A
(2006)
RAxML-VI-HPC:
maximum
likelihood-
based
phylogenetic
analyses
with
thousands
of
taxa
and
mixed
models.
Bioinformatics
22:2688-2690
Swofford
DL
(2001)
PAUP:
phylogenetic
analysis
using
par-
simony
(and
other
methods),
Version
4.
Sinauer
Associ-
ates,
Sunderland,
MA
Thelohan
P
(1895)
Recherches
sur
les
Myxopsporidies.
Bull
Sci
Fr
Belg
26:100-394
)0.
Thompson
JD,
Gibson
TJ,
Plewniak
F,
Jeanmougin
F,
Higgins
DG
(1997)
The
Clustal
X
windows
interface:
flexible
strategies
for
multiple
sequence
alignment
aided
by
quality
analysis
tools.
Nucleic
Acids
Res
25:
4876-4882
Waluga
D
(1983)
Preliminary
investigations
on
carp
spha-
erosporosis.
Medycyna
Veterynaryjna
39:399-403
)0.
Whipps
CM,
Adlard
RD,
Bryant
MS,
Kent
ML
(2003)
Two
unusual
myxozoans,
Kudoa
quadricornis
n.
sp.
(Multi-
valvulida)
from
the
muscle
of
goldspotted
trevally
(Carangoides
fulvoguttatus)
and
Kudoa
permuliicapsula
n.
sp.
(Multivalvulida)
from
the
muscle
of
Spanish
mack-
erel
(Scamberomorus
commerson)
from
the
Great
Barrier
Reef,
Australia.
J
Parasitol
89:168-173
Editorial
responsibility:
Dieter
Steinhagen,
Submitted:
November
20,
2012;
Accepted:
February
4,
2013
Hannover,
Germany
Proofs
received
from
author(s):
April
5,
2013