Four Myxobolus spp. (Myxosporea: Bivalvulida) from the gill lamellae of common carp (Cyprinus carpio) and Japanese silver crucian carp (Carassius langsdorfii) in the western part of Japan, with the description of three new species (M. tanakai n. sp., M. paratoyamai n. sp., and M. ginbuna n. sp.)


Kato, E.; Kasai, A.; Tomochi, H.; Li, Y.C.; Sato, H.

Parasitology Research 116(9): 2427-2441

2017


Approximately three dozen Myxobolus spp. (Myxozoa: Myxosporea: Bivalvulida) have been described to parasitize the gills of carp of the genera Cyprinus and Carassius. Hitherto, these fish were often introduced to temperate waters worldwide as food and ornamental fish from Asia, their place of origin. The present study examined the myxosporean infection of seven common carp (Cyprinus carpio) and seven Japanese silver crucian carp (Carassius langsdorfii) collected from the Fushinogawa River around the university in Yamaguchi City, Japan, during the period April 2015 to October 2016. In total, four Myxobolus spp. were detected in the gill lamellae of Cy. carpio and Ca. langsdorfii, i.e., two species in each species of fish. The four species were characterized morphologically and genetically based on the 18S ribosomal RNA gene (rDNA). A new species, Myxobolus tanakai n. sp., from four individuals of Cy. carpio had an elongated pyriform spore (15.4-18.6 μm by 6.3-8.4 μm), resembling the spore shape of Myxobolus koi from Cy. carpio or Carassius auratus in Japan, China, and the USA, but bigger than it (13.2-15.6 μm by 6.6-7.8 μm). The new species formed a clade with M. koi but was distinct from any of the isolates of this species (nucleotide identities less than 98.6%). The second new species, Myxobolus paratoyamai n. sp., from a single Cy. carpio with its one prominent and one rudimentary polar capsule closely resembled the spore morphology of Myxobolus toyamai from Cy. carpio or Carassius gibelio in Japan, China, and the USA. However, the isolate formed a clade with Myxobolus longisporus from Cy. rubrofuscus in China rather than with M. toyamai isolates (nucleotide identities less than 97.9% with known species). Another new species, Myxobolus ginbuna n. sp., from two individuals of Ca. langsdorfii had similar-shaped spores to Myxobolus wulii, but the dimensions were smaller (11.7-13.9 μm by 8.5-9.8 μm vs. 17.6-18.5 μm by 8.9-10.0 μm). This new species formed a clade with M. wulii but was distinct from any of the M. wulii isolates from Ca. gibelio in China (nucleotide identities less than 99.1%). An additional species, Myxobolus pyramidis, from six individuals of Ca. langsdorfii was morphologically and genetically similar to the previous record from Ca. gibelio in China (99.6% nucleotide identity of the 18S rDNA). Two of these six individuals were mix-infected with M. ginbuna n. sp. This is a new host and geographical distribution record for M. pyramidis.

Parasitol
Res
CrossMark
DOI
10.1007/s00436-017-5545-4
ORIGINAL
PAPER
Four
Myxobolus
spp.
(Myxosporea:
Bivalvulida)
from
the
gill
lamellae
of
common
carp
(Cyprinus
carpio)
and
Japanese
silver
crucian
carp
(Carassius
langsdorfii)
in
the
western
part
of
Japan,
with
the
description
of
three
new
species
(M.
tanakai
n.
sp.,
M.
paratoyamai
n.
sp.,
and
M.
ginbuna
n.
sp.)
Eigo
Kato
l
Aldhiro
Kasai
l
'
2
Hisayuki
Tomochi
l
Ying-Chun
Li
2,3
Hiroshi
Sato
1
'
2
Received:
9
April
2017
/Accepted:
21
June
2017
©
Springer-Verlag
GmbH
Germany
2017
Abstract
Approximately
three
dozen
Myxobolus
spp.
(Myxozoa:
Myxosporea:
Bivalvulida)
have
been
described
to
parasitize
the
gills
of
carp
of
the
genera
Cyprinus
and
Carassius.
Hitherto,
these
fish
were
often
introduced
to
temper-
ate
waters
worldwide
as
food
and
ornamental
fish
from
Asia,
their
place
of
origin.
The
present
study
examined
the
myxosporean
infection
of
seven
common
carp
(Cyprinus
carpio)
and
seven
Japanese
silver
crucian
carp
(Carassius
langsdorfii)
collected
from
the
Fushinogawa
River
around
the
university
in
Yamaguchi
City,
Japan,
during
the
period
April
2015
to
October
2016.
In
total,
four
Myxobolus
spp.
were
de-
tected
in
the
gill
lamellae
of
Cy.
carpio
and
Ca.
langsdorfii,
i.e.,
two
species
in
each
species
of
fish.
The
four
species
were
char-
acterized
morphologically
and
genetically
based
on
the
18S
ribosomal
RNA
gene
(rDNA).
A
new
species,
Myxobolus
tanakai
n.
sp.,
from
four
individuals
of
Cy.
carpio
had
an
elon-
gated
pyriform
spore
(15.4-18.6
um
by
6.3-8.4
pm),
resem-
bling
the
spore
shape
of
Myxobolus
koi
from
Cy.
carpio
or
Carassius
auratus
in
Japan,
China,
and
the
USA,
but
bigger
than
it
(13.2-15.6
pm
by
6.6-7.8
pm).
The
new
species
formed
a
Glade
with
M
koi
but
was
distinct
from
any
of
the
isolates
of
this
species
(nucleotide
identities
less
than
98.6%).
The
second
RI
Hiroshi
Sato
sato7dp4@yamaguchi-u.ac.jp
Laboratory
of
Veterinary
Parasitology,
Joint
Faculty
of
Veterinary
Medicine,
Yamaguchi
University,
1677-1
Yoshida,
Yamaguchi
753-8515,
Japan
2
United
Graduate
School
of
Veterinary
Science,
Yamaguchi
University,
1677-1
Yoshida,
Yamaguchi
753-8515,
Japan
3
Present
address:
Faculty
of
Agricultural
Science,
Guangdong
Ocean
University,
Mazhang,
Zhanjiang,
Guangdong
524088,
China
new
species,
Myxobolus
paratoyamai
n.
sp.,
from
a
single
Cy.
carpio
with
its
one
prominent
and
one
rudimentary
polar
cap-
sule
closely
resembled
the
spore
morphology
of
Myxobolus
toyamai
from
Cy.
carpio
or
Carassius
gibelio
in
Japan,
China,
and
the
USA.
However,
the
isolate
formed
a
Glade
with
Myxobolus
longisporus
from
Cy.
rubrofuscus
in
China
rather
than
with
M
toyamai
isolates
(nucleotide
identities
less
than
97.9%
with
known
species).
Another
new
species,
Myxobolus
ginbuna
n.
sp.,
from
two
individuals
of
Ca.
langsdorfii
had
similar-shaped
spores
to
Myxobolus
wulii,
but
the
dimensions
were
smaller
(11.7-13.9
um
by
8.5-9.8
um
vs.
17.6-18.5
um
by
8.9-10.0
um).
This
new
species
formed
a
Glade
with
M
wulii
but
was
distinct
from
any
of
the
M
wulii
isolates
from
Ca.
gibelio
in
China
(nucleotide
identities
less
than
99.1%).
An
additional
species,
Myxobolus
pyramidis,
from
six
individuals
of
Ca.
langsdorfi
was
morphologically
and
genetically
similar
to
the
previous
record
from
Ca.
gibelio
in
China
(99.6%
nucleotide
identity
of
the
18S
rDNA).
Two
of
these
six
individuals
were
mix-infected
with
M
ginbuna
n.
sp.
This
is
a
new
host
and
geographical
distribution
record
for
M
pyramidis.
Keywords
Myxosporea
Myxobolus
tanakai
n.
sp.
Myxobolus
paratoyamai
n.
sp.
Myxobolus
ginbuna
n.
sp.
Myxobolus
pyramidis
Cyprinus
carpio
Carassius
langsdorfii
Japan
Introduction
Bivalvulid
myxosporeans
classified
in
the
genus
Myxobolus
Biltschli,
1882,
are
distinctly
speciose
in
the
systematics
of
Myxozoa
(Lom
and
Dykova
2006),
approaching
nearly
900
Published
online:
05
July
2017
4t
Springer
Parasitol
Res
nominal
species
(Landsberg
and
Lom
1991;
Eiras
et
al.
2005,
2014;
Abdel-Ghaffar
et
al.
2017;
Abdel-Gaber
et
al.
2017).
The
diminutive
spores
are
simple
in
morphology
without
ex-
ternal
projections,
ellipsoidal,
ovoid
or
rounded
in
valvular
view
and
biconvex
in
sutural
view,
and
contain
two
polar
capsules
mostly
pyriform,
equal
or
unequal,
closely
set
in
the
anterior
part
of
the
spore
on
the
sutural
plane
(Lom
and
Dykova
2006;
Fiala
et
al.
2015).
Despite
specific
differentia-
tion
based
solely
on
the
morphology
of
the
bivalvulid
spore,
i.e.,
shape
and
dimensions,
being
rather
difficult,
Myxobolus
spp.
have
been
demonstrated
to
show
highly
narrow host,
organ,
and
tissue
specificities
(Molnar
1994,
2002a,
2002b;
Cech
et
al.
2012;
Molnar
and
Eszterbauer
2015).
In
other
words,
the
development
of
plasmodia
mostly
occurs
in
a
well-defined
part
of
an
organ
in
a
certain
category
of
fish,
e.g.,
only
in
one
host
species
or
in
closely
related
fish
(Eszterbauer
2004;
Molnar
and
Eszterbauer
2015).
In
the
last
decade,
reliable
specific
differentiations
of
myxozoans,
including
Myxobolus
spp.,
have
been
dramatical-
ly
advanced
by
the
aid
of
molecular
techniques
(Andree
et
al.
1999;
Abdel-Ghaffar
et
al.
2012,
2016;
Morsy
et
al.
2012),
particularly
nucleotide
sequencing
of
the
ribosomal
RNA
gene
(rDNA).
The
critical
differentiation
and
phylogenetic
relationship
of
Myxobolus
spp.
with
morphologically
similar
spores
have
been
successfully
achieved
(Salim
and
Desser
2000;
Molnar
et
al.
2002;
Bahri
et
al.
2003;
Ferguson
et
al.
2008;
Liu
et
al.
2010, 2012,
2013;
Zhang
et
al.
2010a;
Zhao
et
al.
2013;
Liu
et
al.
2016a,
b).
The
taxonomic
relationships
of
nominal
species
showing
different
tissue
tropism in
the
same
host
or
those
having
the
same
phenotypic
features
but
from
different
fish
hosts
have
also
been
clearly
elucidated
(Eszterbauer
2002,
2004;
Molnar
et
al.
2002,
2006,
2011;
Cech
et
al.
2012;
Liu
et
al.
2012;
Zhai
et
al.
2016).
From
cyprinid
fish
of
the
genera
Cyprinus
and
Carassius,
which
are
distributed
in
temperate
waters
worldwide
partly
due
to
their
introduction
as
food
or
ornamental
fish,
at
least
90
nominal
Myxobolus
spp.
have
been
recorded,
of
which
36
species
parasitize
the
gills
only
(19
species)
or
multiple
organs
including
the
gills
(17
species)
(Landsberg
and
Lom
1991;
Eiras
et
al.
2005,
2014;
Liu
et
al.
2016b,
2016c).
In
the
present
study,
seven
common
carp
(Cyprinus
carpio)
and
seven
Japanese
silver
crucian
carp
(Carassius
langsdorfii)
fished
in
the
mainstream
of
the
Fushinogawa
River
or
its
branch
around
Yamaguchi
University
in
Yamaguchi
City,
Japan,
during
the
period
April
2015
to
October
2016,
were
examined
to
disclose
their
myxosporean
infection.
Four
Myxobolus
spp.
were
de-
tected
in
their
gills
(two
from
four
common
carp
and
two
from
six
Japanese
silver
crucian
carp)
and
characterized
morpho-
logically
and
genetically
based
on
the
18S
rDNA.
Although
these
fourMyxobolus
spp.
appeared
superficially
to
have
sim-
ilar
myxospore
morphologies
of
well-known
myxobolids
par-
asitizing
the
gills
of
Cy.
carpio
or
Ca.
auratus,
i.e.,
Myxobolus
koi
Kudo,
1919,
Myxobolus
toyamai
Kudo,
1917,
Myxobolus
wulii
(Wu
et
Li,
1986)
Landsberg
et
Lom,
1991,
and
Myxobolus
pyramidis
Chen,
1998,
three
species
were
differ-
entiated
as
new
myxobolids.
Materials
and
methods
Fish
samples
and
parasitological
examination
During
the
period
April
24,
2015,
to
October
12,
2016,
seven
individuals
of
Cy.
carpio
and
seven
individuals
of
Ca.
langsdorfii
were
collected
by
hand
net
at
two
localities
in
Japan:
the
mainstream
of
the
Fushinogawa
River
in
the
Kurokawa
area
and
its
branch,
the
Kudengawa
River,
around
Yamaguchi
University
in
the
Hirakawa
area,
respectively
(Table
1).
Using
the
naked
eye
and
a
dissection
microscope,
a
thorough
inspection
was
conducted
for
the
presence
of
myxosporeans
in
organs
such
as
skin,
fins,
gills,
brain,
mus-
culature,
and
viscera.
Tissues
parasitized
with
myxosporean
plasmodia
were
placed
in
physiological
saline
and
pressed
between
slides
and
cover
glasses
to
release
the
spores.
Released
spores
were
examined
under
a
microscope
at
a
mag-
nification
of
x800,
and
transformed
into
photographs
with
Adobe
®
Photoshop
®
ver.
11.0
(Adobe
Systems,
San
Jose,
CA,
USA).
Photographs
were
then
printed
at
a
high
magnifi-
cation.
Measurements
were
conducted
on
multiple-printed
photographs
following
the
guidelines
of
Lom
and
Arthur
(1989).
All
measurements,
range
followed
by
average
in
parentheses,
are
expressed
in
micrometers
unless
otherwise
stated.
Following
removal
of
a
portion
of
the
spores
for
DNA
extraction,
the
parasite
was
fixed
separately
in
10%
neutral-buffered
formalin
solution
or
70%
ethanol.
A
por-
tion
of
formalin-fixed
spores
was
treated
with
Lugol's
io-
dine
stain
and
Diff-QuikTM
stain
to
observe
iodinophilous
vacuoles
in
the
sporoplasm
and
polar
filaments,
respective-
ly.
Specimens
collected
in
the
present
work
were
deposited
in
the
Meguro
Parasitological
Museum,
Tokyo,
Japan,
un-
der
collection
nos.
21319-21324.
DNA
extraction,
polymerase
chain
reaction
(PCR),
and
sequencing
Parasite
samples
in
1.5-ml
plastic
tubes
were
first
freeze-
dried
using
a
freeze
dryer
(EYELA
FD-5N;
Tokyo
Rikakikai
Co.,
Bunkyo-ku,
Tokyo,
Japan),
then
homoge-
nized
with
individual
clean
plastic
pestles.
Parasite
DNA
was
extracted
from
these
homogenized
samples
using
an
IllustraTM
tissue
and
cells
genomicPrep
Mini
Spin
Kit
(GE
Healthcare
UK,
Buckinghamshire,
UK)
according
to
the
instructions
of
the
manufacturer.
PCR
amplification
of
overlapping
fragments
of
the
18S
rDNA
was
performed
in
a
20-µL
volume
containing
a
DNA
polymerase,
Blend
Taq-
Plus-
(TOYOBO,
Dojima
Hama,
Osaka,
Japan),
and
41?!
Springer
Parasitol
Res
Table
1
Fish
samples
examined
in
the
present
study
Host
fish
Fish
lot.
Date
of
collection
No.
of
fish
Body
standard
length
(cm)
Body
weight
(kg)
Parasite
recovery
Common
carp
1
June
2015
6
45.7-54.2
2.2-3.4
M
tanakai
n.
sp.
(3/6)
(Cy.
carpio)
2
June
2016
1
20.4
0.13
M
tanakai
n.
sp.
(1/1)
M
paratoyamai
n.
sp.
(1/1)
Japanese
silver
crucian
carp
3
April
to
May
2015
4
28.0-29.6
0.59-0.61
M
ginbuna
n.
sp.
(2/4)
M.
pyramidis
(4/4)
(Ca.
langsdorfit)
4
May
2016
2
33.0,
36.5
0.61,
0.74
M.
pyramidis
(1/2)
5
October
2016
1
20.9
0.12
M.
pyramidis
(1/1)
different
combinations
of
new
primers
(Myxo185_05F,
Myxo185_575F,
Myxo185_887F,
Myxo185_794R,
and
Myxo185_1009R)
and
a
universal
eukaryotic
primer
(SSU18R)
(Table
2).
The
new
primers
were
designed
using
the
online
software
Primer3web
ver.
4.0.0
(Untergasser
et
al.
2012)
and
referring
to
an
18S
rDNA
sequence
of
M.
pyramidis
(DDBJ/EMBL/GenBank
accession
no.
HQ613411)
as
well
as
an
18S
rDNA
sequence
of
Cy.
carpio
(JN628435).
The
PCR
cycling
protocol
was
3
min
at
94
°C,
then
40
cycles
of
45
s
at
94
°C,
1
min
at
63
or
65
°C,
and
1
min
at
72
°C,
followed
by
a
final
extension
at
72
°C
for
7 min.
The
PCR
products
were
purified
using
a
FastGene
Gel/PCR
Extraction
Kit
(NIPPON
Genetics
Co.,
Tokyo,
Japan)
and
directly
sequenced
as
previously
described
(Li
et
al.
2012).
The
nucleotide
sequences
obtained
in
the
present
study
are
available
from
the
DDBJ/EMBL/
GenBank
databases
under
the
accession
nos.
LC228235—
LC228239.
Phylogenetic
analysis
For
phylogenetic
analysis,
the
newly
obtained
18S
rDNA
nu-
cleotide
sequences
of
the
four
species
in
the
present
study
and
closely
related
Myxobolus
nucleotide
sequences
retrieved
from
the
DDBJ/EMBL/GenBank
databases
were
aligned
using
the
CLUSTAL
W
multiple
alignment
program
(Thompson
et
al.
1994),
with
subsequent
manual
adjustment
The
accession
numbers
of
the
sequences
analyzed
in
the
pres-
ent
study
are
given
in
the
figure
showing
phylogenetic
trees.
Regions
judged
to
be
poorly
aligned
and
characters
with
a
gap
Table
2
Primers
used
to
amplify
overlapping
segments
of
the
18S
rDNA
ofMyxobolus
spp.
in
the
present
study
Segment
no.
Primer
name
Nucleotide
sequence
Position
of
5'-end
s
For
amplifying
For
sequencing
F:
Myxo18S_05F
5'-ACCTGGTTGATCCTGCCAGTG-3'
—21
R:
Myxo18S_794R
5'-CGCCTGCTTTGAGCACTCTGT-3'
814
R:
Kud6r/R
5'-TCCAGTAGCTACTCATCG-3'
532
2
F:
Myxo18S_05F
(see
above)
R:
Myxo18S_1009R
5'-CGCATCTGTTAGTCCTTGGC-3'
1028
R:
Kud6r/R
(see
above)
R:
Myxo18S_794R
(see
above)
3
F:
Myxo18S_575F
5'-CGCGGTAATTCCAGCTCCAG-3'
556
R:
SSU18R
5'-TGATCCTTCYGCAGGTTCAC-3'
2028
F:
Myxo18S_1028F
5'-GCCAAGGACTAACAGATGCG-3'
1009
F:
Myxo18S_1217F
5'-GGGAGAGTATGGTCGCAAGT-3'
1198
4
F:
Myxo18S_887F
5'-AATGGTCGAGGGCAACTTTG-3'
868
R:
SSU18R
(see
above)
F:
Myxo18S_1028F
(see
above)
F:
Myxo18S_1217F
(see
above)
F
forward,
R
reverse
a
Relative
position
of
the
5'-end
of
each
primer
in
an
rDNA
sequence
of
M
pyramidis
from
a
Japanese
silver
crucian
carp
(DDBJ/EMBL/GenBank
accession
no.
LC228239)
4t
Springer
Parasitol
Res
in
any
sequences
were
excluded
from
subsequent
analyses:
1495
characters,
of
which
326
were
variable,
remained
for
analysis
A
(molecular
phylogeny
of
representative
Myxobolus
spp.
recorded
in
cyprinid
fish;
31
isolates);
870
characters,
of
which
85
were
variable,
remained
for
analysis
B
(molecular
phylogeny
focusing
on
a
Glade
containing
all
currently
recorded
isolates
of
M
koi,
M
toyamai,
M
wulii,
and
M
pyramidis;
20
isolates);
and
1879
characters,
of
which
211
were
variable,
remained
for
analysis
C
(molecular
phy-
logeny
focusing
on
a
Glade
containing
M
koi,
M
toyamai,
M
wulii,
and
M
pyramidis
isolates
with
long
nucleotide
se-
quences;
14
isolates).
Maximum
likelihood
(ML)
analysis
was
performed
with
the
program
PhyML
(Guindon
and
Gascuel
2003;
Dereeper
et
al.
2008)
provided
on
the
phylogeny.fr
web
site
(http://www.phylogeny.fr/)
.
The
probability
of
inferred
branch
was
assessed
by
the
approximate
likelihood-
ratio
test,
an
alternative
to
the
non-parametric
bootstrap
esti-
mation
of
branch
support
(Anisimova
and
Gascuel
2006).
Results
Occurrence
of
plasmodia
A
thorough
inspection
of
seven
Cy.
carpio
and
seven
Ca.
langsdorfii
found
myxosporean
cysts
only
in
the
gill
lamellae
of
10
fish
samples
(Table
1).
Three individuals
of
Cy.
carpio
collected
in
June
2015
had
one
or
two
myxosporean
cysts
containing
M
koi-like
spores
in
the
gill
lamellae:
a
total
num-
ber
of
four,
0.37-0.82
(0.55)
mm
by
0.26-0.63
(0.42)
mm
[n
=
3].
One
fish
collected
in
June
2016
had
19
myxosporean
cysts
of
two
species
in
the
same
organ
(11
cysts
ofM.
koi-like
spores
and
eight
cysts
of
M.
toyamai-like
spores).
Representative
cysts
were
measured:
M
koi-like,
0.20-0.39
(0.32)
mm
by
0.15-0.34
(0.25)
mm
[n
=
4],
and
M.
toyamai-
like,
0.23
mm
by
0.20
mm
and
0.32
mm
by
0.29
mm
[n
=
2].
Four
individuals
of
Ca.
langsdorfii
collected
in
April
2015
had
nine
to
91
myxosporean
cysts
in
the
gill
lamellae
(geomean
27.3
in
number;
0.10-0.19
(0.16)
mm
by
0.09-
0.16
(0.12)
mm
[n
=
10]),
and
two
Ca.
langsdorfii
individuals
collected
in
May
and
October
2016
had
22
and
five
myxosporean
cysts,
respectively,
in
the
gill
lamellae.
Two
Ca.
langsdorfii
individuals
fished
in
2015
were
parasitized
with
two
species,
i.e.,
M
wulii-like
and
M
pyramidis-like,
with
ratios
of
3:1
and
1:4.
All
the
other
individuals
were
par-
asitized
solely
by
the
M
pyramidic-like
species.
All
the
myxosporean
cysts
mentioned
above
were
classi-
fied
as
the
"intralamellar-vascular
type
(LV
3
)"
of
Molnar
(2002a)
and
situated
at
any
height
from
the
base
of
the
gill
filaments.
Myxospores
from
cysts
in
the
gills
of
Cy.
carpio
were
M
koi-like
and
M
toyamai-like
in
shape
but
not
dimen-
sions.
Those
from
the
gills
of
Ca.
langsdotfii
were
differenti-
ated
into
two
morphotypes,
M
wulii-like
but
with
different
dimensions
and
M
pyramidis-like.
As
shown
later
in
this
re-
port,
the
nucleotide
sequences
of
the
18S
rDNA
of
these
M
koi-like,
M
toyamai-like,
and
M
wulii-like
myxobolids
from
Cy.
carpio
and
Ca.
langsdorfii
clearly
demonstrated
their
taxonomic
independence
from
known
species.
Consequently,
we
have
erected
three
new
myxobolid
spp.
for
them,
Myxobolus
tanakai
n.
sp.,
Myxobolus
paratoyamai
n.
sp.,
and
Myxobolus
ginbuna
n.
sp.,
respectively,
based
on
both
morphological
and
genetic
criteria.
The
other
myxobolid
from
Ca.
langsdotfii
was
identified
as
M
pyramidis.
Description
M
tanakai
n.
sp.
(Myxosporea:
Bivalvulida).
(Fig.
la,
Fig.
2(a,
a'),
and
Fig.
3a).
Ellipsoidal
plasmodia,
0.37-0.82
(0.55)
mm
by
0.26-0.63
(0.42)
mm
[n
=
3]
or
0.20-0.39
(0.32)
mm
by
0.15-0.34
(0.25)
mm
[n
=
4],
formed
in
the
intralamellar
tissue
adjacent
to
the
vessels
in
the
gill
filaments.
No
accumulation
of
inflam-
matory
cells
was
seen
around
the
plasmodia.
Polysporic,
and
synchronized
development
of
spores.
Spores
elongated
pyriform
in
valvular
and
sutural
views,
measuring
15.4-18.6
(17.2)
in
length,
6.3-8.4
(6.8)
in
width,
and
5.9-6.8
(6.3)
in
thickness
[n
=
40].
Two
elongated
pyri-
form
polar
capsules,
generally
equal
but
sometimes
unequal
in
length,
situated
in
the
anterior
2/3-3/4
of
spores;
the
posterior
end
extending
to
more
or
less
the
anterior
70%
of
spores,
measuring
7.6-9.4
(8.7)
in
length
by
2.0-2.7
(2.4)
in
width.
The
anterior
end
of
the
polar
capsules
directed
towards
the
apex
of
the
spore,
but
not
close
to
it,
packing
8-10
spirals
of
the
polar
filament
Neither
mucous
coat
around
the
spores
nor
intercapsular
appendix
visible.
Iodinophilous
vacuole
in
the
sporoplasm.
Two
nucleotide
sequences
(2010
bp
in
length)
covering
almost
the
full
length
of
the
18S
rDNA,
except
for
the
primer-flanking
5'
and
3'
termini,
have
been
deposited
in
the
DDBEEMBL/GenBank
databases
(accession
nos.
LC228235
and
LC228236
from
two
plasmodia
collected
from
different
fish
samples).
These
two
sequences
showed
99.35%
(1997/2010)
identity
with
each
other.
Taxonomic
summary
Type
host:
Cy.
carpio
(Linnaeus,
1758),
common
carp
(Actinopterygii:
Cypriniformes:
Cyprinidae:
Cyprininae)
Site
of
infection:
gill
filament,
intralamellar
tissue
adjacent
to
the
blood
vessel
Type
locality:
Kurokawa
(Fushinogawa
River),
Yamaguchi
City,
Japan
Specimen
deposited:
hapantotype
no.
21319,
Meguro
Parasitological
Museum,
Tokyo,
Japan.
Additional
speci-
mens,
no.
21320
in
the
same
museum
4?!
Springer
Parasitol
Res
a
d
Fig.
1
Microscopic
view
of
myxospores
of
Myxobolus
tanakai
n.
sp.
from
Cyprinus
carpio
(a),
Myxobolus
paratoyamai
n.
sp.
from
Cyprinus
carpio
(b,
c),
Myxobolus
ginbuna
n.
sp.
from
Carassius
Etymology:
The
species
is
named
after
Professor
Emeritus
Shuhei
Tanaka,
Faculty
of
Agriculture,
Yamaguchi
University,
who
was
a
valued
research
collaborator
for
many
years,
providing
excellent
cooperation
with
the
scanning
elec-
tron
microscopy
of
myxosporeans
(Matsukane
et
al.
2010,
2011;
Li
et
al.
2013;
Kasai
et
al.
2016a,
b).
Prevalence:
57.1%
incidence
(4
of
7)
Intensity:
a
few
to
several
plasmodia
per
host
Remarks
The
present
new
myxobolid
species
from
the
gill
lamellae
of
Cy.
carpio
in
the
western
part
of
Japan
(Yamaguchi)
has
similar
myxospore
morphology
to
M.
koi
from
the
gill
lamellae
of
Cy.
carpio
or
Myxobolus
longisporus
Nie
et
Li,
1992,
from
the
gill
lamellae
of
Cy.
rubrofuscus
(syn.
Cy.
carpio
haematopterus)
in
China
(Yokoyama
et
al.
1997;
Chen
and
Ma
1998;
Dykova
langsdorfii
(d),
and
Myxobolus
pyramidis
from
Carassius
langsdoi
(e).
Valvular
view
(a,
b,
d,
e)
and
sutural
view
(c).
All
photographs
at
the
same
magnification
et
al.
2003).
As
shown
in
Table
3,
spores
of
M
tanakai
n.
sp.
are
consistently
longer
than
those
of
M
koi
isolates
in
Japan,
China,
and
the
USA
(15.4-18.6
um
vs.
13.2-15.6
um).
The
spore
morphology
including
dimensions
is
much
closer
to
M
longisporus
except
for
bending
of
the
apical
end
of
spores,
evident
in
sutural
view,
and
smaller
spore
thickness.
Apart
from
these
two
species,
M
tanakai
n.
sp.
can
be
differen-
tiated
from
other
myxobolids
infecting
the
gill
lamellae
of
cyprinid
fish
by
spore
dimensions
such
as
length
or
width,
or
both.
Genetically,
M
tanakai
n.
sp.
has
18S
rDNA
nu-
cleotide
sequences
that
are
distinct
from
those
of
any
M.
koi
isolate
(DDBJ/EMBL/GenBank
accession
nos.
FJ710800,
KT240127,
FJ841887,
KJ725077),
with
low
identities
ranging
from
97.76%
(1916/1960)
to
98.56%
(1922/1950).
These
18S
rDNA
nucleotide
sequence
iden-
tities
with
M
koi
isolates
are
followed
by
M
longisporus
(AY364637)
with
95.53%
(1924/2014)
identity
and
b'
Fig.
2
Stylized
illustrations
of
Myxobolus
tanakai
n.
sp.
(a,
a'),
Myxobolus
paratoyamai
n.
sp.
(b,
b'),
Myxobolus
ginbuna
n.
sp.
(c,
c'),
and
Myxobolus
pyramidis
(d,
d').
Valvular
view
(a,
b,
c,
d)
and
sutural
view
(a',
b',
c',
d').
All
illustrations
at
the
same
magnification
-
7
--
It%
4:
5pm
d
4t
Springer
a
Parasitol
Res
Fig.
3
Microscopic
view
of
myxospores
of
Myxobolus
tanakai
n.
sp.
(a)
and
Myxobolus
paratoyamai
n.
sp.
(b,
c).
Using
Lugol's
iodine
stain
(a,
b),
an
iodinophilous
vacuole
(asterisk)
is
clearly
visible
in
the
sporoplasm.
Using
Diff-QuikTM
stain
(c),
undeveloped
polar
filaments
(arrows)
are
visible
in
the
rudimentary
polar
capsules.
Valvular
view
(a,
c)
and
sutural
view
(b).
All
photographs
at
the
same
magnification
M.
pyramidis
(HQ613411)
with
95.08%
(1915/2014)
identity.
M
paratoyamai
n.
sp.
(Myxosporea:
Bivalvulida)
(Fig.
lb,
c,
Fig.
2(b,
b'),
and
Fig.
3b,
c).
Ellipsoidal
plasmodia,
0.23
mm
by
0.20
mm
and
0.32
mm
by
0.29
mm
[n
=
2],
formed
in
the
intralamellar
tissue
adjacent
to
the
vessels
in
the
gill
filaments.
No
accumulation
of
inflam-
matory
cells
was
seen
around
the
plasmodia.
Polysporic
and
synchronized
development
of
spores
Spores
elongated
pyriform
in
valvular
and
sutural
views,
with
asymmetry,
measuring
14.7-16.4
(15.4)
in
length,
5.5-
6.8
(6.3)
in
width,
and
5.6-6.4
(6.1)
in
thickness
[n
=
42].
Particularly
in
valvular
view,
anterior
end
slightly
bent
Of
two
polar
capsules,
only
one
prominent
polar
capsule
func-
tional,
occupying
most
of
anterior
half
of
spores,
and
the
other
rudimentary;
the
prominent
polar
capsule
measuring
5.9-7.1
(6.5)
in
length
by
3.1-4.2
(3.7)
in
width,
packing
5
or
6
spirals
of
the
polar
filament
Neither
mucous
coat
around
the
spores
nor
intercapsular
appendix
visible.
Large
iodinophilous
vacu-
ole
in
the
sporoplasm.
A
nucleotide
sequence
(2006
bp
in
length)
covering
almost
the
full
length
of
the
18S
rDNA,
except
for
the
primer-
flanking
5'
and
3'
termini,
has
been
deposited
in
the
DDBEEMBL/GenBank
databases
(accession
no.
LC228237).
Taxonomic
summary
Type
host:
Cy.
carpio
(Linnaeus,
1758),
common
carp
(Actinopterygii:
Cypriniformes:
Cyprinidae:
Cyprininae)
Site
of
infection:
gill
filament,
intralamellar
tissue
adjacent
to
the
blood
vessel
Type
locality:
Hirakawa
(Kudengawa
River,
a
branch
of
the
Fushinogawa
River),
Yamaguchi
City,
Japan
Specimen
deposited:
hapantotype
no.
21321,
Meguro
Parasitological
Museum,
Tokyo,
Japan
Etymology:
myxospores
of
the
present
species
closely
re-
semble
those
of
M
toyamai
Kudo,
1917,
from
Cy.
carpio
in
Japan,
China,
and
the
USA
with
their
distinctive
spore
morphology
of
one
prominent
and
one
rudimentary
polar
cap-
sule.
Irrespective
of
this
morphological
resemblance
to
one
another,
this
new
species
is,
however,
genetically
distinct
from
M
toyamai.
The
new
species
is
named
with
reference
to
these
fmdings.
Prevalence:
14.3%
incidence
(1
of
7)
Intensity:
several
plasmodia
per
host
Remarks
The
present
new
myxobolid
species
from
the
gill
lamellae
of
Cy.
carpio
in
the
western
part
of
Japan
(Yamaguchi)
has
sim-
ilar
myxospore
morphology
to
M
toyamai
from
the
gill
lamel-
lae
of
Cy.
carpio
in
Japan,
China,
and
the
USA
(Kudo
1917;
Chen
and
Ma
1998;
Griffin
and
Goodwin
2011;
Yokoyama
and
Ogawa
2015).
As
shown
in
Table
4,
myxospores
of
M
paratoyamai
n.
sp.
have
similar
morphometric
values
to
M
toyamai
isolates
except
for
a
different
number
of
polar
fila-
ment
spirals
(5
or
6
vs.
7
or
8).
Genetically,
M
paratoyamai
n.
sp.
has
the
highest
18S
rDNA
nucleotide
sequence
identity
with
M
longisporus
(AY364637)
with
98.61%
(1982/2010)
identi-
ty,
followed
by
M
toyamai
isolates
(DDBJ/EMBL/GenBank
accession
nos.
FJ710802,
HQ338729,
LC010115,
LC010116)
with
identities
ranging
from
97.25%
(1908/1962)
to
97.88%
(1943/1985).
Intraspecific
nucleotide
variation
of
M
toyamai
using
the
four
isolates
from
Japan,
China,
and
the
USA
is
extremely
low,
showing
more
or
less
99.80%
(1948/1952
or
1995/1999)
nucleotide
identity
in
the
18S
rDNA
sequences.
As
shown
later,
the
present
isolate
forms
a
Glade
with
M
longisporus
from
Cy.
rubrofuscus
in
China
rather
than
with
M
toyamai
isolates
(nucleotide
identities
less
than
97.9%
with
this
species),
indicating
an
independent
status
ofM.
paratoyamai
n.
sp.
from
known
myxobolid
species
including
M
toyamai
and
M
longisporus.
Myxobolus
ginbuna
n.
sp.
(Myxosporea:
Bivalvulida)
(Fig.
1d
and
Fig.
2(c,
c')).
Rounded
plasmodia,
0.10-0.19
(0.16)
mm
by
0.09-0.16
(0.12)
mm
[n
=
10],
formed
in
the
intralamellar
tissue
adjacent
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ID
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M
Paras
ite
sp
ec
ies
ob
00
CO
I
S
a,
1
/4
0
°C?
tri
t
7
f
'01
00
tr;
tri
o
'11
1
/4
0
tf
00
0
O
N
1 /4
r,
te-)
1 /4
6
'
1
1
c4;
1 /4
6
14.
5-
16.
5
(
15.
4)
13.
2-
15.
6
(
14.
4)
12.
0-
15.
0
(
13.
5)
15.
4-
18.
6
(
17.
2)
.00
t
r?
t
r?
1
C
hen
an
d
Ma
1998
N
ie
an
d
L
i
1992
co
rei
4
r
1 /4
i
o
W-1
t
1-
1
rV
Parasitol
Res
to
the
vessels
in
the
gill
filaments.
No
accumulation
of
inflam-
matory
cells
was
seen
around
the
plasmodia.
Polysporic
and
synchronized
development
of
spores
Spores
pyriform
in
valvular
view
and
lemon
shaped
in
sutural
view,
measuring
11.7-13.9
(12.5)
in
length,
8.5-9.8
(9.2)
in
width,
and
6.7-7.3
(7.0)
in
thickness
[n
=
15].
Two
pyriform
polar
capsules,
slightly
unequal
in
size,
situated
in
the
anterior
2/3
of
spores,
measuring
6.6-7.6
(7.2)
in
length
by
2.7-3.2
(2.9)
in
width
for
the
bigger
one
and
6.1-7.1
(6.7)
in
length
by
2.4-3.4
(2.9)
in
width
for
the
smaller
one.
The
anterior
end
of
the
polar
capsules
directed
towards
the
apex
of
the
spore,
packing
8
or
9
spirals
of
the
polar
filament.
Thin
rod-like
intercapsular
appendix
extending
from
the
inside
wall
of
the
apex,
and
an
apparent
iodinophilous
vacuole
in
the
sporoplasm.
No
mucous
coat
around
the
spore
A
nucleotide
sequence
(2005
bp
in
length)
covering
almost
the
full
length
of
the
18S
rDNA,
except
for
the
primer-flanking
5'
and
3'
termini,
has
been
deposited
in
the
DDBJ/EMBL/
0
GenBank
databases
(accession
no.
LC228238).
This
nucleo-
tide
sequence
showed
98.95%
(1984/2005)
identity
with
M.
wulii
(HQ613412)
with
no
gaps.
Amongst
other
myxobolids,
M
pyramidis
(HQ613411)
showed
the
highest
nucleotide
identity
of
95.22%
(1891/1986),
but
with
seven
nucleotide
gaps.
Other
species
showed
equivalently
low
or
even
lower
nucleotide
identities
with
several
nucleotide
gaps.
Taxonomic
summary
Type
host:
Ca.
langsdorfii
(Temminck
et
Schlegel,
1846),
Japanese
silver
crucian
carp
(Actinopterygii:
Cypriniformes:
Cyprinidae:
Cyprininae)
Site
of
infection:
gill
filament,
intralamellar
tissue
adjacent
to
the
blood
vessel
Type
locality:
Hirakawa
(Kudengawa
River,
a
branch
of
the
Fushinogawa
River),
Yamaguchi
City,
Japan
Specimen
deposited:
hapantotype
no.
21322,
Meguro
7:3
Parasitological
Museum,
Tokyo,
Japan.
Additional
speci-
mens,
no.
21323
in
the
same
museum.
Etymology:
The
species
is
named
after
the
Japanese
name
u5'
-4
1
for
the
host,
ginbuna.
Prevalence:
28.6%
incidence
(2
of
7)
§
Intensity:
At
least
10
of
20
cysts
and
approximately
18
of
91
cysts
(an
estimation
based
on
counting
10
formalin-fixed
cysts)
in
two
infected
individuals.
These
two
individuals
were
mix-infected
with
M
pyramidis
mentioned
later.
Remarks
The
present
new
myxobolid
species
from
the
gill
lamellae
of
6
Ca.
langsdorfii
in
the
western
part
of
Japan
(Yamaguchi)
has
§
similar
myxospore
shape
but
not
dimensions
(11.7-13.9
1.1.m
vs.
17.6-18.5
µm)
to
M
wulii
Landsberg
et
Lom,
1991
(syn.
Myxosoma
magna
Wu
et
Li,
1986),
which
has
been
reported
4t
Springer
Paras
ite
sp
ec
ies
.4
g
.4
g
.4
g
a
a a
3.
4-
4.
6
(
4.
2)
8
I I
2.
3-
4.
5
(
3.
5)
7
F-4
ID
n
N
ID
00
6
g
;
3
4
o
ID
c5
Nag
ano,
Jap
an
EL• EL•
E
EL• EL•
a
A
O
3.
1-
4.
2
(
3.
7)
5-
6
2.
4-
3.
6
(
2.
8)
7-
8
1
1
1
/
4
0
1
14.
7-
16.
8
(
16.
2)
14.
7-
16.
4
(
15.
4)
13.
5-
15.
8
(
14.
3)
1
13.
2-
15.
6
(
14.
0)
71-
r
1
/
4
1
N
C.
,
N
C.
,
II II
0 0
C
1
/ 4
.
C
1
/ 4
.
I I I I
0
0
Parasitol
Res
to
parasitize
the
gills,
hepatopancreas,
spleen,
and
abdominal
cavity
of
various
cyprinids
including
Ca.
auratus,
Carassius
gibelio,
Hypophthalmichthys
molitrix,
Pelteobagrus
brashnikowi,
and
Opsariichthys
bidens
(Chen
and
Ma
1998;
Zhang
et
al.
2010b).
Myxobolus
macrocapsularis
Reuss,
1906,
recorded
from
the
gills,
kidneys,
intestine,
and
urinary
bladder
of
various
cyprinid
fish
including
Cy.
carpio,
shows
a
superficial
similarity
in
spore
morphology
but
has
a
slimmer
spore
body
and
shorter
polar
capsules
(Table
5).
Based
on
the
18S
rDNA
nucleotide sequence,
M
ginbuna
n.
sp.
shows
closest
genetic
affinity
with
M
wulii
isolates
in
China
with
98.98%
(1560/1576)
to
99.09%
(871/879)
identi-
ties
(HQ613412,
EF690300,
KJ725081,
KP642131,
KP642132),
followed
by
M
pyramidis
(HQ613411)
with
95.12%
(1911/2009)
identity
and
seven
nucleotide
gaps,
then
M
longisporus
(AY364637)
with
94.70%
(1913/2020)
iden-
tity
and
25
nucleotide
gaps.
Three
of
the
Chinese
isolates
of
M
wulii
show
at
least
99.80%
(1525/1528)
identity
of
the
18S
rDNA
sequence
with
each
other,
with
only
a
few
nucleotide
substitutions.
Although
these
fmdings
indicate
a
close
genetic
relationship
between
M
ginbuna
n.
sp.
and
M
wulii,
the
dis-
tinct
dimensional
differences
between
their
spores
separate
them
as
independent
species.
M
pyramidis
(Myxosporea:
Bivalvulida).
(Fig.
le
and
Fig.
2(d,
d')).
Rounded
plasmodia
of
the
present
species
grew
in
the
in-
tralamellar
tissue
adjacent
to
the
vessels
in
the
gill
filaments,
and
no
clear
differences
in
size
from
plasmodia
ofM.
ginbuna
n.
sp.
Plasmodia
contained
many
spores,
but
their
develop-
ment
was
not
synchronized.
Spores
rounded
in
valvular
view
and
lemon
shaped
in
su-
tural
view,
measuring
9.9-11.7
(11.0)
in
length,
9.4-11.1
(10.3)
in
width,
and
6.5-7.4
(6.9)
in
thickness
[n
=
20],
with-
out
mucous
coat
Two
elliptical
polar
capsules,
slightly
un-
equal
in
size,
situated
in
the
anterior
2/3
of
spores,
measuring
5.3-6.9
(5.9)
in
length
by
2.9-4.0
(3.6)
in
width.
The
anterior
end
of
the
polar
capsules
directed
towards
the
apex
of
the
spore,
packing
5-7
spirals
of
the
polar
filament.
No
intercapsular
appendix.
A
visible
iodinophilous
vacuole
in
the
sporoplasm.
A
nucleotide
sequence
(2008
bp
in
length)
covering
almost
the
full
length
of
the
18S
rDNA,
except
for
the
primer-flanking
5'
and
3'
termini,
has
been
deposited
in
the
DDBEEMBL/
GenBank
databases
(accession
no.
LC228239).
This
nucleo-
tide
sequence
showed
99.60%
(1997/2005)
identity
with
a
Chinese
M
pyramidis
isolate
from
the
gills
of
Ca.
gibelio
(HQ613411),
excluding
three
variable
nucleotide
sites,
and
no
nucleotide
gaps
were
present
between
the
two
sequences.
The
next
closest
18S
rDNA
nucleotide
sequence
to
our
Japanese
M.
pyramidis
isolate
was
that
of
M.
wulii
(HQ613412)
with
95.18%
(1914/2011)
identity
and
seven
nu-
cleotide
gaps,
followed
by
M
koi
(KT240127)
showing
94.53%
(1884/1993)
identity
and
seven
nucleotide
gaps,
then
41?!
Springer
.
3
t
,..
00
k
00
0••••
I I
0••••
-.a
o
g
L
ID
N
1
/ 4
0
a,
a,
'
1
1
00
ID
11.
7-
13.
9
(
12.
5)
1
0
Ct
0
Ct
0
Paras
ite
sp
ec
ies
0
1 / 4
1
0•11
011
cV
(7
-1
rn
O
12.
0-
12.
6
(
12.
1)
6.
6-
7.
6
(
7.
2)
C
hen
an
d
Ma
1998
n
=
?
C
hen
an
d
Ma
1998
n
=
?
Parasitol
Res
M
longisporus
(AY364637)
showing
94.30%
(1902/2017)
identity
and
14
nucleotide
gaps.
As
having
been
indicated
by
Liu
et
al.
(2016b),
Myxobolus
sheyangensis
in
the
gill
lamellae
of
Ca.
gibelio
in
China
showed
an
extremely
similar
spore
morphology
in
both
shape
and
dimensions
to
M
pyramidis,
but
lower
identities
of
thel8S
rDNA
nucleotide
sequence
with
M
pyramidis
(95.12%
[1639/1723]
with
five
gaps),
M
koi
(95.36%
[1645/1725]
with
seven
gaps),
M
wulii
(95.02%
[1640/1726]
with
10
gaps),
or
other
Myxobolus
spp.
including
M
tanakai
n.
sp.
and
M
ginbuna
n.
sp.
Taxonomic
summary
Type
host:
C.
langsdorfii
(Temminck
et
Schlegel,
1846),
Japanese
silver
crucian
carp
(Actinopterygii:
Cypriniformes:
Cyprinidae:
Cyprininae)
Site
of
infection:
gill
filament,
intralamellar
tissue
adjacent
to
the
blood
vessel
Type
locality:
Hirakawa
(Kudengawa
River,
a
branch
of
the
Fushinogawa
River),
Yamaguchi
City,
Japan
Specimen
deposited:
hapantotype
no.
21324,
Meguro
Parasitological
Museum,
Tokyo,
Japan.
Additional
speci-
mens,
no.
21323
in
the
same
museum
Prevalence:
85.7%
incidence
(6
of
7)
Intensity:
In
four
Ca.
langsdotfii
individuals
parasitized
solely
with
M
pyramidis,
3-34
(geomean,
10)
cysts
were
found.
Molecular
phylogenetic
analysis
Confined
phylogenetic
trees
based
on
the
18S
rDNA
nucleo-
tide
sequences
ofMyxobolus
spp.
parasitizing
the
gill
lamellae
of
cyprinid
fish
are
presented
in
Fig.
4.
Myxobolids
distribut-
ed
in
different
geographical
areas
(China
and
Japan,
Europe,
and
India)
were
clearly
divided
into
independent
groups.
Regardless
of
the
length
of
the
18S
rDNA
nucleotide
se-
7:3
quences
examined
here,
morphologically
different
Myxobolus
spp.
collected
from
cyprinid
fish
tended
to
form
separate
clades
(Fig.
5).
In
other
words,
four
myxobolid
iso-
lates
from
Cy.
carpio
and
Ca.
langsdorfii
in
the
western
part
of
Japan
(Yamaguchi),
i.e.,
M
tanakai
n.
sp.,
M.
paratoyamai
n.
§
sp.,
M
ginbuna
n.
sp.,
and
M
pyramidis,
formed
well-sup-
ported,
separate
clades
with
M
koi
isolates,
M
toyamai
iso-
lates,
M
wulii
isolates,
and
an
M
pyramidis
isolate,
respec-
tively.
Intriguingly,
the
addition
of
M
tanakai
n.
sp.
to
a
phy-
logenetic
Glade
of
M
koi
isolates
revealed
a
degree
of
corn-
§
plexity
regarding
the
currently
deposited
18S
rDNA
nucleo-
tide
sequences,
i.e.,
the
isolation
of
"M
koi"
from
the
gill
filaments
of
Ca.
auratus
in
Japan
(FJ710800)
from
other
6
M
koi
isolates
from
the
gills
of
Cy.
carpio
in
China
and
the
§
USA
(KT240127,
FJ841887,
and
KJ725077).
In
addition,
the
initial
86%
from
the
5'-terminus
of
the
18S
rDNA
nucleotide
sequence
of
"Myxobolus
sp.
TJA-2014"
from
the
gills
of
4t
Springer
100
r
62
Parasitol
Res
M.
paratoyamai
n.
sp.
ex.
Cy.
carpio:
JP
(LC228237)
75
100
96
100
r
M.
longisporus
ex.
Cy.
rubrofuscus:
CN
(AY364637)
721M.
toyamai
ex.
Ca.
gibelio:
CN
(FJ710802)
M.
toyamai
(syn.
Thelohanellus
toyamai)
ex.
Cy.
carpio:
US
(HQ338729)
100
96
M.
tanakai
n.
sp.
ex.
Cy.
carpio
#6:
JP
(LC228236)
M.
koi
ex.
Ca.
auratus:
JP
(FJ710800)
86
M.
wulii
ex.
Ca.
gibelio:
CN
(EF690300)
100
M.
wulii
ex.
Ca.
gibelio:
CN
(HQ613412)
M.
ginbuna
n.
sp.
ex.
Ca.
langsdorfii:
JP
(LC228238)
100
r
M.
pyramidis
ex.
Ca.
gibelio:
CN
(HQ613411)
L
M.
pyramidis
ex.
Ca.
langsdorfii:
JP
(LC228239)
79
M.
ampullicapsulatus
ex.
Ca.
auratus:
CN
(DQ339482)
M.
ampullicapsulatus
ex.
Ca.
auratus:
CN
(JQ690373)
85
M.
ampullicapsulatus
ex.
Ca.
auratus:
CN
(JQ690361)
100
M.
ampullicapsulatus
ex.
Ca.
auratus:
CN
(KC425224)
M.
honghuensis
(syn.
M.
pharynae)
ex.
Ca.
gibelio:
CN
(JQ726700)
M.
honghuensis
ex.
Ca.
gibelio:
CN
(HM188545)
84
M.
honghuensis
ex.
Ca.
auratus:
CN
(KJ725074)
M.
honghuensis
ex.
Ca.
gibelio:
CN
(JF340216)
M.
tambroides
ex.
Tor
tambroides:
MY
(JX028236)
M.
alavarezae
ex.
Leuciscus
idus:
HU
(FJ716096)
M.
toyamai
ex.
Cy.
carpio:
JP
(LC010115)
100
M.
koi
ex.
Cy.
carpio:
US
(FJ841887)
M.
tanakai
n.
sp.
ex.
Cy.
carpio
#2:
JP
(LC228235)
91
95
100
1
100
0.08
substitutions/site
M.
obesus
ex.
Alburnus
alburnus:
HU
(AY325286)
M.
hungaricus
ex.
Abramis
brama:
HU
(AF448444)
100
M.
intimus
ex.
Leuciscus
idus:
HU
(JX390689)
L
M
.
intimus
ex.
Leuciscus
idus:
HU
(FJ716098)
73
M.
intimus
ex.
Rutilus
rutilus:
HU
(AY325285)
M.
kalavatiae
ex.
Cirrhinus
cirrhosus:
IN
(KM029973)
M.
basuhaldari
ex.
Labeo
rohita:
IN
(KM029974)
Fig.
4
ML phylogenetic
tree
based
on
the
18S
rDNA
sequences
of
representative
Myxobolus
spp.
recorded
in
cyprinid
fish
(31
isolates,
1495
characters),
including
isolates
collected
in
the
present
study
(highlighted
with
gray
background).
Species
name
is
followed
by
the
Cirrhinus
mrigala
in
India
(KF796619)
corresponded
well
with
the
18S
rDNA
sequences
of
M
koi
isolates
from
China
and
the
USA;
however,
the
remaining
14%
of
the
former
sequence
was
completely
distinct
from
any
organisms
includ-
ing
myxosporeans,
suggesting
a
contaminated
nucleotide
se-
quence
from
an
unknown
source.
Discussion
Molecular
data
combined
with
spore
morphology,
host
and
organ
specificity,
and
tissue
tropism
are
now
strongly
recom-
mended
for
the
taxonomic
identification
of
Myxobolus
spp.
and
other
myxozoan
parasites.
In
accordance
with
this
taxo-
nomic
strategy,
we
endeavored
in
the
present
study
to
identify
four
Myxobolus
spp.
from
the
gill
filaments
(secondary
gill
lamellae)
of
common
carp
and
Japanese
silver
crucian
carp
in
the
natural
freshwater
of
western
Japan.
Our
investigations
determined
the
following:
three
new
species,
M
tanakai
n.
sp.
and
M
paratoyamai
n.
sp.
from
Cy.
carpio,
and
M
ginbuna
n.
name
of
the
fish
host,
country
of
collection,
and
DDBJ/EMBL/
GenBank
accession
number.
Abbreviations
of
country
names:
CN
People's
Republic
of
China,
HU
Hungary,
IN
India,
JP
Japan,
MY
Malaysia,
US
United
States
of
America
sp.
from
Ca.
langsdotfii;
and
M
pyramidis
from
the
latter
fish
host
as
a
new
host
and
geographical
distribution
record.
As
shown
in
Figs.
4
and
5,
myxobolids
with
spores
showing
a
morphological
similarity
(for
this
instance,
only
shape
but
not
dimensions)
tended
to
form
a
Glade:
M
tanakai
n.
sp.
with
M
koi;
M
paratoyamai
n.
sp.
with
M
toyamai;
M
ginbuna
n.
sp.
with
M
wulii;
and
Chinese
and
Japanese
isolates
of
M
pyramidis.
This
finding
has
also
been
observed
in
other
studies
where
limited
numbers
of
Myxobolus
spp.
with
the
same
tissue
tropism
from
closely
related
host
fish
were
phy-
logenetically
analyzed
(Eszterbauer
2004;
Zhao
et
al.
2008;
Zhang
et
al.
2010a;
Liu
et
al.
2016a).
The
differentiation
of
M
tanakai
n.
sp.
from
M
koi
based
on
distinct
myxospore
morphometrics
(bigger
spore
length)
and
substantial
nucleotide
substitutions
of
the
18S
rDNA
se-
quence
(low
identities
ranging
from
97.76
to
98.56%
with
one
to
seven
nucleotide gaps)
revealed
a
paraphyletic
status
of
the
registered
nucleotide
sequences
of
M.
koi,
i.e.,
isolates
from
Cy.
carpio
in
China
and
the
USA
versus
an
isolate
from
Ca.
auratus
in
Japan
(Fig.
5).
Between
these
two
4?!
Springer
Parasitol
Res
88
88
88
M.
paratoyamai
n.
sp.
ex.
Cy.
carpio:
JP
(LC228237)
90
M
koi
ex.
Cy.
carpio:
CN
(KJ725077)
M.
koi
(syn.
Myxobolus
sp.
TJA-2014)
ex.
Cirrhinus
mrigala:
IN
(KF796619)
97
M.
tanakai
n.
sp.
ex.
Cy.
carpio
#2:
JP
(LC228235)
79
4
4-
M.
tanakai
n.
sp.
ex.
Cy.
carpio
#6:
JP
(LC228236)
M.
ginbuna
n.
sp.
ex.
Ca.
langsdorfii:
JP
(LC228238)
M.
wulii
actinospore
ex.
Branchiura
sowerbyi:
CN
(KP642131)
M.
wulii
ex.
Ca.
gibelio:
CN
(HQ613412)
55
84
M.
wulii
actinospore
ex.
Branchiura
sowerbyi:
CN
(KP642132)
-
M.
wulii
ex.
Ca.
gibelio:
CN
(KJ725081)
M.
wulii
ex.
Ca.
gibelio:
CN
(EF690300)
a
9
M.
longisporus
ex.
Cy.
rubrofuscus:
CN
(AY364637)
M.
toyamai
(syn.
Thelohanellus
toyamai)
ex.
Cy.
carpio:
US
(HQ338729)
[
95
M.
toyamai
ex.
Ca.
gibelio:
CN
(FJ710802)
M.
toyamai
ex.
Cy.
carpio:
JP
(LC0101
15)
M.
koi
ex.
Cy.
carpio:
US
(F
J841
887)
M.
koi
ex.
Cy.
carpio:
CN
(KT240127)
97
M.
koi
ex.
Ca.
auratus:
JP
(FJ710800)
99
0.04
substitutions/site
97
7
M.
pyramidis
ex.
Ca.
langsdorfii:
JP
(LC228239)
L
M.
pyramidis
ex.
Ca.
gibelio:
CN
(HQ613411)
b
100
100
M.
paratoyamai
n.
sp.
ex.
Cy.
carpio:
JP
(LC228237)
M.
longisporus
ex.
Cy.
rubrofuscus:
CN
(AY364637)
71
M.
toyamai
ex.
Ca.
gibelio:
CN
(FJ710802)
t
100
M.
toyamai
(syn.
Thelohanellus
toyamai)
ex.
Cy.
carpio:
US
(HQ338729)
M.
toyamai
ex.
Cy.
carpio:
JP
(LC010115)
M.
tanakai
n.
sp.
ex.
Cy.
carpio
#2:
JP
(LC228235)
M.
tanakai
n.
sp.
ex.
Cy.
carpio
#6:
JP
(LC228236)
100
1—
M.
ginbuna
n.
sp.
ex.
Ca.
langsdorfii:
JP
(LC228238)
1
M.
wulii
ex.
Ca.
gibelio:
CN
(HQ613412)
73
r
--
92
M.
koi
ex.
Cy.
carpio:
US
(FJ841887)
9
M.
koi
ex.
Cy.
carpio:
CN
(KT240127)
56
M.
koi
ex.
Ca.
auratus:
JP
(FJ710800)
96
99
100
M.
pyramidis
ex.
Ca.
langsdorfii:
JP
(LC228239)
C
M.
pyramidis
ex.
Ca.
gibelio:
CN
(HQ613411)
0.04
substitutions/site
Fig.
5
Unrooted
ML
phylogenetic
trees
based
on
the
18S
rDNA
sequences
of
representative
Myxobolus
spp.
from
cyprinid
fish,
including
isolates
collected
in
the
present
study
(highlighted
with
gray
background):
a
all
currently
recorded
isolates
of
M
koi,
M
toyamai,
genetic
groups
of
isolates,
nucleotide
sequences
showed
96.99%
(1900/1959)
to
97.35%
(1876/1927)
identities
with
six
or
seven
nucleotide
gaps.
M
koi
was
originally
described
by
Kudo
(1919)
using
specimens
from
cysts,
measuring
0.23
mm
in
diameter,
locat-
ed
in
the
connective
tissue
of
the
gill
filaments
of
Cy.
carpio
in
Tokyo,
Japan.
Later,
however,
Nakai
(1926)
and
Yokoyama
et
al.
(1997)
recorded
M
koi
spores
from
two
different-sized
cysts
(less
than
0.18
mm
in
the
secondary
gill
lamellae,
or
1-
7
mm
in
diameter
in
the
gill
filaments),
which
occurred
prev-
alently
during
different
months
(August
vs.
July,
respective-
ly).
Additionally,
two
M
koi
spore
morphotypes,
thicker
and
thinner
ones,
have
been
simultaneously
recorded
(Yokoyama
et
al.
1997;
Camus
and
Griffin
2010).
A
further
complication
M
wulii,
and
M
pyramidis
(20
isolates,
870
characters)
and
b
selected
M
koi,
M.
toyamai,
M
wulii,
and
M
pyramidis
isolates
with
long
nucleotide
sequences
(14
isolates,
1879
characters).
See
Fig.
4
legend
for
the
list
of
abbreviation
used
is
that
with
the
exception
of
one
nucleotide
sequence
for
M
koi
from
large
cysts
in
the
gill
filaments
of
Cy.
carpio
(FJ841887;
Camus
and
Griffm
2010),
three
nucleotide
se-
quences
of
a
Japanese
isolate
(FJ710800)
and
two
Chinese
isolates
(KT240127,
KJ725077)
deposited
in
the
DDBEEMBL/GenBank
databases
do
not
provide
any
de-
tailed
biological
information
on
the
myxobolid
isolates,
such
as
the
morphological
characters
of
the
myxospores
used
for
the
rDNA
amplification
and
sequencing.
In
addition,
the
de-
posited
sequence
of
"Myxobolus
sp.
TJA-2014"
from
the
gills
of
Cirrhinus
mrigala
in
India
(KF796619),
which
might
be
a
composed
sequence
of
two
biological
sources,
could
be
a
partial
18S
rDNA
sequence
of
"M
koi,"
because
the
initial
86%
from
the
5'-terminus
is
highly
similar
to
those
of
M
koi
4t
Springer
Parasitol
Res
isolates
from
China
and
the
USA.
When
considering
the
cur-
rent
status
of
the
genetic
characterization
ofM.
koi,
we
should
carefully
contemplate
the
following
concerns:
(1)
at
present,
no
validated
18S
rDNA
nucleotide
sequences
for
M.
koi
have
been
deposited
in
the
DDBJ/EMBL/GenBank
databases
and
(2)
"M
koi"
at
present
could
be
a
composed
species
of
at
least
two
different
myxobolids
parasitizing
different
tissues
(gill
filament
vs.
gill
lamella),
forming
two
different
cysts
(macro-
scopic
vs.
microscopic),
and
showing
different
spore
morphotypes
(thicker
and
thinner).
Similar
situations
of
taxo-
nomic
confusion
have
previously
been
experienced
in
the
field
of
myxobolid
research,
e.g.,
M.
rotundus
vs.
M
bramae
(Molnar
and
Szekely
1999;
Molnar
et
al.
2009),
M
pyramidis
vs.
M
sheyangensis
(Liu
et
al.
2016b),
etc.
Molnar
(2011)
has
emphasized
the
importance
of
using
well-identified
samples
collected
from
adequate
organs
of
the
original
hosts
for
the
specified
taxa
when
depositing
nu-
cleotide
sequences
in
DNA
databases.
A
new
elongated
pyriform
myxobolid
species
with
one
prominent
and
one
rudimentary
polar
capsule,
M
paratoyamai
n.
sp.
from
Cy.
carpio,
has
an
apparent
phenotypical
similarity
with
M
toyamai
recorded
from
Cy.
carpio
and
Ca.
gibelio
with
an
Asian
origin.
Surprisingly,
however,
the
present
new
species
showed
a
close
genetic
relationship
with
not
only
M
toyamai
but
also
M
longisporus
with
two
equally
well-developed
polar
capsules.
Its
nucleotide
identity
with
the
former
species
was
slightly
lower
(97.25-97.88%)
than
with
the
latter
species
(98.61%).
Upon
consideration
that
the
known
intraspecific
18S
rDNA
nucleotide
variation
of
M
toyamai
isolated
in
Japan,
China,
and
the
USA
is
extremely
low
(nucleotide
identi-
ties
around
99.80%),
the
present
new
isolate
from
Cy.
carpio
in
the
natural
freshwater
of
Japan
should
be
regarded
as
a
distinct
species
from
all
other
known
myxobolid
species.
Although
M
ginbuna
n.
sp.
from
the
gill
lamellae
of
Ca.
langsdorfii
in
Japan
is
well
differentiated
from
M
wulii,
which
parasitizes
the
gills,
hepatopancreas,
spleen,
and
abdominal
cavity
of
various
cyprinids
including
Ca.
auratus
and
Ca.
gibelio
(Chen
and
Ma
1998;
Zhang
et
al.
2010b),
these
two
species
formed
a
well-supported
Glade
in
the
ML
phylogenetic
tree
based
on
the
18S
rDNA
nucleotide
sequences
(Figs.
4
and
5).
Parasitism
of
M
wulii
in
goldfish
Ca.
auratus
in
Japan
demonstrates
two
types,
i.e.,
limited
to
the
gill
lamellae
or
lim-
ited
to
the
hepatopancreas,
and
the
18S
rDNA
nucleotide
se-
quence
identity
of
these
two
infection
types
ofM.
wulii
in
Japan
is
99.87%
(1574/1576)
according
to
Zhang
et
al.
(2010b).
Excluding
the
"M
wulii"
isolates
from
Hypophthalmichthys
Pelteobagrus
brashnikowi,
and
Opsariichthys
bidens
as
its
hosts,
which
have
not
been
examined
genetically
(Chen
and
Ma
1998),
M
wulii
from
either
the
gills
or
hepatopancreas,
or
both
organs
of
Ca.
auratus
and
Ca.
gibelio,
has
been
vali-
dated
to
be
a
single
species
with
fluctuating
organ
preference,
probably
due
to
different
modes
of
infection
from
the
alternate
invertebrate
hosts
(Zhang
et
al.
2010b).
The
nucleotide
sequence
of
the
18S
rDNA
of
the
present
Japanese
isolate
of
M
pyramidis
in
the
gill
lamellae
of
Ca.
langsdorfii
showed
a
high
identity
(99.60%)
with
that
of
a
Chinese
isolate
of
M
pyramidis
from
the
gill
filaments
of
Ca.
gibelio
(HQ613411),
i.e.,
eight
nucleotide
substitutions
over
a
length
of
2005
bp.
As
reported
by
Liu
et
al.
(2016b),
M
sheyangensis
parasitizing
the
gill
lamellae
of
Ca.
gibelio
in
China
showed
a
highly
similar
spore
morphology
to
M
pyramidis,
but
these
two
species
could
be
differentiated
by
molecular
analyses
based
on
a
low
nucleotide
identity
of
95.12%
with
five
nucleotide
gaps.
The
highest
identity
of
the
18S
rDNA
nucleotide
sequence
was
seen
with
M
koi
(95.36%
[1645/1725]),
which
was
deemed
low
enough
to
separate
these
two
species
in
addition
to
clear morphological
differ-
ences
between
their
spores.
The
present
report
is
a
new
host
and
geographical
distribution
record
for
M.
pyramidis.
As
mentioned
above,
Myxobolus
spp.
having
identical
spore
morphologies
and
similar
host
and
organ
specificities
have
been
successfully
differentiated
by
tissue
tropism
and
genetic
charac-
terization
of
the
18S
rDNA
nucleotide
sequences.
However,
one
exception
is
muscle-parasitizing
Myxobolus
spp.
such
as
Myxobolus
pseudodispar
Gorbunova,
1936,
which
parasitizes
several
cyprinid
fish
(rudd
Scardinius
erythrophthalmus,
com-
mon
bleak
Albumus
albumus,
common
bream
Abramis
brama,
white
bream
Blicca
bjoerkna,
and
common
roach
Rutilus
rutilus)
and
exhibits
high
intraspecific
nucleotide
variation,
up
to
5%
differences,
in
the
18S
rDNA
sequences
(Forro
and
Eszterbauer
2016).
At
present,
no
such
high
intraspecific
genetic
variations
have
been
observed
in
other
Myxobolus
spp.,
even
in
other
muscle-parasitizing
myxobolids
such
as
Myxobolus
cyprini
and
Myxobolus
musculi.
Following
the
unequivocal
demonstration
by
Wolf
and
Markiw
(1984)
that
the
life
cycle
of
Myxobolus
cerebralis
involves
alternating
fish
and
annelid
hosts,
those
of
approxi-
mately
50
myxozoan
species
(of
more
than
2200
nominal
taxa)
containing
22
Myxobolus
spp.
have
been
elucidated
(Yokoyama
et
al.
2012;
Eszterbauer
et
al.
2015).
Regarding
Myxobolus
spp.
parasitizing cyprinid
fish
of
the
genera
Carassius
and
Cyprinus,
at
least
90
nominal
species
as
men-
tioned
above,
the
actinospore
stages
have
been
demonstrated
for
only
three
species,
Myxobolus
cultus,
Myxobolus
dispar,
and
Myxobolus
lentisuturalis
(Yokoyama
et
al.
1995;
Molnar
et
al.
1999;
Holzer
et
al.
2004;
Eszterbauer
et
al.
2006;
Caffara
et
al.
2009);
all
are
raabeia-type
actinospores
grown
in
oligo-
chaetes
Branchiura
sowerbyi
or
Tubifex
tubifex.
To
demon-
strate
the
life
cycle
of
myxozoans,
either
experimental
studies
or
genetic
homology
research
through
some
DNA
nucleotide
sequence
characterization
is
applicable.
In
this
sense,
not
only
is
phenotypical
characterization
critical
but
also
ge-
netic
characterization,
particularly
nucleotide
sequences
of
the
18S
rDNA
when
considering
a
feasible
comparison
with
multiple
myxosporean
congeners
to
speculate
the
oth-
er
life
stage
of
a
species.
41?!
Springer
Parasitol
Res
For
a
better
understanding
of
the
great
biodiversity
of
the
genus
Myxobolus
or
other
myxosporean
species,
further
re-
search
on
both
unknown
and
known
species
is
required.
As
evidenced
here,
although
major
advances
have
been
made,
we
are
still
a
long
way
from
fully
understanding
the
true
biodi-
versity
and
nature
of
myxozoans.
Acknowledgements
We
are
indebted
to
Minoru
Tanaka,
Fushinogawa
Fisheries
Cooperative
Association,
for
his
kind
help
with
the
collection
of
fish
samples.
This
study
was
supported
in
part
by
Grant-in-Aid
for
Scientific
Research
2015
and
2017
from
The
Towa
Foundation
for
Food
Science
and
Research
(HS),
Grant-in-Aid
for
International
Collaboration
Research
in
Asia
2016
from
the
Heiwa
Nakajima
Foundation
(HS),
and
JSPS
ICAICENHI
Grant
Number
15K07722.
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