Iridovirus infection in terrestrial isopods from Sicily (Italy)

Abstract

During our researches on systematics and ecology of terrestrial isopods, carried out in western Sicily, some specimens showing a blue-purple coloration were collected; they belonged to four species: Armadillidium decorum Brandt, 1833, Trichoniscus panormidensis Montesanto et al., 2011, Philoscia affinis Verhoeff, 1908, Porcellio siculoccidentalis Viglianisi et al., 1992. We hypothesized that such coloration could be due, as reported in literature, to characteristic paracrystalline arrays of virions inside the tissues of blue colored specimens. Ultrastructural observations by transmission electron microscopy, on tissues of A. decorum, showed the presence of electron-dense viral particles, with a diameter of nearly 0.12μm. Dual-axis tomography, performed on specimens of A. decorum, evidenced an icosahedral structure of viral particles matching with that of Isopod Iridescent Virus (IIV). Molecular analysis, on 254bp portion of the major capsid protein (MCP) gene, allowed to place the virus into IIV-31 group, already known for other oniscidean species. The symptoms of infected individuals and the course of the disease were followed in laboratory, indicating similarities with other studies on Isopod Iridoviruses. Moreover, some notes on reproduction of infected ovigerous females are reported. Our data support unequivocal and direct evidences for the first case of IIV infection in terrestrial isopods reported in Italy.

Full text

Tissue
and
Cell
45 (2013) 321–
327
Contents
lists
available
at
SciVerse
ScienceDirect
Tissue
and
Cell
jou
rn
al
hom
epage:
www.elsevier.com/locate/ti
ce
Iridovirus
infection
in
terrestrial
isopods
from
Sicily
(Italy)
Pietro
Lupettia,
Giuseppe
Montesantob,∗,
Silvia
Ciolfia,
Laura
Marria,
Mariangela
Gentilea,
Eugenio
Paccagninia,
Bianca
Maria
Lombardob
aDipartimento
di
Scienze
della
Vita,
Università
di
Siena,
Siena,
Italy
bDipartimento
di
Scienze
Biologiche,
Geologiche
e
Ambientali,
Università
degli
Studi
di
Catania,
Catania,
Italy
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
23
April
2013
Received
in
revised
form
2
May
2013
Accepted
2
May
2013
Available online 10 June 2013
Keywords:
Iridovirus
Crustacea
Isopoda
Oniscidea
Infection
Electron
microscopy
DNA
sequencing
a
b
s
t
r
a
c
t
During
our
researches
on
systematics
and
ecology
of
terrestrial
isopods,
carried
out
in
western
Sicily,
some
specimens
showing
a
blue–purple
coloration
were
collected;
they
belonged
to
four
species:
Armadillidium
decorum
Brandt,
1833,
Trichoniscus
panormidensis
Montesanto
et
al.,
2011,
Philoscia
affinis
Verhoeff,
1908,
Porcellio
siculoccidentalis
Viglianisi
et
al.,
1992.
We
hypothesized
that
such
coloration
could
be
due,
as
reported
in
literature,
to
characteristic
paracrystalline
arrays
of
virions
inside
the
tissues
of
blue
colored
specimens.
Ultrastructural
observations
by
transmission
electron
microscopy,
on
tissues
of
A.
decorum,
showed
the
presence
of
electron-dense
viral
particles,
with
a
diameter
of
nearly
0.12
␮m.
Dual-axis
tomography,
performed
on
specimens
of
A.
decorum,
evidenced
an
icosahedral
structure
of
viral
particles
matching
with
that
of
Isopod
Iridescent
Virus
(IIV).
Molecular
analysis,
on
254
bp
portion
of
the
major
capsid
protein
(MCP)
gene,
allowed
to
place
the
virus
into
IIV-31
group,
already
known
for
other
oniscidean
species.
The
symptoms
of
infected
individuals
and
the
course
of
the
disease
were
followed
in
laboratory,
indicating
similarities
with
other
studies
on
Isopod
Iri-
doviruses.
Moreover,
some
notes
on
reproduction
of
infected
ovigerous
females
are
reported.
Our
data
support
unequivocal
and
direct
evidences
for
the
first
case
of
IIV
infection
in
terrestrial
isopods
reported
in
Italy.
© 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
The
type-species
of
Iridovirus
was
firstly
isolated
by
Xeros
(1954)
from
the
crane
fly
Tipula
paludosa
Meigen
1835.
Afterwards,
different
Iridoviruses
have
been
recorded
for
other
Insects,
mainly
Diptera,
Coleoptera
and
Lepidoptera
(Kelly
and
Robertson,
1973;
Carey
et
al.,
1978).
In
the
terrestrial
isopods,
the
virus
was
firstly
isolated
in
1980
(Federici,
1980;
Cole
and
Morris,
1980)
and
named
Isopod
Iridescent
Iridovirus
(IIV).
The
most
evident
feature
of
the
infection
is
a
clear
blue–purple
coloration,
distributed
on
the
body
and
on
the
appendages
of
infected
specimens.
Nowadays
is
well-
known
that
the
coloration
is
produced
by
paracrystalline
arrays
of
virions
inside
the
parasitized
cells
(Federici,
1984).
According
to
Federici
(1984)
the
presence
of
purple–blue
color
can
be
taken
as
a
quite
definitive
evidence
of
Iridovirus
infection.
Before
Iri-
doviruses
were
identified,
the
distinct
purple
to
blue
iridescent
coloration
produced
in
infected
individuals
was
reported
for
Ligid-
ium
hypnorum
(Cuvier,
1792)
and
Philoscia
muscorum
(Scopoli,
1763)
in
France
(Lereboullet,
1843,
1853;
Legrand,
1948;
Vandel,
∗Corresponding
author.
Tel.:
+39
095
7306048.
E-mail
addresses:
g.montesanto@unict.it,
gipo.montesanto@gmail.com
(G.
Montesanto).
1962),
and
for
Trichoniscus
pusillus
(Brandt,
1833)
in
England
(Standen,
1917)
and,
many
other
samples
of
infected
Oniscidean
were
reported
from
several
locations
in
Europe
and
North
Amer-
ica
(Williams,
2008).
A
complete
list
of
species
and
“varieties”
described
in
older
literature
which
are
certainly
infected
with
Iridovirus,
together
with
a
list
of
terrestrial
isopods
carrying
Iri-
dovirus
infection,
is
reported
by
Wijnhoven
and
Berg
(1999)
and
Williams
(2008).
The
authors
cited
a
total
of
19
species,
includ-
ing
newly
reported
samples.
Recently,
eight
species
of
terrestrial
isopods
from
Japan
have
been
found
infected
with
IIV
(Karasawa
et
al.,
2012);
two
of
them
are
new
reports
of
Iridovirus
infec-
tion.
In
a
wider
context
of
researches
on
ecology
and
systematics
of
terrestrial
isopods,
recently
carried
out
on
Mt.
San
Giuliano
(Erice)
in
western
Sicily,
new
species
and
new
records
of
terrestrial
isopods
were
reported
(Montesanto
et
al.,
2011).
During
those
samplings
some
specimens
belonging
to
four
species:
Armadillidium
decorum
Brandt,
1833,
Trichoniscus
panormidensis
Montesanto
et
al.,
2011,
Philoscia
affinis
Verhoeff,
1908,
Porcellio
siculoccidentalis
Viglian-
isi,
et
al.,
1992,
have
been
collected
showing
a
clear
blue–purple
coloration,
distributed
on
the
body
and
on
the
appendages.
The
aim
of
this
study
was
verifying
the
presence
of
Iridoviridae
in
terrestrial
isopods
from
Sicily,
and
to
compare
the
detected
Isopod
Iridescent
Virus
(IIV)
with
other
IIV
already
known
for
0040-8166/$
–
see
front
matter ©
2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tice.2013.05.001

322 P.
Lupetti
et
al.
/
Tissue
and
Cell
45 (2013) 321–
327
Fig.
1.
Sampling
site
in
western
Sicily
(Italy);
WGS84
coordinates:
38◦0213N,
12◦3535E.
Oniscidean
species
but
not
yet
detected
in
Italy.
Another
goal
of
this
study
was
also
to
briefly
describe
the
symptoms
and
the
course
of
the
disease
in
lab
reared
isopods
specimens.
Moreover,
some
facts
about
the
biology
of
reproduction
were
taken
into
account,
trying
to
asses
a
notation
by
Hess
and
Poinar
(1985):
“it
would
be
interesting
to
determine
if
these
individuals
are
infected
with
an
Iridovirus
and
if
so,
if
they
are
still
able
to
mate
and
reproduce
before
being
destroyed
by
the
disease.
If
so,
this
would
represent
a
remarkable
tolerance
to
the
disease”.
2.
Materials
and
methods
2.1.
Terrestrial
isopods
sampling
and
breeding
The
animals
were
collected,
using
entomological
forceps,
in
the
litter
and
under
stones
of
holm-oak
woods
at
the
top
of
Mount
San
Giuliano,
in
Trapani
province
(western
Sicily).
Sampling
site
is
indicated
in
Fig.
1.
In
laboratory,
appropriate
breedings
were
car-
ried
out
in
plastic
boxes
of
35
cm
×
60
cm,
with
soil
taken
from
the
sampling
sites
and
previously
sterilized.
The
specimens
were
fed
with
sterilized
and
rehydrated
plate-tree
leaves,
and
with
slices
of
potatoes
and
carrots.
The
breeding
substrate
was
periodically
moistened
by
nebulized
water,
and
kept
at
constant
temperature
of
20 ◦C
(±1◦C).
2.2.
Electron
microscopy
Nervous
tissue
of
A.
decorum,
T.
panormidensis,
and
P.
siculoc-
cidentalis
dissected
in
0.1
M
phosphate
buffer
(PB),
pH
7.2,
was
fixed
for
1
h
at
4◦C
in
2.5%
glutaraldehyde
in
PB.
Samples
were
then
rinsed
in
PB
and
postfixed
for
1
h
in
1%
osmium
tetroxide
water
solution.
Tissues
were
then
rinsed
in
PB,
dehydrated
in
ascending
alcohol
series
and
embedded
in
Epon-Araldite
epoxy
resin.
Ultrathin
sections
(60–70
nm)
obtained
with
a
Reichert
Ultracut
IIE
ultramicrotome
were
stained
with
uranyl
acetate
and
lead
citrate
according
to
Reynolds
(1963).
Thin
sections
were
then
observed
and
imaged
by
a
Philips
CM10
transmis-
sion
electron
microscope
operating
at
an
electron
accelerating
voltage
of
80
kV.
From
the
resin-embedded
samples
were
also
obtained
120–250
nm
thick
sections;
after
the
above
described
staining
procedure,
they
were
treated
on
both
sides
with
a
solu-
tion
of
colloidal
gold
particles
of
10
nm
and
examined
using
a
Philips
CM
200
FEG
TEM
equipped
with
a
digital
camera
(TVIPS
TemCam-F224HD).
The
tomographic
series
were
acquired
by
a
computer
assisted
routine
with
the
software
TVIPS
EMMENU4
and
EMTool
controlling
TEM
settings,
stage
movements,
magni-
fications
and
defocus
along
image
collection.
Tomographic
series
were
acquired
from
regions
of
the
sample
that
remained
vis-
ible
at
least
throughout
a
tilting
excursion
ranging
from
+55◦
to
−55◦.
Images
were
collected
at
incremental
tilting
steps
of
1◦and
with
dual
axis
tilting
strategy.
Tomograms
were
gener-
ated
from
dual
axis
tomographic
series
of
images
using
the
open
source
software
IMOD
(http://bio3d.colorado.edu/imod/).
All
ren-
derings
were
produced
by
the
open
source
software
Chimera
(http://www.cgl.ucsf.edu/chimera/).
2.3.
Molecular
analysis
Genomic
DNA
was
extracted
from
blue
colored
specimens
of
A.
decorum,
T.
panormidensis,
and
P.
siculoccidentalis
using
the
Wizard®Genomic
DNA
Purification
kit
(Promega).
To
amplify
a
region
of
254
bp
of
the
major
capsid
protein
(MCP)
gene,
specific
primers
were
designed
on
the
basis
of
the
highly
con-
served
regions
of
sequences
identified
in
other
isopods,
especially
those
from
A.
vulgare
and
P.
scaber
(GenBank
accession
num-
bers:
AF042337.1
and
AF297060.1,
respectively).
The
primers
were:
IIVfor:
5-ATTGGTAACATTTCGGCTCTTATC-3;
IIVrev:
5-
GCACCAACTACAGGTACAACAGAC-3.
About
100
ng
of
genomic
DNA
was
used
in
each
PCR
reaction
following
the
protocol
of
Webby
and
Kalmakoff
(1998).
A
contamination
control
(reaction
without
tem-
plate)
and
a
template
control
(reaction
with
primers
for
the
actin
gene)
were
set
up
for
each
amplification
experiment.
PCR
products
were
visualized
on
a
1.5%
agarose
gel
in
TAE
1%,
and
the
expected
band
of
254
bp,
corresponding
to
a
portion
of
the
MCP
gene,
was
purified
using
the
Wizard®SV
Gel
and
PCR
Clean-Up
System
kit
(Promega)
and
sequenced
on
both
strands
(Bio-Fab
Research).
The
sequences
have
been
submitted
to
GenBank
database
under
the
accession
numbers:
JX847599
(T.
panormidensis);
JX847600
(A.
decorum);
JX847601
(P.
siculoccidentalis).
2.4.
Data
analysis
The
254
bp
nucleotide
and
the
corresponding
deduced
amino
acid
sequences,
obtained
by
PCR
from
the
three
Sicilian
iso-
pod
species,
were
compared
with
other
MCP
gene
sequences
by
means
of
BLASTn
and
BLASTp
tools
at
NCBI
website
(http://www.ncbi.nlm.nih.gov/).
Nucleotide
and
deduced
amino
acid
sequences
were
aligned
using
ClustalW
with
default
sett-
ings
(http://www.ebi.ac.uk/Tools/msa/clustalw2/).
Phylogenetic
tree
was
inferred
by
the
Neighbor-Joining
(NJ)
method,
performing
a
1000-replicates
bootstrap
test,
using
MEGA
version
5
(Tamura
et
al.,
2011).
The
list
of
Iridoviruses
and
host
species
involved
in
this
study,
together
with
sites
of
isolation
and
Genbank
accession
numbers,
are
reported
in
Table
1.

P.
Lupetti
et
al.
/
Tissue
and
Cell
45 (2013) 321–
327 323
Table
1
List
of
Iridoviruses,
host
species,
collection
sites
and
GenBank
accession
numbers
used
in
this
study.
Virus
type
Host
of
isolation
Sites
of
isolation
Accession
no.
IIV-31
Trichoniscus
panormidensis
Erice,
Sicily,
Italy
JX847599
IIV-31
Armadillidium
decorum
Erice,
Sicily,
Italy
JX847600
IIV-31
Porcellio
siculoccidentalis
Erice,
Sicily,
Italy
JX847601
IIV-31
Armadillidium
vulgare
Inzai,
Chiba,
Japan
AB686457
IIV-31
Armadillidium
vulgare
Hitachi,
Ibaraki
1,
Japan
AB686459
IIV-31
Armadillidium
vulgare
Hitachi,
Ibaraki
4,
Japan
AB686463
IIV-31
Porcellio
scaber
Hitachi,
Ibaraki
2,
Japan
AB686460
IIV-31
Porcellio
scaber Hitachi,
Ibaraki
3,
Japan AB686461
IIV-31
Burmoniscus
kathmandia
Naha,
Okinawa,
Japan
AB686462
IIV-31
Ligidium
koreanum
Chikuzen,
Fukukoka,
Japan
AB686458
IIV-31
Armadillidium
vulgare
USA
AAC97170
IIV-1
Tipula
paludosa
UK
AAA46245
IIV-2
Sericesthis
pruinosa
Australia
AAC97168
IIV-3
Aedes
taeniorhynchus USA
ABF82044
IIV-6
Chilo
suppressalis
Japan
AAK82135
IIV-9
Wiseana
cervinata
New
Zeland
AAB82568
IIV-16
Costelytra
zealandica
New
Zeland
AAB82569
IIV-22
Simulium
sp. Wales
AAA66585
IIV-23
Heteronynchus
arator
South
Africa
AAC97175
IIV-24
Apis
cerana
India
AAC97173
IIV-29
Tenebrio
molitor
USA
AAC97172
IIV-30
Helicoverpa
armigera
USA
AAC97169
AeIV
Aecetes
erythraeus
Madagascar
EF467167
LDCV-China
Paralichthys
olivaceus
China
AAS47819
FV-3
Rana
pipiens USA
AAB01722
IIV:
Invertebrate
Iridovirus;
AeIV:
Aecetes
erythraeus
Iridovirus;
LCDV:
Lymphocistis
Disease
Virus;
FV:
Frog
Virus.
3.
Results
and
discussion
3.1.
Observations
in
laboratory
The
course
of
the
disease
was
monitored
by
daily
visual
obser-
vations
on
the
laboratory
rearings
of
infected
A.
decorum
(12
specimens)
and
P.
siculoccidentalis
(5
specimens).
Under
labora-
tory
rearing
conditions,
the
discoloration
normally
appeared
at
the
ventral
side
of
specimens
from
both
species.
In
early
stages
of
infection,
it
was
possible
to
observe
a
blue
bloom
on
the
unpig-
mented
sternites,
more
evident
at
the
margins
level.
After
2–3
weeks,
in
a
middle
stage,
the
discoloration
moved
slowly
toward
the
dorsal
side.
Clear
light-blue
to
violet
spots
appeared
locally
on
the
epimera,
the
sides
of
pleonites,
the
backsides
of
the
pereion-
ite,
and
on
the
cephalothorax.
After
4–5
weeks,
the
color
of
the
individuals
changed
to
completely
iridescent
blue–violet
(Fig.
2).
The
epidermic
cells
were
also
infected.
After
having
reached
this
advanced
stage,
the
infected
specimens
normally
died
in
6–8
days.
Behavior
of
the
infected
specimens
was
also
monitored
in
the
laboratory.
In
the
early
stage
of
infection
animals
showed
a
nor-
mal
behavior.
In
the
intermediate
infection
stage,
it
was
possible
to
observe
a
slower
response
to
exogenous
stimuli,
like
light,
touch
or
water
contact;
specimens
also
displayed
slower
movements
in
the
rearing
box.
In
the
advanced
stage
of
the
infection,
the
specimens
with
a
totally
discolored
body
showed
a
strong
decrease
in
photo-
tactic
response
and/or
no
response
at
all
when
brought
into
contact
with
water.
Moreover,
the
amount
of
feces
released
by
infected
animals
was
less
abundant
than
the
one
ejected
by
healthy
ani-
mals,
indicative
of
a
reduced
food
consumption
in
infected
animals.
All
the
above
obervations
are
in
accordance
with
those
reported
previously
by
Wijnhoven
and
Berg
(1999).
Longevity
of
infected
animals
was
also
monitored.
On
average
they
lived
30
days,
one
specimen
of
A.
decorum
died
after
79
days.
The
observed
longevity
is
much
longer
than
reported
by
Federici
(1980)
but
is
the
same
reported
by
Wijnhoven
and
Berg
(1999).
Reproduction
of
infected
individuals
was
never
reported
in
the
literature.
We
followed
three
infected
ovigerous
females
of
P.
sicu-
loccidentalis.
These
specimens
were
immediately
isolated
after
field
samplings.
Two
of
them
died
before
the
mancas
were
released;
but
one
female
reproduced,
releasing
nearly
30
mancas,
all
show-
ing
no
symptoms
of
infection.
Some
of
these
larval
stages
were
isolated
and
followed
until
they
became
adult,
still
showing
no
infection.
This
is
the
first
reported
case
of
reproduction
of
an
ani-
mal
infected
by
Iridovirus,
in
laboratory.
Generally,
the
number
of
Fig.
2.
Specimens
of
laboratory
reared
terrestrial
isopods,
showing
Iridovirus
infec-
tion
in
an
advanced
stage,
compared
to
not-infected
specimens.
(A)
Armadillidium
decorum;
(B)
Porcellio
siculoccidentalis.
Scale
bar:
1
cm.

324 P.
Lupetti
et
al.
/
Tissue
and
Cell
45 (2013) 321–
327
Fig.
3.
TEM
micrographs
of
A.
decorum
nervous
tissues.
(A)
General
view
of
nervous
tissues;
(B)
the
cytoplasm
of
perineurium
cells
is
invaded
by
numerous
viral
particles.
(C)
Higher
magnification
of
viral
particles
with
an
electron-dense
axial
body
of
about
100
nm
surrounded
by
a
capsid
with
hexagonal
and/or
pentagonal
contours.
postmarsupial
larvae
released
by
the
females
of
this
species,
ranges
approximately
from
20
up
to
115
(see
Fig.
4
in
Montesanto
et
al.,
2012).
We
also
observed
that
the
time
necessary
to
these
mancas
to
become
adult
is
about
60
days,
the
same
time
already
recorded
dur-
ing
rearing
observations
on
P.
siculoccidentalis
(Montesanto
et
al.,
2012).
3.2.
Ultrastructure
While
dissecting
the
nervous
system
out
of
fixed
samples,
we
noticed
that
the
whole
nervous
system
of
A.
decorum
was
covered
by
a
blue
layer,
which
remained
visible
also
after
chemical
fixation
for
electron
microscopy.
The
tissue
survey
we
performed
on
thin
Fig.
4.
3D
renderings
of
a
tomogram
from
a
sample
section
containing
several
viral
particles.
(A)
Side
view
of
the
tomographic
density
map;
false
colors
indicate
the
position
of
particles
along
Z
axis
with
red
associated
to
the
top
of
the
map
and
blue
to
its
bottom.
This
rendering
shows
that
viral
particles
are
staggered
in
parallel
plans.
(B)
Tilted
view
of
the
same
tomogram
showing
the
distribution
of
viral
particles
in
paracrystalline
pattern.
(C)
High
magnification
of
the
tomogram
shown
in
A
and
B.
Yellow
lines
mark
the
distance
vectors
among
mass
centers
(represented
by
red
spheres)
of
six
viral
particles
(three
for
each
of
the
two
layers
of
particles).
(For
interpretation
of
the
references
to
color
in
this
figure
legend,
the
reader
is
referred
to
the
web
version
of
the
article.)

P.
Lupetti
et
al.
/
Tissue
and
Cell
45 (2013) 321–
327 325
Fig.
5.
Serial
virtual
sections
from
a
tomographic
reconstruction
showing
different
sectioning
levels
of
five
viral
particles.
Notice
the
transition
from
pentagonal
to
hexagonal
contour
in
the
two
boxed
particles.
Such
a
variation
is
compatible
with
the
icosahedral
geometry
of
Iridoviridae
viral
particles.
sections
from
nervous
system
revealed
that
the
cytoplasm
of
many
perineurium
cells
was
almost
completely
invaded
by
viral
parti-
cles
at
different
levels
of
organization
(Fig.
3A–C)
indicative
for
the
ongoing
replicative
cycle
of
viral
particles.
Each
viral
particle
has
a
diameter
of
almost
120
nm
with
an
electrondense
core,
surrounded
by
a
thin
layer
of,
less
electron-dense
material
(Fig.
3C).
Analy-
sis
of
tomographic
reconstructions
of
thick
sections
from
infected
nervous
tissues
(Fig.
4A
and
B),
revealed
that
the
viral
particles
Fig.
6.
Phylogenetic
analysis
of
MCP
genes.
Neighbor
Joining
(NJ)
tree
inferred
from
the
deduced
amino
acid
sequences
of
a
region
of
the
major
capsid
protein
(MCP)
gene
from
23
invertebrate
and
2
vertebrate
Iridoviruses
(see
Table
1).
The
tree
was
computed
using
the
p-distance
method
and
was
validated
by
1000
bootstrap
repetitions.
At
nodes
bootstrap
values
are
indicated.
Branch
lengths
are
drawn
to
scale,
and
scale
bar
is
shown.

326 P.
Lupetti
et
al.
/
Tissue
and
Cell
45 (2013) 321–
327
Fig.
7.
World
map
of
terrestrial
isopods
species,
which
are
certainly
hosts
of
Iridoviruses
IIV
(data
from:
Wijnhoven
and
Berg,
1999;
Williams,
2008;
Karasawa
et
al.,
2012;
present
study).
were
positioned
along
regularly
staggered
planes
at
a
distance
of
190
nm
one
from
another,
(see
yellow
lines
in
Fig.
4B
and
C),
in
paracrystalline
mode
(Fig.
4A–C).
Virtual
sections
of
0.6
nm
along
tomographic
reconstructions
of
viral
particles
revealed
almost
pen-
tagonal
or
hexagonal
contour,
depending
on
the
sectioning
level
(Fig.
5).
This
is
in
good
accordance
with
the
icosahedral
symmetry
demonstrated
for
viral
particles
of
Iridoviridae.
3.3.
Molecular
analysis
In
an
attempt
to
identify
the
virus
type
infecting
the
Sicilian
isopods,
a
molecular
analysis
was
performed
using
the
major
cap-
sid
protein
(MCP)
gene
as
a
marker.
It
has
been
shown
that
the
MCP
gene
is
characterized
by
highly
conserved
domains
but,
at
the
same
time,
this
gene
shows
peculiarities
suitable
to
discriminate
closely
related
Iridovirus
isolates
(Tidona
et
al.,
1998).
For
this
purpose
gene
specific
primers
were
designed
on
the
A.
vulgare
and
P.
scaber
nucleotide
sequences
to
amplify
a
region
of
254
bp
of
the
MCP
gene.
The
genomic
DNA
extracted
from
the
tissues
of
A.
decorum,
P.
sicu-
loccidentalis
and
T.
panormidensis,
along
with
the
dsDNA
of
viral
particles,
was
used
as
a
template
in
PCR
reactions,
yielding
a
clear
single
band
of
254
bp,
as
expected.
The
nucleotide
and
deduced
amino
acid
sequences
of
the
MCP
gene
from
the
three
species
listed
above
were
identical
(Supplementary
data
Fig.
1,
deduced
amino
acid
sequence
alignment
not
shown).
The
BLASTn
and
BLASTp
anal-
ysis
of
the
sequenced
MCP
gene
regions
of
viruses
infecting
the
three
Sicilian
isopod
species,
showed
high
degree
of
identity
with
the
MCP
genes
of
the
IIV-31
family,
isolated
from
several
terrestrial
isopod
species.
In
particular,
we
obtained
a
98%
of
nucleotide
iden-
tity
and
100%
amino
acid
identity
with
the
MCP
gene
sequences
of
the
IIV-31
infecting
Armadillidium
vulgare
(Webby
and
Kalmakoff,
1998)
and
Burmoniscus
okinawaensis
(Karasawa
et
al.,
2012);
97%
of
nucleotide
identity
and
100%
of
amino
acid
identity
with
Ligid-
ium
koreanum
and
A.
vulgare
isolated
in
Japan
(both
by
Karasawa
et
al.,
2012).
In
addition
to
isopods
the
three
analyzed
sequences
showed
a
98%
of
nucleotide
identity
and
100%
amino
acid
identity
with
the
coleopteran
Popillia
japonica
IIV-31
MCP
gene
(Webby
and
Kalmakoff,
1998).
Furthermore,
the
254
bp
nucleotide
sequences
from
the
three
Sicilian
terrestrial
isopods
were
aligned
with
the
corresponding
region
of
23
Invertebrate
Iridescent
viruses
and
2
Vertebrate
Irides-
cent
viruses
MCP
genes
(alignment
showed
in
Fig.
2,
Supplementary
data;
species
listed
in
Table
1).
The
distance
tree
in
Fig.
6,
produced
by
Neighbor
Joining
(NJ)
method,
showed
the
phylogenetic
rela-
tionship
between
translated
amino
acid
sequences
of
the
same
MCP
region
from
the
species
listed
in
Table
1.
The
phylogenetic
analysis,
supported
by
high
bootstrap
values,
groups
the
viruses
infecting
the
three
Sicilian
blue-colored
isopods
within
the
IIV-31
family,
thus
confirming
that
A.
decorum,
P.
siculoccidentalis,
and
T.
panormiden-
sis
were
all
infected
by
a
IIV-31
family
member
transmitted
by
an
unknown
factor
to
different
isopod
species
collected
in
the
same
sampling
site
(Erice,
Sicily).
4.
Conclusion
Detection
of
Iridovirus
in
the
four
species
of
terrestrial
isopods
A.
decorum,
T.
panormidensis,
P.
affinis,
P.
siculoccidentalis,
is
the
first
record
for
Italy
on
animals
infected
by
Iridovirus
IIV-31.
Our
observations
raises
to
25
the
total
number
of
known
species
of
terrestrial
isopods
infected
by
Iridovirus
spp;
other
isopod
species
from
western
Europe
and
North
America
are
supposed
to
be
infected
by
iridoviruses,
since
many
records
of
“blue”
isopods
have
been
reported
in
the
past;
the
real
presence
of
the
virus,
however,
was
not
assessed
by
ultrastructure
stud-
ies.
As
shown
in
Fig.
7,
the
virus
type
IIV-31
is
widespread
around
the
world,
in
terrestrial
isopod
species.
So
probably,
there
is
a
wider
distribution
of
this
infection
with
respect
to
what
have
been
already
reported.
Possible
lacks
are
due
to
few
samplings
and
records,
in
this
research
avenue.
A
well-known
biogeographical
hypothesis
illustrates
how
most
of
the
cosmopolitan
species
of
terrestrial
isopod
came
under
a
pas-
sive
transport
from
Europe
to
North
America
(Vandel,
1962).
One
example
is
Porcellionides
pruinosus,
one
of
the
species
that
has
been
spread
most
by
man
across
the
world,
and
can
now
be
considered
as
“synanthropically
cosmopolitan”
(Schmalfuss,
2003).
Moreover,
as

P.
Lupetti
et
al.
/
Tissue
and
Cell
45 (2013) 321–
327 327
reported
by
Hess
and
Poinar
(1985)
about
a
hypothesis
of
infection
spread
from
Europe
to
America
“[.
.
.]
it
is
interesting
to
speculate
whether
the
isopod
iridovirus
was
always
widespread
throughout
the
world
where
isopod
occour
or
whether
is
originated
in
Europe
[.
.
.]”.
Such
a
question
is
hard
to
address
on
the
basis
of
our
current
knowl-
edge
and
available
experimental
evidences.
A
connection
between
Mermithid
nematods
and
Iridovirus
infection
has
been
reported
by
Hess
and
Poinar
(1985)
and
we
have
evidences
that
Mermithids
infect
the
same
populations
of
woodlice
in
the
same
sampling
site
on
the
top
of
Mt.
San
Giuliano
(unpublished
data).
Nevertheless,
it
is
not
possible
yet
to
determine
whether
these
nematods
are
inter-
mediate
hosts
for
Iridovirus.
It
will
be
very
important
to
perform
further
studies,
in
order
to
understand
the
transmission
mecha-
nisms
of
Iridovirus
among
individuals
and
populations
of
terrestrial
isopods.
Our
observations
seem
indicative
of
the
fact
that
the
presence
of
the
virus
does
not
interfere
with
the
reproductive
fitness
of
the
females,
but
further
and
more
extended
research
is
needed
to
improve
our
knowledge
about
potential
influence
of
IIV
infection
on
reproductive
biology
of
terrestrial
isopods.
Appendix
A.
Supplementary
data
Supplementary
data
associated
with
this
article
can
be
found,
in
the
online
version,
at
http://dx.doi.org/10.1016/j.tice.2013.05.001.
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leatherjacket
Tipula
paludosa.
Nature
174,
562–563.