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Crustaceana 85 (10) 1269-1278

REPORT ON IRIDOVIRUS IIV-31 (IRIDOVIRIDAE, IRIDOVIRUS)


INFECTING TERRESTRIAL ISOPODS (ISOPODA, ONISCIDEA) IN JAPAN
BY
S. KARASAWA1,4 ), J. TAKATSUKA2 ) and J. KATO3 )
1 ) Faculty of Education, Fukuoka University of Education, Munakata, Fukuoka 811-4192, Japan
2 ) Forest Entomology Division, Forestry and Forest Products Research Institute,

Matsunosato 1, Tsukuba, Ibaraki 305-8687, Japan


3 ) Takezono, Tsukuba, Ibaraki 305-0032, Japan

ABSTRACT
We conducted a comprehensive review of blue isopods in Japan by collecting isopod species
from eight sites located on three Japanese islands (Honshu, Kyushu and Okinawajima). We also
interviewed Japanese soil-fauna researchers and retrieved data from the Internet detailing isopod
collections in Japan. From analysis of the complete data set, we conclude that a total of eight
species from six families (i.e., Ligiidae, Alloniscidae, Philosciidae, Armadillidae, Porcellionidae
and Armadillidiidae), excluding two unidentified species, have been observed at 18 Japanese sites.
The distributional range of the blue isopods covered the whole of Japan (from Okinawajima
Island to Hokkaido). Examination of transmission electron microscope (TEM) images revealed
non-enveloped virions in the epithelial cell cytoplasm of the blue coloured Armadillidium vulgare
(Latreille, 1804) from Japan. We designed primers for amplifying partial sequences from the major
capsid protein (MCP) gene from invertebrate iridescent viruses (IIVs). The predicted amino acid
sequences of the MCP gene from IIV isolated from the blue coloured isopods, A. vulgare, Porcellio
scaber Latreille, 1804, Burmoniscus kathmandia (Schmalfuss, 1983), and Ligidium koreanum
Flasarov, 1972, collected from Japan, were completely congruent with IIV-31 from the U.S.A.
However, nucleotide sequence analysis showed that genetic variation exists among the IIV-31 species
collected from the four collection sites in Japan and the North American IIV-31 species.

RSUM
Nous avons conduit une tude exhaustive des isopodes bleus au Japon en chantillonnant les
espces disopodes de huit sites localiss sur trois les japonaises (Honshu, Kyushu et Okinawajima).
Nous avons aussi interrog des chercheurs japonais spcialistes de la faune du sol et rcupr des
donnes disponibles sur internet dtaillant les prcdentes collection disopodes au Japon. A partir de
lanalyse complte des donnes, nous concluons qu un total de huit espces provenant de six familles
(cest--dire, Ligiidae, Alloniscidae, Philosciidae, Armadillidae, Porcellionidae et Armadillidiidae),
en excluant deux espces non identifies, ont t observes sur 18 sites japonais. Laire de rpartition

4 ) e-mail: karashi@fukuoka-edu.ac.jp

Koninklijke Brill NV, Leiden, 2012

DOI:10.1163/15685403-00003116

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S. KARASAWA, J. TAKATSUKA & J. KATO

des isopodes bleus couvre tout le Japon (de lle de Okinawajima Hokkaido). Lobservation des
images de microscopie lectronique transmission (TEM) rvle des virions sans enveloppe dans
le cytoplasme des cellules pithliales dArmadillidium vulgare (Latreille, 1804) du Japon. Des
amorces ont t dfinies pour amplifier les squences partielles du gne de la principale protine
de la capside (MCP) partir de virus iridescents dinvertbrs (IIVs). La squence prdite dacides
amins du gne MCP de IIV isole partir des isopodes, A. vulgare, Porcellio scaber Latreille, 1804,
Burmoniscus kathmandia (Schmalfuss, 1983), et Ligidium koreanum Flasarov, 1972, collects au
Japon a t compltement conforme avec IIV-31 des U.S.A. Cependant, lanalyse des squences des
nuclotides a montr quune variation gntique existe parmi le IIV-31 entre les espces collectes
partir des quatre sites japonais et les IIV-31 des espces nord amricaines.

INTRODUCTION

Purple-blue isopods have been collected from terrestrial isopod communities


in some regions of the world. In 1980, Cole & Morris (1980) and Federici (1980)
found that the striking coloration of the isopods was caused by infection with invertebrate iridescent viruses (IIVs). IIVs are large ichosahedron-shaped DNA viruses
belonging to the family Iridoviridae; there are currently two genera, Iridovirus and
Chloriridovirus, recognized in this family (Chinchar et al., 2005). The genus Iridovirus contains viruses that have been isolated from several different orders of
invertebrates and have particles (in ultrathin section) of around 120-130 nm in diameter. Due to limited genome sequence data, only two viruses in this genus, IIV-1
and IIV-6, currently have species status. Another 11 viruses are regarded as tentative species within this genus. In contrast, the genus Chloriridovirus comprises a
single viral species that has a particle size of about 180 nm diameter (in ultrathin
section); within this genus, IIV-3 was originally isolated from mosquitoes.
The virus studied in 1980 by Federici had a diameter of about 140 nm (negative
stain) and icosahedral symmetry. The same study designated the viruses isolated
from Armadillidium vulgare (Latreille, 1804) and Porcellio dilatatus Brandt, 1833
as IIV-31 and IIV-32, respectively. More recently, however, the virus infecting
terrestrial isopods is considered a tentative species designated IIV-31 within the
genus Iridovirus (cf. Chinchar et al., 2005). Infected isopods are light blue to
violet in colour, have decreased photo and water responsiveness, and a shorter life
span than non-infected individuals (Federic, 1980; Wijnhoven & Berg, 1999). Viral
infection can also affect intra- and inter-specific interactions in terrestrial isopods
(Grosholtz, 1992). Williams (2008) compiled a list of the natural host species for
IIVs and established that 108 invertebrate host species had been reported in the
literature, and that 19 of these species were terrestrial isopods. Isopods with the
blue coloration that is the characteristic sign of IIV infection have been reported in
the United States, United Kingdom, Netherlands, France, former Czechoslovakia
and the Russian Federation (Hess & Poinar, 1984; Wijnhoven & Berg, 1999;

IRIDOVIRUS IIV-31 INFECTING TERRESTRIAL ISOPODS IN JAPAN

1271

Williams, 2008); however, blue isopods have not been reported previously in Asia.
Moreover, because there are no records of IIV-31 isolated from blue isopods in
Japan, almost nothing is known about the genetic and morphological characters of
the Japanese isopod IIVs.
The aim of this study was, therefore, (1) to catalogue the species and collection
sites of blue isopods from Japan, and, (2) to classify the species of iridoviruses
collected in Japan using a combination of morphological and molecular genetic
analyses.

MATERIAL AND METHODS

Morphological study
Before examination by transmission electron microscopy (TEM), epithelial
tissues removed from blue coloured Armadillidium vulgare collected from Ibaraki
were pre-fixed in half Karnovsky fixative for 2 h at 4C, post-fixed in 1% OsO4 for
1 h at 4C, dehydrated using a graded series of ethanol washes and embedded
in Epon 812 resin. Ultrathin sections were cut using an ultramicrotome (Om
U3 Ultramicrotome; Reichert Technologies, U.S.A.) with a sapphire knife. The
sections were stained with 2% uranyl acetate followed by lead citrate. Specimens
were examined with a JEM-2000EX transmission electron microscopy (JEOL,
Japan) at 100 kV. Images from the microscopic view field were acquired using
the CCD camera system, KeenView (Soft Imaging System, Germany).
Molecular analysis
The following specimens were used for molecular analysis: three A. vulgare,
two Porcellio scaber Latreille, 1804, one Burmoniscus kathmandia (Schmalfuss,
1983) and one Ligidium koreanum Flasarov, 1972 (table I). Small tissue samples
(approximately 10 mg) from the blue coloured individuals were homogenized in
TE buffer (pH 8.0; 10 mM Tris-HCl, 1 mM EDTA) using a hand homogenizer for
30 s, prior to DNA extraction using a PUREGENE Tissue DNA Purification kit
(Qiagen, Japan). Polymerase chain reactions (PCRs) were performed to amplify
partial sequences from the major capsid protein (MCP) gene. Degenerate primers
MCP_F (5 -acHtggttcacYcaRgtacc-3 ) and MCP_R (5 -gtNagYttRccRtaRttggt-3 )
were designed for this purpose. Amplifications were conducted in 50 l volumes
containing 1 unit of MightyAmp DNA polymerase (Takara Bio, Japan), 0.3 M
each of the forward and reverse primers, 1 MightyAmp PCR buffer (Takara Bio)
and 10 ng of template DNA. PCR conditions were as follows: 1 cycle at 98C
for 2 min, 37 cycles at 98C for 10 s, 53C for 15 s, 68C for 1 min, and then 1

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S. KARASAWA, J. TAKATSUKA & J. KATO

TABLE I
Host species and collection sites for the specimens used in phylogenetic analysis and their
corresponding DDBJ/EMBL/GenBank accession numbers
Virus type

Host of isolation

Sites of collection or isolation

Accesion no.

IIV-31
IIV-31
IIV-31
IIV-31
IIV-31
IIV-31
IIV-31
AeIV
IIV-1
IIV-2
IIV-3
IIV-6
IIV-9
IIV-16
IIV-22
IIV-23
IIV-24
IIV-29
IIV-30
IIV-31
LCDV-China
FV-3

Armadillidium vulgare
Armadillidium vulgare
Armadillidium vulgare
Porcellio scaber
Porcellio scaber
Burmoniscus kathmandia
Ligidium koreanum
Acetes erythraceus
Tipula paludosa
Sericesthis pruinosa
Aedes taeniorhynchus
Chilo suppressalis
Wiseana cervinata
Costelytra zealandica
Simulium sp.
Heteronychus arator
Apis cerana
Tenebrio molitor
Helicoverpa armigera
Armadillidium vulgare
Paralichthys olivaceus
Rana pipiens

Inzai, Chiba, Japan


Hitachi, Ibaraki, Japan
Hitachi, Ibaraki, Japan
Hitachi, Ibaraki, Japan
Hitachi, Ibaraki, Japan
Naha, Okinawa, Japan
Chikuzen, Fukuoka, Japan
Madagascar
U.K.
Australia
U.S.A.
Japan
New Zealand
New Zealand
Wales
South Africa
India
U.S.A.
U.S.A.
U.S.A.
China
U.S.A.

AB686457
AB686459
AB686463
AB686460
AB686461
AB686462
AB686458
EF467167
AAA46245
AAC97168
ABF82044
AAK82135
AAB82568
AAB82569
AAA66585
AAC97175
AAC97173
AAC97172
AAC97169
AAC97170
AAS47819
AAB01722

IIV, invertebrate iridovirus; AeIV, Acetes erythraeus iridovirus; LCDV, lymphocystis disease virus;
FV, frog virus.

cycle at 68C for 7 min. PCR products were electrophoresed through 1.5% agarose
gels. Products of the expected sizes (1100 bp) were purified using a QIAquick
Gel Extraction kit (Qiagen, Japan) prior to sub-cloning into a p3T cloning vector
(MoBiTec GmbH, Gttingen, Germany). DNA sequence analysis for both strands
was performed using a capillary sequencer (ABI 3130XL; Applied Biosystems,
U.S.A.). Three clones from each sample were sequenced on each DNA strand.
In addition to the eight sequences from Japan, sequences from 15 other viruses
were used for phylogenetic analyses (table I). Phylogenetic trees were constructed
based on nucleotide coding sequences and their putative corresponding amino
acid sequences. Nucleotide sequence analysis was performed on the IIV-6 and
IIV-31 data only (table I). Amino acid and nucleotide sequences were aligned
using the default setting of Clustal X (Thompson et al., 1997). Phylogenetic trees
were estimated by neighbour-joining (NJ) methods using MEGA 5 (Tamura et al.,
2011). Bootstrap support was assessed using 1000 replicates. Alignment gaps were
excluded from the analyses.

IRIDOVIRUS IIV-31 INFECTING TERRESTRIAL ISOPODS IN JAPAN

1273

RESULTS AND DISCUSSION

Geographical and phylogenetic constraints on blue isopods


We collected eight species of blue isopods from eight sites located on three
Japanese islands (i.e., Honshu, Kyushu and Okinawajima Island). We also interviewed soil-fauna researchers in Japan and retrieved Japanese words corresponding to blue isopods and iridovirus from the Internet. From this, we concluded
that four species of blue isopods have been found at 10 sites located on Hokkaido
and Honshu (fig. 1; table II). Therefore, a total of eight species from six families,
excluding two unidentified species, have been found in 18 Japanese sites. Watanabe (2011) stated that most reports of blue isopods tended to be from Kanto (in
the centre of Honshu). The present study indicated that 12 of the 18 sites were
in Kanto. The distributional range of blue isopods covered most of the temperate
zone in Japan, i.e., the subtropical region (Okinawajima Island) to the subarctic
region (Hokkaido). We suggest, therefore, that blue isopods are distributed across

Fig. 1. Map illustrating the sites where blue coloured isopods have been found in Japan. Numbers
refer to table II.

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S. KARASAWA, J. TAKATSUKA & J. KATO

TABLE II
Species of blue coloured isopods and the sites where they have been found in Japan
Species

No.

Site

Identification

Ligiidae
Ligidium japonicum
Ligidium koreanum*
Ligidium koreanum
Ligidium?

6
16
17
9

Inzai, Chiba
Chikuzen, Fukuoka
Ukiha, Fukuoka
Kitamoto, Saitama

By authors
By authors
By authors
Article on the Internet

Hitachi, Ibaraki

By authors

15
18
4

Kama, Fukuoka
Naha, Okinawa
Hitachi, Ibaraki

By authors
By authors
By authors

Yokosuka, Kanagawa

Interview research

Hitachi, Ibaraki

By authors

Alloniscidae
Alloniscus balssi
Philosciidae
Burmoniscus kathmandia
Burmoniscus kathmandia*
Littorophiloscisa nipponensis
Armadillidae
Venezillo obscurus
sensu Kwon (1995)
Armadillidae sp.
Porcellionidae
Porcellio scaber
Porcellio scaber*
Porcellio scaber

1
4
14

Niikappu, Hokkaido
Hitachi, Ibaraki
Kamo-gun, Shizuoka

Based on image on the Internet


By authors
Interview research

Armadillidiidae
Armadillidium vulgare
Armadillidium vulgare
Armadillidium vulgare*
Armadillidium vulgare
Armadillidium vulgare
Armadillidium vulgare*
Armadillidium vulgare
Armadillidium vulgare
Armadillidium vulgare
Armadillidium vulgare
Armadillidium vulgare

2
10
4
3
5
6
7
13
11
12
14

Hakodate, Hokkaido
Nagatoro, Saitama
Hitachi, Ibaraki
Chikusei, Ibaraki
Tsukuba, Ibaraki
Inzai, Chiba
Futtsu, Chiba
Tama, Tokyo
Shinjuku-ku, Tokyo
Meguro-ku, Tokyo
Kamo-gun, Shizuoka

Interview research
Interview research
By authors
By authors
By authors
By authors
Interview research
Interview research
Interview research
Interview research
Interview research

* The host species used for isolation and molecular analysis of the MCP gene of invertebrate

iridescent viruses (IIVs).

the temperate regions of Japan. Historically, the number of studies conducted on


terrestrial isopods in regions outside Kanto has been small, which may account for
why blue isopods have never or rarely been reported in such regions.
Williams (2008) reported that blue isopods had been found in 13 genera of seven
families across the world, but the family Armadillidae and the genus Burmoniscus
were not reported in the study. Hence the present study is the first to report
on identification of the blue isopods belonging to the Armadillidae family and

IRIDOVIRUS IIV-31 INFECTING TERRESTRIAL ISOPODS IN JAPAN

1275

genus Burmoniscus. Phylogenetic analysis of terrestrial isopods by Mattern &


Schlegel (2001), infers that Ligiidae, Philosciidae, Armadillidae, Porcellionidae
and Armadillidiidae are a polyphyletic group. Ligiidae are the basal group for
Oniscidea, while the three families, Armadillidae, Philosciidea and Porcellionidae,
appear to have somewhat derived groups. This result indicates that terrestrial
isopods can become blue colored, indicative of iridovirus infection, regardless of
their phylogenetic relationship.
Identification of the iridovirus IIV-31
TEM images show that non-enveloped virions are present in the epithelial cell
cytoplasm of the blue isopod Armadillidium vulgare collected from Japan (fig. 2).
The virions appeared hexagonal in outline, indicating an icosahedral symmetry;
they measured ca. 130 nm in diameter and had an electron-dense core. The
morphological characters are consistent with those of IIVs belonging to the genus
Iridovirus.
Furthermore, we aligned subsets of sequences comprising 101 amino acids of
the MCP gene isolated from the IIV-31 viruses obtained from seven blue-coloured
specimens belonging to four species (table I, the Japanese samples with accession
numbers AB686457-63). The tree produced by the NJ analysis, as based on the
amino acid sequences of MCP is shown in fig. 3. Inspection of the tree revealed
that the amino acid sequences originating from Japanese iridovirus isolates were

Fig. 2. Transmission electron micrograph (TEM) illustrating IIV-31 particles in epithelial sections of
Armadillidium vulgare (Latreille, 1804).

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S. KARASAWA, J. TAKATSUKA & J. KATO

Fig. 3. Neighbour Joining (NJ) tree based on amino acid sequences encoded by the major capsid
protein (MCP) gene. Nodal values indicate bootstrap supports (1000 replicates). OTU are shown in
detail in table I, and the numbers following specific names correspond to the site numbers shown in
fig. 1.

completely congruent with those of IIV-31 collected from A. vulgare in the


U.S.A.. This result indicates that in Japan four blue-coloured species, A. vulgare,
Porcellio scaber, Burmoniscus kathmandia and Ligidium koreanum, were infected
by IIV-31. The IIV-31 group formed a sister group of IIV-6.
To determine the extent of genetic diversification within IIV-31, we also
analyzed the nucleotide sequences of a 361-bp fragment of the MCP region
collected from eight samples from the IIV-31 group (table I, Japanese samples

IRIDOVIRUS IIV-31 INFECTING TERRESTRIAL ISOPODS IN JAPAN

1277

Fig. 4. Neighbour Joining (NJ) tree based on nucleotide sequences encoding the major capsid protein
(MCP) gene. Nodal values indicate bootstrap supports (1000 replicates). OTU are shown in detail in
table I, and numbers following specific names correspond to the site numbers shown in fig. 1.

with accession numbers AB686457-63 and AAC97170 (U.S.A.)) and one sample
from IIV-6 (table I, Japan, AAK82135) (fig. 4). A monophyletic group for all
of the IIV-31 samples was not supported by a high bootstrap value, but four
species collected from four sites in Japan formed a monophyletic group supported
by a bootstrap value of 62. Our findings show, therefore, that genetic variation
exists among specimens collected from four of the sampling sites (Ibaraki, Chiba,
Fukuoka and Okinawa). However, no genetic differences were found between
the two species collected from the Ibaraki site. The data indicate that IIV-31
has evolved to infect a diversity of isopod species at each of the collection sites
in Japan. Intriguingly, the IIV-31 transmission route remains unclear. Cole &
Morris (1980) showed that the virus was infectious through ingestion of virus
contaminated diets. The virus may also infect isopods via a damaged exoskeleton
(Grosholz, 1992). The results suggest that cannibalism, coprophagy and interspecific aggression in the isopod species observed in natural populations could
be important transmission routes for the virus (Cole & Morris, 1980; Grosholz,
1992; Kautz et al., 2002). Moreover, it is possible that nematodes could serve as
a vector for viral transmission to isopods (Hess & Poiner, 1985). Few studies,
however, have examined parasitism by nematodes in relation to isopod species
or the route for IIV-31 transmission in Japan: thus it is unclear which infection
routes might play an important role in transmission of the virus among Japanese
isopods.

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S. KARASAWA, J. TAKATSUKA & J. KATO

ACKNOWLEDGEMENT

This research was partially supported by a Grant-in-Aid for Scientific Research


from the Ministry of Education, Science, Sports and Culture (No. 21310025).

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First received 27 December 2011.


Final version accepted 14 April 2012.

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