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
DOI:10.1163/15685403-00003116
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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
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.
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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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
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
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.
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Fig. 1. Map illustrating the sites where blue coloured isopods have been found in Japan. Numbers
refer to table II.
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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
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
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Fig. 2. Transmission electron micrograph (TEM) illustrating IIV-31 particles in epithelial sections of
Armadillidium vulgare (Latreille, 1804).
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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.
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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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ACKNOWLEDGEMENT
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