フィールドサイエンス（J. Field Science）15：29‒34，2017

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研究資料
DNA barcoding identified the exotic terrestrial isopod Porcellio scaber(Crustacea, Isopoda, Oniscidea) on the Kyushu mainland, western Japan
Shigenori KARASAWA＊1, Tomoki IWASAKI＊2, Jun NAKAJIMA＊3

DNA バーコーディングを用いた九州からの外来性ワラジムシ類 Porcellio scaber の発見
唐沢重考＊1・岩崎

朝生＊2・中島淳＊3

Porcellio scaber Latreille, 1804 was found on the Kyushu mainland for the first time. The specimens wereidentified based on DNA barcoding and the following five morphological characteristics: 1) posterior marginof pereonite 1 is almost straight, 2) epimerons 1–4 have obvious sinuous posterior margins, 3) exopodites ofpleopods 1 and 2 bear monospiracular lungs, 4) the posterior tip of the telson tapers, and 5) the flagellum of
the second antenna is composed of two segments. Moreover, the specimens collected from a park in Fukuoka
City showed carapace color variation.
Keywords：Color variation, Fukuoka, Invasive species, Porcellionidae
九州本土において初めてワラジムシ Porcellio scaber を確認した。本研究で採集した標本は， 1 ）第 1 胸節背板の後縁中央部がほぼ直線であること， 2 ）第 1 – 4 胸節背板の側部後縁が S 字になること， 3 ）第 1 ，2 腹肢外肢が 1 個の呼吸穴を持つこと， 4 ）腹尾節の先端が尖ること， 5 ）第 2 触角の鞭部が 2 節で構成されること，および，DNA バーコードによる類似度検索により P. scaber であると判断した。また，福岡市
内で採集された標本には体色に変異が認められた。
キーワード：体色変異，福岡，外来種，ワラジムシ科

Introduction
Exotic species are species that were transferred

However, if they have invaded a new site, it is important to detect them early so they can be eradicated or controlled (Simberloff et al., 2013).

from their native distribution to other areas by

Precise identification of species often requires

human activities (Ontario, 2013). The number ofprofessional skill and specimens with sufficient

exotic species is rising with the global increase ofmorphological characters to identify species. For

human travel, and these species have the potential

example, adult male morphological characteristics

to cause extinction of native species (Gurevitch

are usually important for identifying terrestrial

and Padilla, 2004). The most effective management

isopods (e. g., Kashani, 2015; Taiti and Wynne,of exotic species is to prevent invasion of species.

2015), it implies that female specimens are difficult

２０１6．7 ．7 受付；２０１6．11．15受理＊1〒 680-8551 鳥取市湖山町南4丁目101番地 Faculty of Regional Sciences, Tottori University, 4 -101  Koyamamachi, Minami, Tottori, 680-8551 Japan＊2〒 819-0005 福岡県福岡市西区内浜 1 -13-33-101 Uchihama 1 -13-33-101, Nishi-ku Fukuoka-shi, Fukuoka, 8190005 Japan＊3〒 818-0135 福岡県太宰府市向佐野39 福岡県保健環境研究所 Fukuoka Institute of Health and EnvironmentalSciences, Mukaizano 39, Dazaifu, Fukuoka, 818-0135 Japanフィールドサイエンス

30

15号

to precisely identity. DNA barcoding is a powerful

Fukuoka Prefecture (N33.6494, E130.4148) on 14

tool that can resolve this issue. This approach is aOctober 2015 by Tomoki Iwasaki. One female wasway to identify species using a short genetic se-collected from under leaf litter at the Akama Kita-

quence and enables species identification of speci-

guchi Mini Park in Munakata City, Fukuoka

mens without sufficient morphological characters(N33.80879, E130.56780) on 1 December 2015 by(Hebert et al., 2003). There is some possibility of

Shigenori Karasawa. The specimens were pre-not collecting specimens with full taxonomic char-served in 99.5% ethanol until morphological obser-acteristics at early stages of invasion because of avation and molecular analysis.

small number of individuals. Thus, the DNA barcoding is expected to be able to identify exotic
species in early stage of invasion.

Morphological observation
Photographs were obtained based on multifo-

The aim of this study was to report the first

cused montage images using a digital microscopecollection of exotic terrestrial isopods, Porcellio

VHX-2000 (KEYENCE Corporation, Japan). The

scaber Latreille, 1804, from the Kyushu mainland,specimens for scanning electron microscopy werewestern Japan. All of the specimens were female,dried at room temperature, coated with gold, andand it is difficult to identify P. scaber individuals

photographed using a JCM-5100 scanning electron

based on female morphological characters, there-microscope (JEOL, Japan).

fore we used DNA barcoding to identify species.
Moreover, we describe morphological characters,

Molecular analysis

including color variation, and determine DNA se-One P. scaber collected from the Minato-quences of additional genetic regions for future

100-Nen-Park in Fukuoka City was used for mo-DNA-barcode studies.

lecular analysis. One P. scaber found in Tokyo

Materials and Methods(N35.728269, E139.715181) was also analyzed inorder to determine the genetic difference between
specimens in Tokyo (Kanto region) and Kyushu. A

Examined specimens
Two females were collected from under leaf lit-

partial region of the mitochondrial cytochrome cter at the Minato-100-Nen-Park in Fukuoka City,

oxidase subunit I (COI) gene was sequenced forTable 1. Species, localities, and DDBJ accession numbers.
SpeciesLocality

Porcellio scaber
Porcellio scaber
Porcellio scaber
Porcellio scaber
Porcellio scaber
Porcellio scaber
Porcellio scaber
Porcellio scaber
Porcellio scaber
Porcellio scaber
Burmoniscus ocellatus
Ligidium ryukyuense
Mongoloniscus koreanus
Mongoloniscus vannamei
Porcellio laevis
Porcellio laevis
Porcellionides pruinosus
Porcellionides pruinosus

Fukuoka, JapanTokyo, Japan
British Columbia, Canada
British Columbia, Canada
British Columbia, Canada
British Columbia, Canada
British Columbia, Canada
Massachusetts, USA
Poitiers, FranceGdansk, Poland
Iriomotejima Is., Japan
Amami-Oshima Is., Japan
Fukuoka, Japan
Fukuoka, Japan
Siracusa, Italy
Siracusa, Italy
Siracusa, Italy
Australia

Accession no.
COI

12S

LC126629
LC126628
KM611741
KM611704
KM611660
KM611577
KM611545
HQ978726
LK052885
DQ305142
AB626177
AB626262
LC017826
LC017827
FN824122
FN824123
FN824140
KR424607

LC126631
LC126630
-

18S

28S

LC126633 LC126635
LC126632 LC126634-First record of Porcellio scaber in Kyushu（KARASAWA）

31

DNA barcoding analysis. In addition, we se-Hopkin, 1991; Schmidt, 2003): there were threequenced three genetic regions for future study;

lobes on the anterior margin of the cephalon (Fig.

nuclear 18S and 28S ribosomal RNA (rRNA) genes

1A), the posterior margin of pereonite 1 is almost

and the mitochondrial 12S rRNA gene. DNA ex-straight (Fig. 1A), epimerons 1–4 have obvious sin-

traction protocol, PCR conditions, and primers

uous posterior margins (Fig. 1A), exopodites ofused were described in Karasawa et al. (2014). The

pleopods 1 and 2 bear monospiracular lungs (Fig.

sequence data have been deposited in DDBJ/

1B and C), the posterior tip of the telson tapers

EMBL/GenBank (COI: LC126629, 12S: LC126631,(Fig. 1A), and the flagellum of the second antenna

18S: LC126633, 28S: LC126635).

is composed of two segments (Fig. 1D). The speci-

Identification of species based on DNA barcod-mens examined had all of these morphologicaling was carried out using the BOLD Identification

characters. However, some of these characters

System for COI (http://www.barcodinglife.org/in-were shared with congeneric species (e.g.,dex.php/IDS_OpenIdEngine) on 2 May 2016. Se-Schmidt, 2003).

quence data were input into the system, which in

To ensure proper species identification, we used

turn showed candidate species that geneticallythe DNA barcoding approach which can identify

matched the input data. We used the All Barcode
Records on BOLD search database.
To visualize the validity of DNA barcoding, weconstructed a phylogenetic tree with two exotic
species (Porcellio laevis Latreille, 1804 and Porcellionides pruinosus (Brandt, 1833)), three morphologically resembling species found in Japan (Burmoniscus ocellatus (Verhoeff, 1928), Mongoloniscus
koreanus Verhoeff, 1930 and M. vannamei (Arcangeli, 1927)), and six P. scaber specimens in other countries based on the COI sequence data. The
sequence data of these specimens were obtained
from the GenBank database (Table 1). Multiple sequence alignment was carried out by MUSCLE inSeaView 4. 5. 4 (Gouy et al., 2010), and all gaps
were excluded from subsequent analysis. Maximum Likelihood (ML) analysis was performed using MEGA 7 (Kumar et al., 2016). The best-fit models of sequence evolution determined by the Bayes
information criterion (BIC) in the program MEGA
7 was HKY+G model. Bootstrap support was assessed using 100 replicates.
Results and Discussion
Precise species identification of P. scaber requires male morphological characteristics (Vandel,1962; Gruner, 1966), but unfortunately we cannot
collect male specimens. Both male and female P.
scaber are known to have the following morphological characteristics (Vandel, 1962; Gruner, 1966;

Fig. 1. P hotographs and scanning electron micrographs of Porcellio scaber collected from
Fukuoka, Kyushu. A: Dorsal surface, female
collected from Minato-100-Nen-Park; scale bar,
2 mm. B: Ventral side of the abdomen, female
collected from Akama Kitaguchi Mini Park;
scale bar, 1 mm. C: Ventral side of exopodite 1,
the upper white square indicates monospiracular photographed from a different angle, female
collected from Minato-100-Nen-Park; scale bar,
0.3 mm. D: Second antenna, female collectedfrom Minato-100-Nen-Park; scale bar, 1 mm.

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3215号

Fig. 2. ML phylogenetic tree based on combined COI
sequence data. Bootstrap values exceeding 70%
are shown at each relevant node.

female specimens because genetic data are used
(Hebert et al., 2003). The BOLD Identification System selected the top 99 candidate specimens withmore than 98% similarity to specimens collected

Fig. 3. Map of Porcellio scaber collection sites in Japan.
Circles indicate sites that have been reported
in the Japanese terrestrial isopod distribution
database (http://isopoda.sakura.ne.jp/map/map.
php), and squares indicate new collection sites.

from Kyushu, and all except for eight unidentified
specimens in the database were P. scaper. In addi-Honshu and Hokkaido. Later, P. scaber were col-tion, the phylogenetic tree revealed that P. scaber

lected from Tottori, Osaka, Nara, and Hyogo inin Fukuoka Prefecture was included in a clade

western Honshu, and Tokushima in Shikoku

composed of eight specimens collected from four

between 1992–1998 (Nunomura, 1998). In 2012,countries (Fig. 2). Therefore, we identified theseHayashi (2012) found this species in Shimane,specimens as P. scaber. Porcellio scaber is also

which is located in western Tottori, therefore theknown to show distinctive variation in carapace

western limit of their distribution in Honshu has

coloration (Bhella et al., 2006), and both orange andbeen considered to be Shimane. Nunomura (2011)

gray types were collected from the park inalso collected this species from Tsushima Island,

Fukuoka City.

which is north of the Kyushu mainland. However,Porcellio scaber is distributed worldwide, but in

P. scaber has not been collected on the Kyushu

most areas, outside of Europe, the species is con-mainland until now. The results of the present

sidered exotic (Schmalfuss, 2003); while it has been

study indicate that this species recently spread tosuggested that this species represents six subspe-

the southwest part of Japan. The phylogeneticcies (WoRMS Editorial Board, 2016), and the taxo-tree indicated that P. scaber in Fukuoka Prefec-

nomic treatment remains unclear. Solving the tax-

ture genetically differed from P. scaber in Tokyo

onomic confusion is important for precise(Fig. 2), which implied that P. scaber in Fukuoka

identification, but it requires further analysis be-

Prefecture might be introduced from anotheryond the subject of this present study. In Japan,

country and not Honshu.

the species is also thought to have been intro-

Taxonomic status of terrestrial isopods is usual-duced by human activity (Nunomura, 2007). Japanly delimited using male morphological characterscomprises four main islands: Hokkaido, Honshu,(e.g., Kashani, 2015; Taiti and Wynne, 2015), andShikoku, and Kyushu (Fig. 3). Nunomura (1998)

some of the taxonomical characters may changereported that P. scaber was not found in the Kan-

as individual growth (Tanaka and Karasawa, 2016).

sai region of western Honshu during 1967–1977,

Thus, identification based on immature and female

and its distribution might be limited to Easternspecimens is difficult and has the potential toFirst record of Porcellio scaber in Kyushu（KARASAWA）

33

cause misidentification, and the present study

DNA markers for identification. Edaphologia,

showed that DNA barcoding is useful for identif-

93: 11-27.
Kumar, S, Stecher, G. and Tamura, K., 2016.

ing these specimens.
Acknowledgement

MEGA7: Molecular Evolutionary Genetics
Analysis version 7.0 for bigger datasets.

This research was partially supported by theMolecular Biology and Evolution (in press).

Ministry of Education, Culture, Sports, Science andNunomura, N., 1998. Occurence of Porcellio scaber

Technology, a Grant-in-Aid for Young Scientists (B)in western Japan. Edaphologia, 61: 63-64 (In(no. 26830145).

Japanese).
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