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ue t wt Science 64 (3): 123-127, November 2013

Distinguishing the externally similar imagines of firacola and T aureata whose forewing lengths were shown to overlap (Lepidoptera, )plagiata

i) i}･2) Daisuke WasABIKI and Shin-ichi YosHJMMsu

i)Graduate School ofAgriculture, Tbkyo University ofAgriculture, Funako 1737, Atsugi, Kanagawa, 243-O034 Japan 2) National Institute for Agro-Environmental Sciences, Kannondai 3-1-3, Tsukuba, Ibaraki, 305-8604 Japan

Abstract Two species of the genus firacola Moore known from Japan, T plagiata and T aureata, have very similar wing markings and very variable coloration of the forewings. They have been considered to be distinguishable easily from each other by the lengths of the forewings. Usually T aureata has been betieved to be 1arger than Z plagiata. However, by measurement of thc forewings, we have found that some specimens of Z plagia ta are larger than T aureata. We have therefOre developed discrimination methods for them using the female genitalia and standard DNA barcoding. As a result, we could distinguish them by the characteristics of the antrum of the female genitalia. Furthermore, wc could distinguish them successfully using standard DNA barcoding (COD.

Key words DNA barcoding, female genitalia, mtCOI,nracola aureata, n'racola plagiata.

Introduction Materials and methods

The genus 7iracola Moore, 1881 comprises 13 species MeasurementojYbrewings worldwide 1989; Holloway, 1989): two species, (Poole, For the measurement of lengths of forewings of the T plagiata (Walker) and T aureata Holloway, have been specirnens, we used a total of 37 adults from Japan, Vietnam, recordedfromJapan(Sugi,2001;Ybshimatsu,2011).The Laos, Thiwan, Philippines, Malaysia, Indonesia and India. larva of T plagiata seems to be highly polyphagous and Their fbrewing Iengths were measured by digital scales is known as a pest of 71Pieobroma cacao (Cacao), Hevea (MITUTOYO, ABS Digimatic Caliper CD-15CX) spp. (Para rubber tree) and Pinus merkusii (Merkus pine) individually. In addition, we also examined the significant in Indonesia and Laan, 1981) and Citrus spp. (Kalshoven difference of length between forewings of both species in Taiwan (Sugi, 1982). But, as mentioned by Holloway using the Mann-Whitney U test. (1989), host records of T plagiata should be carefu11y checked because they may include those of T auieata As a first step a]1 individuals including males and females also.Both were dissected and we could identify all males easily. Secondly, we examined several smaller female specimens species have very simi1ar wing markings and very (S20.9 mm) which we regarded as incontrovertibly T variable coloration of the fbrewings (Figs 1-4), but they ptagiata and from this obtained an understanding of its areeasilydistinguishablebythemalegenitaliaasillustrated female genitalia. Similarly, we also examined several by Holloway (1989). Additionally, it was thought to be larger female specimens ()26.0 mm) which we considered easy to distinguish them because T plagiata had been to be T a"reata and hence obtained an understanding of believed to be usually smaller than T a"reata as mentioned its female genitalia. In this way, we established the by Sugi (2001). However, from our detailed study we characteristics of the female genitalia of both species. On discover that while T plagiata is generally significantly this basis we could identify the medium sized female smaller than T a"reata, the lengths of their forewings individuals (>20.9 mm and S25.9 mm) by the female overlap in some specimens. genitalia. Therefore, to establish an exact identification method for D?NLtl euperiments both species, we investigate in this paper the morphological differences between their female genita]ia and we also A total of ten adult specimens collected from Vietnam in were used fbrexperiments. consider the possibility of distinction between them by 2012 and 2013 at Light Trap standard DNA barcoding. All legs on the right side of the body of all specimens collected were removed and one leg was put into 99.5% ethanolfbrsubsequentDNAextraction;DNAwasextracted using a DNeasy Blood & Tissue Kit (Qiagen, Hilden,

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124 D. WrmBIKI and S, YOSHIMATSU

f'

4 3 -

Figs ]4. n'tucola spp. (Scale bars = 1e mm; LF = Lengths of forewings) 1: T plagiata, 8 {LF 23.9 mm), 2: Ditto, 4 (LF 24,5 mm), 3: T aureata. 8 {LF 222 mm), 4: Ditto, ? (LF 22.3 mrn).

Numbers individuals

43210of Ol2B4 s67 7i'65 'Iilli]j1:ll<19)]

Ii d]ll,iil(18129)iI(ieE l 27.0mm zz.fiuE S26,9mmS2S,9mm524,9mmS23,9mmS iliiIiiliill].

£ ℃ l'lg)il1] 22.9mmS21.9rnm5

r84.-}:oA (2di/]?) '(2e19)---[---

20,9mmi.1.?i?.1.M. 1 '''' tt(2819) ttttttttttt...... !.t..,1 ktpiab}'ata Maureata

Fig. 5. Lengths of forewings and numbers of individua]s of T ptagiata and T aureatct fa1]ing within each band, Males and females are distinguished and their numbers are also shown.

Tlable.1. Numbers of indiividuals examined and ]engths of forewings of T plagiata and T attreata.

Lengths ef of'duals Species nameinNodivi forewings z p (mm, MeantSE)

Tplagilats 17 21,6±L6 4.604p(O.OOOI Maureata 20 25,Ul.5

*Lengths of forewings of two species are significantly difTerent by Mann-Whitney U tesl,

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12S Distinguishing nracola plagiata and T aureata

1985) was Germany). Usually the remaining two legs were kept in the bootstrap method (Felsenstein, performed OC -30 1 replications. We used v5 and a freezer at using a 99.5 % ethanol soaked tube for with ,OOO DNAsp (Librado

further study of other regions of DNA, The leg was Rozas, 2009) to search for sequence divergence between

the two species. homogenized in 200 yl ML-Buffer with 20 yl proteinase OC K and incubated at 55 for one hour. After incubation,

total DNA was extracted following the genomic Specimens examined manufacture's instructions, A DNA fragment of the mitochondrial cytochrome oxidase subunit I (COD gene nracola plagiata (Walker) was amplified using the polymerase chain reaction (PCR) Bong District,Chu 1 9 , Dak Lak Prov., Krong '-GGT [Vietnam] wi th the LCO1490: (5 CA ACAAATCMAAA primers Yang Sin National Park, alt. 733m, 12. iii. 2013, T. Mita GATTGG-3')andHC02198i(5'-TAAAC[I'IrCAGGGTGA et al. (Accession No; AB827314); [Philippines] 1g, CCAAAAAA TCA-3') et aL, 1994). The template (Folmer 14, Okinawa-Is., 21. ii. 2000, S. OC Negros,1991;[Japan] was as fo11ows: 94,O for 300 sec, 40 cycles at profile [Ibminaga; 1 8, ditto, 28. xi. ] 999, S. [[bminaga; 1 8, ditto, OC OC 94.0 for 30 sec, 47.0 for two examples; (except 13. xi. I999, S. Tominaga; 1?, ditto, 17. xi, 1999, S. AccessionNumberAB827313andAB827314weredone 1 Okuni-Rindou, Kunigami-vill., Okinawa., Tbminaga; 4 , OC) OC at 46 fbr 3O sec, 72,O for 60 sec; with final extension 27. v. 2000, A. Sasaki;1S, Kin-town, Okinawa, 28. v. OC at 72.0 fbr 300 sec. PCR was in a reaction preformed 2000, A. Sasaki; 19, ditto, 8. viii, 1995, K. Kudo; 1S, volume of 40 using, 3,2 of dNTP Mixture, 4.0 yl of pl yl Tengan riv., Okinawa, 22. vii. 1998, K, Kudo; 18, each 4.0 of 10xEX faq Buffer primer(10ymol/1), yl Gushikawa, Okinawa, 14, vi. 1997, K. Kudo; 19, Mt. rfaKaRa Bio, [[bkyo, Japan), O,4 vl of EX [faq (Takara Banna, Ishigaki-jima, Okinawa, 3. vi. 1973, YL Kurosawa; Bio), 2.0 of template DNA, and 22.4 yl of SDW. (fakara yl 1 \, Chichi-jima, Ogasawara, Tbkyo, 19. v. 1995, K. We asked Takara Bio Inc. to do the analysis of nucleotide Takeuchi; [Malaysia] 28, 1laiping, viii. 1973; 18, ditto, sequences by direct sequencing using a Big Dye [[bminator vii. 1973; [Indonesia] 1 X, Berastagi, N. Sumatra, 27. iv v3.1 Cycle sequencing kit Biesystems) and an (Applied - 4. v. 1988, S. &A. Saito. ABI 3730-XL genetic analyzer (Applied Biosystems, Foster City, CA, USA). nracola aureata Holloway MEGA5 software et at,, 201 1) to align We used (famura Tram [[bn, Sapa, 1926m, [Vietnam]4 83 9 , Lao CaiProv,, sequencing and to construct a neighborjoining nucleotide 22-23, vi. 2012, S. Ybshimatsu & D. Watabiki (Accession [[b (NJ; Saitou and Nei, 1987) tree using p-distance. No:AB827305-AB827306,AB827308.AB827312);1?, estimatetheconfidenceprobabi1ityfbreachinteriorbranch,

n

Figs 6-7. Distal half of female genitalia of Tl plagiata (Fig. 6) and Z aureata (Fig. 7) in ventral view. Scale bars = 1 mm. Following abbreviations as in Komai (201 1) areused.ap;apephysisposterioris, aa; apophysis anterioris, an; antrum, db; ductus bursae.

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126 D. WMABIKI and S. YOSHIMArSU

B827305) B827306) B827308) B827309)

B82731 O)

B82731 f ) B8273a2) B8273a3) B827307)

ta{AB73523G)

- O.02

Fig. 8.Neighbor-joining tree of nraeola spp. based on 616 bp ofthe mitochondrial COI gene. S))odopteraexemptaquotedfromGenbankwasusedasanoutgroupspecies.Thebootstrap method was perfOrmed with 1,OOO replications,

Lao Cai Prov., Tla Phin,1296m, 25.vi. 2012,S.Ybshimatsu and protrudes to produce an angle in the middle of both &D. Watabiki (Accession No: AB827307); ] 8,Dak Lak sides (Fig. 6), while that of T aureata is narrow and never Prov.,Krong Bong District, Chu Yang Sin National Park, protmding at the sides (Fig. 7). alt 733 m, 13. iii. 2013, T. Mita et aL (Accession No; Additionally, the male genitalia of both species are AB827313); [Laos] 14, Xieng Khouang Prov,, Phou obviously different. The most reliable distinguishing Samsoum, alt 1940m, S. Ybshimatsu & D. Watabiki; "In feature is mentioned as fbllows by Holloway (1989) [Indonesia] 38, Bali, west, ix. 1994; [Taiwan] 1g19, aureata the cucullus is triangular, the ventral angle (with Chiayi, 12-13. vii. 1964, H. Inoue; [Malaysia] 29 Thiping, spur or digitus) approximately level with the dorsal one, vii. 1987; [India] 18, Dacieeling, v. 1975, T. Hasegawa; whereas in plagiata the dorsal angle is more rounded and 1 e 1 \ , Sabah, Mt. Kinabalu, 1 6-21, lii. 1977, N. Kashiwai. strongly produced relative to the ventral one (though this Vbucher specimens are deposited in the Natural Resources is less pronounced in Pacific populations ofplagiata) inventoryCenter,NationalInstitutefbrAgrQ-EnvironmentaletcY.In Sciences (NIAES). addition, we also developed a discrimination method using standard DNA barcoding, As a result, we could Results and Discussion distinguish them successfully by this method also. We

registered their nucleotide sequences in the DNA Data The results of measurement of the length of fbrewings are Bank ofJapan (DDBJ) and we show the accession numbers shown in Fig. 5. The results apparently show that there is of those registered specimens in Fig. 8. There was 5.1% no significant difference between the length of forewings sequence divergence between the two species. This ofmaleandfemale.However,asignificantdifferencewas percentage of sequence divergence is comparable to the observed between the respective lengths of the forewings average numerical value, 5.6% of six Japanese Spodbptera of each species (fable 1). pest species as mentioned in Watabiki et at. (2013). The male genitalia of T aureata were illustrated by Holloway (1989) and Kononenko and Han (2007). However, thefemalegenitaliaofTplagiatahavenotbeenillustratedAcknowledgments andnodistinguishablecharacteristicsofthefemalegenitalia We thank Dr T, Mita of Tbkyo Uniyersity of Agriculture of thesetwo species have been gjven unti1 now, Therefbre, for providing us materials, Dr Yl Baba of NIAES for his here we show discrimination methods fbr both species kind information, Ms E. Tanaka of NIAES for DNA using the female genitalia. The most reliable character to experiments and Mr S. Sugi of [Ibkyo for kind donation distinguish them is that the antrum of T plagiataiswide of his collection to NIAES. This work was partially

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127 Distinguishing plagiata and 7：aureata

supported by JSPS KAKENHI Grant Number 24405028． 摘 要

前翅 長 が 重複す る こ とが 判明 した酷似 種 ノ コ バ ヨ ト ウ References （Tiracola ptagiata）と オ オ ノ コ バ ヨ トウ （Tiracola aureata ） 一 を 識 別す る （綿引大 祐 ・吉松 慎 ） FelsensteinJ．，1985 ． Confidence limits on phylogenies：an appr 〔｝ach − using bootstrap． E レoiution 38： 16 24 ， Tiracola 属 〔ヤ ガ 科 ： ヨ トウ ガ 亜 科 ） に は 世 界 か ら 13種 が Folmer 0 ．M ． Black W ． Hoeh R ， Lutz and R ，Vrijenhoek1994 ． の で ， ， ， ， ， 知 ら れ て い る ．日本 か ら は 束南 ア ジ ア 広 域 分 布種 もあ DNA primers for amplification of mitochondrial cytochrome る ノ コ バ ヨ ト ウ Tplagiata （Walker） と オ オ ノ コ バ ヨ ト ウ T coxidase subunit I from diverse metazoan invertebrates． aureata Holloway の 2 種 が 記 録 さ れ て い る （sugi，2001； ルfolec． Man Biot． Biotechnol．3 ：294 −297． Yoshimatsu，201D ．ノ コ バ ヨ トウ は非常 に広食性 の 様 で ，イ Holloway J． D ．1989． The of Borneo Part l2 ．4e4 figs． ， ， ， ， ， ン ドネ シ ア で は カ カ オ や パ ラ ゴ ム ノ キ 属 8pls．， Malayan nat ．ノ．42 ： 226 pp， The Malayan Nature H6 レ印 等 の 害 虫 と して の 記録 が あ り （Kalshoven alld Laan， Society， Kuala LumpuL 1981），台 湾 か ら は 柑 橘 類 の 害虫 と して 記録 さ れ て い る が Kalshoven， L． G ． E ． and P A ． van der Laan l 98L Revised and 〔Sugi，1982），両 種 は 近 年 ま で 混 同 さ れ て き た た め に translated by P A ． van der Laan with cooperation of G ． H ． （Holloway，1989），寄主植物 に つ い て は 今後 の 再検 討 が 必 要 L ．Rotschild． Ichtiar Bam −Van Hoeve ， Pests of crops in で あ る． Indonesia，720 PP． Jakarta， Indonesia． and Y ． Yoshiyasu 2011．1． Morphology Komai ，E ，S，Hashimoto ， 両種 の 翅 の 斑 紋 は 類 似 す る の に 加え て ，色彩 は 共 に 非常 に and Biology 1．Morphology In Komai F． Yoshiyasu， Y．， ， ， ， ， 変異 に 富 み （Figs 14 ），外見 的 に 両種 を 識 別 す る こ と は 非 Nasu Y and Saito T eds ， A Guide to the Lepidoptera of ’ ， ， （ ） い オ オ ノ コ バ ヨ トウ は ノ コ バ ヨ ト ウ よ り常 に − 常 に 難 し が ， Japan；3 36， Tokai University Press， Tokyo．（ln Japanese） 大 き い こ と か ら両種 の 識別 は 可 能 だ と さ れ て き た （Sugi． Kononenko ，V． S ，and H ， L ． Han ，2007． In Park， K ．T （ed ．）， Atlas 2001 ）．と こ ろ が ，筆者 らが 手元 に あ る 両種 の 標本 の 雌 雄 交 genitalia of the Noctuidae in Korea （Lepidoptera）Jnsects 尾 器 と 前 翅 長 を 詳細 に 比 較検 討 し た と こ ろ ，両 種 の 前 翅 長 of Koroa （ll），464pp，， Jungheng Publishing Co 吟SeouL コ バ ヨ に は 有意差 が あ っ た もの の （Table D ，ノ ト ウ の 中 に L 孟brado P and J． Rozas ，2009． DnaSP v5 ： asoftware for は オ オ ノ コ バ ヨ トウ よ り前翅長 の 長 い もの や 同等 の 前翅 長 comprehensive analysis of DNA polymorphism data． − を 持 つ も の が 存 在 す る こ と が 判 明 し た （Fig．5）．両 種の 雄交 Bioinformatics 25 ： 1451 1452． は Holloway 1989 に よ っ て 明瞭 な識別点 が 図示 さ れ Poole R ． W ．，1989， Lepidopterorum Catalogus（New Series） 尾器 ， （ ） こ で れ て Fascicle l 18Noctuidae Part1−3 ， L314pp ，， E ． J． BrilL て い る が ，両種 の 雌 交 尾 器 に 関 し て は れ ま 検討 さ − こ の 尾 を 細 に 観 察 Saitou，N ．and M ． Nei，1987．The neighbor joining method ： anew こ な か っ た ．そ で 筆 者 らが 両 種 雌交 器 詳 method for reconstructing phylogenetic trees． M ∂lec， Biol ． し た と こ ろ ，antrum は ノ コ バ ヨ トウ で は 幅広 く，側方 に 三 − コ バ 石ソol ．4：406 425． 角形状 に 突 出す る の に対 し ，オ オ ノ ヨ トウ で は 幅 が 狭 Sugi， S ．，1982 ． Noctuidae， In lnoue， H ． et al ．（eds ）， Moths ofJapan く，突 出 し な い こ と で 両 種 は 簡 単 に 識 別 で きる こ と が 今 回 − − − l：699 935，2：344 408，pls．146 226．（In Japanese） 新 た に 判明 し た （Figs 6，7）．ま た ，今 回 ベ トナ ム 産 の 両種 の SugiS．2001． Occu【Tence ofTiracolaplagiata Walker （Noctuidae）・ 一 ，， ， ’ 新鮮 な標本が得 られ た こ と か ら，ミ ト コ ン ド リ ア CO ノの species ofthe in Japan・Japan Heterocerists ー ー asecond genus 部 領域 の 塩基配列 を 解 読 し ，標 準 的 な DNA バ コ デ ィ J．212 ： 217−219．In Japanese with English summary ） 〔 ） 〔 ン グ に よ る 両 種 の 識別 を 試 み た と こ ろ ，有効 で あ る こ とが Tamura K ，， D ． Peterson， N ． Peterson， G ． Stecher， M ． Nei and S． 示 さ れ た ．さ ら に ，Watabiki et al ．（2013）に お い て 著名 な 害 Kumar ，2011 ．MEGA5 ： Molecular evolutionary genetics 虫 ハ ス モ ン ヨ ト ウ 等 を含 む 日本 産 ヤ ガ 科 勘 o の ρ艀 α 属 6 種 analysis using maximum like】ihc）od ， evolutionary distance， ー ー の DNA バ コ デ ィ ン グ 領 域 の 種 間 の 平 均 の 塩基置換率 and maximum parsimony methods ． Molec ， Biで冫♂． Evot．28： は 5．6％ で あ る こ と が 示 さ れ て い る が ，本 研 究 に お い て ノ 2731 −2739 ． コ バ ヨ トウ と オ オ ノ コ バ ヨ トウ の 種 問 の 塩 基 置 換 率 を 算 出 Watabiki， D ．， S． Ybshimatsu ， H ． Ybshitake， Y Baba， T， Uesato， の し た と こ ろ お よ そ 5．19 。 で あ り，日 本 産 Spodoptera OS 6 種 M ．Shimatani， H ． Ibusuki and T． Yuda，2013，Discrimination 種 間 の 平均 の 塩基置換率 と類似 の 値 を 示 した の で ，参 考ま methods for Japanese pest species of Spodoptera Guen6e で に こ こ で 触 れ て お く Fig ．8 ． （Lepidoptera：Noctuidae）lured by traps using a synthetic （ ） sex pheromone for Spodoptera exempta ．ノpn・J・ ApPl ・ EntomoL 一 な お ，本 研 究 の 部 は 科学研究 費補 助 金 （No ．24405028） Z∂o ’．57 ： 19−26 ．（In Japanese with English Summary） の 研 究 助 成 に よ り 行 わ れ た ． Ybshimatsu， S ．，2011 ．Noctuidae ， In KishidaY （ed ．）， − 2011，The Standard（ゾハ40 漉 ∫ in ／dpan 2：368 384，Gakken （Received June 28 ，2013 ． Accepted September 20，2013） Education Publishing， Tokyo ．（ln Japanese ）

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