The LepidopterologicalSocietyLepidopterological Society of Japan

ut U re Lapidoptetu Science 65 (1): 44-49, Apri1 2014

Wing color change by sunlight irradiation in the spotless grass yellow , laeta betheseba (, )

i), 2), 3) Ichiro TANAHAsHI [[bushi MIFuNE Norio HIRAI and Minoru IsHII3)

i) Department of Applied Chemistry, Faculty of Engineering, Osaka Institute of Technology, 5-16-1 Omiya, Asahi-ku, Osaka,535-8585Japan 2)YbshidaFutagozuka-aza,Nakajima-mura,Nishi-shirakawa-gun,Fukushima,961-O106Japan

3) Entomological laboratory, Graduate School of life and Environmental Science, Osaka Prefecture University, Sakai,

Osaka, 599-8531 Japan

Abstract Wing color change of the spotless grass yellow butterfiy, Eurema laeta betheseba, was compared between specimens exposed to sunlight and others kept in the dark for one month, and between specimens exposed to ultraviolet (wavelength of254 nm) and green light (S32 nm) for 8 h, The results showed that the color of the ventral surface of the hind wings was changed frem yellow to yellowish brown by sunlight irradiation. Irradiation of ultraviolet light had more effect on wing coloration than irradiation with green Iight. The color change in the wings was more marked in males than in females. Wing color did not change in specimens kept in the clark. Microscopic observations showed that the color change was derived from changes in pigrnents in wing scales. The light reflectance increased in the wavelength region of 400-500 nm, whereas it decreased in the region of 500-650 nm ufter sunlight irradiation. In the photoluminescence (PL) spectra of the wings measured at room temperature, a broad emission around 500 nm was observed, suggesting the presenceofpterinpigmentsinthescales.IhepeakintensityofPLspectrabeearneslightlysmaileraftersunlightirradiation in both sexes. These results indicate that the wing color change in E"retna laeta betheseba was due te deterioration of the pterin pigments in the scales as a result of ultraviolet light irradiatjon.

Key words Euiema laeta betheseba, photoluminescence, pterin pigment,refiectance spectrum, seasonal form, wjng colon

Introduction The spotless grags yellow, Eutema laeta betheseba, is distributed in Honshu, Shikoku and Kyushu in Japan have a wide variety of beautiful wing colors, (Kawazoe and Wakabayashi, 1 976). This coliadine butterfly which have attracted scientific and technologicaHnterest. is listed in the Red Data List of Japan as one of the most Their vividly colorfu1 wings are either due to the selectiye endangered species due to the degradation of habitats with absorption of light by pigments in the wing scales or to the 1arval fbod plant, Chamaecrista nomame (Leguminosae) the refiection of light by regularly arranged scales and (Ministry of Environment, Japan, 2006). This species has nanometer-sized structures in the scales (Ghiradella, 1972; a multivo]tine life cycle with several generations per year Kinoshita and Ybshioka, 2005; Rutowski et al., 2007; and passes the winter in the adult stage (Shir6zu, 2006). Wijnen, 2007). It is known in some species that the The adults show two distinct seasonal forms determined coloration of the body and wings of is initiated by by larval photoperiods, the summer and the autumn forms the stimuli of light, temperature and humidity (Kato and induced by long and short days respectively, and the latter Yhmada, 2001; Kato et al. 2010; Yamamoto et al., 201 1), adult forrn passes the winter (Yata, 1974). The dorsal and The two rnain subfamilies, Co]iadinae (sulfurs) and Pierinae ventral surfaces of adult wings are yellow with black (whites), of the Pieridae are distinguished from each other markings in both fbrms. by their wing colors; those of coliadine species are yellow It is known, however, that the ground color of the ventral to orange, and those ofpierine species are white. 1[Ihe wing surface of the hind wings in the autumn form varies from coloration of pierid butterflies is mainly derived from yellowish brown to reddish brown (Shir6zu, 2006). It has pigments called pterins consisting in granules or beads been reported that this color is yellow just after adult studded at the scale crossribs (Yagi, 1954; Rutowski et emergence but gradually turns yellowish brown with al., 2005; Edward, 1968; Morehouse et al,, 2007). It is elapsed time in the field (Hasegawa, 1989; Mifune, 2009). also known that the ultraviolet (UV) refiection patterns However, it is unclear what factors are responsible for this on the male wings are important for species recognition phenomenon. and sexual identification in the mating behavior of pierid this study, effects of sunlight, UV and light species (Obara and Hidaka, 1968). In green

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Wing color change of E. Iaeta 45

irradiation on the ground color change of the ventral surface IVietograpity and speetroseqpo,for wiug suijTace observadon of the hind wings of E, laeta betheseba were investigated in both Experiment I and II, the difTUse refiectance spectra by spectroscopic measurements and microscopic of intact wings were measured with a spectrophotometer observations. In addition the difference in wing coloration (Shimazu, UV3600 equipped with an integrating sphere, was compared betwecn male and female adults of E. taeta ISR-3100) with the of the wing betheseba, plane approximately perpendicular to the probe in the range from 200 to 800 nm. A white reflectance standard (BaS04) was used as a Materials and methods reference. The ventral surface of the left hind wing cut off from thebody was used to InsectsLast analyze the color change of the wings, In order to examine the pigments in the wing instar larvae of E, laeta betheseba were collected scales, the photoluminescence (PL) spectra of the wings from C nomame at Shirakawa City, Fukushima Prefecture were measured using a He-Cd laser (325 nm, 50 mW) as in the [[bhoku region ofJapan, and reared on leaves of C. an excitatien source, The emitted light from the specimens nomame under room conditions in Fukushima City late was detected by a photon-counting system with a in summer of 2008. Eighteen adults (12 males and 6 photomultimeter through a monochromater. females) of the autumn form obtained from the rearing

were ki11ed immediately after emergence, and used in two Microscopy

series of laboratory experiments, Experiments I and II, The morphology and the color of the wings were observed from November to December, 2008, withalaserscamimgmicroscopewithabluelaser(Keyence VK95OO) and with a scanning electron microscope (SEM, Experiment I: ELtllect ofsunlight exposure KeyenceVE8800) at an acceleration voltage of 8 kVl For Male and female adult specimens were prepared with the SEM observations, wing specimens were gold coated fblded wings and the wings cut off from the body at the by sputtering for 30 s (Sanyu Electron SC-701). The base with fine scissors, In order to explore the effect of sputtered specimens were attached to the sample holder sunlight exposure on the wing color change, the specimens using carbon double-sided tape. The coloration of the

were placed near the window and kept in the dark, wings was also observed by the naked eye. respectively for one month in the laboratory of Osaka

Prefecture University, Sakai City in Osaka Prefecture, The ResultsExperiment specimens were placed horizontally on their right side on a desk near the window so that the ventral surface of the I

left hind wings received sunlight through the window, The The ground color of the ventral surface of the left hind duration of sunshine during the experiment (from Nov. wing gradually changed from yellow to brown 6th to Dec. 6th, 2008) was calculated to be 170 hours frQm yellowish in male specimens subjected to one-month exposure to the data of Japan Meteorological Agency, Sakai City, 2008. sunlight, whereas that of both hind wings remained yellow As a control for the experiment, the other specimens were in male specimens kept in the dark fbr one month (Fig, put into a light-tight box and placed next to the ones which 1), The color change of the wings as a result of sunlight were exposed to sunlight, in:adiationstoppedwi'thinonemonth.Comparingspecimens of both sexes exposed to sunlight, the resultant ground Experiment U; ELCfect of UV light imadiation color ofthe ventral hind wing sumbce was datker in males

In order to explore the effect of UV light irradiation on thaninfernales(Fig.2).Microscopicobservationsshowed

the wing color change, the ventral surface of the left hind that the change in wing color as a result of sunlight exposure

wings cut off from the body of male and female adults was derived from that in scale color, but there was no was irradiated with UV light of 254 nm using a 15-W difference in the morphology of scales (Fig, 3). The density low-pressure mercury lamp (a germicidal lamp) with a ofthe scales of the males was slightly higher than that of poweT density of 1 .0 mWcm'2 for 8 h at room temperature the females, (around 25DC) in the laboratory of Osaka Institute of In the diffuse reflectance spectra from the ventral hind- [lbchnology, Irradiation of green light (532 nm) was also wing surface, the reflectance increased in the wavelength carTied out fbr the wings of male and female adults using region of 400-500 nm and decreased in the region of 500- a semiconductor laser with a power density of 1 .3 mWcm'2 650 nm after the one-month sunlight irradiation for both for 8 h at room temperature, The right hind wings cut off sexes (Fig. 4). The sunlight irradiation did not affect the from the body were put into a light-tight box, reflectance of the ventral hind wing surface in visible

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46 I. TANAHAsHI et aL

wavelengths longer than 650 nm (about 65% for both

sexes) and in the ultraviolet range shorter than 400 nm (about 5% in female and a few percent in male),

The SEM observations showed that the scaies on the ventralsurfaceofthehindwingconsistofthicklongitudinal

ribs at intervals of approximately l.7-1,8um with a series ofsmallercross-ribsseparatedbyO.9-1.1um(Fig.5).The sca]es were densely packed with beads: numerous oval- shaped beads were seen hanging down from the 1ongitudinal

ribs and cross-ribs into the scale interior, The density of

beads was higher in males than in females. Fig,1. The ventral surface of the ]eft wings of ma]e E. Iaeta

betheseba adults exposed to sun1ight{a) and kept in the dark

Experiment II (b) forone month.

The irradiation of 254 nm light had a similar effect on the

reflectance of the hind wing surface: it increased in the the same photoluminescence spectra as shown in Fig, 7, wavelength region of 400-500 nm and decreased in the

region of 500-650 nm after the irradiation when compared with that of a hind wing kept in the dark (Fig. 6). On the Discussion other hand, the refiectance of the hind wing after the In this study the ventral surface of hind wings in the autumn irradiation at 532 nm was almost the same as that of the form of E iaeta betheseba gradually changed from yellow hind wing kept in the dark. to yellowish brown when the wings were exposed to Abroademissionpeakingataround500nmwithashoulder sunlight, It was also proved in this study that irradiation around 450 nm was observed in all the photoluminescence by UV light (254 nm in wavelength) has an efTect similar spectra of the ventral surfaces of hind wings for both sexes to that of sunlight, Thus the results of this study demonstrate (Fig, 7), For both sexes, the intensity of emission was that a primary factor responsible for the color change of siightiy smaller in hind wings exposed to sunlight than in ventral hind wings in the autumn form adults after

those kept in the dark, Another specimen showed almost emergence (Hasegawa, 1989; Mifune, 2009) is UV light

't'- ' ,.'t't'/./t .,,zv. ,..,. .t t-/.t-.t.t

'

"w .v,tlt. ww-igasth "" Fig, 2. The dorsa] (left) and ventral (right) surfaces of the wmgs ot' male Ctop) and female (bottom) E, ･' laeta betheseba adults ttt/t"1tt "tt"tt a-2 tttt/tttt t/t. placed horizontally on tttt t , .. t/t t'./･maS't-g.t.' tttt ittt .. ",-.v.{as,ktssdithsde:t,t.... ''{', theirrightsideonadesk

near the window so that

the ventral surface of the

t'/ left hind wing was exposed to sunlight for awtp onc month. a-1; dersal ' 'tss surface of male wings,

a-2; ventral surface of

mu[e wings. b-1; dorgal suri'aceoffemalewings, b-1 b-2 b-2; vcntral surface of female wings.

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Wing color change of E. Iaeta 47

Fig. 3. Microseopic views of

the ventral surfaee of the right and left hind wings ef rnale (top) and femaie (bottom) E. Iaeta betheseba adults. The same specimens as shown in Fig, 2 were used. a-1; male right hind wing without sunlight irradiation,a-2:maleleft hind wing with sunlight irradiation. b- lt fema]e right hind wing without sunlightirradiation.b-2; femalc left hind wing with sunlight irradiation,

706050403e20too-tnp RH female(dark) tttt"-tr""tt ... r'.1!.- × Fig. 4. Refiectance spectra of the ventrai "'' ii × surface of thc hind wings of male and ges.ee=stov ,s' LH rnale (light) LH female(ljght) female E. taeta betheseba adults. The ,xtijY' x RH male 11 (dark) samespecimensasshowninFig.2were 'lft/tttti't ¢ used. RH and LH stand for right and oet left ventral hind wings, respectively, `'light" C`dark" and stand for sunlight irradiation and without sunlight irradiation.respectively. 200300400 500 600 700800

Wavelength / nrn

(a)Fig. (b)ind

5. SEM photographs ofa scale on thc vcntral h wing oi' male (a) and female (b) E. Iaeta betheseha adults.

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48 I. TANAHASHI et al.

70605040302010o2eo3004oo humidity. It is inferred from the results of this study that

the browning of ventral hind wings in the Australian

gex8g8v subspecies is induced by UV Iight in sunlight.

The measurement of diffuse refiectance spectra in this

study showed that the color change ofventral hind wings

¢ eet in E. Iaeta hetheseha occurred at a wavelength between 400 and 650 nm: it increased at 400-5OO nm and decreased

at 500-650 nm after the one-month sunlight irradiation. The results also demonstrate that the ¢ olor change of the see 6oo 700800 ventral surface of the hind wing by sunlight is caused by SNlavelength f nrn UV light. In pierid butterflies wing reflectance at the wavelength of 400-500 nm correlates with the Fig. 6. Reflectance spectra of the ventral surfhce of the hind wings yellowish color(StavengaandLeertouwer,2007)derivedfrompterins of male E. taeta betheseba adults exposed to irradiation of in scales 1954; Rutowski et al,, 2005). From the 254 and 532 nm light for8h and kept in the dark at room (Yagi, temperature for more than one month. refiectance spectra, light in wavelengths of less than 400 nm is absorbed in the wings, The absorption peak of zanthopterin was reported to be about 390 nm (Wijnen et al,, 2007), Therefore, light less than 400 nm in wavelength

is effective in creating color change on the ventral surface ca･--=?fi ofthehindwings.Photoluminescencespectraoftheventral

surface of hind wings measured in this study showed a broad emission peaking at around 5OO nm with a shoulder around 450 nm. The spectra are considered similar to those )sS･. of already reported pterin pigments (Parker et al., 1979). Since the intensity of emission slightly decreased after

sunlight irradiation, it seems that the color change of s-pt ventral hind wings is due to the deterioration of pterin pigments in the scales in E. iaeta betheseha.

The SEM observation revealed that each scale has numerous

300400 500 600 oval-shapecl beads hanging down from longitudinal ribs

Wavelength 1nrn700800 and cross-ribs into the scale interior in this species. It is possible that these beads consist of pterin pigments as in Fig. 7. Photoluminescence spectra of the ventral surface of the other pierids (Yagi, 1954; Rutowski et at,, 2005), The hind wings of male and female ofE, laeta betheseba adults. ground color of the ventral hind wings became darker in The same specimens as shown in Fig. 2 were used. RH and "light" males than in females after sunlight irradiation in this LH stand for right and reft hind wings, respectively. species, which may be related to the finding that the bead and"dark"standforsunlightirradiationandwithoutsunlight density was higher in males than in females. irradiation,respectively. Tanaka et al. (2005) inferred fi:om field observations that this species has two peaks of adult emergence in autumn

with individuals emerging later having brownish ventral in sunlight. The results are consistent with the wide color hind wings, Moreover, the color ofthe ventral hind variation of ventral hind wings of the autumn fbrm adults ground wings in the autumn fbrm adults collected in the Yheyama found in the field. It should be noted that the color change Islands, southwestern Japan and Wakabayashi, of ventral hind wings by sunlight occurs even in dead and (Kawazoe 1976) and Shimane Prefecture, western Japan (Mishima, dried specimens in this species. Jones et al, ( 1985) clarified 1989) was sometimes extremely dark brown, and these by rearing experiments that the Australian subspecies, E. were therefore considered to be a djfferent subspecies Iaeta lineata, has two seasonal fbrms similar to those of and Wakabayashi, 1976; Inomata, 2005). It is the Japanese subspecies, E, laeta betheseba, However, (Kawazoe also inferred from the results of our study that these Jones et al, (1985) and Jones (1992) failed to find factors individuals had been exposed to UV light in sunlight causing the ground color of ventral hind wings to change intensely andror for a long time after emergence. from yellow to brown in the autumn form of E. Iaeta tineata by controlling temperature, photoperiod and

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Wing color change of E． laeta 49

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colors natlIre ： KinoshitaS．and S．Yoshieka2005．Structural in ， ツ マ グ ロ キチ ョ ウEuremalaetabethesebaの 腹側翅に太 陽光， Ihe role of regularity and irregularity in the structure・ 254nm お よび532nm の 波長 の 光 を室 温 で 照射 した と きの ChemPh isGhem 6： 1−19． ） 翅 の 色彩変化 に つ い て 検討 し た ．雌雄 の ツ マ グ ロ キ チ ョ ウ Mifune T．，2009， E ” rema laeta living in the no 曲 em limit of its − − − の 翅 の 反射 ス ペ ク ト ル お よ び フ ォ トル ミ ネ ッ セ ン ス ス ペ ク 「ange （II）・Fukushimano mushi 27 ： 1 7，（ln Japanese） ト ル を 測定 し ，雌雄 で の 翅 の 色彩変化の 違 い に つ い て も検 Mishima S，1989，A record of Eurema laeta with dark brown hind 討 した ．腹側翅 は ，太陽光と254nm の 光照射に よ り次第 に wings collected in Shimane Peninsula． Choken Field〔44）： 一 黄色 か ら黄褐色 に 色彩が変化 した， 方，暗所 に 保存 した 31．（In Japanese） 翅 や 532nm の 光を照射 した 翅 に は 色彩 の 変化が 見 られ な Morehouse N ．1．，RVukusic 勧nd R ， Rutowski，2007．Pterin pigment か っ た．翅 の 反射ス ペ ク トル にお い て ，太陽光照射後の 反 granules are responsible for both broadband light scattering 射率 は，照射前 と比 べ て ，400−500nm の 波長領域 で 大きく amd wavelength selective absorption in the wing scales of − − こ っ た ．こ の よ pierid butterfiies， Pn ｝c ． R ， Soc． B 274： 359366， な り，500650nm で 小 さ くなる とが分か う ペ and of on ス ル の は の の の よ か っ ObaraY T ，Hidaka， 1968．Recognition thefemale， the な ク ト 変化 雄 翅 方が雌 翅 りも大 き ー ー basis of ultra−violet re 且ection ， in the white cabbage butter月y， た ．色彩 が 変化す る 前後 の 翅 を レ ザ 顕微鏡 に よ り観察 Pieris rapae crucivo 砌 BoisduvaL Proc ． JpnL Acad ．44 ：829− した と こ ろ ．翅 の 色彩変化は 鱗粉の 色彩変化 に 起因 し て い

832， る こ と が 分 か っ た ．ツ マ グ ロ キ チ ョ ウ の 翅 の 黄色 か ら黄褐 Free E ． M 鋤 d R ． B ．Dunlap ー ParkerP．T．，R ．S． 旦ander ， ．Schulman ， 色 は ，鱗粉 に 存在 し て い る 顆粒 （ビ ズ）中の プ テ リ ン 系 1979．Room temperature PhQsphorescence of sclected 色素 に よ る と考え ら れ る ．雄 の 鱗粉 に は 雌の 鱗粉 に 比 べ ， ．Anal． Chem ．51； 1921−1926． pteridines ビーズが 高密度 で 存在 し，こ の こ とは ，雄の 翅の 色彩変化

RutowskiR．L．J．M ．Macedonia」．W ．Merry， N ．Morehouse， ， ， よ い で る と る， に る − が雌 りも大 き 要因 あ 考えられ 室温 お け K ．Yturralde， L． TaylorTaft， D ． Gaalema， D ．」． Kemp and 腹側翅 の フ ォ トル ミ ネ ッ セ ン ス ス ペ ク トル か らプ テ リン 系 R ，S，Papke，2007，Iridescent ultraviolet signal in the orange 色素の 存在が 分 か り，太陽光 に 含まれ る 紫外線 に よ りプ テ sulphur butterfly（Colias eurytheme ）： spatial ， temporal and − リ ン 系色素 が 変質 し翅 の 色 彩変化 が 起 こ る もの と考 え られ sPect 「al properties． Biol．ノ1 Linn．∫oc ，90： 349 364， る ． Rutowski R ． L ，」． M ， Macedonia， N ． Morehouse and L． Taylor−

Taft，2005． Pterin arnplify iridescent ultraviolet pigments （Received November 12，2013、 Accepted January 17，2014） signal in 皿 ales of the orange sulphur buttorfly Cotias − e ”りithe 〃 te ， Proc． R ， Soc． B 272： 2329 2335．

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