Fluorescent pupal secretion of the papilionid butterfly, Luehdorfia japonica

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TThhee LLeepipdiopdteorpoltoegircaollSoocgieitycal Society ooff JJaapapnan
utEwh 7>'ans.tepidS.oc,Japan 59 (2):165-170,March2008

Fluorescent pupal secretion of the papilioni dbutterfl Lyu,ehdoijia japonica
KeiichiHoNDAb*,HiroshiHoNDA2) Hisashi6MuRAi)
and
"'Graduate Schoo lof Biosphere Science, Hiroshima University ,Higashihjroshi m73a9,-8528 Japan
]) Graduat eSchoo lof Li fe and Environmcnta Slcience sU,niversit yof Tsukuba, Tsukuba, 305-8572 Japan
Abstract Pupac of the papilioni dbutterfl yL,uehdocfia j'aponic ase,crete a slightly viscous ljquid
immediately after pupatio nfrom thc dorsa lsurface of the head and thorax, mainly from pro- and
meso-thoraces. The secretion is colored pale yellowish-bro wannd emits greenish-blu efluores-
cence under ligh tirradiati oTnh,e fiuid ,which gradually covers the dorsa lsurface of almost the
whole body within 2 hours of 1arval-pu peacdlysis, solidifies a]ong with the melanization and scle-
rotization of the pupal cuticle. The solidified exudate was almost insolubl ein water, but was found
to absorb a considerable quantLt yof water, suggcsting that the secretion serves as a moisturizer
against desiccati oAnI.though the secretion showed no proniinen tantimicrobial activity, it seerned
to huve a weak bacteriostat ietcfect, Possibl eecological roles of the secretion are discussed in re-
gard to the survival strategy of the butterfly.
Key words Pupal secretion, Lttehdoijiajopon iPcapai,lionidae ,fluorescen ccheem,ical defense.
Introduction
Luehdocf7a,icrpon ianc aA,ristolochiaceae-fee dpiapniglioni dendemic to Japan ,is a univol-
tine butterf lwiyth a unique lif ecycle. The butterf leymerges in early spring (i nApril in
many localiti eofs Japan) and Iarva egrow int epupae in about 2 months (mi dJune),
Pupation usually takes place on dead twigs or stones in a dar kand somewhat wet environ-
ment close to the ground ,Pupae pass summer and autumn in diapausin gstate, and hiber-
nate as pharat eadults until eclosion in the foIlowin gspring (Hidak eat al., 1971; Ishi iand
Hidaka, 1982). Pupae have a thick and hard cuticle and this appears te be very important
for them to survive hot and cold seasons lastin gas long as 10 months. While the cuticle of
pupae fres hfrom ecdysis is very flexibl eand colored creamy-white, it turns black and tough
within about 12 hours due to melanization and sclerotization (Fi gI.A). We have found that
shortly after the commencement of larval-pup eacldysis, pupae begin to exude a liquid col-
ored yellowish-brow fnrom the dorsa lintegument ,As far as we know, no publication shave
so far referred to pupal secretion, at leas tin Lepidoptera. We report here on the pupal exu-
date of L. y'aponic aand discus sit spossible role in the survival strategy during the long
pupal period of the butterfly.
Materials
and methods
Insects
Eggs of L. japoni cweare obtained from female scollected in Kanagawa and Hiroshima
Prefecture sJ,apan .Larvae fed with fres hleave sof Heterotrop ablumei or H. nilu)onica
(Aristolochia cweeraee r)eared at 18eC on a 16: 8 hours ligh td:ark photo regime.
"Correspondingauthor:honce@hiroshima-u,ac,jp
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TThhee LLeepipdiopdteorpoltoegircaollSoocgieitycal Society ooff JJaapapnan
166 Keiich iHoNDix ,Hiroshi HoNDA and Hisashi 6MLrRA
Collection of pupal secretion
Two hours after larval-pup aecldysis, the secretion was rinsed out of the integumen tby
soaking a pupa in distill ewadter with continuous swirling for 30 sec. The secretion was
collected from a total of 80 pupae using 100 ml of water. The resultant aq, solution of the
secretion was filter etdo rcmove miscellaneous insoluble matter, concentrated in vacb{o
below 500C to ca 50 ml, and stored in a refrigerator (a t50C) until use,
Fractionatio nand chemical analysis of the secretien
An aliquot of the concentrate of the secretion was once lyophilize dan,d extracted with
methanol to atford an insolubl esubstance (A) and a yel]ow methanolic solution. The
methanol-soluble substances were subjected to thin-lay echrromatography (TLC )on silicic
acid (Merck TLC plat eSilic agel 60) with methano]lethyl acctate (20/8 0as) a developer.
Spots were detected wi th a UV lamp.
Fluorescenc sepectra were measured in methanol with a Hitachi F-2000 spectrofluorometer.
IR spectra werc recorded on a JASCO IR-810 infrare sdpectrophotometer, FAB-MS spectra
were recorded on a JEOL JMS-DX 303 mass spectrometer using glyceri ans a matrix.
Inspection of exudation site
Immediatel yafter larval-pup aecldysis, the whole body of the pupa was washed with water
fbr several seconds. Tb make the site of secretion visible, the dorsal surface of the pupa,
which was held either horizontall yor perpendicularly ,was uniformly painted with a com-
mercial white watercolor (Holbe Wionrks, Ltd,) .The developmcnt of the yellowish-brown
color of the exudate was observed 2 hours later.
Moisturizing efft:ct of the secretion
In this experiment, we used pupae of 2 cohorts consisting of 10 individua lesach.
Immecliate layfter larval-pup aecldysis, pupae of one group were washed with a large
amount of water under ultrasonication and those of the other were lef tuntreated.
Approximately 1 day after pupation, treated and intac tpupae were transferred into an incu-
bator and kept at 200C and 90% RH for 24 hours. Sum tota lof the pupal weight of each
group was measured befbre and after incubation .The tota lweights of IO treate dand lO in-
tact pupae at ca 50% RH (befb irnecubatio nwe)re 5312.9 mg and 5500,3 mg, respectively,
Antimicrobial
activity
Since preliminary experiments revealed that the pupal secretion exerted neither fungicidal
nor bactericid aactlivities, it santimicrobial activity was briefl yexamined. Antifungal activ-
ity of the secretion was tested against AspeJgillt{ nsig. er, which was precultur eadt 320C in a
SCD broth medium for 72 hours .The 96-wel lplate swere prepare dby dispensin ignto each
well 1OO ul of serial dilutio nofs a test sample (sol imadterial of the secretion), 1O ut of the
inocu]um ,and 90 "l of the nutrient broth or sterilized water. Content sof each well were
thoroughly mixed with a pipett eand the plate sincubated at 32eC for 72 hours .The sample
was tested at three doses ;430 "g/ml, 215 uglmt, and 108 uglml. Any growth of the fungus
was assessed by microscopically monitoring mycelial growth. Antibacteria lactMty was
tested against two organisms, Escherichia coli and Staphytococcu saureus, The bacteria
were precultur eaderobically at 320C in a SCD broth medium for 24 hours ,A mixture con-
sisting of 1 ml of a test sample and 1O ml of the inoculum was incubated at 32"C. After 72
hours and 7 days ,the number of viable cells was counted, The sample was tested at i55 ugl
mL
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Pupal Secretio nof a Butterfly 167
Results discussion
and
General
observation
The pupal secretion, which gradually covered the dorsa lsurface of almost the whole body
within 2 heurs of larval-pup eacldysis, solidified along with the melanization and sclerotiza-
tion of the pupal cuticle within about 12 hours ,The solidified exudate was almost insoluble
in water, and therefor reemained on the surface of the pupal cuticle throughout.
Chemical nature of the secretion
Brief examination of th eexudate revealed tha tthe secretion contained solid materials at an
average of 1.28 mglindividual, and was composed of at least three major substances, viz, a
methanol-insoluble substance (1 )and two fluoresce ncotmpounds (2 and 3; 725 "glindivid-
ual in total) ,Compound 2 was detecte dat Rf O,3 on TLC, but compound 3 was not devel-
eped with the solvent system used. Substanc e1 (pal yeellow amorphous powder) was con-
sidered to be a non-proteineous substance, because a ninhydrin test of it shydrolysi psroduct
(10% aq, HCI at 80'C for 2 hours) was negative, Although the substance appeared to be
c･. ･, f-'i
A
1 2 3 4
Fig. 1. A: L. japoni pcuapae ;upper: 1-day-old pupa, lower: pupa fresh from ecdysis, Bl: A pupa
of which dorsa lsurface was painte din -'hite and kept horizontall yafter larval-pupa lecdy-
sis. A brownish tin tdue to the exudate can be seen developin ognly over the upper half
(hea dand thoraces )of the body; B2: A pupa of which dorsal surface was painte din white
and kept perpendicularl yafter ecdysis. YelLowish-bro wcnolor is spread as far as the ab-
domen; B3: A pupa washed with water immediatel ayfter ecdysisl B4: An intac ptupa ,C:
Intac ptupae under UV irradiati sohnow,ing tha ttheir dorsal surface is covered with fluores-
cent substances. D: A methanolic solution of compound 2 ernitting greenish-blu efluores-
cence under UV irradiation.
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j68 Keiich iHoNDA, Hirosh iHoNDA and Hisash i6MuRA
ro:ut
m-
300400500600700800
nm
Fig. 2, Fluorescence spectrum of compound 2 in methanol (excit eatd 265 nm).
contaminated with small amounts of miscellaneo ucsompounds, substance 1 was presumed
to be a salt of carboxylated polysaccharid eb,ased on it sIR spectra (327 6m,; 2937, w;
1599, s; 1390, s; 1118, s; 1048 cm'L, m) and elementary analytical data (C :37,7%, H: 6.3%,
N: 3.3%). Compounds 2 and 3 exhibited similar greenish-bl ufeiuorescen cuneder UV irra-
diatio nin methanol (Fi gI.D). The fluoresce nspceectrum of compound 2 (emissi moaxni-
mum at 512 nm; excitation at 265 nm in methanol) is shown in Fig. 2. Compound 2 exhib-
ite da quasi-molecul airen [(M+H) ･at] mlz 265, suggesting a molecular weight of 264.
Since purificati oofn compound 3 was not successfu1, the compound was not pursued fur-
ther.The
site of exudation
As Fig .IB clearly shows, the secretion is exuded from the dorsa lsurface of both the head
and the thorax, mainly from the prothora xand mesothorax, No secretion was exuded from
the abdomen. We tried to fin dthe outlet of the secretion by means of scanning electron mi-
croscopy, but several attempts faile dto find any pores or similar architecture on the dorsal
surface likel yto exude a liquid .Further histologica linvestigation osn the epidermal tissue
of pupae are needed to locat ethe secretary organ and clarify the origin of the exudate.
Hygroscopic nature of the secretion
After incubatio nt,he total weight gain of 10 treated (washe dp)upae was only 2.6 mg
(O,05 %of the original weight), whereas that of 10 intac tpupae amounted to 48,3 mg
(O.88% )T.his unequivocally implies that the pupal exudate absorbs a significant quantity
of water, and thus strongly suggests that the exudate would serv ¢ as a moisturizing agent.
Antimicrobial activity
The secretion exerted no fungicidal activity against A. niger even at the highest concentra-
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PupalSecretion Butterfly 169
of a
Table 1. Tests for antibacterial activity of
pupal secretion ofL.japonica.
Nbfll(D p
Bacteriam
w Oh 72h 7d
E. c,oli 1×10i 1×10i ×loT4
S. 1.4102 5.710i1 10
aureus × × ×
tion (43 0gglml). However, mycelial growth was not observed in simple water media. This
is suggestive of a weak suppressive effect of the secretion on the proliferat iofo nthe fungus.
Since an aq, solution of the secretion (O.1 w7lv% of the solid material) was alkaline (pH:
9.2 at 200C), the possibili ctanynot be ruled out that the inhibiti oofn mycelial grewth might
have been, in part ,due to the alkalinity of the secretion. Similarl yno, bactericida lactivity
was found againstE. coli and S. aunetts ([fab l1)e .However, the secretioll seemed to have a
weak bacteriostat iecffect against S, at{ne"s at the concentration tested. We were not able to
measure accurate quantit yof the secretion one larv aexudes, but per ccrpita quantit ywas es-
timated at a few tens of micro liter sat most. Since one pupa exudes 1.28 mg of secretion
(sol imadterial) on average, the actual concentration of the solute on the pupal surface
would be much higher than that employed fbr the antimicrobial assays.
Possibl eecological role of pupal secretion
Larvae of Lttehdor: fsipeacies have been reported to exude, from the cervical fbrked ex-
ocrine glands (osmeter aina o)do,riferous liqui d(Hond a1,980) ,which has been demonstrat-
ed to be biosynthesi dzee ndovo (Hond a1,900) and to play a defensiv reole against predato-
ry enemies (Honda ,1983). [[b the best of our knowledge, however ,no publication shave
hithert roeferred to exocrine substances secreted by lepidoptero upsupae.
As describe dabove, since L, J'aponica larvae usually pupate in dark and somewhat wet
place sclose to the ground and pupae pass the summer in the diapausing state, and hibernate
as pharat eadults, the pupae most likel yhave some defensiv emaneuvers to protec tthem-
selves from considerable climatic changes, microorganjsms and predator yenemies. We
propose the fo11owin gdefensive roles of the pupal secretion: The secretion may function as
(1 )a moisturizer that prevent sthe pupae from desiccati odnuring hot and dry cold seasons,
(2 )an antimicrobial agent that inhibi ttshe preliferat iofo nfungi andlor bacteri oan cuticular
surface, and (3 )a visual threa t(fluoresc eemniscsieon) to small predator yenemies like
Iizard osr rats. It is feasibl tehat the secretion of pupae in combination with their hard cuti-
cle may provid tehem with survival benefit dsuring the long immotile stage,
Aeknowledgments
We thank Tttkash iA. Inoue at the Japanes eNationa lInstitu otfeAgrobiological Science sfor
SEM observations.
References
Hidaka ,T. ,Ishizuk aY,. and Y. Sakagami ,1971 .Contro lof pupal diapause and adult differentiati ionn a uni-
voltine papilioni dbutterfl yL,uehdodiajaponica. J. j}tsec Pthysiol .17: 197-203.
Honda, K,, 1980, Osrneteria lsecretions of papilion ilcairvae in the genera, Luehdor:fi a,Grcrphiu mand
Atrophaneura (Lcpidopte rInas)e.ct Biochem. 10: 583-588.
, 1983. Defensive potenti aefl components of the larva losmeterial secretion of papilioni dbutterfiies
against ants. Ph.vsio tE.nt. 8: 173-179.
1990 .GC-MS and i]C-NMR studies en the biosynthesi sof terpenoid defensive secretions by the
,
larvae ofpapiliunid butterfli e(sLuehdo, zanfdi Paapilio) .Ihsec tB.iochem ,20: 245-250.
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TThhee　 LLeepipdiopdteorpoltoegiroaollogical　 SSoooiceityety 　ooff　 JJaapapnan
170 Keiich　iHoptDA，　Hirosh　iHoNDA　and 　Hisash　iO“｛uRA
1　shi，ノ　iaMp ‘．　）naincda 　（TLc．　pHjiddoapktae，ra1，　9P8a2pi．　hCQhnairdaacet）e．　rKiosntiり　c漉sof　5p0u：6p1a0l　−d6i2a0p．aus　ei旧 he　univoltine 　papilioni，d　LL‘8鰄o頑α
摘 要
一 ・ ・
ギフ チ ョ ウ蛹の蛍光性分泌物 （本田計 本田　洋 大村　尚）
・ ・
ギフチ ョ ウの蛹は蛹化直後に頭 胸部背面，主に前 中胸部の 背面か らやや粘性の液体を分泌する，
分泌物は淡黄褐色を呈 し，特に長波長紫外線照射下で緑青色の蛍光を発する （極大波長：512・1m ）．分
泌液は脱皮 （蛹化）後約2時間でほぼ全身の背面を覆い，蛹クチクラの着色 （黒化）と硬化の進行に伴っ
て固化する．い っ たん固化 した分泌物は水にほとん ど不溶となり，蛹の期間中ずっ と体表面に残る，し
か し分泌物は顕著な吸水 （保水）性を示し，乾燥に対する保湿剤 として機能するこ とが示唆された．分
泌液は弱アリカリ性を示し，顕著な抗菌性は示さなかっ たものの弱い 静菌作用が認められた．これ らの
こ とをふ まえて，本種蛹の生存戦略に関わる分泌物の生態学的な機能につ い て 考察した．
（Accepted　November 　22，2007）
Pub］Lshe　dby　the　Lcp弖dopterologicu且Society　of　Japan，
5−2〔｝，Mot yokoyama 　2，　Hとしchiqii ，　Tokyo，192−OO63　Japan
一
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