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Runn ing head : Hairpencil se cretion of bu tter f ly
Major components i n the hairpencil sec retion of a butterfly,
Euploea m ulc iber (Le pi doptera, Danaidae) : Their origi ns
and mal e behavioral responses to pyrrol i zidine alkal oids
Yasuyuk i Honda, Keiich i Honda* , Hisas h i Ômura
Department o f Bio funct iona l Scie nce and Techno log y, Gr adua te School
o f Biosphere Science, H ir osh ima Un iv ersity , Higash ih irosh ima 739-8528 ,
J apan
*Correspond ing au tho r . Tel. : +81-82 -424- 6501 ; fa x: +81-82- 424-0758.
E- ma il addr ess: honce@hir osh ima-u .a c.jp ( K. Honda)
Manuscript
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Abst ract
Two compounds, 9 ,10- epoxytetr ahydroedu lan ( ET) and v ir id if lo r ine
ß - la ctone (VL) , we re iden tif ie d as majo r c omponen ts f rom the
ha irpencils o f f ield - caugh t males o f a danaid bu tter f ly, Eup loea mu lciber .
By con tras t, laboratory-reared ma les en tir ely lacked VL, bu t possessed a
s ign if ican t quan tity o f ET. Var ious feed ing e xper ime n ts with lar vae
a nd indoor adu lt males s trongly suggested that ET is b io syn thes ized de
novo on ly af ter eclo sion from nu tr ien ts inge ste d dur ing the larv al
developmen t. S ince VL was suspected to be der ived from pyrro li zid ine
a lkalo ids (PAs) acqu ired as an adu lt, te s ts for fe ed ing response to and
o ral admin is tration of four PAs (a 4:1 mixtu re of
in te rmed ine /lycopsa mine, helio tr ine, monocro taline, and re tronecine)
wer e c onducted . When the ta rs i or proboscis wer e s timu la ted w ith PA
s o lu tions , ma les sho we d pos itive feed ing responses (p roboscis ex tens ion
a nd suck ing movemen ts) to in ter me d ine/lycopsamine, he lio tr ine, and
r etronecine in decr easing o rder o f re sponsiveness , thereby prov id ing
e v iden ce that male adu lts ar e endowed with tas te recep tor (s) spe cif ic to
PAs on the legs as we ll as on the proboscis . Dif f eren tly f rom gus tato ry
r espon sive ness , on ly males fed with in termed ine/lyco psamine produced
a s ign if ican t quan tity o f VL (ca. 35 µg/ma le) , whereas those th at
inges ted helio tr ine or monocro taline hyd roch lor ide pr oduced tr aces of
VL (<0.18 µg/ma le) . Up take o f retronec ine d id no t lead to VL
fo rmation at al l. In behav ioral b ioass ays to tes t the attra ctiv ity o f PAs
to males , al l ind iv idua ls tes ted were attracted exclusiv ely to
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in te rmed ine /lycopsa mine. Th is shows that cer tain PA(s) per se serve
a s attr actan t( s) for ma les in loca ting PA sou rces , and fur th er suggests
that in the f ield , ma les w il l seek par tic u lar PA(s) that are ind ispensab le
a s pr ecursors for the eff icien t b io syn thesis of VL.
Key words: Biosyn thesis ; Sex pheromone ; Pyr ro liz id ine a lkalo id s;
At tra ctiv ity ; Gusta to r y re cep tion
1 . I ntroduct ion
Che mical s ignaling by males is no les s impor tan t tha n by females in
the c our tsh ip of some lep idop ter ans (e.g. Eisner and Me inwald , 1987 ;
Birch et al. , 1990) . In bu tter f lies , vo latile chemicals d isseminate d by
males from the and rocon ial o r gans have been r epor ted to gener ally p lay a
much more s ign if ican t ro le than those of fe males du r ing the sequ ence of
mating be hav io r (Boppr é, 1984 ; Honda, 2005) . Among the mos t
c onsp icuous androcon ial ar ch itectu res bu tter f lies possess ar e the
s cen t-producin g organs found in the family Dana idae . Ma le adu lts o f
most danaids have two types o f scen t-producing or gans, i.e. a pa ir of
a bdominal ha irb rushes (hairpenc ils) and alar pa tch - or pou ch- like glands
( sex brand ) (P liske and Salpeter , 1971 ; Boppr é and Vane-Wr igh t, 1989) .
The hairpencils , though ord inar ily r etr acte d in s ide the a bdomen , a re
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e xtr uded and sp layed nearby th e female a t a species-spec if ically
p rogra mmed timing dur ing the p recopu laro ty pursu it o r hover ing. The
ha irpencil chemica ls are perceived b y f ema les at the time o f
ha irpencilling (Brower et a l. , 1965 ; Mo to fu ji, 1976) .
Ex te nsive inve stiga tions conducted of a number o f lep idop ter ans
have r evealed the occur rence of d iverse compounds in the and rocon ial
s ecre tions, wh ich are though t to be h igh ly responsib le f or sexu al
c ommunication . D ihydropyrro liz ine der iva tives such as dan aidon e,
hydroxyd anaidal, and dana idal are the componen ts most frequen tly
e ncoun tered in danaid secr etions ( e.g. Me inwald et al. , 1966, 1974 ;
Edgar et al. , 1971, 1973 ; Koma e e t al. , 1982 ; Honda et al., 1995 ; Schu lz,
1998 ; Schu lz e t a l. , 2004) . In so me danaids , thes e con stitue n ts are
a ccompan ied by terpeno ids, aliphatic acid s , es ters , and o ther
miscel lane ous compounds ( Bellas et a l. , 19 74 ; Sc hu lz et a l. , 1988a,b ,
1993b ; Schu lz and Nish id a, 1996) , thus probab ly fu rn ish ing a
s pecies-specif ic bouque t that ser ves a s an arr esta n t o r an aphro d is iac
e licit ing the r ecep tiv e response from females ( Meinwald et a l. , 1969 ;
P lis ke a nd Eisner , 1969; N is h ida et al. , 1996) .
C lose as sociations o f some lep idop ter an ta xa w ith pyrro li zid ine
a lkalo ids ( PAs) have long d rawn atten t ion and have been well
documen te d in regard to pharmacophagy ( Boppré , 1990 ; Ha r tmann and
Ober , 2000) . For exa mple, larvae of several mo th species in the family
Ar cti idae are known to take up and s tore PAs from PA- con ta in ing hos t
p lan ts (Weller et al ., 1999) and adu lts , pa r ticu lar ly males , o f mos t
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bu tter f ly spe cie s of the Dan aidae and I thomiidae als o congregate on
de caying o r dead p lan ts con ta in ing PAs in the f amilies Bo raginacea e,
Astera ceae, and Fabaceae , from wh ich the y imb ibe the alkalo ids (P lisk e,
1975a ; Boppr é, 1981) . PAs are also inciden tally imb ibed together w ith
the nectar of PA- con ta in ing in f lorescenc e (P liske , 1975b) .
P lan t-acqu ired PAs are seques ter ed , of te n chemic ally mod if ied , and
s ystemical ly s to red (e.g. Edgar and Cu lvenor , 1974; Tr igo e t a l. , 1994,
1996 ; Brückmann et a l., 2000 ; Har tmann et a l., 2005) . In Le p idop ter a,
inges ted PAs have been shown to be u tilized as p r ecur sors for
pheromone b iosyn thesis and als o for che mical p ro tec tion a gains t
p redators (Boppré a nd Schneider , 1985 ; Orr et al., 1996 ; Con ner et al. ,
2000 ; Ros sin i e t a l. , 2003) . The regu lato ry actio n o f PAs on the
developmen t o f cor emata ( tubu la r abdominal scen t or gans of arctiid s)
ha s also been repor ted for ar ctiid mo ths (Boppré and Schneider , 1985;
Ege lhaa f e t a l. , 1992) . Ano ther PA- insec t relationsh ip is f ound in the
e gg- layin g by a p r imitive O ld Wor ld danaid bu tter f ly , I dea l eucono e ,
wh ich exp lo its mac rocyclic PAs p resen t in its host p la n t ( Parsons ia
l aeviga ta ) a s ov ipos ition s timu lan ts (Honda et al., 1997) .
I t is fu lly es tab lis hed that hyd roxydanaida l, an impor tan t ma le
pheromone of some arc tiid mo th s, is b iosyn thes ize d from var ious PA
p recur sors through sp ecif ic chemical tr ansformations (Schu lz et a l. ,
1993a ; Ha r tmann et a l. , 2003 , 2005) . I n bu tter f lies , however , v ery
l ittle is k nown abou t the b iosyn thetic mecha n isms invo lved in the
p roduction o f androcon ial sub stances includ ing d ihydropyrro li zines .
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Since ear lier work has repor ted that in Danaus bu tterf lies , danaidone
was der ived from PAs (Schneider et al., 1975) , it s eems to be a gener al
no tion that dana id ma les ar e capab le o f hand ling a wide var iety o f PAs to
p roduce d ihydropyrro lizine der iva tives . Recen tly , however , we have
de monstr ated that male s o f Parantica s it a , an Asia n danaid bu tter f ly, can
on ly successfu lly conv er t lim ited ch emical types of PAs in to danaidone
( Honda et a l. , 2005) . Apa r t f rom the der iva tion of and rocon ial
s ubstances , on ly a fe w stud ies have exper imen tally subs tan tia ted their
pheromonal activ i ty. Therefor e, the pher omone system of dana id
bu tter f lie s , on the who le, r emains poo r ly understood .
We chos e as a rese arch ob jec t the s tr ipe d b lue crow bu tte r f ly,
Eup loea mu lciber (Lep idop te ra, Danaidae) , wh ich be longs to the same
s ub tr ibe Eup loeina as does I . leuconoe (Acker y and Vane-Wr igh t, 1984) .
The genus Eup loea is a r ela tively lar ge g roup compr is ing more than 50
s pecies . E. mu lciber , a sub trop ical o r trop ical bu tter f ly occurr ing from
the Sou thwes ter n I s la nds of Japan to Indo -Australian re gions , u tili zes as
host p lan ts a great var iety o f species in the families Mor acea e,
Apoc ynacea e, and Asclep iadacea e. The ir major host p la n ts in J apan
a re Ficus microcarpa , F. b en jamina (Mora ceae) , and Nerium ind icum
( Apocynac eae) . Ano ther milkweed , As clep ias curassavica
( Ascle p iadaceae) , is also a food p la n t very su ita b le for the larval growth .
Al l these p lan ts are devo id of PAs. Presumab ly for that reason , ma le
a du lts o f the bu tterf ly as we ll as o ther danaids are of ten observed in the
f ield to v is it f lowers and withered o r d is eased tissue of PA-con tain ing
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p lan ts . E. mu lciber males , un like Danaus male s , never exh ib it con ta ct
behav ior between the two scen t or gans p r io r to cour tsh ip , wh ich , in
c er tain danaid species , is believed to be a p rer equ is ite fo r the production
o f a ndrocon ial s ubstances (Brower and Jones , 1965 ; Boppr é et al. , 1978) .
Al though Francke et al. ( 1989) have alr eady r epor ted a terpeno id
c omponen t ( 9,10 -epoxy tetrahydroedu lan) from the hair pencils o f E.
mu lcibe r and two o th er Eup loea spe cies (E. klug ii a nd E. leucost ic tos) ,
a lmost no inf ormation is ava ilab le on o ther constituen ts , the ir o r igin s , or
a bou t how bu tter f lie s man ipu la te p la n t-a cqu ired PAs .
I n the pres en t work , we investiga ted vo lat ile compound s in the
ha irpencils o f E. mu lcibe r and attempted to elucidate the der ivation of
the majo r componen ts . Fu r thermore, the attr activ i ty o f and gus tato ry
r espon se to sever al PAs in adu lt ma les were also examined by beha v ior al
b ioassays.
2 . Mate ria l and methods
2 .1 . In sect s
E. mu lc iber adu lts cap tured in the Sou thwes tern I s lands (Ok inawa
a nd Kagosh ima p refectu res , Japan) and their progen y were u se d in th is
s tudy. Larv ae f ed with fr esh lea ves o f F. microcarpa , N. ind icum or A.
c urassavic a were raised under s tandard labor ator y cond it ions (16L:8D,
24-26 º C) . Occasionally, larva l rear ing was conduc ted on po tted p lan ts
in a g reenhouse ( 23-27 º C and under a natural pho toper iod regime) .
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Adults were sexed immed ia te ly af ter eclo sion and ma les were kep t in
transparen t p las tic chamber s at 25 º C under a 16L:8D re gime ( ca. 100
lux) . Throu ghou t th e exper imen ts , they we re fed w ith 15% a q . sucrose
s o lu tion once a da y, and exposed daily to incandescen t l igh t ( ca. 3500
lux) for at least 8h dur ing the pho tophase , un les s o therwise no te d . To
main ta in labo rato r y cu ltu r es , several young adu lts o f bo th sexes we re
r ele ased in to a n ou tdoor cage equ ipped with f lowers and PA-con tain ing
p lan ts (Eupa torium spp .) and allowed to ma te. Af te r copu lation , grav id
f emales were transfer red to the laboratory to co lle c t eggs.
2 .2 . Prepara t ion o f ha irpencil ext racts and ch emica l ana lysis
The hairpencils were ar tif icia lly p ro truded with for ceps , excise d ,
s oaked ind iv idually in 100 µ l o f pur if ied d ich lo rome than e, and s tor ed at
- 20ﾟC un til u se. For labora tory -reared males , the hairpencils we re
d isse cte d on a pr edetermined day af ter emergence. The hairpencil
e xtr acts o f w ild males , ca p tu red in October , 2001 a nd 2005, were
p repar ed with in 24 h of co llec tion .
Che mical analyses of hairpencil extracts were performed by gas
c hromatography (GC) and gas chromatogr aphy-ma ss spectrometry
( GC- MS) . GC ana lyse s were car r ied ou t on a Sh imadzu GC-14A gas
c hromatograph equ ipped with a f lame- ion iza tion detecto r , us ing a
EQ UITY-1 fus ed-s il ica cap illary co lumn ( Supelc o , 0.25 mm I .D. x 15 m,
0 .25 µ m f ilm th ickn ess) . Samples were in jected sp lit les s at 250°C
us ing N2 as the car r ier gas ( f low; 1 ml/m in ) and the oven te mpe ratu re
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was p rogrammed from 50°C ( held in it ial ly for 5 min ) to 300°C (held for
10 min ) a t 10°C /min . EI -MS spe ctra were reco rded a t 70 eV on a
Sh imadzu GCMS-QP5000 ma ss spectromete r coup led with a Sh imadzu
GC-17A gas chroma togr aph , using a DB-1 fused-silica cap illary co lumn
( J&W, 0.25 mm I .D. x 15 m, 0.25 µ m f ilm th ick ness) and He as the
c arr ier gas (f low; 1 ml /m in ) . The GC system was operated w ith the
s ame temp erature prog ram as th at employed in GC analyses .
Iden tif ica tion of the componen ts was based on a c omp ar ison of their GC
r ete n tion data and mass spec tra with those of au then tic samples or
r epo r ted data.
Quan tif ic ation of the componen ts was perfo rmed b y GC u sing benzyl
a lcoho l a s the in ternal s tandard . The amoun t of each componen t was
e stima ted from its peak area in terms of benzyl a lcoho l.
2 .3 . PAs
He l io tr ine ( Latoxan , pur ity : 98%) and monocro talin e ( Ald r ich , 99%)
wer e commerciall y availab le. S inc e mon ocro taline was spar in gly
s o lub le in water , its HC l s alt ( monocro tal ine hydr och lor ide) was
p repar ed when necess ary. Retr onecine ( 98% by GC and NMR) was
p repar ed from monocro taline by hydro lysis w ith aq . NaOH. A PA
mix tu r e composed of in te rmed ine and lycopsamine ( ca. 4:1) was
ob tained f rom a methano l extrac t o f the under ground par t of Eupa torium
c h inen se ssp . sacha linense (Astera ceae) co lle cted in Hirosh ima
p refe ctur e, and pu r if ied by ion-exchange chromatogr aphy on Ac cell P lu s
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C M and QMA resin s (Wa ter s) . Their s tructu re s , ratio , and pu r ity (c a.
95% a s a who le) we re de termined by GC- MS and 1 H- and 1 3 C-NMR
a nalys es (Roeder , 1990 ; Lo gie e t al. , 1994) . NMR spe ctr a we re
measured in D2 O with a JEOL J NM-A400 FT-NMR spectrometer w ith
te tramethy ls ila ne as the ex ter nal s ta ndard . The chemica l s tr uctu res of
these PAs are shown in F ig . 1.
2 .4 . Feed ing response to PAs
Se ven-da y-o ld males (N=10) were tes ted by a s imi la r method of
Honda et al . ( 2005) . Befo re the b ioassay, each ind iv idual was given
water and 15% aq . sucrose to satiety. Th is e xper imen t was designed to
e xamine the elic ita tion o f a sequen tia l be hav ior that consis ted of two
phases ; p robos cis extension by s timu lat ion of ta rs i w ith PA so lu tions and
a ctua l f eed ing on them. An ind iv idual was p laced on a f ilter paper
s tr ip ( 20 x 20 mm) impregna ted with a 2.5% w /v (ne t c oncen tration of
PA) aq . so lu tion of a PA. F ir s t, the nu mber of ind iv iduals that
s pon ta neously unco iled and extended the proboscis w ith in 5 s af ter tar s al
c on tac t w i th a tr eate d paper was reco rded . If an ind iv idual was no t
induced to unco il the proboscis by tar sal s t imu lation , 1 µ l o f the same
s o lu tion was app lied w ith a m icr osyr inge to the c o iled p roboscis .
F inally, the number of ind iv iduals that exh ib ited a positive feed ing
behav ior compr is ing proboscis e xtens ion and suck ing for more than 30 s
was re corded . Unre spons iveness of males to water was conf irmed ju s t
befo re ea ch tr ial. The s ame test was rep lic a ted w ith o ther o lder males
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( 26 to 40 days o ld , N=10) to e xamine th e a ge dependence of
r espon sive ness . The response was exp re ssed as the percen tage of
ind iv iduals that r esponded positiv ely .
2 .5 . Ora l admin is t ra t ion o f PAs
Af ter 7 da ys o f eclosion , ind iv idual ma les were fed daily w i th 0.2
mg (net weigh t o f PA) of any one of the four PA s amp les fo r 7 days (1 .4
mg in to tal for each ma le) . For PA sample s th at hard ly s timu la ted
vo lun tary feed ing, these were given to males toge ther w ith 15% sucrose
s o lu tion . Con tro l males wer e fed w ith p lain sucrose so lu tion on ly.
On day 14 o f e mergence , the hairpenc ils wer e d issecte d ind iv idually and
e xtr acte d as des cr ibed above.
I n add ition to PA- admin is tr ation exper imen ts , chemic al ana lys is
was also conducted o f the hairpenc il secr etion o f male adu lts der ived
f rom larv ae that had been reared on P. l aeviga ta , the one hos t p lan t o f I .
l euconoe , dur ing the las t (5th ) larva l in star . S inc e th is p la n t has been
r epo r ted to con tain macro cyclic PAs (e.g. Abe and Yamauch i, 1987 ; Abe
e t a l., 1991) , th is exper imen t wa s aime d to tes t whether or no t the
bu tter f ly ca n u tili ze PAs acqu ired dur ing the larva l s tage . A lthough
most larva e failed to per form well on th is non-host p lan t, sever al lar vae
developed in to ad u lts . The se males were fed w ith 15% sucrose so lu tion
a lone fo r 14 days .
2 .6 . In vest iga t ion o f th e or ig in o f 9 ,10-epoxyt etrahydr oedu lan
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To examine whe ther 9,10-e poxytetr ahydroedu la n was b iosyn thesized
de novo o r der ive d f rom s econdary metabo lite( s) taken up as lar vae from
the host p la n ts , 3 groups of la rvae were rear ed on any one of the food
p lan ts , F. m icrocarpa , N. ind icum and A. curassavica , and the males
thereof were fed daily w ith e i ther 15% a q . sucr ose or wa ter alone fo r 14
da ys af te r ec lo sion . Those that were give n wate r alone wer e always
kep t in d im ligh t un ti l u se so as to rep ress their energy consu mption to a
low leve l. Che mical constituen ts of the abo ve 3 p lan ts we re also
e xamined for the pr esenc e of 9,10 -epoxy tetrahydroedu lan and its
possib le pre cursor , d ihydroe du lan : Fresh leaves (10g) o f each p la n t we re
e xtr acte d w ith isopen tan e ( 200 ml) below 15°C for 1 we ek . The ex tra ct
was f iltered and concen tr ated in va cuo at R.T. to c a. 1 ml. Af ter
r emov ing the precip itates ( so lid hydrocarbons) by cen tr ifuga tion and
a dd ing 1 ml o f d ich loromethane to the supernata n t, th e re su lting c lear
s o lu tion was sub je cted to GC a nd GC- MS analyse s under s imilar
operationa l cond it ions to those u se d for the ana lysis of hairpencil
e xtr acts .
2 .7 . Age dependence o f 9 ,10-epo xytetrah ydroedu lan fo r ma tion
I nd iv idual larva e wer e r eare d on one or two of the th r ee food p lan ts
de scr ibed above . Af ter eclo sion , male a du lts wer e fed d aily w ith 15%
s ucros e so lu tion un til d isse ction . On post-emergence days 0 (w ith in 12
h ) , 3, 7, 11 , 14, 20, and 30, the amoun t of 9,10 -epoxy tetrahydroedu lan
p roduced in the hairpencils was dete rmined .
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2 .8 . Att ract ion t es t to PAs
The attra ctiv ity to adu lt ma les o f 4 PAs, i.e.
in te rmed ine /lycopsa mine, h elio tr ine, mo nocro taline, and re tron ecin e,
was tes ted a t 25°C in the laboratory w ith good ven tila tion . A methano l
s o lu tion of a PA was un iformly app lie d to a f ilter paper d isc (4 c m in
d iameter ) so that the PA conc en tr ation was ad justed at 410 nmol/cm2 .
Four pa per d iscs tr eated w ith d if feren t PAs a nd a c on tro l d isc of the
s ame s ize tr eated w ith an equal amoun t o f the so lven t alone were held by
a w ire arm a t 1 m h igh f rom the f loor and p laced at regu lar in tervals
( abou t 30 c m apar t f rom each o the r) on a c irc le 60 cm ac ross in the
e xper imen tal a rena (150 x 70 cm) . The f loor of the aren a was covered
with gree n p lastic p la tes to imi ta te gree n ve geta tio n . Af ter evapo rating
the so lven t, ea ch d isc was mo iste ned b y spr aying water to facilita te PA
vapor izat ion . Subsequen tly, 10 naïve males o f 14 to 34 days o ld that
had never ha d an acces s to PAs were r ele ased and allo wed fr ee f ligh t
over the arena under il lumina tion with f luores cen t la mps (3500 lux) .
Thr ee h befo re the b ioass ay, ma les were fed w ith water and 15% a q .
s ucros e to satie ty. The d isc that received th e f ir s t v is it f o llowed by
p robosc is e xtens ion and suck ing movemen ts by each male dur ing a
30-min assa y per iod was recorded .
3 . Res ult s
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3 .1 . Id en tifica tion o f ma jor ha irpencil vo la tiles o f wild ma les
Rep resen tat ive gas ch romatog ram of a hairpencil e xtr act f rom a
f ield -caugh t male is shown in F ig. 2 (A) . Two major componen ts , A1
a nd A2, were detected . Compound A1, on GC-MS, showed d ia gnostic
f ragmen t ions at m/z 100 (18%) , 85 (23) , 72 ( 12) , 71 (21) , 67 ( 15) , 57
( 79) , 55 (18) , 45 ( 49) , 43 (100) , and 41 (32) . Although its mo lecu lar
ion was no t observed , the fr agmen tation patte rn of the compound was
qu ite s im ilar to tha t o f v ir id if lor ine ß- lactone, wh ich has p rev iously been
iden tif ied f rom the hairpencils o f ano ther danaid bu tter f ly, I . leuconoe
( Nish ida et a l., 1996) . The mass spe ctrum and GC re ten t ion time o f A1
c o incided well w ith those of the ß - la ctone of v ir id if lo r ic acid , one o f the
two poss ib le la ctones tha t ca n be pr epared by the reaction between
methanesu lfonyl ch lo r ide an d nec ic acid s [ (+) - tr achelan th ic and
( -) -v ir id if lor ic] (Schu lz and Nish ida, 1996) der ived from
in te rmed ine /lycopsa mine (1) by alkaline hydro lysis . The iden tity was
fu r ther co r roborated by compar ison with the repor ted componen t prese n t
in a hairpen cil extrac t o f I . leuconoe . Ac co rd ingly, compound A1 was
iden tif ied as v ir id if lo r ine ß- lactone [ (2S ,3S)- o r (2R ,3R)-
2 -hydroxy-2- isopropylbu tano-3- lac tone, hereaf ter abbr ev ia ted VL] .
Th is compound ha s 2 ch iral cen ters at C2 and C3 positions, however ,
their a bso lu te conf igur ations remained undetermine d .
Pe ak A2, giv ing f ra gme n t ions at m/z 195 ( 8%) , 177 ( 8) , 153 (38) ,
135 ( 27) , 123 (20) , 111 ( 26) , 95 (34) , 83 ( 28) , 69 (46) , 55 ( 51) , and 43
( 100) , was assumed to be 9,10 -epoxy tetrahydroedu lan , wh ich h as alr eady
15
be en iso la ted and iden tif ied f rom some Eup loea species includ ing E.
mu lcibe r (Fr ancke et a l. , 1989) . Whe n measu re d at 25 e V, the
c ompound gave a weak M+ a t m/z 210 ( 5%) , o f wh ich mo lecu lar for mu la
was def in itely de ter mined , by exact mas s meas uremen t (h igh - reso lu tion
MS) , to be C13 H2 2O 2 (Calc d . fo r C13 H2 2O 2 , 210.16198 ; Obsd .
210.16555) . Compound A2 was, thus , conc luded to be
9 ,10- epoxytetr ahydroedu lan (ET) . The chemical s tructures o f the 2
iden tif ied compounds ar e shown in F ig. 3.
3 .2 . Feed ing response o f labora to ry-r eared ma le s to PAs
Bo th in termed in e/lycopsa mine (1) and helio tr ine (2) s trongly e voked
f eed ing (100% r esponse) as shown in F ig. 4. Several ind iv iduals
s pon ta neously unco iled and e xtended the p roboscis seek ing fo r PAs
immed iate ly af ter con tacting some of th e PAs with mid - and h ind- legs .
In th e exper imen t w ith 7-da y-o ld male s (F ig. 4A) , the p ropor tio n of
ind iv iduals that exh ib ited p roboscis extension upon tarsa l c on tac t w ith
c ompounds 1 and 2 were 70% and 10%, resp ectively. I n con tras t, no
males r esponded positively to HC l s a lt o f monocro ta line (3 ) , wh ile
r etronecine (4 ) elic ite d a n in ter med iate feed ing response (60%) ,
a lthough tarsal con tact d id no t s timu la te p robosc is ex tens ion . All
those th at extended the proboscis even tually s ucked PA so lu tions.
The o lder males (F ig. 4B) a lso showed a s imilar responsive p rof ile
fo r the 4 PAs, bu t proboscis extension appeared to b e more r ead ily
tr iggere d by tars al con tact with PAs (100%, 50%, and 10% for
16
c ompounds 1 , 2 , and 4 , r espec tively) . However , compound 3 altogether
f ailed to elicit a ny f eed ing r esponse.
3 .3 . Effe ct o f up tak e o f PAs and the ir s truc tures on th e chemic al
c omposit ion o f t he ha irpencil s ecret ion
Rep resen tat ive gas chromatograms o f ha irpencil extrac ts prepared
f rom males that inge sted compound 1 (B) and a con tro l male (C) a re
s hown in F ig. 2. All c on tro l males s ecreted ET, bu t la cked a peak
c orre spond ing to VL, wherea s those tha t ingested 1 possess ed bo th ET
a nd VL. Fur ther mor e, F ig. 5 clear ly ind ica tes that males of ev ery
g roup , regard less of the tr eatme n ts , secreted ET at averages rang ing
f rom 2.4 to 9.2 µg /ma le. Th is sugges ts that E. mu lciber males have
mor e or less the in tr in sic ab il ity to p roduce ET by themse lves . On the
o ther hand , a s ign if ican t qua n tity ( 35 µg /ma le on average) of VL was
p roduced when males f ed on compound 1 . Howe ver , the up take of 2 or
3 resu lted in the formation o f very small amo un ts of VL (0.18 and 0 .09
µg /ma le , r espe ctively ) and no tra ce o f VL was detected f rom the males
that we re given 4 . Males that o r ig inated f rom larvae ra ised on a
PA- con ta in ing p lan t, P. laev iga ta , also p roduced VL at an avera ge of 5.4
µg /ma le . Th is ind ica tes tha t c er tain PAs con tain ed in th e larval d iet
wer e ef f icie n tly incorporated and conver ted to VL.
I n add it ion to ET and VL, some ind iv id uals of bo th f ield -co llec ted
a nd PA-fed ma les were found to have small amoun ts of danaidone and /or
hydroxyd anaidal, wh ic h ar e well-known d ihydropyrro lizines pres en t in
17
the a ndrocon ial se cretions of man y danaids . The r ough e stima tes o f
per cap ita quan tit ie s of danaidone and hydroxydanaidal found in the
ha irpencils , as determined a s descr ibed prev ious ly (Honda et al., 1995) ,
r anged from 0 to 0 .31 and from 0 to 9.1 µg, r espec tively. Apparen tly,
no t all ind iv iduals that a ctua lly had or were su spected to have had acce ss
to PAs possess ed thes e compounds, wh ile con tro l males p roduced ne ither
o f the m. A ll ma le s , independen t o f the ir f eed ing h is to r y, had in
c ommon small quan tit ies of o ther mis cellaneous compounds that we re
c onsidered to be ub iqu itous hydroc arbons, es te rs , and aliphatic ac id s ,
bu t they were no t pursued fur ther .
3 .4 . Effect o f lar va l and adu lt d ie ts on the p roduct ion o f
9 ,10- epoxytetrahydroedu lan and the age dependenc e o f its quan t it y
Ma les of every cohor t, ir r espec tive of their larval d iets , produced
s imilar amoun ts (5 .3- 5.9 µg /ind iv idual) of ET (Fig. 6) . S ince e ven the
males that had been give n wate r on ly e ver s inc e their emer gence we re
a b le to produce ET in an amoun t comparab le to those found in males fed
w ith a q . sucros e, it is ev ide n t that adu lt d ie t is no t essen tial f o r ET
fo rmation . These f ind ings suggested th at c er tain second ary
metabo lites o r nu tr ien ts ac qu ir ed from food p lan ts dur ing the larv al
developmen t we re d ir ectly o r ind irectly responsib le fo r ET for mation .
The n , we examin ed vo la ti le componen ts of 3 f ood p la n ts , F.
m icrocarpa , N. ind icum , and A. curassavica , to as cer tain whether o r no t
9 ,10- epoxytetr ahydroedu lan or d ihydroedu lan was con tained in th ese
18
p lan ts . However , ne ither compound was detected f rom any of the
p lan ts .
Ma les f resh f rom eclosion ( day 0) were found to have an e xtr eme ly
s ma ll amo un t of ET (0.08 µg/ma le) (F ig . 7) . However , its quan tity
g radually inc reas ed with age, re ach ing a ma ximum at an aver age of 5.3
µg /ma le on day 14 and thereaf te r s howed a downward tendency , turn ing
ba ck to the leve l o f day 7 a f ter 30 days. Surpr is ingly, 60- day-o ld
males (N=3) s till r eta ined near ly the same quan tity o f ET ( 3.5 µg /male)
a s that pos sessed by 30-day-o ld ma les .
3 .5 . Att ract iv it y o f PAs to ma le adu l ts
As shown in F ig. 8 , a ll male s tes te d were attrac ted exclusively to
c ompound 1 , wh ereas the o ther PAs d id no t rece ive even temporar y v is its .
Ea ch male al igh ted on d isc 1 af ter shor t-duration aer ial se arch ing with
no s ign of confusion . Th is ind ica tes that males ar e endowed with a
r ef ined o lfac to r y sens e to detect and r ecogn ize pa r ticu lar PA( s) they
p refer .
4 . D isc uss ion
4 .1 . Componen t s o f the ha irpenc il secret ion o f E. mu lciber
To the best o f o ur knowledge, ET is the so le componen t so f ar
r epo r ted from the hairpencils of E. mu lciber (Fr ancke et al., 1989) . We
r econf ir med its occur renc e as one of the ma jo r componen ts in the
19
ha irpencil s ecretio n . In th is s tudy, we found an add itional domina n t
c omponen t, VL, f rom wild males . Th is compound migh t ha ve po ssib ly
be en mis sed in the ear lier s tudy, bu t we th ink that th is inconsis tency
a rose fr om the d if ference in age o f ind iv idual ma les exa mined , because
VL was detecte d on ly f rom ma les tha t ingested PAs (F ig . 5) and our
observ ations in an ou tdoor ca ge r evealed that ma les u sually began to
s eek for and feed on PAs several days af te r emergence. VL is a
pe cu liar compound with a ß- la ctone s tructu re , wh ich was iden tif ied for
the f ir s t time f rom th e hairpencils o f ano ther da naid bu tter f ly, I .
l euconoe , as one of the male sex pheromones (Nis h ida et a l., 1996) . A
r ecen t pap er a lso repor ts on its p resence in the alar f r inges of a few
i thomiid bu tte r f lies (Schu lz et al. , 2004) . Al though the
s ter eochemistr y of VL p roduced by E. mulciber is unknown , the
c ompound most likely has (2S ,3S) -conf igur ation , because bo th
in te rmed ine and lycopsamine have S- conf igu r ation a t C2’ ( see
d iscussion below) and I . leuconoe s ecre tes (2S ,3S )- VL ( Schu lz and
Nish ida, 1996) . Tha t E. mu lciber and I . l euconoe shar e VL as a
p redomin an t componen t is congruen t w ith their clo s e phylogenetic
r ela tionsh ip ( sub tr ibe Eup loeina) argued by Acker y and Vane-Wrigh t
( 1984) . However , no re por ts have ever ref err ed to the p rese nce o f ET
in the s ecre tion of I . leuconoe . To da te, dan aid hairpencil secr etions
have be en examined for more than 30 s pecies cover ing most genera of
the family ( see references c ited) . S ince ET has on ly been found from
Eup loea spe cies ( Francke e t al. , 1989) and is a lso suspected to be prese n t
20
in o ther Eup loea species (Edgar , 1975, 1982) , th is compound appears to
be sp ecif ic to th e ge nus Eup loea .
4 .2 . D if fer en tia l fe ed ing re sponse to PAs
Ma les s howed s trong feed in g respons es to compounds 1 and 2 , and
e ngorged them (Fig. 4) . Th is co incides well w ith p rev ious repor ts that
PAs ac t as phagostimu lan ts (P lis ke, 1975a ; P liske et al. , 1976) .
However , al l PAs d id n o t s timu la te f eed ing b y males . Whi le compound
4 elic ite d a moderate response, comp ound 3 , a d iester - typ e PA, d id no t at
a ll. Th is means that cer tain PA(s) may be inactive to gus tato ry sens e,
o r per haps even act as an tif eedan ts , because males seemed some wh at
unwill ing to imb ib e a sucrose so lu tion admixe d with 3 . As co mpared
with males o f ano ther danaid , P. s ita , wh ich e xh ib ite d a ver y s tr ict
f eed ing b ehav ior , pos itively r espond ing to 1 on ly (Honda et a l. , 2005) , E.
mu lcibe r ma les appear to have a broade r spectrum of gus tato ry
r espon sive ness to PAs. In con trast to thes e danaids , ma les of Danaus
g ilippus and some arc ti id mo ths vo lun tar ily imb ibe monocro taline (3)
( e.g . Eisne r and Me inwa ld , 1987 ; Co nner et al., 2000) .
Bu t ter f lies have b een shown to posses s gus tato ry sensilla on the
tars i that s ensitively res pond to sugars ( Chapman , 1995) . I mmed iately
when the mid - and h ind - legs ( the s tub- like fo relegs are normally h eld
t igh tly to the bod y) were b rough t in to con tact w ith a so lu t ion of 1 , mos t
males were observ ed to spon taneously unco il and exte nd the proboscis
s eek ing for the PA. Co mpound 2 also re leas ed s imi la r beha v ior al
21
r espon ses f rom sev eral males . S ince no ind iv iduals , even at clo se rage,
tr ied unco il ing the pr oboscis a s long as their legs wer e kep t ou t of
c on tac t wi th PA so lu tions, we th ink that the re seems no possib ility that
the probos cis extension response wa s e voked by some o lfactory s timu li,
a lthough o lfac tion wou ld pro found ly be invo lved in their a ttract ion to
PAs ( se e 4.5) . There fore , thes e r esu lts p rov ide the f ir s t ev idence th at
male adu lts possess che motactile (gus tato ry) recep tor (s) spec if ically
r espon sive to PAs on the mid - and /o r h ind- legs a s well as on the
p robosc is . La rvae of some arctiid s have been shown to posse ss
s pecif ic tas te re cep tors for PAs (e.g. Bernays et a l. , 2002 , 2004) .
Al though PA r espons iveness of the proboscis d id no t seem to vary w ith
a ge, tha t o f the tars i a ppeared to be enhanc ed as male s go t o lder (F ig.
4B) . We hyp o thes ize tha t th is phenomenon was caus ed by PA-def icit
s tre ss wh ich augmen ted their appetite f or PAs .
4 .3 . Sel ect ive u t iliza t ion o f part ic u lar PA(s ) fo r the p roduction o f VL
S ince PA-unfed (con tro l) male s en tirely fa iled to p roduce VL, i t is
obv ious th at they need to inges t s ome spec ial compound(s) o ther than
nu tr ien ts to b iosyn thesize VL. Of the 4 PAs a dmin is ter ed , on ly
c ompound 1 served as a s ign if ica n t pr ecursor f or VL b ios yn thesis ,
though 2 and 3 also affo rded VL in tra ces (F ig . 5) . No s tud ies have
h ith er to elucida ted the b iosyn thetic mech an is ms under lying the
fo rmation of VL in in sects . By v ir tue o f the s tru ctur al s im ilar ity
be tween VL and the nec ic a cid mo iety o f 1 , VL is s upposed to be a
22
c ycliza t ion produc t d irec tly der ived f ro m the necic acid o f 1 . Given
that, it is qu ite co nv incing that no trace of VL wa s fo rmed from 4 , bu t we
c anno t determine, at p r esen t, wh ich of th e two PAs, i.e. lycops amine and
in te rmed ine , serv ed as a p recu rso r for VL. S ta r ting f rom lycops amine
( 2’S ,3’S ) , VL wou ld be r ead ily p roduc ed e ither by the hydro lysis of an
e ste r group , fo llowed by la cton izat ion of the necic acid ther eof
(v ir id if lo r ic ac id) or s imp ly by in tra molecu lar transe ste r if ica tion .
Ano ther pos sib ili ty remains that in termed in e ( 2’S ,3’R ) a lso p layed a par t
in VL p roduc tion . Th is p rocess , however , en tails e p imer iza tion at C3’
to S for m. In fac t, th is type o f s ter ic invers ion at C3’ [ tr ansf ormation
f rom (+)- trac hela n th ic a cid to (- ) -v ir id if lor ic acid ] ha s be en r epor ted to
oc cur for PAs s eques tered by an ithomiid bu tter f ly (Tr igo e t a l. , 1994)
a nd an ar ctiid mo th (Tr igo et al. , 1993) . The inab ility o f E. mu lciber to
u s e compound 2 , o f wh ich nec ic ac id is s imilar in s tru ctur e to
in te rmed ine bu t methoxylated at C3’, may, in par t, be due to the lack of
e nzymes invo lved in th e cleavage or d isp lacemen t of the ether bond .
On the o ther hand , the for ma tion of VL f r om 3 , albeit in trace, is v ery
c ur ious, bu t we have no ide a abou t that. Al though the pre sen t resu lts
do no t p rov ide d ir ect ev idence fo r the transformation of the necic ac id
in to VL, it seems tenab le that the c arbon framework of
2 ,3-d ihydr oxy-2- is opropy lbu tano ic a cid tha t constitu tes the necic ac id of
c er tain PAs is inco rpo ra ted in to VL.
The f ind ing that male adu lts der ived f rom the larvae fed w ith a
non-hos t PA-con tain ing p lan t ( P. l aevig ata) p roduced a small bu t
23
s ign if ican t quan tit y o f VL (Fig. 5) , undoub ted ly de mons tr ates th at the
larva e are ab le to assimila te, s to re, and retain PAs up to adu lts .
Conc ern ing the man ipu lation of PAs by immatu r es of bu tter f lie s ,
e v iden ce is alr eady given in many dana ids and ithomiids that larvae s till
main ta in the capacity fo r ass imila ting and s tor ing PAs (Ro thsch ild and
Edgar , 1978 ; Tr igo a nd Motta, 1990) . The q uestion is wha t k inds of
PAs in P. l aevigata were availab le fo r VL b iosyn thesis . Four major
PAs are known from th e p lan t (e .g. Abe and Yamauch i, 1987 ; Abe e t al. ,
1991) . Of these , 2 PAs cou ld be enumer ate d as pos sib le cand idate s for
p recur sors ; parson ine, a un ique keto-d ihydropyrr o lizine es ter if ied w ith
v ir id if lor ic acid and 17- me thylparsonsian id ine, a macroc yclic PA with a
trac helan th ic acid mo iety. The same s itua tio n wou ld be true of I .
l euconoe th at s ecre tes VL as the majo r componen t. In add it ion , E.
t reitschkei aenea r ear ed o n P. sp iral is , its ho st p la n t con tain ing PAs , has
be en r epor ted to selectively p roduce and s tor e lycops amine in the body
that is be lieved to have ar isen by selective in v ivo hydr o lys is of some of
the food p lan t macrocyclic PAs w ith a v ir id if lo r ic acid mo iety (Edgar ,
1982) .
4 .4 . Derivat ion o f ET and the varian ce o f its quan tity with age
The data pr esen ted in F ig. 6 s tr ongly suggest de novo b iosyn thesis
o f ET f rom nu tr ien ts ta ken up fro m lar val d iet. Ev idence for th is is
that the 3 lar val food p lan ts proved to con ta in no trace s of eithe r ET or
d ihydroedu lan , and that a nu tr ie n t ( sugar) acqu ired as adu lts exer ted no
24
s ign if ican t ef fect on the a moun t o f ET formed . Although on e shou ld
e xpect the invo lvemen t o f o th er p lan t s econdary metabo lite s in ET
p roduction , it is very un likely that the 3 food p la n ts of d ifferen t fa milies
s hare te rpeno id p recur sor (s) w ith edu lan or relate d ske letons. In fact,
de novo b ios yn thesis of terp erno ids is w idely known in many in sects
( Mor gan , 2004) .
S inc e day-0 a du lts had no appr eciab le quan tity o f ET, i t is appare n t
that ET b iosyn thes is s tar ts on ly a f te r e clo s ion , pr ogresses fu r the r in the
imag ina l s ta ge, and ther eaf ter dec lines in some de gree w ith a ge ( F ig. 7) .
Ba sed on the c on te n t o f ET sto red by male adu lts , they seem to s exually
matu re approximate ly two weeks af ter eclo sion .
4 .5 . PAs as a ttractants fo r male adu lts
With r espec t to PA-Lep idop te ra in terac tions, one of the fundamen tal
c oncerns has been the nature of chemical s ignal( s) that med ia te
o r ie n tat ion to PA s ources . PAs thems elves are though t to bar ely at tra ct
bu tter f lie s due to their low vo la tility. Ex tens ive tr app ing exper imen ts
w ith var ious baits inc lud ing wither ed p lan ts , PAs, and r elated
c ompounds and an e lec trophysio logical s tudy have reveale d that vo lati le
p roducts e manating f rom degraded PAs ar e key subs tanc es that enab le
in sec ts to loc ate PA s ources: Un iden tif ie d vo latile ( s) der ived from nec ic
a cid s ar e suspected to be a powerf u l a ttractan t for ithomiid bu tter f lie s ,
wh ile necine base-der ived hydroxydana idal a ttracts arct iid mo th s (P liske
e t a l. , 1976 ; Kr asnof f and Dussou rd , 1989 ; Bogner and Boppré, 1989) .
25
However , our resu lts (F ig. 8 ) de monstr ate tha t cer tain PAs composed of
par ticu lar necic acid s do serve as attrac tan ts at least for E. mu lciber
males . The pos sib ili ty that as yet un iden tif ied tr ace b reakdown
p roducts o f PAs worked as attr actan ts canno t ab so lu tely be ru led ou t.
However , hydr oxydana idal a nd f ree necic acid s a s lik ely a ttrac tan ts we re
no t detec ted from compound 1 , th ough it was con ta minated w ith a small
a moun t ( les s th an 5%) of retron ecine (4) . Acco rd in gly, in termed ine
a nd /or lyc opsamine per se cou ld be regarded as th e real o lf actory
p r inc ip le to lu re the bu tter f ly. Compound 1 also s eemed attractiv e to P.
s it a males , bu t i t appears that in genera l, a cons ider ab le con cen tration of
a PA is requ ired to attr act danaids (Honda et al., unpub lished) .
4 .6 . Adap t ive s ign if ic ance o f the linkage between sensor y r ecep t ion and
u t il iz a tion o f PAs
Our prev ious paper ha s shown that ma les o f P. s it a on ly showed a
positive feed ing response to compound 1 wh ich , among the PAs tes te d ,
they c an conver t most eff icien tly to danaidone (Honda e t a l., 2005) .
Th is trait seems very adap tive in v iew o f their repr oductive s tr ate gy.
In con trast, desp ite the ve ry poor av ailab il ity o f 2 , E. mu lciber males
positive ly responded in f eed ing to th e compoun d and even to 4 th at is
a pparen tly u se les s fo r VL b iosyn thesis . The males may perhaps take
up such PAs for defensive u se. In tere stingly enough , however , the
o lfac tion o f males is f ine ly tuned to par ticu lar PA(s ) that a re
ind ispensab le as p recur sors for the eff icien t b io syn thesis o f VL.
26
Therefore, they c ou ld successfu lly f ind a nd ac qu ir e nec essa ry PA( s) in
the f ield . The clo se r ela tionsh ip with a nd s trong requ ir emen t for
in te rmed ine /lycopsa mine were unusua lly p rominen t in P. s it a . The
s ame appears to ho ld true of E. mu lc iber .
Obv ious ly males o f E. mulciber p roduc e and rocon ial chemic als o f
two d if fer en t b io syn thetic o r ig in s: One (VL) is h igh ly likely to be
der ived from par ticu lar PAs with v ir id if lo r ic o r tr achelan th ic a cid
mo iety wh ich ma les acqu ire as adu lts f rom p lan ts , and the o ther (ET) ,
f rom nu tr ien ts taken o ver from larva e by wa y of de novo b iosyn thesis
( F ig. 9) . A lthough no th ing is known o f the ro le s of ET and VL in the
s exual communication o f E. mu lc iber , it is fe asib le that these elabora te
a nd pr edominan t componen ts act as the se x pheromones.
The details o f the s tereochemic al pathway of VL b ios yn thesis and
the b io logic al func tion of ET and VL are under investigation .
Ac knowle dge me nts
We thank M. Fukush ima and K. Kohno who he lped co lle cting bu tter f lie s .
We a re indeb ted to Dr . N. Hayas h i a t Fukuyama Un ivers ity for hav ing
in it ia ted u s in to th is s tudy. Th anks are a lso due to M. Sakamoto at
Hirosh ima Ci ty Fo res t Park for help in rear ing E. mulciber larvae.
27
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2267-2271.
Be rnays, E.A., Chapman , R.F ., Har tmann , T. , 2002. A h igh ly sens itive
tas te rec ep to r cell fo r pyrro li zid ine alkalo ids in the la teral ga le al
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39
(Fig. 1)
40
(Fig. 2)
41
(Fig. 3)
42
(Fig. 4)
43
(Fig. 5)
44
(Fig. 6)
45
(Fig. 7)
46
(Fig. 8)
47
(Fig. 9)
48
( Legends to F igures )
Fig.1. Che mical s tructure s o f pyr ro lizid ine a lkalo ids fed to E. mu lciber
males . 1: in te rmed ine ( 3’R ) /lycopsamine (3’S ) , 2: helio tr ine, 3:
monocro taline, 4: re tron ecin e.
F ig.2. Rep res en ta tive GC c h romatograms of the hairpenc il extrac ts o f E.
mu lcibe r males . A: f ie ld -c augh t male ; B: male that ingested compound
1 ( see F ig.1 ) ; C : con tro l ( PA-unfed ) male. IS : in ter nal s tand ard
(benzy l a lcoho l) ; Peaks A1 a nd B1: v ir id if lo r ine ß- lactone; Peaks A2, B2 ,
a nd C2: 9,10-e poxytetr ahydro edu lan .
F ig.3. Majo r vo lat ile componen ts iden tif ie d from the hairpenc ils o f E.
mu lcibe r males . VL is a (2S ,3S) -for m or its enan tiomer .
F ig.4. Feed in g response to PAs. A: 7- day-o ld males (N=10) . B: 26-
to 40 -day-o ld ma les (N=10) . Compound numbers cor respond to those
o f PAs shown in F ig. 1 . Fo r compound 3 , its HC l salt ( monocro taline
hydroch lor ide) was u sed in th is tes t. Black bar : ind iv iduals that
a ctua lly fed on PA so lu tio ns, Gr ay b ar : ind iv iduals that spon taneous ly
e xtend ed the p roboscis upon tarsa l con ta ct w ith PAs.
F ig.5. Amoun ts of VL and ET ( mea n + SD) in the hairpencils o f E.
mu lcibe r males sub je cted to PA-admin is tration tes ts or c ap tu red in the
49
f ield . PA numbers co r respond to those of PAs shown in F ig. 1. For
c ompound 3 , its HC l sal t was u s ed . Ind iv idual males excep t w ild ones
wer e der ived f rom larvae rear ed on nor ma l host p lan ts (PA- lack ing) , i.e.
A. cur assav ica , F. microcarpa , or N. ind icum o r on a PA-con tain ing
non-hos t, P. laeviga ta . Indoor male s were fed da ily wi th 15% a q .
s ucros e af te r e clo s ion .
F ig.6. Ef fect o f larva l a nd adu lt d iets on the produc tion of ET ( mean +
SD) in the hairpen cils o f E. mu lc iber males . Four teen-day-o ld
ind iv iduals that wer e f ed da ily w ith 15% aq . sucrose (S) or water alone
( W) af ter eclo s ion . Each ind iv idua l f ed w ith water a lone or iginated
f rom larvae re ared on any one of the 3 f ood p lan ts g iven above. The
r esu lt sugges ts that ne ither s econdary metabo lites p resen t in the larv al
food p lan ts nor adu lt d ie ts are responsib le for ET formation .
F ig.7. Age dep endence of ET formation (mean ± SD) .
The number o f ind iv id uals examined is g iven in paren thes es . For the
va lue of day 14, data p resen ted in F igs . 5 and 6 ar e co llectively sh own .
F ig.8. Attr action tes t o f E. mu lcibe r males to PAs.
A to tal o f 10 ma les were used . Compound numbers c orrespond to those
o f PAs shown in F ig . 1. The p ho togr aph shows males gather ing and
s uck ing on a paper d isc tre ate d w ith in te rmed in e/lycopsamine (1) .
50
Fig.9. Proposed b iosyn thetic p rocess o f ET and VL in male adu lts o f E.
mu lcibe r .
