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Journa l o f C hem ica l Eco log y , VoL 22 , N o . 6 , 1996

S E X P H E R O M O N E O F Stenoma cecropia M E Y R I C K( L E P I D O P T E R A : E L A C H I S T I D A E )

P I E R R E Z A G A T T I , I ' * P H I L I P P E L U C A S , I P H I L I P P E G E N T Y , 2S A N D R A A R A N G O , 3 C H R I S T I A N M A L O S S E , 1 a n d

F R I ~ D I ~ R I Q U E T E L L I E R j~ N RA U nit~ de P hy toph arm ac ie e t M #dia teurs C h im iques78026 Versa i l l e s C edex , France

' l n d u p a l m a S AA . A . 6 2 2 6 B o g o t 6 , C o l o m b i a'aPalmas de Tum aco Ltda.A . A . 4 3 9 0 6 B o g o t d , C o l o m b i a

(R ece ived M ay 12 , 1995 ; accep ted Janua ry 29 . 1996)

Abstract--(Z,E)-9,1 l - te t radec adien al (Z9 .E 11 - 14 : Aid; I 1%), (Z ,E )-9 , 1 1 , 1 3 - t e t r a d e c a t d e n a l ( Z 9 , E I 1 , 13 -14 : A id ; 67 % ) , (Z ,E ) -9 ,1 l - t e t r adeca -dieny l aceta te (Z9 ,E 11-1 4 : Ac . 5 .5 %) . and (Z ,E)-9~ 11,13- te tradecatr ienyIace ta t e (Z 9 ,E 11 ,13-1 4 : A c : 16 .5 % ) w ere iden ti fi ed in the ex t r ac t s o f f em a lep h e r o m o n e g l a n d s o f S t e n o m a c e c r o p i a (Lepidoptera: E lach is t idae) . Fro me l e c t r o a n t e n n o g r a m s a n d s i n g l e s e n s i l l u m r e c o r d i n g s , r e c e p to r s t o Z 9 , E 1 1 , 1 3 -1 4 : A l d a n d Z 9 , E I 1 - 1 4 : A l d w e r e f o u nd o n m a l e a n t e n n a . B e h a vi o ra l d a t awere obta ined f rom ol fac tom etr ic tes ts in the laboratory and f ie ld t rappinge x p e r i m e n t s in C o l o m b i a . I t a p p e a r e d t h a t a b l e n d o f Z 9 , E I 1 , 1 3 - 1 4 : A l d(8 3 % ) a n d Z 9 , E 1 1 - 1 4 : A t d ( 1 7 % ) w a s a t tr a c ti v e t o m a l e s . T h e s e a l d e h y d e sa re a s s u m e d to b e c o m p o n e n t s o f t h e s e x p h e r o m o n e o f S. cecropia , w h e r e a st h e a c e t a te s f o u n d in g l a n d e x t r a ct s m i g h t b e p r e c u r s o r s o f t h e p h e r o m o n e ,K e y W o r d s - - L e p i d o p t e r a , E l a c h i s t i d a e , S tenom a cecrop ia , s e x p h e r o m o n e ,iden t if i ca tion , e l ec tm an te nno grap hy , s ing le sens i l l um record ing , f ie ld t r app ing(Z ,E ) -9 ,1 l - t e t r adecad iena l , (Z .E )-9 ,11.13-tetradecatrienal.

I N T R O D U C T I O N

T h e m o t h S t e n o r n a c e c r o p i a M e y r i c k b e l o n g s to a n e o t r o p i c a l s u b f a m i l y ( S te n -o m i n a e ) o f t h e G e l e c h i o i d e a . T h e s e m o t h s w e r e c o n s i d e r e d a s O e c o p h o r i d a e b y

* T o w h o m c o r r e s p o n d en c e s h o u l d b e a d d r e ss e d .

1 1 0 3

0 0 t ) 8 - 0 3 3 1 / 9 6 / 0 6 0 0 1 1 0 3 5 0 9 . 5 0 / 0 ( cj 1 9 9 6 P l e n u m Pu b l i s h i n g C o rp t~ r at io n

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1104 P. ZAGATT!ET AL.H o d g e s ( 1 9 74 ) . M o r e r e c e n t l y , M i n e t r e v i s it e d t h e t a x o n o m i c s t at u s o f S t e n -o m i n a e a n d i n c l u d e d t h e m in th e f a m i l y E l a c h i s t id a e o n t h e b a s is o f t h e a b d o m -i n a l a r t i c u l a t io n o f t h e n y m p h ( M i n e t , 1 9 8 9 ). T h e a d u l t s o f S, cecropia l o o kl i k e c o l o r f u l t o r t r i c i d s , b u t t h e l a r v a e l i v e i n f i x e d b a g s a n d f e e d o n s e v e r a lf o r e s t t r e e s , i n c l u d i n g th e l e a f l e t s o f P a l m a c e a e . I n o i l p a l m p l a n t a t i o n s o f c e n t ra lC o l o m b i a , t he p o p u l a t i o n s o f th i s i n s e ct c a n r e a c h h u g e l e v e l s , a n d t h e t h r e s h o l df o r in s e c t i c i d a l t r e a t m e n t i s 8 0 l a r v a e p e r l e a f ( G e n t y , 1 9 7 8) . S e v e r e d e f o l i a t i o n sb y th i s i ns e c t m a y d e c r e a s e t h e y i e l d o f o il p a l m t r e e s b y a s m u c h a s 1 5 - 2 0 % .W e u n d e r t o o k th e i d e n t i fi c a ti o n o f th e s e x p h e r o m o n e o f S. cecropia t o p r o v i d ea u s e fu l t o o l fo r m o n i t o r i n g t h e p o p u l a t i o n s o f th e m o t h .

T h e c h e m i c a l a s p e c t s r e l a t i n g t o t h e s y n t h e s i s o f p h e r o m o n e c o m p o n e n t sh a v e b e e n p a r tl y p u b l is h e d ( R a m i a n d r a s o a a n d T e l l i e r , 1 9 90 ; T e t l i e r a n d D e s -c o i n s , 1 9 9 0 ). W e p r e s e n t h e r e t h e f u ll i d e n t i f i c a t i o n s t u d y t o g e t h e r w i t h b i o l o g -i c a l d a t a .

METHODS AND MATERIALSInsec ts

P r e l i m i n a r y t ri a l s t o s e n d l i v e i n s e c ts t o F r a n c e w e r e v e r y u n s a t i s f a c t o r y ,b e c a u s e 9 0 % o f th e l a r v a e o r p u p a e d i e d . I t w a s t h u s d e c i d e d i n 1 9 8 7 to e x t r a c tt h e p h e r o m o n e i n c e n t r a l C o l o m b i a b y u s i n g a f i e l d l a b o r a t o r y i n a 9 0 0 0 - h ap l a n t at i o n (S a n A l b e r t o , C e s a r ) . T h e p u p a e w e r e c o l l e c t e d d a i l y in t h e ir b a g sa n d s e x e d i n t h e l a b o r a t o ry . H a l f t h e p u p a e a n d e m e r g i n g a d u l t s w e r e t h e n k e p tu n d e r n at u ra l c o n d i t i o n s ( t e m p e r a t u r e v a r y i n g b e t w e e n 2 8 a n d 4 0 C , 1 0 0%r e la t iv e h u m i d i t y , a n d d a y l i g h t f r o m 0 6 : 3 0 t o 1 8 :3 0 h r ) . T h e o t h e r h a l f o f t hei n se c ts w e r e k e p t i n a r o o m u n d e r s i m i l a r c o n d i t i o n s e x c e p t t h a t l ig h t w a sp r o v i d e d f r o m 0 4 : 3 0 t o 1 6 : 3 0 h r .

T h e i n s e c t s u s e d f o r o l f a c t o m e t r i c t e s t s , e l e c t r o a n t e n n o g r a p h i c ( E A G )r e c o r d in g s , a n d s i n g l e - s e n s i l lu m r e c o r d i n g s w e r e c o l l e c t e d a s y o u n g p u p a e int h e fi e ld a n d b r o u g h t b a c k t o F r a n c e c o u r t e s y o f t r a v e l in g c o l l e a g u e s . T h e y w e r es e x e d a n d k e p t a t 2 7 C w i t h 7 5 % r e l a ti v e h u m i d i t y u n d e r a 1 2 - 1 2 h r p h o t o -p e r i o d u nt il e m e r g e n c e o f t h e a d u l ts . T h e m a l e m o t h s u s e d in th e s e l a b o r a t o r ys t u d i e s w e r e 1 o r 2 d a y s o l d .Pheromone Ex trac t ion

A s s h o w n b y o b s e r v a t i o n o f tr a p s b a i t e d w i th v i r g i n f e m a l e s , t h e p e r i o d o fs e x u a l a c t i v i t y o f S. cecropia b e g i n s h a l f a n h o u r b e f o r e d u s k a n d l a s ts 2 h rs .I n S a n A l b e r t o , t h e p h e r o m o n e w a s e x t r a c t e d b e t w e e n 1 8 : 3 0 an d 2 0 : 3 0 h r( in s e c ts u n d e r n a t u r a l p h o t o p e r i o d ) o r b e t w e e n 1 6 : 3 0 a n d 1 8 : 3 0 h r ( in s e c t s u n d e rs h i f t e d l i g h t r e g i m e ) .

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SEX PHEROMONEOF Stenom a cecropia 1105T h e l a s t t h r e e a b d o m i n a l s e g m e n t s o f 1 o r 2 - d a y o l d v i rg i n f e m a l e s w e r e

c l i p p e d a n d s o a k e d f o r 12 h r in 1 m l h e x a n e ( C a r l o E r b a C o d e x D e p u r a t o ) a t- 5 C . T h e e x t r a c t t h en w a s f i lt e re d t h r o u g h g l a s s w o o l , r e d u c e d to c a . 1 0 0 / z lu n d e r a N z f lo w a n d k e p t a t - 3 0 C u n t il u s e d . T h r e e e x t r a c ts o f , r e s p e c t i v e l y ,6 8 4 , 7 5 9 , a n d 5 5 7 f e m a l e a b d o m i n a l t i p s w e r e m a d e u s i n g t h i s m e t h o d f r o mF e b r u a r y 2 7 t o M a r c h 1 8, 1 9 87 .Pheromo ne Analys is

T h e s e x p h e r o m o n e e x t r a c t s w e r e a n a l y z e d b y g a s c h r o m a t o g r a p h y ( G C )a n d g as c h r o m a t o g r a p h y - m a s s s p e c tr o m e t ry ( G C - M S ) . G C a n a ly s e s w e r e p e r -f o rm e d o n a H e w l e t t - P a c k a r d 5 8 9 0 c h r o m a t o g r a p h w i th f la m e i o n i z a t io n d e t e c t o r( F I D ) a n d a R o s s i n j e c t o r h e a t e d a t 2 4 0 C , e q u i p p e d e i t h e r w i th a p o l a r c o l u m n( 30 -m W C O T C P S i l -8 8 C B , 0 .2 2 m m I D ; C h r o m p a c k , M i d d l e b u r g , T h e N e t h -e r l a n d s ) o p e r a t e d i s o t h e r m a l l y a t 1 8 0 C , o r w i th a n o n p o l a r c o l u m n ( 2 5 - mW C O T C P S i l -5 C B , 0 . 2 2 m m I D ; C h r o m p a c k ) h e a t e d f ro m 1 60 t o 2 2 0 C a t5 C / m i n . G C - M S w a s p e r f o r m e d o n a G i r d e l 3 2 g a s c h r o m a t o g r a p h l in k e d toa N e r m a g R 1 0 -1 0 C q u a d r u p o l e m a s s s p e c t r o m e t e r c o n t r o ll e d b y a S I D A R d a t aa c q u i s i ti o n sy s t e m . T h e c h r o m a t o g r a p h w a s f it te d w i th a f u s e d s i li c a c a p i l l a r yc o l u m n (2 5 -m W C O T C P S i l - 5 C B , 0 .2 2 m m I D ; C h r o m p a c k ) a n d o p e r a te di s o t h e r m a l l y a t 1 8 0 C . M a s s s p e c t r a w e r e o b t a i n e d b y e l e c t ro n i m p a c t ( E l) a t7 0 e V s c a n n i n g f r o m 4 0 t o 3 50 a m u , a n d b y c h e m i c a l i o n i z a t i o n ( C I ) at 9 2 . 5e V w i t h a m m o n i a ( N H 3) a n d i s o b u t a n e ( i- C 4 H 10) a s t h e r e a c t a n t g a s . P h e r o m o n ec o m p o u n d s w e r e i d e n t if ie d b y c o m p a r i n g t h e i r G C r e te n t io n ti m e s a n d m a s ss p e c t ra w i t h t h o s e o f c h e m i c a l s s y n t h e s i z e d in th e l a b o r a t o r y (s e e b e l o w ) .Synthesis of Chemicals

W e r e p o r t h e r e i n t h e s y n t h e s i s o f ( Z , E ) - 9 , 1 1 , 1 3 - t e t r a d e c a t r i e n - l - a l . F o rs o m e y e a r s , s e v e r a l l a b o r a t o r i e s h a v e b e e n i n te r e s te d i n s y n t h e s i s o f 1 , 3 , 5 -u n d e c a t r ie n e s ( N / if e t a l . , 1 9 7 5; R a t o v e l o m a n a n a a n d L i n s t r u m e l l e , 1 9 87 ;A n d r e i n i e t a l ., 1 9 8 7) , w h i c h a r e r e p o r t e d t o h a v e o d o r s h i g h l y a p p r e c i a t e d inp e r f u m e r y . T h e n e e d f o r r e g i o - a n d s t e r e o c h e m i c a l t y p u r e c o m p o u n d s f o r b i o -l o g i c a l t e s ts i s w e l l r e c o g n i z e d . A n u m b e r o f s t e re o s e l e c t i v e m e t h o d s ( R a t o -v e l o m a n a n a a n d L i n s t r u m e l l e , 1 9 87 ; A n d r e i n i e t a l . , 1 9 87 ) f o r o b t a i n i n g th e s en o n f u n c t i o n a l i z e d tr ie n e s h a v e b e e n d e s c r i b e d . I n o u r c a s e t h e a p p r o a c h t h a t h a sg i v e n t h e b e s t r e s u l t s w a s b a s e d u p o n t h e m e t h o d o f R o s s i ( A n d r e i n i e t a l . ,1 9 87 ), w h i c h i n v o l v e d t w o s e q u e n t i a l p a l l a d i u m - c a t a l y z e d c r o s s - c o u p l i n g re a c -t io n s . A f ir st c o u p l i n g b e t w e e n a m o l a r e x c e s s o f c o m m e r c i a l E / Z 1 , 2 - d i -b r o m o e t h y l e n e a n d a d e q u a te a l k y n y l z i n c b r o m i d e s l e d t o ( E ) - l - b r o m o - l - e n - 3 -y n e d e r iv a t i v e s w i t h a h ig h s t e r e o i s o m e r i c p u r i ty , s i n c e ( E ) - l , 2 - d i b r o m o e t h y l e n er e a c ts p r e f e r e n t i a l l y w h e n i t i s b l e n d e d w i th t h e Z s t e r e o i s o m e r . T h e s e i n t e r -m e d i a t e b r o m o e n y n e s w e r e t h e n c o u p l e d w i t h v i n y l z i n c b r o m i d e t o af fo r d t h e

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1 1 0 6 P . Z A G A T T I E T A L.

c o r r e s p o n d i n g 1 , ( E ) - 3 - d i e n - 5 - y n e s . T h e l a t t e r w e r e a l l o w e d t o u n d e r g o a Zs t e r eo se l ec tiv e r ed u c t io n to g iv e 1 , (E ) -3 , (Z ) -5 - t r i en e s . T h e ac tu a l s t ep s fo r sy n -th e s i z in g th e d e s i r ed tr i en a l a r e sh o w n in F ig u re 1 . IH an d ' 3 C N M R sp ec t r aw e r e r e c o r d e d o n a B r u k e r A C 2 0 0 s p e c t r o m e t e r [ C D C I 3 ; ( 5 ( p p m ) f r o m T M S ,J ( H z ) ] . I n f r a r e d s p e c t r a w e r e m e a s u r e d o n a P e r k i n - E l m e r 3 9 7 s p e c t r o m e t e r[ n e a t, 3' c m - ' ) ] . S t e r e o i s o m e r i c p u r i ty o f a ll p r o d u c t s w a s d e t e r m i n e d b y g a sc h r o m a t o g r a p h i c a n a l y s e s p e r f o r m e d o n a m o d e l 2 9 0 0 C a r l o E r b a i n s t r u m e n te q u ip p e d w i th s i li ca c ap i ll a ry p o l a r c o l u m n ( 2 5 - m W C O T F F A P 0 . 3 2 I D , H 2ca r r i e r g a s f lo w 2 5 ml /min , 1 . 2 b ) .

(E)-1-Terbutoxy-12-bromo-9-dodecyn-11-ene. T o a so lu t io n o f 1 - t e rb u -t o x y - 9 - d e c y n e (T e l li e r a n d D e s c o i n s , 1 9 90 ) [ 1 0 .5 g ( 0 . 0 5 m o l ) i n 2 5 m l E t 2 0a n d 2 5 m l T H F ] a t 0 C w e r e a d d e d 3 1 m l ( 0 . 0 5 m o l ) o f 1 . 6 M n - B u L i i nh e x a n e . A f t e r 3 0 m i n a t 2 0 C , a l k y n y l l i th i u m w a s o b t a in e d . T o t h i s r ea g e n tw a s a d d e d a t 0 C a n a n h y d r o u s Z n B r 2 s o lu t io n [ 1 2 . 4 g ( 0 . 0 5 5 m o l ) / 5 0 m l T H F ] .T h e s t ir re d m i x tu r e w a s w a n n e d t o 2 0 C f o r 3 0 m i n ; t h e n a t 0 C w e r e a d d e ds u c c e s s i v e ly s o l u t io n s o f P d ( P P h 3 ) 4 ( 3 % ) [ 1 .7 g ( 1 . 5 m m o l ) / 2 0 m l T H F ] a n d4 6 . 5 g ( 0 . 2 5 m o l ) o f E /Z ( 4 0 / 6 0 ) 1 , 2 - d i b r o m o e t h y l e n e . T h e r e ac t io n m i x t u rew a s a l l o w e d t o w a r m t o r o o m t e m p e r a t u r e f o r 1 6 h r a n d t h e n h y d r o l y z e d b yH 2 S O4 so lu t io n (1 N ) . A f t e r ex t r ac t io n wi th e th e r an d ev ap o ra t io n o f t h e so lv en t ,t h e c r u d e m i x t u r e w a s p a s s e d t h r o u g h a s m a l l s i l i c a g e l c o l u m n i n o r d e r t or e m o v e t h e p a l l a d i u m c a t a l y s t ( e l u t i n g w i t h c y c l o h e x a n e ) . T h e s o l v e n t s w e r ee v a p o r a t e d a n d t h e r e s id u e d i s ti ll e d t o a f f o rd 7 . 9 g o f b r o m o y n e n e ( 5 0 % y i e l d );E p u r i ty _> 9 9 % ; b p , 1 2 0 C /0 . 1 T o r r ; m/z: 2 9 9 , 3 0 1 (M - 1 5 ) ; I R : 2 2 5 0 , 1 2 0 0 ;tH N M R : 1 . 1 8 ( s , 9 H ) , 1 . 3 3 (m, 1 2 H) , 2 . 2 2 (m , 2 H ) , 3 . 3 2 ( t, 2 H ) , 6 . 1 3 (d t,H I1) , 6 . 56 (d , H 12) JH,~/H,~ = 14, JH~m,, = 2; '3C N M R : 19 ~5 , 61 .6 , 72 .3 ,7 7 . 3 , 9 3 . 2 , 1 t 6 . 8 , 1 1 8 .1 . A n a t . c a l c d , f o r C , 6H 2 7 B r O : C , 6 0 . 9 5 ; H , 8 . 6 3 .F o u n d : C , 6 1 . 2 0 ; H , 8 . 7 7 .

(E)-1-Acetoxy-12-bromo-9-dodecyn-11-ene. A c 2 0 , 4 . 7 m l ( 0 . 0 5 m o l ) , a n d0 . 4 g (2 . 5 m m o l ) o f FeC 13 we re su cc es s iv e ly ad d ed to a so lu t io n o f 7 . 9 g (0 . 0 2 5

t-BuO(CH2)sC~CH 1) BuLL ZnBr2 = t 'B u O (C H :D sC ~ C S B r A c20, FeC}~2) BrHC--CHB r, Pd

///------Br CH;t__CHZnBr pd o A cO (C H 2)s C ~C ///.. .. .ffA c O ( C H 2 ) s C ~ C . . _ . ~ ~"/ /

1) KO H, MeOH2) Zn, KC N, PrO'H

HOCH~ PDC,. OHC(CH2) __/CH2CI2

FIG. 1. Synthesis of (Z,E)-9,11,13-tetradecatrien-l-al.

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SEX PHEROMONEOF Stenorna cecropia 1107m ol) t -bu ty l e the r in Et2 0 (50 ha l ) . Th e s t i r red so lu t ion w as le f t a t room te m -pera ture overn igh t . A sa tura ted aqueou s so lu t ion o f Na2HPO4 (50 ml) was addedand the mix ture w as s t ir red for 1 hr . T he so l id prec ip i ta te was rem ove d byf il trat ion . A fte r ex t rac t ion w i th e ther and evapo ra t ion o f the so lvent , 6 .9 g oface toxyb rom oynen e were ob ta ined (92 % c rude y ie ld ) ; E pu r i ty (99 %) ; re~z: 221(M-80) ; IR : 2250 , 1740; ~H NM R: 1 .2 -1 .7 (m , 12H ) , 2 .03 ( s , 3H ) , 2 .25 (m,2H ), 4 .0 4 (t , 2H ), 6 .1 6 (dt , H it) , 6 .57 (d , H 12) JH,,H,-" = 14, JH"/H" = 2; ~3CNMR: 19 .4 , 64 .45 , 77 .4 , 93 .1 , 116 .9 , 118 .1 , 170 .6 .

( E ) - 1 - A c e t o x v - 1 1 , 1 3 - t e t r a d e c a d i e n - 9 - y n e . A solu tion of ZnB r2 [24.8 g (0.11m o l ) / 1 0 0 m l T H F ] w a s a dd e d t o a I M T H F s o lu t io n o f v i n y l m a g n e s i u mbromide (100 ml , 0 .1 mo l ) coo led a t 0 C . The reac t ion mix tu re was s t i r red a t20C fo r 30 m in , and a f t e r coo l ing to 0 C , 0 .7 g (0 .6 m m ol ) o f Pd(PPh3)4 inT H F (20 m l ) fo l lowed by 6 g (0 .02 m o l ) o f b rom oyn ene were added . T he s tir redmix tu re was a l lowed to wa rm to room tem pera tu re fo r 12 h r and then hyd ro lyzedby H 2SO4 so lu tion (1 N) . A f te r ex trac t ion wi th e the r and evapora t ion o f theso lvent the c rude mix ture was passed th rough a smal l packed s i l ica ge l co lumn(e lu t ing wi th cyc lohexane) . The so lvent was evapora ted and the res idue d is t i l ledto a fford 3 .7 g of ace tox ydien yne (75% yie ld) ; E pur ity (96% ); b p 115C/0 .1T o r r ; m / z : 248 (M + , 3% ), 91 (100% ); IR: 2250 , 1740 , I62 0 , 1245 , 1000 , 940 ,905 , 840 , 745 , 720 , 700 ; ~H N M R : 1 .33 (m, 12H), 2 .02 (s , 3H), 2 .30 (m,2H ) , 4 .04 ( t, 2H) , 5 .09 (dd , HI4 ) , 5 .21 (dd , HI4 ' ) , 5 .59 (d , H i l l 6 .25 (dd ,HI2), 6 .45 (ddd, H 1 3 ) J H I 3 / H I 4 " = 16.5, JH"/I-I'-"= 14.4, JM"-/H'~ = 9.9, JH'~m'~= 9, JH,4/H,,. = 2; 13C N M R : 64 .5, 79.6 , 93 .4, 112.7, 1 18.4 , 136.4, 140.9,17I . Anal . ca lcd , fo r C 1 6 H 2 4 0 2 : C , 77 .38 ; H , 9 .74 . Found : C , 77 .33 ; H , 9 .62 .

( E ) - 1 1 , 1 3 - T e t r a d e c a d i e n - 9 - y n - l - o l . This p roduc t was p repa red by s apon-if ication o f the co r re spond ing ace ta te wi th 2 N K O H in M eO H ; C rude y ie ld(98%); E pu r ity (96%); m / z : 206 (M + , 4% ), 91 (100% ); IR: 3350 , 2 200 , 1620 ,1580, 1000, 940 , 90 5 ,8 40 , 740 , 695 ; ~ H NM R: 1 .34 (m, 12H) , 2 .32 (m, 2H) ,3.0 (m , 1H), 3 .57 (t , 2H ), 5 .08 (dd, HI4), 5 .2 0 (dd, H I4 ') , 5 .58 (d , HI1), 6 .2 4(dd, H I2), 6.45 (ddd , H 13) JH,,/H,4. = 16.5, JH'~/H~-' = 14.4, Jn'-'m '~ = 10.8,JH'~/H'4 = 9, Jn,,m,~. = 2. 13C N M R : 62 .5, 79 .6, 9 3.5 , 112.7, 118.4, 136.3,140.8.

( Z , E ) - 9 , 1 1 , 1 3 - T e t r a d e c a t r i e n - 1 - o L A m ix tu re o f d ienyno l (2 .9 g , 0 .014mol ) , 1 -p rop ano l -w a te r (1 /1 ) (600 m l ) , zinc powd er (200 g ) , and po ta s siumcyan ide (10 .4 g , 0 .16 too l ) was s t i r red a t room tempera tu re fo r 24 h r unde rargon in the dark. The reaction product was f i l tered through celi te , and thef il trate was ex t rac ted wi th E t20 . A fte r evap ora t ion o f the so lvent , 2 .6 g of tr ienolwere ob ta ined (90% crude y ie ld) ; Z E puri ty (98%); m / z : 208 (M , 11% ), IR:3340, 1620, 1570, 1000, 935 , 895, 740 , 720, 695; ~H N M R : 1.33 (m , 12H ),1 .99 (m, IH ) , 2 .16 (m , 2H ), 3 .60 ( t , 2H ) , 5 .07 (dd , H14) , 5 .20 (dd , H14 ') , 5 .46(dt, Hg), 6.0 0 (d dt, H m), 6.1 9 (dd, H t2), 6.41 (ddd, HI3), 6.51 (dd, H jl)JH,3m ,~ = 16.8, JH,,m,z = 14.6, J H ~ / H , ~ , = 11, J H I t , / t t , l = 10.6, JH"-/H'~ = 10.6,

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1108 P. ZAGATTIET AL.Jn , sm , , = 10 , JHs /n9 = 7 .7 , JH,,/Hh4"= 2 , JHS/H,O = 1 .5 ; t3C N M R : 62 .4 , 116 .6 ,1 2 8 . 4 , 1 2 8 . 6 , 1 3 2 . 9 , 1 3 3 . 2 , 1 3 7 . 2 . An a l . c a l cd , fo r C 1 4 H2 4 0 : C , 8 0 . 7 1 ; H ,1 1 . 6 1 . F o u n d : C , 8 0 . 9 4 ; H , 1 1 . 4 5 .

(Z,E)-1-Acetoxy-9,11,13-tetradecatriene. A c ~ O , 1 . 4 m l ( 1 5 m m o l ) , w a sa d d e d t o a st ir re d m i x t u r e o f 1 g ( 5 m m o l ) o f a l c o h o l a n d 1 . 2 m l o f p y r id i n e .S t ir ri n g w a s m a i n ta i n e d f o r 3 h r a t r o o m t e m p e r a t u r e , a n d t h e m i x t u r e w a sp o u r e d o n c r u s h e d i ce . A f t e r e x t r a c ti o n w i t h e t h e r a n d e v a p o r a t i o n o f t h e s o l v e n t ,1 .1 g o f a ce ta t e we re o b ta in e d (9 0 % c ru d e y i e ld ) ; Z E p u r i t y ( 9 8 % ) ; m/z: 2 5 0( M , 1 0 % ) , 7 9 ( 1 0 0 % ) ; I R : 1 7 4 5 , 1 2 3 5 ; ~H N M R : 1 .3 2 (m , 1 2 H ) , 2 . 0 2 ( s,3 H ) , 2 . 1 6 ( m , 2 H ) , 4 . 0 4 ( t, 2 H ) . D a t a o f t h e d o u b l e - b o n d s y s t e m a r e t h e s a m eas th o se o f t r i en o l. 13CN M R : 6 4 . 6 , 1 1 6 . 8 5 , 1 2 8 . 7 , 1 2 8 .8 , 1 3 3 . 2 , 1 3 3 .3 , 1 3 7 . 4 ,1 7 0 .8 . A n a l . c a l c d , f o r C 16 H 26 02 : C , 7 6 . 7 5 ; H , 1 0 . 4 7 . F o u n d : C , 7 6 . 5 0 ; H ,10 .36 .

(Z,E)-9 ,11,13-Tetradecatr ien- l -al . T o a s o l u t i o n o f a l c o h o l , 1 g ( 5 m m o l )i n C H 2C 12 ( 2 0 m l ) , w a s a d d e d 5 . 6 g ( 1 5 m m o l ) o f P D C ( p y r i d i n iu m d i c h r o m a t e ) .T h e r e a c ti o n m i x t u r e w a s s ti rr e d a t r o o m t e m p e r a t u r e f o r 8 h r , m i x e d w i th E t 2 0 ,an d f i l t e r ed th ro u g h a sma l l co lu mn p ack ed wi th F lo r i s i l t o g iv e , a f t e r ev ap o -r a ti o n o f s o lv e n t s , th e d e s i re d c o m p o u n d , 0 . 9 g ( 9 0 % c r u d e y i e ld ) ; Z E p u r i ty( 9 8 % ) ; re~z: 2 0 6 (M + , 6 % ) , 7 0 ( 1 0 0 % ) ; I R : 2 7 0 0 , 1 7 2 0 ; JH N M R " 1 .3 1 ( m ,1 0 H) , 2 . 1 5 (m , 2 H) , 2 . 4 0 ( td , 2 H ) , 9 . 7 2 ( t , 1 H) ( J = 1 . 7 5 ) . Da ta o f t h e d o u b le -b o n d sy s t em a re id en ti c a l to t h o se o f t r ien o l . ~3C N M R : 4 3 . 8 , 1 1 6 . 7 , 1 2 8 . 5 ,1 2 8 .6 , 1 3 3 .0 , 1 3 3 . 1 5 , t 3 7 . 2 , 2 0 3 . 3 . A n a l . ca l c d , f o r C I 4 H 2 2 0 : C , 8 1 . 5 0 ; H ,1 0 . 7 5 . F o u n d : C , 8 1 . 7 1 ; H , 1 0 . 9 1 .

Other Chemicals. T h e d i e n i c c o m p o u n d s a n d ( Z ) - 9 , 1 1 - t e t r a d e c e n y n - l - a l( Z 9 , Y n e l 1 - 1 4 : A l d ) w e r e p r e p a r e d a s d e s c r i b e d i n R a m i a n d r a s o a a n d T e l l i e r( 1 9 9 0 ). T h e o t h e r g e o m e t r i c i s o m e r s o f tr ie n ic c o m p o u n d s , u s e d i n t h e i d e n ti -f i c a t io n wo rk , we re sy n th e s i zed a s d e sc r ib ed in T e l l i e r e t a l . ( 1 9 9 1 ) an d T e l l i e r(1991) .Electrophysiology

E t e c t r o a n t e n n o g r a m s w e r e r e c o r d e d o n m a t e S. cecropia secu red in a s ty -ro fo am b lo ck . A f t e r a f ew an ten n a l s eg m en t s h ad b een sn ip p ed o f f , th e di s ta le n d o f o n e a n t e n n a w a s i n se r te d i n to t h e t ip o f th e r e c o r d i n g e l e c t r o d e . T h en eu t ra l e l e c tro d e w as imp lan ted in t h e n eck o f t h e in sec t. B o th e l ec t ro d es w e reg la s s c ap i l l a r i e s f i l l ed wi th R o ed e r ' s s a l in e (NaC I : 9 . 0 g / l i t e r ; KC I : 0 . 2 g / l i t e r ;g l u c o s e : 4 . 3 6 g / l it e r) . E l e c t r o d e s w e r e c o n n e c t e d t o a W P I p r e a m p l i fi e r t h r o u g hch lo r id i zed s i l v e r w i r e s . T h e f i l t e r ed s ig n a l (DC to 3 0 0 Hz ) was f ed in to ano s c i l l o s c o p e a n d m o n i t o r e d d i r e c t l y o n t h e s c r e e n .

T o s t imu la t e t h e an ten n a , a i r was p u f f ed (0 . 5 s ec ; 1 . 5 l i t e r s /min ) o n to th ean ten n a th ro u g h a g l a s s c a r t r i d g e co n ta in in g a p i ece o f f i l t e r p ap e r imp reg n a tedw i th 5 0 0 n g o f sy n t he t ic c o m p o u n d . T e n c o m p o u n d s w e r e t e st ed , i n c lu d i n g

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SEX PHEROMONEOF Stenoma cecropia 1109t h o s e f o u n d i n g l a n d e x t r a c t s o f f e m a l e S. cecropia, a n d a n u m b e r o f a n a l o g s.A l l w e r e s y n t h e s i z e d i n t h e l a b o r a t o r y , w i t h a n i s o m e r i c p u r it y b e t t e r t h a n 9 8 % .T h e r e s p o n s e p r o fi le s t o s y n t h e ti c c o m p o u n d s w e r e r e c o r d e d o n 10 m a l e s ( fi ver e p l i c a t e s o n l y f o r t h e t ri e n i c a l d e h y d e , a c e t a t e , a n d a l c o h o l t h a t h a d n o t b e e ns y n t h e s i z e d a t t h e b e g i n n i n g o f th e E A G s c r e e n i n g ) .

S i n g l e s e n s i l l u m r e c o r d i n g s w e r e c a r r i e d o u t o n e x c i s e d a n t e n n a e , u s i n gt h e t i p r e c o r d i n g t e c h n i q u e ( K a i s s l i n g a n d T h o r s o n , 1 9 8 0; L u c a s a n d R e n o u ,1 9 8 9) . T h e b a s e o f th e a n t e n n a w a s i n s e r t e d in a g la s s m i c r o e l e c t r o d e fi ll e d w i t hh e m o l y m p h s a l i n e ( K a i s s l in g a n d T h o r s o n , 1 9 80 ). T h e t i p o f a fe w s e n s i l l at r ic h o d e a w e r e c u t o f f w i t h s h a r p e n e d f o r c e p s , a n d t h e r e c o r d i n g e l e c t r o d e , f il le dw i t h r e c e p t o r l y m p h s a l i n e , w a s s l i p p e d o v e r th e c u t e n d o f a s i n g l e h a i r . T h ea c t io n p o t e n t i a ls w e r e f il te r e d ( 1 5 0 - 5 0 0 0 H z ) , a m p l i f ie d ( 1 0 0 0) , a n d r e c o r d e do n a n a u d i o ta p e r e c o r d e r ( R e v o x B 7 7 ) . T h e r e c o r d i n g s w e r e t h e n p l a y e d b a c ka n d d i g i ti z e d , w i t h a 1 0 - k H z c o n v e r s i o n r a t e , b y a n A / D c o n v e r t e r b o a r d ( D a s ht 6 o f M e t r a b y t e ) i n t e g r a t e d i n a P C m i c r o c o m p u t e r . T h e r e s p o n s e s w e r e a n a -l y z e d u s i n g a sp e c i fi c p r o g r a m w r i t te n i n A s y s t ( M a c m i l l a n S o f t w a r e C o m p a n y )l a n g u a g e (L u c a s a n d R e n o u , 1 9 89 ).

T h e o u t l e t o f a Y - s h a p e d g l a s s t u b e w a s p o s i t i o n e d 2 c m f r o m t h e a n t e n n a ,a n d a f lo w o f h u m i d i fi e d p u re a i r ( 1 .5 l i te r s / m i n ) w a s c o n t i n u o u s l y b l o w n o v e rt h e a n t e n n a t h r o u g h o n e o f th e l a t e r a l b r a n c h e s . T h e s t im u l a t io n c a r t ri d g e c o n -s i st e d o f a P a s t e u r p i p e t c o n t a i n i n g a p i e c e o f f i lt e r p a p e r ( 0 . 5 c m 2) o n w h i c hh e x a n e e x t r a c ts o r h e x a n e s o l u t io n s o f c o m p o u n d s w e r e d e p o s i t e d . T h i s c a r t ri d g ew a s i n tr o d u c e d i n th e m a i n b r a n c h o f t h e Y t u b e . S t i m u l a t i o n w a s a c h i e v e d b yb l o w i n g a p u f f o f a i r ( 0 . 5 s e c ; 0 . 5 l i t e r s / m i n ) t h r o u g h t h e c a r t r i d g e . T h e s a m ec o m p o u n d s a s th o s e u s e d f o r E A G w e r e a s s a y e d o n 14 s e n s i l l a t r ic h o d e a b e l o n g -i n g t o s i x d i f f e r e n t m a l e m o t h s .Olfactometr ic Tes t s

T h e a v a i l a b i l i ty o f t en s o f l iv i n g p u p a e i n th e s p r i n g o f 1 98 9 e n a b l e d u s tod e v e l o p a l a b o r a t o r y - b a s e d b i o a s s a y o f p h e r o m o n e s . A s w e h a d i n s u ff ic i e n t t i m et o c h e c k t h e o p t i m u m c o n d i t i o n s f o r w i n d - t u n n e l t e s t s , w e c h o s e a d y n a m i c ,l i n e a r o l f a c t o m e t e r ( S o w e r e t a l . , 1 9 7 3 ; Z a g a t t i e t a l . , 1 9 8 7 ), c o n s i s t i n g o f ag l a s s t u b e ( 7 0 c m l o n g , 3 . 5 c m d i a m e t e r ) . A i r w a s h u m i d i f ie d b y b u b b l i n g in t ow a t e r a n d h e a t e d t o 2 5 C , t h e n f l o w e d i n t o th e tu b e a t 1 .5 l i t e r s / m i n . T h eo l f a c t o m e t e r w a s s i tu a t e d in a r o o m h e a t e d a t 2 5 C a n d l i g h t e d b y t h r e e i n c a n -d e s c e n t r e d b u l b s ( 4 0 W e a c h ) .

T h e t e s t s ta r t e d j u s t a t t h e b e g i n n i n g o f t h e s c o t o p h a s e a n d l a s t e d 1 h r .E i g h t v ir g i n m a l e s (1 o r 2 d a y s o l d ) w e r e p l a c e d a t th e d o w n w i n d e n d o f t h et u b e . T h e n t h e b l e n d o f s y n th e t i c c o m p o u n d s t o b e te s t e d w a s d e p o s i t e d (1 /zl)o n a g la s s a p p l i c a t o r a t t h e u p w i n d e n d o f th e t u b e . T h e m a l e s w e r e s c o r e dd u r in g 3 m i n a c c o r d i n g t o w h e t h e r t h e y e x h i b i t e d a n y o f t h e f o l lo w i n g b e h a v i o r

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1110 P. ZAGATT!ET AL.p a t t e r n s : ( 1 ) w i n g f a n n i n g , ( 2 ) o r i e n t a t i o n t o w a r d t h e s o u r c e b y w a l k i n g t h r o u g ho n e h a l f o f t h e o l f a c t o m e t e r le n g t h , a n d ( 3 ) c o n t a c t w i t h t h e s o u r c e o f a t tr a c t a n tw i t h o r w i t h o u t c o p u l a t i o n a t t e m p t s . T h e s e b e h a v i o r p a t t e r n s a l w a y s o c c u r r e di n t h is o r d e r i n a h i e r a r c h i c a l s e q u e n c e . E a c h m a l e w a s t e s t e d o n l y o n c e , a n d1 1 2 m a l e s w e r e t e s t e d i n 1 4 s e t s .

T o t e st s y n th e t i c a t t ra c t a n t s , t h e c o m p o u n d s w e r e d i l u t e d i n h e x a n e ( C a r l oE r b a A n a l y t i c a l s R P E ) a n d t h e b l e n d s w e r e p r e p a r e d t o o b t a in t h e d o s e s ( in 1~1 h e x a n e ) s h o w n in T a b l e 1. T h e s e s i x b l e n d s w e r e q u a l i t a t i v e l y t h e s a m e a st h o s e u s e d d u r i n g t h e 1 9 89 f i e ld t r a p p i n g e x p e r i m e n t i n S a n A l b e r t o ( T a b l e 2 ) .I n a d d i t i o n , th e e f fe c t o f n a t u r a l p h e r o m o n e w a s a s s a y e d b y c l i p p i n g o f f t h ep h e r o m o n e g l a n d s o f tw o c a l l in g f e m a l e s a n d c r u s h i n g t h e m i m m e d i a t e l y t o b et e s t e d o n t h e g l a s s a p p l i c a t o r .

T h e r e s u lt s w e r e a n a l y z e d b y a 7 2 c h i - s q u a r e t e st f o r g l o b a l h e t e r o g e -n e i ty ( P < 0 . 0 5 ) , a n d th e n a ll b l e n d s o r fe m a l e g l a n d s w e r e c o m p a r e d p a i r w i s eu s i n g a 2 2 c h i - s q u a r e te s t . A s t h is m e t h o d m u l t i p l i e s t h e d e g r e e s o f f r e e d o m ,t h e th r e s h o l d o f s ig n i f ic a n c e fo r t h e p a i r w i s e c o m p a r i s o n s w a s f ix e d at P 00e

oSCaohvgnemaeaWic

samizoeomacp

bFge3ocoocda

" N t

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SEX PHEROMONEOFStenoma cecropia 1113TABLE 3. TOTAL CATCHES OF ADULT S. cecropia IN TRAPS BAITED WITH DIFFERENT SYNTHETIC

BLENDS FROM JUNE 6 TO JUNE 20, 1992 (TUMACO, COLOMBIA)

Compounds (gg) Male Mean FemaleBlend Z9,EI ,13-14 : Aid Z 9 , E I1-14 : Aid Z9-14 Aid catches" catch/day/trap catchesSC5 250 50 27b 0,6 0SC7 250 0c 0 0SC8 50 0c 0 0SC9 250 50 10 9bc 0.2 0SCI0 250 250 14bc 0.3 0SCll 1000 200 l13a 2.7 0SC12 250 5 0c 0 0

"Numbers followed by the same letter are not significantlydifferent at P = 0.05 (two-way ANOVA on threeblocks, after transformationof data to x/~ + 0.5) and Newman-Keuls est).

dispensers were hung inside the trap in front of the windows. The trap catcheswere recorded daily for one month, with three replicates of each blend. Thepheromone dispensers were changed once, 18 days after the beginning of theexperiment.

A second trapping campaign was scheduled in June 1992 in Tumaco(Narifio, south of Colombia) in a 3500-ha plantation (estate of Palmas de TumacoLtda.). Synthetic chemicals, dispenser, and trap design were the same as in SanAlberto. Seven blends were compared (Table 3) with three replicates in a com-pletely randomized block design. The trap catches were recorded daily for twoweeks.

The results were submitted to one-way (San Alberto) or two-way (Tumaco)analysis of variance on data transformed by " , ~ + 0.5) to reach homoscedas-ticity. The means were then compared by Newman-Keuls test with P < 0.05(Dagnrlie , 1975). As some traps caught both males and females, the differencesbetween male and female catches were compared using Wilcoxon's random-ization test for matched pairs (Siegel, 1956).

RESULTSP h e r o m o n e I d e n i f i c a t i o n

GC-MS analyses of the 557, 684, and 759 female gland extracts revealedthe same chromatographic pattern. In addition to saturated and unsaturatedhydrocarbons and fatty acids, GC-MS data (in electron impact mode) indicated

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1114 P. ZAGATTIET AL.two 14-carbon a ldehydes , one of them diunsatura ted [m/z 41, 55, 67 (100%),82, 9 5, 105, 208 (M "; confirme d by CI -N H 3 with ions at m/z 209 and 226)]and the other t r iunsatura ted [m/z 41, 55, 67, 79 (100%), 80, 91, 107, 206( M + - ; i on s a t m/z 207 and 224 in CI-NH3) ] . The cor resp ondin g 14-carbonaceta tes were a l so ident i f ied [one of them was diunsatura ted wi th ions a t m/z43, 55, 67 (100 %), 79, 82, 93, 110, 121, 192 (M -CH 3C OO H) and 252 (M +'"ions at m/z 253 and 270 in CI-NH 3) and the other tr iunsatu rated w ith ions atm/z 43, 55, 67, 79 (100 %), 80, 91, 105, 119, and 250 (M + ' ; ions a t m/z 251and 268 in CI-NH3)] . T he diunsatura ted a ldehy de was ident i f ied by chem icalioniza t ion analys i s us ing i sobutane to de term ine the po s i t ion of the double bo nds(Einhom et a l . , 1985). T he mass spect rum ex hibi ted var ious molec ular species :M +" (m/z 208, 2 2% ), (M + C4H9) (m/~ 265 , 4%) , MH + (m/z 209 , 91%) ,( M H - H 2 0 ) + (m/z 191, 39% ), and revealed the presence o f abundant ions a t m~z 71 (33 %), 169 (100% ), and 151 (60 % ), which w ere clea r ly related to a 9,11pos i t ion of the d ie thylenic sys tem. The s tereochemis t ry was ass igned to (Z,E)-9,11 by comp ar ison o f the GC re tention t imes wi th s tandards . Data on the tr ienicsys tem were comp lete ly lacking a t the beginnin g of our work. The mass spec-t rum could be ass igned e i ther to a t r iene or to a t r ip le bond, A9,Y ne I l -eny ne.The t r iene hypothes is of a conjugated sys tem wi th a te rminal double bond wassugges ted by compar ing the mass spect rum of the natura l product wi th spect raof severa l chemicals shar ing the same t r ienic mot i f , a l ready prepared in ourlaboratory as react ion intermediates during studies on m oth pherom ones . Fin al ly,a ll poss ib le isomers o f (A ,A) -9 ,11 ,13 and A 9. Y ne l l 14-ca rbon a ldehydes andacetates were synthes ized, and com par ison of GC re tention times and m assspect ra proved tha t the natura l products were (Z ,E)-9 ,1 l , 13- te tradecat rienal and(Z,E)-9,1 t , t3- te t radecat r ieny l ace ta te. The ext rac ts of ph erom one glands of S .cecropia con ta ined : (Z ,E ) -9 ,11- t e t r adecad iena l (Z9,E 11-14 : A ld ; 11%) , (Z,E)-9,11,13- te t radecat r ienal (Z9,EII,13-14:AId; 67%) , (Z ,E ) -9 ,11- t e t r adeca -d i eny l acet at e (Z 9 , E1 1- 14 :A c ; 5 .5% ) and (Z ,E) -9 ,11 ,13- t e t r adeca t r ieny l ace -tate (Z9,E11,13-14:Ac, 16.5%).Etectrophysiology

The EAG screening demonst ra ted tha t the two a ldehydes found in thefemale gland extracts were act ive on the antenna of male S. cecropia (F igure2 ). T h e m o st a c ti v e c o m p o u n d w a s t he t ri en e a l d e h y d e , Z 9 , E l t , 1 3 - 1 4 : A I d ,which el ici ted an EA G response of 1.9 +_ 0.7 mV (mean _+ 95% confid enceinterval ) . The dienic a ldehyde, Z9,E11-14 :A ld , a l so e l i c i t ed h igh-ampl i tudeEA G ( 1.5 _+ 0 .5 mV). Respon ses to o ther com pou nds assayed , and in par t icularto the two acetates ident i f ied in the gland extracts , were al l infer ior to 0.7 inV.

The ac t iv i ty of 14 sens i lla t r ichodea w as recorded f rom s ix d i fferent malesin response to 10 pherom one co mp oun ds and phero mo ne ana logs . Sens i l la t r i-chodea could be separa ted in to four d i f ferent groups based on the responseprofi les of their sensory n eurons (Figu re 2) . Se nsi l la 1-6 presented a f i r ing

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S p i k e s / l s . - m Y

n t _ _

a t _ _

Zg,E11-14:AId o.

Z 9 ,E 1 1 ,1 3 -1 4 :A Id ~ m ~ m ~ [ ~ ~ o ~ o2Z 9 -1 4 :O H 1 . . . . . n t - ] _ ! n t

Zg , E 1 1 , 1 3 o1 4 : O Ho - - _ _ m . . . . [ - - ] r E ] oS e n s i l l u m n u m b e r l l2 3 4 5 6 7 ~ 9 1 0 1 1 1 2 1 3 1 4 EAGS e n s illu m ro u p I VF IG . 2 . E l e c t r o a n t e n n o g r a m s ( E A G ) a n d s i n g l e s e n s i l l u m r e c o r d i n g s o b t a i n e d f ro m m a l e

Stenoma cecropia a n t e n n a e s ti m u l a te d b y s y n t h e t i c c o m p o u n d s . E A G a re e x p r e ss e d a st h e m e a n ( in m i l l iv o l t s ) o f 5 - 1 0 r e p l ic a t e s. T h e v e r t ic a l b a r s r e p r e s e n t th e 9 5 % c o n f i d e n c ei n t e r v a l o f t h e m e a n . T h e s i n g l e s e n s i l l u m r e c o r d i n g s w e r e o b t a i n e d f r o m 1 4 s e n s i l l at r ic h o d e a b e l o n g i n g t o s ix d i f f e r e n t m a l e m o t h s . B l a c k b a r s : A c e l l a c t i v i ty ; o p e n b a r s :B c e l l a c t i v i t y ( g r o u p s I l l a n d I V ) . N a m e o f c o m p o u n d s w e r e a b r i d g e d a s f o l l o w s :Z9-, g e o m e t r y a n d p o s i t i o n o f t h e d o u b l e b o n d ; Y n e l 1 - , t r i p l e b o n d ; - 1 4 , c h a i n l e n g t h ;O H , A i d , A c , a l c o h o l , a l d e h y d e , a n d a c e t a t e , r e s p e c t iv e l y ; n t: c o m p o u n d n o t t e s te d .

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ac t iv i ty co n s i s t i n g o f a s in g le am p l i tu d e c l a s s o f a c t io n p o ten t i a l s . T h e i r f i ri n ga c ti v it y w a s s ti m u l a te d b y Z 9 , E 1 1 , 1 3 - 1 4 : A l d . R e s p o n s e s t o o t h e r c o m p o u n d sw e r e n o t s i g n if i c an t w h e n c o m p a r e d t o th e a i r c o n t r o l . A s e c o n d g r o u p , s e n s il la7 - 9 , p r e s e n te d a s in g l e c l a ss o f a c ti o n p o t e n t ia l s a n d r e s p o n d e d t o th e o t h e rE A G - a c t i v e c o m p o u n d , Z 9 , E 1 1 - 1 4 : A i d . T h e s e s e n s il la w e r e le s s sp e ci fi c t h ang r o u p I s en sil la s in c e t he y al so w e a k l y r e sp o n d ed t o Z 9 , E l l , 1 3 - 1 4 : A c a n d t oZ 9 , Y n e l l - 1 4 : A l d . T h e f ir in g a c ti v it y o f g r o u p I I I ( s en s il la 1 0 a n d 1 1) a n dg r o u p I V ( s e n s il la 1 2 - 1 4 ) c l e a r l y c o n s is t e d o f t w o a m p l i tu d e c l a s se s o f ac t io np o ten t i a ls , i n d ica t in g th e p re sen c e o f at l e a s t tw o r ecep to r n eu ro n s . In g ro u p sI I I a n d I V , t h e c e ll s f ir in g h i g h - a m p l i t u d e a c t i o n p o t e n t i a l s, u p t o 3 m V , a r ere fe r r ed to a s t h e A ce l l s . Ac t io n p o ten t i a l s emi t t ed b y th e o th e r c e l l s , r e f e r r edt o a s B c e ll s , w e r e m u c h s m a l l e r ( 5 0 % o f t he a m p l i tu d e o f A c e ll a c t i o n p o t e n -t ia ls ). I n g r o u p I I I s e n s il la , A c e l ls c l e a r ly r e s p o n d e d t o Z 9 , E 1 1 - 1 4 : A i d a n d Bce l l s to Z9 , E11 , 13-14: A i d . T h e r e s p o n s e p r o f il e s o f se n s il la f r o m g r o u p I Vw e r e c h a r a c te r i z e d b y s tr o n g r e s p o n s e s f r o m B c e ll s to Z 9 , E 1 1 - 1 4 : A l d a n d t oa l es s er e x en t to Z 9 , E 1 1 - 1 4 : A c , Z 9 , Y n e 1 1 - 1 4 : A c , Z 9 - 1 4 : A l d a n d Z 9 , Y n e 1 1 -1 4 : A i d . I n t w o o u t t h r e e o f t h e s e s en s i ll a , A c e l l s re s p o n d e d w e a k l y t oZ 9 , E 1 1 , 1 3 - 1 4 : A i d .Otfactometric Tests

T h e r e su lt s a re sh o w n in T ab le 1 . I t ap p ea red th a t two g l an d s o f v i rg inf e m a l e s w e r e a b l e to e v o k e w i n g f a n n i n g a n d o r i en t a t io n t o w a r d t h e o d o r s o u r c ein m a t e m o t h s . T h e p e r c e n t a g e o f m o t h s m a k i n g c o n t a c t w i th t h e s o u r c e w a sv e ry lo w ( 1 8 . 8 % ) . T w o b l e nd s o f s y n th e ti c c o m p o u n d s e v o k e d t he s a m er e s p o n s e s b y v i r g i n m a l e s a s d i d f e m a l e g l a n d s : a b l e n d o f Z 9 , E 1 1 - 1 4 : A i d ,Z 9 , E l l , 1 3 - 1 4 : A I d , Z 9 , E I I - 1 4 : A c , a nd Z 9 , E l l , 1 3 - 1 4 : A c an d a b le n d o fZ 9 , E l l - 1 4 : A I d a nd Z 9 , E I 1 , 1 3 -1 4 : A ld .Field Trapping

T h e r e su l ts o f th e f ir st t rap p in g ex p e r im en t (F ig u re 3 ) sh o w ed th a t t h ep o p u l a ti o n l e v e ls w e r e v e r y h i g h . T h e n u m b e r o f l iv i n g p u p a e p e r l e a f r ea c h e d3 0 at th e b e g i n n i n g o f t h e e x p e r i m e n t . T h e e m e r g e n c e o f ad u l t s , a s s h o w n b yt h e d e c r e a s e o f p u p a c o u n t s , o c c u r r e d d u r i n g t h e f ir st d a y s o f S e p te m b e r . B yt hi s t im e t he n u m b e r o f u n h a t c h e d e g g s h a d i n c r e as e d a n d r e a c h e d a m a x i m u mo n S ep tem b e r 8 . T h ese d a t a su g g es t t h a t t h e p o p u la t io n o f s ex u a l ly ac t iv e f ema le sw a s a t i t s h i g h e s t b e t w e e n S e p t e m b e r 3 a n d S e p t e m b e r 1 5 , 1 9 8 9 .

T h e a t t ra c t iv en es s o f v i rg in f em a le s a s b a i t wa s n o t co r r e l a t ed w i th th e sed a ta . T h e n u m b e r o f m a l e s c a u g h t i n tr a p s w a s v e r y s m a l l d u r i n g t h e f ir st t w ow e e k s o f S e p t e m b e r , s t ar te d i n c r e a s i n g a s la te a s S e p t e m b e r 1 8, a n d r e a c h e d am a x i m u m o f 3 7 m a l e s / d a y /t r a p o n S e p t e m b e r 2 0 . T h e t h r ee f e m a l e tr ap s a l s o

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SEX PHEROMONEOFStenoma cecropia 1117

40

N u m b e r o f p u p a e f o u n d o n 1 l e a fNu m b er o f eg g s fo u n d o n 10 l ea f le ts

- e - M e a n n u m b e r o f m a l e s c a u g h t in v ir g in f e m a l e t ra p s

30

2O

10

008124 0829 0903 09108 09 /13 09 /18 0923 09128

FIG. 3. Chronological catches of Stenoma cecropia males obtained with traps baited withtwo virgin females (mean of three traps) from September 25 to October 27, 1989 (SanAlberto, central Colombia). The population levels were estimated by counting livingpupae on leaf number 17 and counting unhatched eggs on 10 leaflets (of leaf number 17)on the trees from which the traps were hung (mean of three counts). The scale of the yaxis is the same for the three curves.

caught females regularly throughout the month the experiment lasted, with anaverage of 0.25 females/day/trap.

The other part of this experiment was carried out at the same time and inthe same habitat as virgin female trapping. It consisted of a comparison betweendifferent synthetic pheromone blends. The results (Table 2) showed that all trapscaught female moths (average of 0.2 female/trap/day), with no significant dif-ferences between blends. Male and female catches in the same trap were sig-nificantly different when the traps were baited with blend SC5 (Z9,E 11-14 : Ald,50/zg, and Z9,E 11,13- t 4 :Ald, 250/zg) or virgin females (P = 0.031). BlendSC5 caught significantly more males than the other blends but catches with thisblend were five times lower than those obta ined using the virgin females as bait.The second trapping campaign was conducted in 1992 with a low S. cec-ropia population level that did not exceed one larva per leaf. Mono- or polyun-saturated aldehydes were tested along with the best blend of the 1989 experiment(SC5). The results (Table 3) showed that only males were caught. Blend SC11caught four times more males than SC5 (Table 3). It consisted of the same blendof Z9,Ell-14:AId and Z9,Ell,13-14:Ald at a load four times higher than

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1118 P. ZAGATTIET AL.

S C 5 . W h e n t e s t e d s e p a r a t e l y ( S C 7 a n d S C 8 , T a b l e 3 ) t h e s e a l d e h y d e s w e r e n o ta t t r a c t i v e .

DISCUSSIONSex Pheromone o f S t e n o m a c e c r o p i a

F o u r c o m p o u n d s w e r e i d e n t i fi e d in th e s e x p h e r o m o n e g l a n d e x t r a c t s o fStenoma cecropia f e m a l e s : a d i e n i c a n d a t ri e n i c a l d e h y d e , a n d t h e c o r r e s p o n d -i ng a c e t a te s . T h e e l e c t r o p h y s i o l o g i c a l s t u d y g a v e e v i d e n c e f o r s p e ci fi c r e c e p to r st o th e a l d e h y d e s o n l y . T h e o l f a c t o m e t r i c te s ts s h o w e d a s i m i l a r l e v e l o f r e s p o n s et o f e m a l e g l a n d s a n d t o a b l e n d c o n t a i n i n g t h e f o u r s y n t h e t i c c o m p o u n d s i n ar a ti o c l o s e to t h at o f f e m a l e e x t r a c t s . H o w e v e r , r e m o v a l o f th e t w o a c e t a te s d i dn o t a l te r t h e r e s p o n s e o f m a l e s . I n f i e l d - tr a p p i n g e x p e r i m e n t s , b l e n d s c o n t a i n i n gt h e d i e n i c a n d t r i e n i c a l d e h y d e s a t t r a c t e d m a l e s , w h e r e a s a f o u r - c o m p o n e n tb l e n d , w h i c h i n c l u d e d t h e a c e t a t e s , d i d n o t .

F r o m t h es e b i o l o g i c a l d a t a i t c a n b e c o n c l u d e d t h at (Z,E)-9 ,11,13- te tra-d e c a t r i e n a l a n d (Z ,E) -9 ,11- te tmdecad iena l a r e c o m p o n e n t s o f th e s e x p h e r o m o n eo f Stenoma cecropia. T h e c o r r e s p o n d i n g a c e t a t e s d o n o t s e e m t o b e i n v o l v e din t he p h e r o m o n e c o m m u n i c a t i o n s y s t e m . T h e y a r e p r o b a b l y p r e c u r s o r s o f t h ea l d e h y d e s in a w a y s i m i l a r t o th e b i o s y n t h e t i c p a t h w a y d e s c r i b e d i n Choristo-neura fum iferana. I n t h is to r t ri c i d m o t h , t h e p h e r o m o n e c o m p o n e n t s ( E - a n dZ - l l - t e t r a d e c e n a l ) a r e b i o s y n t h e s iz e d d e n o v o v i a A - 1 1 - t e t r a d e c e n - l - o l a n dA - 1 1 - t e t r a d e c e n - t - o l a c e ta t e . T h e a m o u n t s o f z~-I 1 - t e t r a d e c e n - l - o l a c e t a te inf e m a l e g l a n d s o f C. fumiferana v a r y d u r i n g t h e d a i l y c y c l e a s t h i s a c e t a t e i su s e d a s a s t o r a g e p r e c u r s o r o f th e p h e r o m o n e a n d c o n v e r t e d i nt o t h e c o r r e s p o n d -i n g a l d e h y d e j u s t b e f o r e p h e r o m o n e e m i s s i o n ( M o r s e a n d M e i g h e n , 1 9 84 , 1 9 86 ).

T r a p s b a i t e d w i t h t h e b e s t a t t r a c t a n t s i n t h e p r e s e n t w o r k c a u g h t f e w e ri n s e c ts th a n t r a p s b a i t e d w i th v i r g i n f e m a l e s . T h i s m a y m e a n t h a t a t l e a s t o n ec o m p o n e n t o f t he p h e r o m o n e o f S. cecropia r e m a i n s u n k n o w n .

( Z , E ) - 9 , 1 1 , 1 3 - T e t r a d e c a t r i e n a l h as b e e n i d e n t if ie d a s a p h e r o m o n e c o m -p o n e n t o f th e c a r o b m o t h Ectomyelois ceratoniae ( B a k e r e t a l . , 1 9 8 9 ), w h i c hb e l o n g s t o t h e f a m i l y P y r a l i d a e , a f a m i l y n o t c l o s e l y r e l a t e d t o E l a c h i s t i d a e .T h i s c o m p o u n d w a s a l s o i d e n ti fi e d in a n o t h e r Stenoma s p e c i e s , Stenoma decora ,a m a j o r p e s t o f c o c o a i n B r a z i l ( D . R . H a l l , p e r s o n a l c o m m u n i c a t i o n ) .

T h e p h y l o g e n e t i c re l a t io n s h i p s b e t w e e n f a m i l i e s o f t h e G e l e c h i o i d e a re m a i np u z z l i n g . M i n e t ( 1 9 8 9) i n c l u d e d in th e E l a c h i s t i d a e s e v e r a l g e n e r a f o r m e r l yt h o u g h t to b e l o n g t o th e O e c o p h o r i d a e . S o m e o f t h e m a r e li s te d in a r e c e n tr e v i e w o f se x a t tr a c t a n t s ( A m e t a l . , 1 9 9 2 ): Ethmia s p p . ( E l a c h i s t i d a e : E t h m i -i n a e ) , Depressaria olerelta a n d Agonopter ix s p p . ( E l a c h i s t id a e : D e p r e s s a r i i n a e ) .N o n e o f t h e m u s e a l d e h y d e s as s e x a tt r ac t a n t s. I n s t e a d t h e y u s e m o n o e n i c a c e -t a te s o r a l c o h o l s w it h c h a i n l e n g t h s o f 1 0 - 1 4 c a r b o n s . T h u s , t h e c h e m o t a x o n o m y

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SEX PHEROMONEOF Stenoma cecropia 1119o f se x p h e r o m o n e s d o e s n o t s e e m t o b e p r o m i s i n g f o r c l a r i fy i n g t he s ta tu s o ft h i s g r o u p .

I n fl u e nc e o f In s e c t D e n s i t yT h e r e s u l ts o f t h e f ie l d - t r a p p i n g e x p e r i m e n t s r a i se d q u e s t i o n s a b o u t th e

i n f lu e n c e o f t h e p o p u l a t i o n d e n s i t y o n t h e e f f e c t i v e n e s s o f s e x u a l t r a p p i n g . D u r -i n g t h e 1 9 8 9 s e a s o n , t h e p o p u l a t i o n l e v e l s w e r e v e r y h i g h . I t c a n b e s e e n i nF i g u r e 3 th a t th e m a x i m u m n u m b e r o f se x u a l l y a c t iv e f e m a l e s o c c u r re d b e t w e e nS e p t e m b e r 3 a n d S e p t e m b e r 1 5. T h e n u m b e r o f m a l e s c a u g h t d u r i n g th i s p e r i o db y th e v i r g i n f e m a l e t r ap s w a s v e r y s m a l l ( 3 3 o u t o f a t o ta l c a t c h o f 4 5 8 ) , I tb e g a n t o i n c r ea s e a f t er S e p t e m b e r 1 8, at a t i m e w h e n f e w f e m a l e s w e r e l ik e l yt o b e u n m a t e d a n d c a l l i n g f o r m a l e s .

I t i s g e n e r a l l y a d m i t t e d t h a t c a t c h e s i n t r a p s b a i t e d w i t h s y n t h e t i c a t t r a c t a n t sa r e n o t c o r r e l a t e d w i t h i n s e c t p o p u l a t i o n l e v e l s a t t h e h i g h e s t l e v e l s o f d e n s i t y( C a r d r , 1 9 7 9) . T o o u r k n o w l e d g e , t h i s h a s n o t b e e n r e p o r t e d f o r t ra p s b a i te dw i t h v i r g i n f e m a l e s . T h e s e a r e e x p e c t e d t o b e v e r y a t tr a c t i v e , b e c a u s e t h e c a l l in gp e r i o d o f th e c a g e d f e m a l e s l as ts 1 o r 2 h r w h e r e a s t h e " f r e e " f e m a l e s s t o pc a l l i n g a s s o o n a s t h e y m a t e . T h e l a c k o f m a l e c a t c h e s d u r i n g t h e e a r ly p a r t o fo u r e x p e r i m e n t w i th S . c e c ro p i a s u g g e s t s t h a t t h e i n s e c t s u s e d o t h e r m a t e -s e a r c h in g s y s t e m s t ha n p h e r o m o n e o r i e n ta t io n w h e n d e n s i t y w a s h i g h . T h em o v e m e n t s o f m a n y m o t h s in t h e v ic i n i ty o f t h e t ra p s m a y e x p l a i n t h e n u m b e ro f d r o w n e d f e m a l e s ( T a b l e 2 ). I t m a y a l s o r e s u lt in s e x u a l e n c o u n t e r s a n ds u b s e q u e n t m a t i n g s .

T h e m o n i t o r i n g o f p e st p o p u l a t i o n s w i t h p h e r o m o n e s h a s n o w b e c o m e ar o u t i n e t e c h n i q u e f o r p l a n t p r o t e c t i o n i n t e m p e r a t e c o u n t r i e s . I t i s n o t y e t w i d e -s p r e a d i n t h e t r o p i c s ( Z a g a t t i , 1 9 9 3 ) , w h e r e d i f f e r e n t p e s t c h a r a c t e r i s t i c s m a yr e s u lt in a d i f fe r e n t a p p r o a c h t o p e s t m a n a g e m e n t p r o g r a m s .

Acknowledgments--This w ork was accom plished through the financial and technical supportof lndupalma S.A. We are pleased to thank Dr. E. Andrade, general manager, and Ing. ManoloinAvila for field assistance. Palmas de Tum aco L tda. helped us finish the w ork , and w e are indebtedto Drs . F. Bernal and F. Rod riguez, D. M ariau (CIR AD -CP) provided financial support and per-manent contact between French and Colombian partners. W e also thank D rs. D. R. H all (NRI,Chatham) for results on Stenoma decora, J, M ine t (Mu seum N ational d'Histoire Naturelle. Paris)lor his he lp on systema tics, and B. F rrrot, M, Lettere, F. R amiandm soa, and M. Reno u (INR A)for their help during the work and discussions on the m anuscript. We are indebted to M r. Ph. Butler(INR A, Trad uctions) for his comp lete revision of the En glish text.

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