Insect Pheromone Technology: Chemistry and ... - ACS Publications

5 Recent Pheromone Research in the Netherlands on Muskrats and Some Insects F. J. RITTER, I. E. M. BRÜGGEMANN, J. GUT, and C. J. PERSOONS

Downloaded by PURDUE UNIV on August 28, 2014 | http://pubs.acs.org Publication Date: June 18, 1982 | doi: 10.1021/bk-1982-0190.ch005

Netherlands Organization for Applied Scientific Research TNO, Delft, Netherlands The paper describes research on three p e s t s , intro duced from America i n t o Europe: the muskrat, Ondatra z i b e t h i c u s , the American cockroach, P e r i p l a n e t a americana, and the beet armyworm, Spodoptera e x i g u a . Screening of a variety of muskrat l u r e s in the field and in pens showed that e x t r a c t s of the p r e p u t i a l glands of male muskrats were most attractive. GC/MS a n a l y s i s proved that the composition of the musk i n these glands changes w i t h season. The e x t r a c t s con tain at l e a s t 13 m a c r o c y c l i c ketones, the major ones of which are the (Z)-5 isomers of cycloheptadecenone and cyclopentadecenone, and the corresponding s a t u rated ketones. They are probably biogenetically r e l a t e d to the f a t t y acids found to be present i n the same glands. The identification of p e r i p l a n o n e - A , one of the two sex pheromones of the American cockroach, is r e p o r t ed. I t i s a s e s q u i t e r p e n o i d , C H O, which r e a d i l y isomerizes to a more s t a b l e but biologically inac tive compound. The s t r u c t u r e s of both compounds are deduced from spectroscopic d a t a . P e r i p l a n o n e - A i s 7-methylene-4-isopropyl-12-oxa-tricyclo [4.4.2.0 ] -9-dodecene-2-one: 15

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In Insect Pheromone Technology: Chemistry and Applications; Leonhardt, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

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From females of the beet armyworm, Spodoptera e x i g u a , the ( Z , E ) - and ( Z , Z ) - t e t r a d e c a d i e n y l a c e t a t e s , the ( Z ) - 9 - and ( Z ) - 1 1 - t e t r a d e c e n y l acetates and t e t r a decanyl acetate were i s o l a t e d and identified. Syn thetic mixtures of some of these components a t t r a c t male moths in field t e s t s .


Mammalian Pheromones: An A r e a N e g l e c t e d b y A p p l i e d B i o l o g i s t s and P e s t i c i d e Chemists Legends and o l d h i s t o r i c a l r e c o r d s p r o v e t h a t e v e n i n a n c i e n t t i m e s man h a s shown i n t e r e s t i n o d o u r o u s s e c r e t i o n s o f a n i m a l s , s u c h as t h e musk d e e r and t h e c i v e t , t o u s e i n p r e c i o u s p e r f u m e s . T h i s i s t h e m a i n r e a s o n why c h e m i s t s h a v e a n a l y s e d , s y n t h e s i z e d and m i m i c k e d t h e components o f t h e s e s e c r e t i o n s l o n g b e f o r e a n y i n s e c t pheromone was i s o l a t e d and i d e n t i f i e d . One may t h e r e f o r e wonder why s o l i t t l e a t t e n t i o n h a s b e e n p a i d b y s c i e n t i s t s t o t h e b e h a v i o u r a l r o l e , t h e c h e m i s t r y and t h e a p p l i c a t i o n o f s u c h mammalian c h e m i c a l s i g n a l s . T h i s p a r t l y c a n be a t t r i b u t e d t o g r e a t e x p e r i m e n t a l d i f f i c u l t i e s i n s t u d y i n g mammalian pheromones (J_) . Compared w i t h b e h a v i o u r a l and p h y s i o l o g i c a l b i o a s s a y s u s e d i n i n s e c t pheromone r e s e a r c h , r e l i a b l e , r e p r o d u c i b l e and s i g n i f i c a n t b i o a s s a y s f o r mammalian pheromones a r e h a r d t o d e v i c e . Such a s s a y s a r e e s s e n t i a l f o r c h e m i s t s t o g u i d e t h e i r i s o l a t i o n and i d e n t i f i c a t i o n and f o r b i o l o g i s t s t o d e t e r m i n e t h e b i o l o g i c a l s i g n i f i c a n c e o f a com pound f o r t h e b e h a v i o u r o f t h e a n i m a l . The r e a c t i o n o f mammals t o o l f a c t o r y s t i m u l i i s c e r t a i n l y much more i n f l u e n c e d b y e x p e r i e n c e , e n v i r o n m e n t a l f a c t o r s a n d other sensory i n p u t s than i s the r e a c t i o n o f i n s e c t s t o phero mones. M o r e o v e r , t h e same o d o u r may s e r v e d i f f e r e n t b e h a v i o u r a l f u n c t i o n s and t h e s e may b e d e p e n d e n t on s u c h v a r i a b l e s as s e a s o n , age, e t c . . E v e n t h e w o r d "pheromone" f o r c h e m i c a l s i g n a l s i n mammals h a s b e e n q u e s t i o n e d (2). A l t h o u g h t h e o b j e c t i o n s were r e b u t t e d (3) t h e t e r m i s t o d a y s t i l l a m a t t e r o f c o n t r o v e r s y o r at l e a s t d i s c u s s i o n ( 4 ) . A n o t h e r r e a s o n f o r t h e r e l a t i v e l a c k o f i n t e r e s t i n mammalian pheromones l i e s i n t h e f a c t t h a t - a p a r t f r o m t h e u s e i n p e r fumes - o n l y one a c t u a l a p p l i c a t i o n o f mammalian pheromones i s known so f a r . T h i s i s n o t i n c o n n e c t i o n w i t h a p e s t b u t w i t h a v e r y u s e f u l a n i m a l , t h e d o m e s t i c p i g ( 5 ) . The s a l i v a and sweat g l a n d s o f t h e s e x u a l l y a r o u s e d b o a r c o n t a i n two s t e r o i d s w i t h a musky s m e l l , Δ ~androstenol a n d A - a n d r o s t e n o n e . These a r e a c t u a l pheromones e m i t t e d b y t h e male a n d e l i c i t i n g a c h a r a c t e r i s t i c b e h a v i o u r i n t h e f e m a l e . Sows i n h e a t r e a c t t o t h e s c e n t o f t h e s e compounds b y a s s u m i n g a c h a r a c t e r i s t i c c o p u l a t i n g s t a n c e . The pheromone i s c o m m e r c i a l l y a v a i l a b l e as a n a i d i n a r t i f i c i a l i n s e m i n a t i o n , t o show when a y o u n g sow i s i n o e s t r u s . The m a j o r p a r t o f mammalian pheromone r e s e a r c h , much o f w h i c h i s m e n t i o n e d i n r e c e n t b o o k s a n d symposium p r o c e e d i n g s (5-10) i s n o t d i r e c t e d t o any a p p l i c a t i o n . T r a p p e r s , h o w e v e r , h a v e u s e d mammalian s e c r e t i o n s i n t h e i r l u r e s l o n g b e f o r e t h e 16

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t e r m pheromone was c o i n e d , and t h e p o s s i b i l i t y o f a p p l i c a t i o n o f mammalian c h e m i c a l s i g n a l s has b e e n s u g g e s t e d by s c i e n t i s t s l i k e C h r i s t i a n s e n and D i v i n g f o r c o n t r o l o f s m a l l r o d e n t s (11) and by M a r s h and Howard f o r c o n t r o l o f r o d e n t s i n g e n e r a l ( 1 2 ) . Although they p o s i t i v e l y e v a l u a t e the p o t e n t i a l i t i e s of these a g e n t s i n r o d e n t c o n t r o l , t h e few p u b l i c a t i o n s i n t h i s a r e a r e l a t e r a t h e r to l a b o r a t o r y experiments than to a c t u a l a p p l i c a t i o n i n pest c o n t r o l . T h i s i s p a r t i c u l a r l y s u r p r i s i n g i n t h e c a s e o f common r a t s s i n c e we know t h a t t h e m a l e p r e p u t i a l g l a n d c o n t a i n s a m i x t u r e o f a l i p h a t i c a c e t a t e s w h i c h a p p e a r s t o a c t as an a t t r a c t i v e s e x pheromone t o t h e f e m a l e ( 1 3 ) . T h i s g l a n d a l s o a p p e a r s t o h a v e an important f u n c t i o n i n c o n s p e c i f i c chemical communication i n mice. I t s w e i g h t i s i n v e r s e l y r e l a t e d t o p o p u l a t i o n d e n s i t y (14) and i s l a r g e r i n dominant than i n s u b o r d i n a t e males ( 1 5 ) . E x t r a c t s of t h e s e g l a n d s a t t r a c t f e m a l e m i c e (16) and p r e p u t i a l e c t o m y a f f e c t s male f i g h t i n g b e h a v i o u r ( 1 7 ) . Many o t h e r s k i n g l a n d s o f mammals a l s o p r o d u c e o d o u r o u s s e c r e t i o n s to which b e h a v i o u r a l f u n c t i o n s are a t t r i b u t e d : marking of t e r r i t o r i e s , r e c o g n i t i o n o f s p e c i e s , o f s e x o r i n d i v i d u a l s , e t c . . They may a l s o s e r v e i n f a m i l i a r i z a t i o n w i t h s t r a n g e s u r r o u n d i n g s e.g. " g a i n i n g c o n f i d e n c e " as e x e m p l i f i e d by t h e b e h a v i o r o f r a b b i t s (_1_) . A r e c e n t r e v i e w on t h i s s u b j e c t i s g i v e n by M u l l e r - S c h w a r z e (18) . The M u s k r a t :

A P o p u l a r F u r A n i m a l , A N i g h t m a r e i n The

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I n most c o u n t r i e s i n t h e N o r t h e r n h e m i s p h e r e t h e m u s k r a t , O n d a t r a z i b e t h i c u s , i s m a i n l y a p p r e c i a t e d f o r i t s b e a u t i f u l muskr a t o r " b i s a m " f u r . I t was f o r t h i s r e a s o n t h a t i t was i n t r o d u c e d f r o m A m e r i c a i n t o C z e c h o s l o v a k i a and o t h e r E u r o p e a n c o u n t r i e s e a r l y i n t h e 2 0 t h c e n t u r y . I n t h e N e t h e r l a n d s , h o w e v e r , i t i s now a m a j o r p e s t , damaging t h e b a n k s o f t h e many c a n a l s and d i k e s , so v i t a l f o r a c o u n t r y l y i n g l a r g e l y b e l o w s e a l e v e l . T h i s t h r e a t i s e v i d e n t f r o m t h e s c h e m a t i c d r a w i n g o f F i g u r e 1. I n t h e b e g i n n i n g o f W o r l d War I I t h e r a t s i n v a d e d t h e N e t h e r lands, which f o r b r e v i t y , I w i l l c a l l H o l l a n d , although t h i s i s a p a r t of the Netherlands i n which t h i s rodent i s s t i l l r a r e . U n l i k e t h e German army, h o w e v e r , t h e m u s k r a t f i r s t c r o s s e d t h e B e l g i a n , and l a t e r t h e German b o r d e r . B e t w e e n 1950 and t h e p r e s e n t day t h e m u s k r a t has c o n s t a n t l y e x p a n d e d i t s t e r r i t o r y and i t now occupies the l a r g e r p a r t of the country, i n s p i t e of constant t r a p p i n g by g o v e r n m e n t - e m p l o y e d p r o f e s s i o n a l t r a p p e r s . T h e r e f o r e t h e D u t c h government d e c i d e d t o l o o k f o r a l t e r n a t i v e methods t o s t o p and c o n t r o l t h e i n t r u d e r , and o u r r e s e a r c h group was a s k e d t o l o o k i n t o t h e p o s s i b i l i t i e s o f u s i n g p h e r o mones o r any o t h e r s e m i o c h e m i c a l s . T h e r e i s a v a s t l i t e r a t u r e a b o u t t h e m u s k r a t . A r e v i e w by H o f f m a n n (19) m e n t i o n s 6858 r e f e r e n c e s , 1669 o f w h i c h came f r o m t h e USSR. Not a l l o f t h e s e s o u r c e s a r e r e a d i l y a v a i l a b l e , b u t f r o m t r a n s l a t e d t i t l e s and f r o m t h e E n g l i s h and German l i t e r a t u r e i t



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seems t h a t l i t t l e has b e e n p u b l i s h e d a b o u t c h e m i c a l c o m m u n i c a t i o n i n muskrats. I l l - d e f i n e d c o n c o c t i o n s have f o r c e n t u r i e s been used t o l u r e m u s k r a t s i n t h e USA, b u t we h a v e l o o k e d i n v a i n f o r p u b l i s h e d d a t a w h i c h p r o v e t h e e f f e c t i v e n e s s o f t h e s e l u r e s . O n l y one v e r y b r i e f s t a t e m e n t by W i l l i a m s (20) a b o u t t h e e f f e c t i v e n e s s o f a d d i t i o n o f " m u s k r a t s c e n t " t o c a r r o t - b a i t e d t r a p s was f o u n d i n t h e J o u r n a l o f W i l d l i f e Management o f 1951. A t a b l e showed t h a t t h e " a v e r a g e t r a p p i n g s u c c e s s " i n c r e a s e d f r o m 23.6 t o 4 2 . 8 % , b u t t h e two s e r i e s o f e x p e r i m e n t s ( c o n t r o l s and s c e n t e d c a r r o t s ) w e r e n o t made s i m u l t a n e o u s l y , so t h e s e r e s u l t s h a r d l y p r o v e t h e a t t r a c t a n c y o f t h e scent. One of o u r f i r s t t a r g e t s was t h e r e f o r e t o f i n d o u t w h e t h e r o r n o t a t t r a c t i o n by m u s k r a t musk o r any o t h e r t r a d i t i o n a l l u r e can be d e m o n s t r a t e d . T h i s was i n i t i a l l y i n v e s t i g a t e d by a p p l y i n g t r a p p i n g methods n o r m a l l y u s e d by t r a p p e r s i n H o l l a n d ; t h i s was done i n c o l l a b o r a t i o n w i t h e x p e r i e n c e d t r a p p e r s and u s i n g a v a r i e t y of l u r e s . Methods f o r F i e l d - T r a p p i n g M u s k r a t s I n H o l l a n d t h e m u s k r a t u s u a l l y l i v e s i n b u r r o w s i n dams, d i k e s and b a n k s o f t h e c a n a l s and l a k e s , as d e p i c t e d i n F i g u r e 1. The " m u s k r a t h o u s e s " o r " w i n t e r h u t s " , w e l l - k n o w n i n t h e USA, are r e l a t i v e l y r a r e i n H o l l a n d . They a r e u s u a l l y f o u n d i n w e t l a n d s and marshes w h e r e no d i k e s a r e a v a i l a b l e . They a r e c o n s p i c u o u s and r e l a t i v e l y e a s y t o f i n d . T h i s i s n o t t h e c a s e w i t h t h e b u r rows i n t h e b a n k s and d i k e s . The e n t r a n c e s a r e b e l o w w a t e r l e v e l and c a n o n l y be o b s e r v e d when t h e l e v e l d r o p s . E x p e r i e n c e d t r a p pers have, however, a v a r i e t y of c l u e s t o d e t e c t the p r e s e n c e o f an e n t r a n c e , e.g. by swimming t r a i l s , o r h e a p s of s a n d removed from the burrows. I n H o l l a n d , t h e use o f p o i s o n e d b a i t s w h i c h c o u l d k i l l o t h e r w i l d l i f e , and g r o u n d t r a p s w h i c h w o u l d n o t k i l l t h e a n i m a l immed i a t e l y , a r e p r o h i b i t e d . The d e v i c e s most o f t e n u s e d a r e : - b a i t e d t r a p s , a f l o a t on r a f t s , i n w h i c h a p p l e s a r e o f t e n u s e d as b a i t , - "Conibear t r a p s " , p l a c e d at the entrance below the water s u r f a c e , - metal hoop-nets, e i t h e r p l a c e d at the entrance of a burrow or at the i n t e r c o n n e c t i o n o f waterways o r under a b r i d g e o r , according to a r e c e n t l y developed technique, i n "fake entrances", made o f p l a s t i c t u b e s o r p i p e s ( F i g u r e 2 ) , - a v a r i e t y of f l o a t i n g " v a l v e - t r a p s " or o t h e r c o n t r a p t i o n s , usually baited with apples. A number o f f i e l d t e s t s u s i n g s u c h methods w e r e made i n o r d e r to s c r e e n a v a r i e t y o f l u r e s , e i t h e r o b t a i n e d t h r o u g h t h e h e l p o f P r o f . M u l l e r - S c h w a r z e o f New Y o r k S t a t e U n i v e r s i t y i n S y r a c u s e , w i t h whom we c o l l a b o r a t e , o r made by o u r s e l v e s f r o m c o l l e c t e d glands or commercially a v a i l a b l e f r a g r a n c e s .



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Figure 2. Top: Scheme of 4 rafts with baited traps and 24 funnel traps placed in "artificial burrows" at the Berkel on the German border. Bottom: Funnel trap in plastic pipe placed below the water surface. Λ scented cork is stuck fust above the trap. Results of 2 series of experiments with such traps are shown in Figures 3 and 4.


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R e s u l t s of F i e l d T e s t s w i t h Muskrat Lures F i e l d t e s t s w e r e made a t a v a r i e t y o f l o c a t i o n s n e a r t h e German o r B e l g i a n b o r d e r and i n an a r e a b e t w e e n r i v e r s , s o u t h o f the Rhine. I n t h i s p a p e r we c a n n o t sum up a l l t h e r e s u l t s , many o f w h i c h were i n c o n c l u s i v e . We a t t r i b u t e t h e l a t t e r t o an i n s u f f i c i e n t number o f m u s k r a t s i n t h e t e s t a r e a ( a p a r t f r o m t r a p p i n g , no r e l i a b l e method was a v a i l a b l e t o e s t i m a t e t h e s i z e o f t h e p o p u l a t i o n ) o r t o t h e l a c k o f a t t r a c t i v e n e s s o f t h e l u r e s i n mid-summer, o u t s i d e t h e m a t i n g o r m i g r a t i o n s e a s o n . We h a v e t o s t r e s s , t h e r e f o r e , t h a t t h e f o l l o w i n g r e s u l t s must be c o n s i d e r e d as p r e l i m i n a r y ones and a r e a s e l e c t i o n of o n l y t h o s e r e s u l t s w h i c h seem t o be statistically significant. The most c o n v i n c i n g e v i d e n c e of t h e e f f e c t i v e n e s s o f some l u r e s was o b t a i n e d a t t h e B e r k e l , a l i t t l e b o r d e r r i v e r b e t w e e n Germany and H o l l a n d . H e r e we m a i n l y u s e d t h e method w i t h a r t i f i c i a l e n t r a n c e s , i n which funnel-shaped t r a p s were p l a c e d ( F i g u r e 2 ) . I n t h e s l o p e o f t h e bank 24 h o l e s w e r e dug i n w h i c h p i p e s w i t h t r a p s w e r e p l a c e d . T h r e e d i f f e r e n t l u r e s were t e s t e d i n s i x f o l d a g a i n s t an e q u a l number of c o n t r o l s . I n a d d i t i o n , 4 f l o a t i n g t r a p s , b a i t e d w i t h a p p l e s , and t h e same l u r e s w e r e t e s t e d . F i v e d r o p s o f l u r e w e r e a p p l i e d t o c o r k s p l a c e d on s t i c k s above t h e f u n n e l t r a p s and s i m i l a r c o r k s were u s e d on t h e r a f t s . A t l e a s t once a week t h e t r a p s w e r e c h e c k e d and f r e s h d r o p s o f l u r e s a p p l i e d . The r e s u l t s w i t h t h e f u n n e l s a r e shown i n F i g u r e 3. The s a m p l e s t e s t e d w e r e : A. S c e n t g l a n d e x t r a c t s o f m a l e m u s k r a t s , c a u g h t i n M a r c h i n t h e same v i c i n i t y b u t somewhat more t o t h e N o r t h ( t h e p r o v i n c e o f D r e n t h e ) . The two b a t c h e s t e s t e d (26 and 29 i n F i g u r e 3) were o b t a i n e d by c e n t r i f u g a t i o n o f h o m o g e n i z e d g l a n d s i n e t h a n o l . The s u p e r n a t a n t u s e d r e p r e s e n t e d 5 ml e x t r a c t p e r g l a n d , and t h e 5 d r o p s c o r r e s p o n d e d r o u g h l y w i t h 0.1 ml o r 1/50 o f t h e e x t r a c t f r o m one g l a n d . B. C o m m e r c i a l l y a v a i l a b l e A m e r i c a n " M u s k r a t Musk" ( P e r k i n s H i l l Ltd., C a z e n o v i a , N.Y.) ( s a m p l e 28 i n F i g u r e 3 ) . C. A m i x t u r e o f o i l o f sweet f l a g i n g l y c e r i n e (1 : 2 ) , made a c c o r d i n g t o one o f t h e t r a d i t i o n a l t r a p p e r s r e c i p e s , k i n d l y p r o v i d e d by P r o f . M.M. A l e x a n d e r , S y r a c u s e , N.Y. ( s a m p l e 13 i n F i g u r e 3). As t h e F i g u r e shows, o n l y 4 m u s k r a t s w e r e c a u g h t d u r i n g t h e w h o l e t e s t p e r i o d - w h i c h r a n f r o m September 15, 1980 t o June 5, 1981 i n t h e c o n t r o l s compared w i t h 19 i n t h e t r a p s w i t h t h e home-made g l a n d e x t r a c t s . I n t h e f i r s t two months t h e s e numbers w e r e e v e n 1 and 10. W i t h sweet f l a g and A m e r i c a n musk t h e t o t a l c a t c h e s o v e r t h e w h o l e p e r i o d w e r e 6 and 9, r e s p e c t i v e l y . U n f o r t u n a t e l y , t h e w a t e r l e v e l d r o p p e d c o n s i d e r a b l y d u r i n g t h r e e p e r i o d s , as i n d i c a t e d i n t h e F i g u r e , b u t t h i s does n o t a f f e c t t h e c o n c l u s i o n t h a t t h e g l a n d e x t r a c t was b e s t . I n t o t a l 4 f e m a l e s and 34 m a l e s w e r e caught.



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The b a i t e d t r a p s on t h e r a f t s seemed l e s s s u i t e d f o r s c r e e n i n g a t t r a c t a n t s , as t h e a p p l e s a t t r a c t h u n g r y m u s k r a t s and o n l y s l i g h t , i f any, improvement was o b t a i n e d by a d d i n g t h e l u r e s , w i t h t h e e x c e p t i o n of t h e A m e r i c a n musk w h i c h seemed t o enhance t h e a t t r a c t i v i t y o f t h e b a i t s d u r i n g t h e months M a r c h and A p r i l . An a d d i t i o n a l p r o b l e m w i t h t h e b a i t e d t r a p s i s t h a t t h e y a l s o c a t c h o t h e r a n i m a l s . I n t h e t e s t p e r i o d 37 m a l e and 2 f e m a l e muskr a t s w e r e c a u g h t t h i s way, b u t a l s o 9 common b r o w n r a t s . A t t h e Hegebeek, a n e a r b y l o c a t i o n , a t t h e s l o p e s o f a s m a l l p o o l n e a r a l o c k , t h e l u r e s A, Β and C c o u l d o n l y be t e s t e d s i n g l y , i n t e r s p a c e d b y two n o n - s c e n t e d t r a p s ( b l a n k s ) ( F i g u r e 4 ) . H e r e a g a i n , t h e e x t r a c t s of t h e D u t c h m u s k r a t s came o u t b e s t , a l t h o u g h t h e o t h e r t r a p s and even one o f t h e b l a n k s w e r e n o t i n e f f e c t i v e . A c c o r d i n g to the t r a p p e r s , the predominance of males i s not u n u s u a l a t t h e s e l o c a t i o n s n e a r t h e German b o r d e r . I t i s a t t r i buted t o the o b s e r v a t i o n t h a t d u r i n g m i g r a t i o n the males t r a v e l first. F i e l d t e s t s w i t h b a i t e d t r a p s and t h e same t h r e e l u r e s i n t h e m i d d l e of the c o u n t r y between the branches o f the R h i n e , d i d not a l l o w c o n c l u s i o n s a b o u t t h e a t t r a c t a n c y of t h e l u r e s t o be r e a c h e d , b u t t h e y showed t h a t h e r e a b o u t e q u a l numbers o f m a l e s and f e m a l e s w e r e c a u g h t . A t t h i s l o c a t i o n a s t a b i l i z e d c o l o n y o f m u s k r a t s had s e t t l e d . These p r e l i m i n a r y r e s u l t s seem t o c o n f i r m t h a t e x t r a c t s o f m u s k r a t p r e p u t i a l g l a n d s and p o s s i b l y a l s o o f sweet f l a g c a n i n d e e d enhance t h e c a t c h e s o f m u s k r a t s , e s p e c i a l l y i n s c e n t e d f u n n e l t r a p s i n a r t i f i c i a l p i p e s a t r i v e r b a n k s , where m i g r a t i n g m u s k r a t s p a s s . The a n i m a l s c a u g h t a r e t h e n m a i n l y m a l e s w h i c h p r e f e r e n t i a l l y e n t e r a r t i f i c i a l p i p e s s c e n t e d w i t h e x t r a c t s of musk g l a n d s w h i c h - i t s h o u l d be r e m a r k e d h e r e - a r e p r o d u c e d by other males. Screening Lures w i t h Muskrats

i n Captivity

A number o f m u s k r a t s c a u g h t i n l i v e - t r a p s n e a r t h e B e l g i a n b o r d e r w e r e u s e d f o r s c r e e n i n g l u r e s i n two s e r i e s o f t e s t s . The f i r s t one was made i n a c o n c r e t e b a s i n , 7 m l o n g , 1.5 m w i d e and 1.6 m deep ( F i g u r e 5 ) , i n w h i c h 4 t o 8 m u s k r a t s w e r e k e p t . N e s t b o x e s w e r e made i n t h e c o r n e r s and two c o u n t i n g c a g e s were p l a c e d on a r a f t n e a r t h e m i d d l e of t h e b a s i n . A n i m a l s v i s i t i n g t h e cages w e r e c o u n t e d m e c h a n i c a l l y by a d e v i c e a t t a c h e d t o a f l a p which c o u l d swing i n both d i r e c t i o n s . A v a r i e t y o f l u r e s was s c r e e n e d h e r e i n d u a l c h o i c e t e s t s . A p e r f o r a t e d aluminum c a p s u l e , c o n t a i n i n g f i v e d r o p s o f t h e t e s t s a m p l e on f i l t e r p a p e r , was a t t a c h e d t o t h e b a c k w a l l o f one c a g e ; t h e o t h e r cage c a r r i e d a b l a n k c a p s u l e . The a v e r a g e t e s t p e r i o d was one week, i n t h e m i d d l e o f w h i c h t h e p l a c e s o f t h e cages w e r e i n t e r c h a n g e d and t h e s a m p l e s w e r e renewed. A f t e r e a c h t e s t t h e cages w e r e t h o r o u g h l y r i n s e d w i t h c l e a n w a t e r . The c o u n t s were r e c o r d e d d a i l y and t h e t e s t s were made a t l e a s t i n duplicate.



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Figure 3. Results of the screening experiment at the Berkel on the German border. Key: A, ( ), Dutch muskrat musk 26 and 29; B, (- · - -), American muskrat musk 28; C,( ), sweetflag13; and blank (- · -).

25 Sep

5 Nov

5Dec

5 Jan

5 Feb

5 Mar

~~i

5 Apr

5 May

5Jun

1

1980

1981

Figure 4. Results of the screening experiment at the Hegebeek on the German border. Key: A, ( · ), Dutch muskrat musk 26 and 29; B, (- · - -), American muskrat musk 28; blank 1, ( ); C, (- · -), sweet flag 13; and blank 2, ( ).

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I

t 1.5 m

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t r a p s with counter

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nest-boxes \sliding

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l V////// Figure 5.

j_j_J

_L

"^waterlevel

ν

wall

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1

Schematic representation of the basin for keeping muskrats at Rijswijk. Top shows view from above, and bottom shows view from the side.



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W i t h i n the framework of t h i s paper i t i s not p o s s i b l e to d i s c u s s a l l t h e s c r e e n i n g r e s u l t s . F o r t h o s e who a r e i n t e r e s t e d , a d e t a i l e d r e p o r t (21) ( i n D u t c h ) i s a v a i l a b l e . I t may s u f f i c e h e r e t o r e l a t e t h a t t h e home made g l a n d e x t r a c t A ( b a t c h 29, w h i c h a l s o p e r f o r m e d b e s t i n t h e f i e l d t e s t s o f F i g u r e s 4 and 5) was t h e b e s t a t t r a c t a n t , f o l l o w e d by a m i x t u r e o f t h e o i l s o f sweet f l a g and c a t n i p ( r e c i p e o b t a i n e d t h r o u g h t h e c o u r t e s y o f P r o f . A l e x a n d e r ) and t h e A m e r i c a n m u s k r a t musk, w h i c h was a l s o u s e d i n t h e f i e l d t e s t s . As an e x a m p l e , t h e r e s u l t s o f one o f t h e s e t e s t s i s shown i n F i g u r e 6. The s e c o n d s e r i e s o f t e s t s i s a t p r e s e n t b e i n g c a r r i e d o u t i n an " o l f a c t o m e t e r " , w h i c h c o n s i s t s o f a r o u n d " m u s k r a t p e n " w i t h a d i a m e t e r o f 10 m, c o n t a i n i n g a swimming p o o l ( F i g u r e 7 ) . E i g h t cages w h i c h a r e c o n n e c t e d t o e l e c t r i c c o u n t i n g d e v i c e s and a r e c o r d e r , a r e e q u a l l y s p a c e d r o u n d t h e p e r i p h e r y o f t h e pen (Figure 8). A t p r e s e n t t h e b e h a v i o u r o f t h e m u s k r a t c o l o n y i n t h e pen and t h e i n d i v i d u a l r e a c t i o n s o f t h e m u s k r a t s t o d i f f e r e n t l u r e s i s b e i n g s t u d i e d by s t u d e n t s o f P r o f . Wiepkema i n Wageningen. They w a t c h t h e a n i m a l s f r o m an o b s e r v a t i o n h i d e d u r i n g t h e d a r k and l i g h t p e r i o d s o f g r e a t e s t a c t i v i t y and k e e p r e c o r d o f t h e number and d u r a t i o n of t h e v i s i t s t o s c e n t e d cages and c o n t r o l s . T h i s pen and i t s e q u i p m e n t h a v e o n l y r e c e n t l y b e e n i n s t a l l e d and a r e b e i n g t e s t e d o u t a t p r e s e n t . We hope t h a t t h e y w i l l c o n t r i b u t e t o a b e t t e r u n d e r s t a n d i n g o f t h e g e n e r a l b e h a v i o u r and t h e c h e m i c a l c o m m u n i c a t i o n o f m u s k r a t s and a t t h e same t i m e w i l l a l l o w us t o s c r e e n d i f f e r e n t l u r e s i n a s y s t e m a t i c manner, t o d e t e r m i n e t h e i r o p t i m a l c o n c e n t r a t i o n s , and t o s t u d y t h e e f f e c t o f t h e v a r i o u s components f o u n d i n t h e e x t r a c t s o f t h e s c e n t gland. P a r a l l e l s t u d i e s on t h e b e h a v i o u r a l f u n c t i o n o f t h e musk odour o f m u s k r a t s a r e b e i n g done i n t h e USA by P r o f . M u l l e r Schwarze i n Syracuse, N.Y. P o s s i b l e F u n c t i o n o f Musk G l a n d S e c r e t i o n and S e a s o n a l i t s Chemical Composition

Changes o f

F u n c t i o n . The p r e p u t i a l g l a n d o f t h e m u s k r a t e n l a r g e s g r e a t l y i n t h e b r e e d i n g s e a s o n . I t i s r e a s o n a b l e t o assume t h a t t h i s s t r o n g - s m e l l i n g s e c r e t i o n has a t l e a s t one and p o s s i b l y s e v e r a l f u n c t i o n s i n t h e c h e m i c a l c o m m u n i c a t i o n b e t w e e n members of t h i s s p e c i e s . In muskrats these glands are present i n both sexes, but they a r e much l a r g e r i n m a l e s , i n w h i c h s i z e o f t h e g l a n d s and s t r e n g t h o f t h e o d o u r s e c r e t i o n p a r a l l e l t h e c y c l i c changes i n testes size. The v a r i e t y o f p o s s i b l e f u n c t i o n s o f p r e p u t i o n a l g l a n d s e c r e t i o n s o f mammals h a v e b e e n m e n t i o n e d b e f o r e . I t i s s t i l l n o t c l e a r w h i c h o f t h e s e f u n c t i o n s a p p l y t o m u s k r a t s . They may i n c l u d e s e x a t t r a c t i o n , r e p u l s i o n o f members o f t h e same s e x , marki n g o f n e s t s o r h i d i n g p l a c e s , g e n e r a l a t t r a c t i o n o f young and a d u l t members o f t h e same s p e c i e s by a f a m i l i a r o d o u r , m a r k i n g of




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Figure 6. Results of one of the tests in the basin in Rijswijk (see Figure 5) obtained during screening of a variety of muskrat lures. Attractant No. 29 ("muskoil") was obtained (December 5-11, 1980) by extraction of preputial glands with ethanol.

hide

Figure 7. Schematic representation of the "olfactometer" consisting of a round muskrat pen of iron gauze (diameter 10 m; height 2.20 m). An iron swimming pool was placed in the middle. Key: 1-8, counting cages with encoder box (see Figure 8); and D, door.

box with encoder system

Figure 8.

Cage with encoder box in which time, number and duration of visits of muskrats are signaled and transmitted to a recorder.



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t e r r i t o r y as w e l l as s t i m u l a t i o n ( " p r i m i n g " ) o f e n d o c r i n e p r o c e s ses, e t c . . Among t h e few c l u e s f o u n d i n t h e l i t e r a t u r e w h i c h m i g h t i n d i c a t e a f u n c t i o n , a r e t h o s e o f Akkerman (22) who o b s e r v e d t h a t a m a l e m u s k r a t was " s c e n t - m a r k i n g " a f e m a l e d u r i n g c o u r t s h i p and m a t i n g w h i c h o c c u r s w h i l e t h e y a r e swimming. He a l s o f o u n d t h a t f a e c a l p i l e s a r e h i g h l y i m p o r t a n t i n m u s k r a t c o m m u n i c a t i o n , as Muller-Schwarze r e c e n t l y d e s c r i b e d f o r beavers (23). I n s p r i n g the f a e c a l p i l e s of muskrats a r e found where they l e a v e the w a t e r . These p i l e s a r e produced where p o p u l a t i o n d e n s i t i e s a r e h i g h , which again p a r a l l e l s the s i t u a t i o n i n beavers (23). The m u s k r a t s e c r e t e s i t s musk d u r i n g s c e n t m a r k i n g t h r o u g h t h e p e n i s . The s o l e s o f t h e h i n d f e e t p i c k up t h e f l u i d and t h r o w i t behind the animal (22). C h e m i c a l c o m p o s i t i o n and s e a s o n a l changes o f musk g l a n d s e c r e t i o n The p r e p u t i a l (musk) g l a n d o f t h e m u s k r a t c o n t a i n s a numb e r o f m a c r o c y c l i c k e t o n e s , s u c h as i s o m e r s o f c i v e t o n e ( c i s - 9 c y c l o h e p t a d e c e n o n e ) , t h e m a i n odourous component o f t h e s e c r e t i o n of t h e c i v e t . I n t h e m u s k r a t t h e m a i n m a c r o c y c l i c component i s t h e c i s - 5 - i s o m e r o f c i v e t o n e ( F i g u r e 9 ) . The g l a n d s o f b o t h a n i m a l s a l s o c o n t a i n a number o f o t h e r m a c r o c y c l i c compounds and a v a r i e t y o f f a t t y a c i d s (24,25,26) w h i c h c o u l d be p r e c u r s o r s o f m a c r o c y c l i c k e t o n e s and a l c o h o l s as p r o p o s e d by S t e v e n s i n 1945 (25). The v a s t l i t e r a t u r e o n t h e m u s k r a t m e n t i o n e d b e f o r e , as summ a r i z e d by E r r i n g t o n (27) and H o f f m a n n (19) m a i n l y c o n c e r n s e c o l o g y , taxonomy, anatomy, p a r a s i t o l o g y and economy. O n l y a few c h e m i c a l s t u d i e s h a v e b e e n c a r r i e d o u t and t h e i n t e r e s t i n t h e b i o l o g i c a l f u n c t i o n s o f t h e components f o u n d i n t h e g l a n d s e c r e t i o n seemed t o be n e g l i g i b l e . I n 1973, Van Dorp e t a l . f o u n d a number o f i n t e r e s t i n g m a c r o c y c l i c compounds, i n c l u d i n g some w i t h b o t h d o u b l e and t r i p l e u n s a t u r a t e d bonds (26) , b u t made no a t t e m p t t o d e t e r m i n e t h e i r b i o l o g i c a l s i g n i f i c a n c e . They compared t h e m a c r o c y c l i c k e t o n e s and the f a t t y a c i d s f o u n d i n c i v e t and m u s k r a t g l a n d and c o n c l u d e d t h a t t h e r e i s e v i d e n t l y no c o r r e l a t i o n b e t w e e n t h e m a c r o c y c l i c k e t o n e c o m p o s i t i o n and t h e f a t t y a c i d c o m p o s i t i o n . F o r e x a m p l e , c y c l o h e p t a d e c a n o n e was t h e most p r o m i n e n t peak i n t h e m u s k r a t g l a n d (41% of t h e m a c r o c y c l i c k e t o n e s ) , whereas the s t e a r i c a c i d f r o m w h i c h i t c o u l d h a v e b e e n f o r m e d , was among t h e m i n o r compon e n t s (3%) o f t h e f a t t y a c i d s . We a n a l y s e d e t h a n o l i c e x t r a c t s o f p r e p u t i a l g l a n d s o f m u s k r a t s c o l l e c t e d d u r i n g a f u l l a n n u a l c y c l e by one t r a p p e r , i n ZeeuwschV l a a n d e r e n , n e a r t h e B e l g i a n b o r d e r . The m a j o r i t y o f t h e g l a n d s w e r e t a k e n f r o m a d u l t m a l e s , b u t some g l a n d s o f a d u l t f e m a l e s and y o u n g f e m a l e s and m a l e s w e r e a l s o c o l l e c t e d and a n a l y s e d s e p a r a t e l y (GC and MS; SE-30 c a p i l l a r y c o l u m n ) . T a b l e s I and I I compare t h e compounds w h i c h we h a v e f o u n d so f a r w i t h t h o s e f o u n d b y Van Dorp e t a l . A l t h o u g h we h a v e n o t y e t



118


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b e e n a b l e t o d e f i n i t e l y e s t a b l i s h t h e p o s i t i o n o f most o f t h e d o u b l e o r t r i p l e b o n d s , t h e d a t a show t h a t p r o b a b l y n i n e k e t o n e s w e r e f o u n d by b o t h g r o u p s , w h e r e a s two components i d e n t i f i e d by Van D o r p e t a l . h a v e n o t y e t b e e n f o u n d by us and f o u r o f o u r compounds w e r e n o t f o u n d by t h e o t h e r g r o u p . A g a i n w i t h some r e s e r v e r e g a r d i n g t h e p o s i t i o n o f t h e d o u b l e bonds, T a b l e I I shows t h a t we f o u n d a l l t h e a c i d s m e n t i o n e d by Van Dorp e t a l . , b u t , i n a d d i t i o n , n i n e more a c i d s l a c k i n g f r o m t h e i r list. There i s , however, a g r e a t s i m i l a r i t y w i t h r e g a r d t o the q u e s t i o n w h i c h w e r e t h e m a j o r and w h i c h t h e m i n o r components i n t h e two g r o u p s o f compounds. We c a n n o t , h o w e v e r , s u p p o r t t h e o p i n i o n t h a t t h e r e i s no c o r r e l a t i o n b e t w e e n t h e k e t o n e s and f a t t y a c i d s . A n a l y s e s made o v e r a one y e a r p e r i o d seem t o i n d i c a t e t h a t t h e p e r c e n t a g e o f m a c r o c y c l i c k e t o n e s i s o f t e n h i g h when t h a t o f t h e a c i d s i s low and v i c e v e r s a . T h i s w o u l d s u p p o r t S t e v e n s ' h y p o t h e s i s , w h i c h was c h a l l e n g e d by Van Dorp e t a l . ( 2 6 ) , t h a t t h e k e t o n e s a r e f o r m e d by c y c l i z a t i o n and d e c a r b o x y l a t i o n o f f a t t y a c i d s ( 2 5 ) . As an e x a m p l e , F i g u r e 10 shows t h e s e a s o n a l c h a n g e s i n t h e r e l a t i v e p e r c e n t a g e o f a l l t h e 17-membered r i n g k e t o n e s t o g e t h e r and t h o s e o f t h e C i 8 a c i d s and e s t e r s . T h e s e d a t a n o t o n l y s u p p o r t t h e b i o g e n e t i c r e l a t i o n s h i p b e t w e e n a c i d s and m a c r o c y c l i c k e t o n e s b u t a l s o s u g g e s t t h a t t h e compounds w i t h t h e musk o d o u r a r e m a i n l y p r o d u c e d d u r i n g f a l l and w i n t e r . The p e r c e n t a g e o f t h e musk compounds d r o p s s h a r p l y d u r i n g t h e f i r s t t h r e e months o f t h e y e a r and i n c r e a s e s a g a i n i n s p r i n g . The a n a l y s e s o f t h e f e m a l e g l a n d s showed t h a t t h e s e a l s o c o n t a i n some m a c r o c y c l i c compounds b u t t h a t t h e i r r e l a t i v e p e r c e n t a g e s a r e much l o w e r t h a n t h o s e f o u n d i n t h e m a l e s and t h a t , c o r r e s p o n d i n g l y , the percentage of f a t t y a c i d s i s l a r g e r . We w i s h t o t h a n k U n i l e v e r , V l a a r d i n g e n f o r s u p p l y i n g s a m p l e s o f m a c r o c y c l i c k e t o n e s and Dr. P . E . J . V e r w i e l and I n g . A. L a k w i j k f o r c a r r y i n g o u t GC/MS e x p e r i m e n t s . The w o r k was s u p p o r t e d by a g r a n t f r o m t h e D u t c h D e p a r t m e n t s o f W i l d l i f e Management and P u b l i c Works. We a l s o t h a n k Dr. W.J. Doude v a n T r o o s t w i j k and h i s c o l l a b o r a t o r s o f t h e C o m m i t t e e f o r M u s k r a t C o n t r o l f o r t h e i r v a l u a b l e h e l p and advices. S t r u c t u r e E l u c i d a t i o n of

Periplanone-A

The names p e r i p l a n o n e - A and p e r i p l a n o n e - B w e r e f i r s t c o i n e d by R i t t e r and P e r s o o n s i n 1974 a t t h e I n t e r n a t i o n a l C o n g r e s s o f P e s t i c i d e C h e m i s t r y i n H e l s i n k i , i n a g e n e r a l p a p e r on pheromone r e s e a r c h i n the N e t h e r l a n d s . I n t h i s paper the i s o l a t i o n o f a number of pheromones w e r e d e s c r i b e d , i n c l u d i n g t h e two p e r i p l a n o n e s , b o t h s e x pheromones, p r e s e n t i n t h e e x c r e t a o f t h e f e m a l e A m e r i c a n c o c k r o a c h , P e r i p l a n e t a a m e r i c a n a . The e l e m e n t a r y f o r m u l a e o f t h e s e two s e s q u i t e r p e n o i d s , C15H20O2 and C15H20O35 w e r e g i v e n and t h e c a r b o n s k e l e t o n o f p e r i p l a n o n e - B , a g e r m a c r a n e d e r i v a t i v e , was r e v e a l e d . U n f o r t u n a t e l y , we w e r e i n f o r m e d , one y e a r a f t e r t h e


5.

Pheromone

RiTTER E T A L .

H

(CH )


2

Figure 9.

Research

on Muskrats

H

H

H

I I

I I

C=C

C =C (CH )

7

2

7

(CH ) 2

and Insects

(CH^h

3

Structures of the main macrocyclic ketones of the civet (left) and the muskrat (right).

I x

1

1

1 I l _ l '

•

'

• '

Nov Dec Jan FebrMarApr May Jun J u l AugSept 7 1979

1980

Figure 10. Variation with season of the proportion of macrocyclic C ketones and of their possible precursors (bottom), the C acids and their esters (top). Sum of all compounds detected by the GC analysis is 100%. 17

18


119

120


TECHNOLOGY


Table I MACROCYCLIC KETONES I N THE PREPUTIAL GLAND OF THE MUSKRAT Compound

V. Dorp e t a l . R i t t e r e t a l .

-

Cyclotridecanone

+++ · +++ +

Cyclopentadecanone Cyclopentadecenone, (Z)-5C y c l o p e n t a d e c y n o n e , 5-*

+

Cyclohexadecanone Cyclohexadecenone*

-

C y c l o h e p t adecanone Cycloheptadecenone, (Z)-5Cycloheptadecenone, (Z)-7-* Cycloheptadecadienone, (Z,Z)-5,11-* C y c l o h e p t a d e c y n o n e , 5-* Cycloheptadecen-5-ynone, (Z)-7-* Cyclononadecanone Cyclononadecenone, Cyclononadecenone*

+ +++ +++ + + +

+++ +++ ++ ++ + ++

+++ +++ ++

+

+ + +

-

(Z)-9-*

++

* P o s i t i o n o f double bond(s) n o t y e t e s t a b l i s h e d i n o u r work. +++ ++ +

^ 10% r e l a t i v e o f t h e m a c r o c y c l i c > 1% " " ^ 1 7 " M M M 11

11

ketones 11

M

not found, o r not i d e n t i f i e d


5.

RiTTER E T A L .

Pheromone

Research

on Muskrats

and Insects

Table I I FATTY ACIDS I N THE PREPUTIAL GLAND OF THE MUSKRAT


Fatty Number o f C-atoms

acid Number o f double bonds*

V. Dorp e t a l . R i t t e r e t a l .

0 1

14 14

0 1

++

16 16 16

0 1 1

+++ ++

-

+++ ++ ++

17

0

-

+

18 18 18 18

0 1 1 2

++ +++ ++

++ +++ + ++

19

1

-

+

20 20 20

0 1 2

+

+ + +

-

-

-

* P o s i t i o n of double bond(s) not y e t e s t a b l i s h e d +++ +

+ +

12 12

++ +

i n o u r work.

^ 10% r e l a t i v e o f t h e f a t t y a c i d s > 17 " It II II II < 1% " " " not f o u n d , o r n o t i d e n t i f i e d 11

11



122


TECHNOLOGY

c o n g r e s s , t h a t none o f t h e symposium p a p e r s c o u l d be p u b l i s h e d and t h a t we had t o s e e k p u b l i c a t i o n e l s e w h e r e . T h i s we d i d i n t h e N e t h e r l a n d s J o u r n a l o f Z o o l o g y i n 1975 ( 2 8 ) . M e a n w h i l e we h a d p u b l i s h e d the e x p e r i m e n t a l d e t a i l s i n our communication to the R o y a l D u t c h Academy o f S c i e n c e s ( 2 9 ) . A s i m i l a r p a p e r had b e e n r e j e c t e d f o r p u b l i c a t i o n i n 1973, f i r s t by S c i e n c e , and t h e n by Experientia. We s u s p e c t t h a t t h e r e l u c t a n c y o f a u t h o r i t a t i v e j o u r n a l s o u t s i d e H o l l a n d t o p u b l i s h our d a t a r e s u l t e d from f e a r of r e p e a t i n g the u n f o r t u n a t e events a f t e r the premature p u b l i c a t i o n of a w r o n g s t r u c t u r e f o r t h i s s e x pheromone by e m i n e n t A m e r i c a n s c i e n t i s t s i n 1963 ( 3 0 ) , who h a d t o w i t h d r a w t h e i r s t r u c t u r e two years l a t e r (31). A few y e a r s ago t h e c o m p l e t e s t r u c t u r e o f p e r i p l a n o n e - B , a p a r t f r o m i t s s t e r e o c h e m i s t r y , was e l u c i d a t e d and p u b l i s h e d by our r e s e a r c h team (32,33,34) w i t h P e r s o o n s as t h e p r i n c i p a l i n v e s t i g a t o r , who i n c l u d e d t h i s w o r k i n h i s d o c t o r a l t h e s i s ( 3 5 ) , and s h a r e d t h e R o y a l D u t c h S h e l l P r i z e f o r 1978 w i t h R i t t e r f o r t h i s w o r k and t h e s t r u c t u r e e l u c i d a t i o n o f f a r a n a l , t h e t r a i l pheromone o f t h e P h a r a o h s a n t , Monomorium p h a r a o n i s ( 3 6 , 3 7 ) . The d e t e r m i n a t i o n o f t h e a b s o l u t e c o n f i g u r a t i o n o f p e r i p l a none-B by A m e r i c a n s c i e n t i s t s i n c o o p e r a t i o n w i t h P e r s o o n s (38) and t h e c o n f i r m a t i o n o f t h e s t r u c t u r e by t h e e l e g a n t s y n t h e s i s o f S t i l l a t t h e C o l u m b i a U n i v e r s i t y (39) f o l l o w e d i n 1979. The p l a n a r and s t e r e o s t r u c t u r e o f p e r i p l a n o n e - B a r e shown i n F i g u r e 11. We c a n now r e p o r t t h e s t r u c t u r e e l u c i d a t i o n o f t h e o t h e r i s o l a t e d compound: p e r i p l a n o n e - A . I t i s , as we m e n t i o n e d i n e a r l i e r p a p e r s (28,29) an u n s t a b l e s e s q u i t e r p e n o i d w i t h t h e g e n e r a l f o r m u l a Ci5H2o02» I t s s t r u c t u r e , l i k e t h a t o f p e r i p l a n o n e - B , i s r a t h e r c o m p l i c a t e d compared w i t h t h o s e o f o t h e r known s e x pheromones. I t c o u l d be d e d u c e d f r o m c o m p a r i s o n o f i t s NMR, UV, I R and mass s p e c t r a w i t h t h o s e o f an i n a c t i v e rearrangement product ( F i g u r e 12), the carbon s k e l e t o n of which resembles t h a t of p e r i p l a n o n e - B , apart from b e i n g b i c y c l i c i n s t e a d o f h a v i n g a 10-membered . r i n g . T h i s r e a r r a n g e m e n t p r o d u c t , w i t h t h e same e l e m e n t a r y f o r m u l a as p e r i p l a n o n e - A i s r e a d i l y f o r m e d f r o m i t when k e p t i n NMR c a p i l l a r i e s i n c a r b o n d i s u l f i d e o r h e x a n e . On t h e b a s i s o f NMR c o u p l i n g c o n s t a n t s and i t s o t h e r s p e c t r o s c o p i c a l data i t would have the c o n f i g u r a t i o n shown i n F i g u r e 13. I t c a n be s e p a r a t e d f r o m p e r i p l a n o n e - A by GC, u s i n g DEGS o r OV 101 as t h e s t a t i o n a r y p h a s e . S c r u t i n o u s a n a l y s i s o f t h e NMR s p e c t r a o f m i x t u r e s c o n t a i n i n g d e c r e a s i n g amounts o f p e r i p l a n o n e - A w i t h t h e NMR spectrum o f t h e r e a r r a n g e m e n t p r o d u c t y i e l d e d t h e NMR s i g n a l s a t t r i b u t a b l e to p e r i p l a n o n e - A . Assuming t h a t s i g n a l s w i t h s i m i l a r c h e m i c a l s h i f t s and p a t t e r n s i n t h e s p e c t r a o f p e r i p l a n o n e - A and i t s r e a r r a n g e m e n t p r o d u c t a r o s e f r o m i d e n t i c a l p a r t i a l s t r u c t u r e s and subsequent c o m b i n a t i o n of these p a r t i a l s t r u c t u r e s , t o g e t h e r w i t h a c o n s i d e r a t i o n o f c h e m i c a l s h i f t s and c o u p l i n g c o n s t a n t s , l e a d 1


Pheromone

Research

on Muskrats

and Insects


RiTTER E T A L .

Figure 13.

Expected configuration for the rearrangement product of periplanone-A.


124


TECHNOLOGY

t o t h e s t r u c t u r e f o r p e r i p l a n o n e - A , shown i n F i g u r e 14. A c c o r d i n g t o IUPAC n o m e n c l a t u r e i t i s 7-methylene-4-isopropyl-12-oxat r i c y c l o [4.4.2.0 ]-9-dodecene-2-one. The s t r u c t u r e o f p e r i p l a n o n e - A as w e l l as t h a t o f i t s r e a r rangement p r o d u c t s t i l l r e q u i r e s c o n f i r m a t i o n by s y n t h e s i s , b u t a l l t h e i r s p e c t r a l d a t a a r e i n c o m p l e t e agreement w i t h t h e p r o posed s t r u c t u r e s . A paper i n which a l l these d e t a i l s are g i v e n i s i n press (40). P e r i p l a n o n e - A and p e r i p l a n o n e - B h a v e b e e n f o u n d t o o c c u r i n a r a t i o o f 1:10 i n f a e c a l m a t e r i a l o f P e r i p l a n e t a a m e r i c a n a , b u t o n l y p e r i p l a n o n e - B has b e e n f o u n d i n i n t e s t i n a l t r a c t s . T h i s c o u l d s u g g e s t t h a t p e r i p l a n o n e - B i s a g e n u i n e s e x pheromone, w h e r e a s p e r i p l a n o n e - A c o u l d be a b i o l o g i c a l l y a c t i v e t r a n s f o r m a t i o n p r o d u c t , w h i c h i n t u r n c a n i s o m e r i z e i n t o a more s t a b l e b u t i n a c t i v e compound. We h a v e g i v e n h y p o t h e t i c a l schemes f o r s u c h a t r a n s f o r m a t i o n elsewhere (32,40). T h i s would i n v o l v e a r e d u c t i o n of the 1(2)-epoxigroup of p e r i p l a n o n e - B , f o l l o w e d by a t r a n s a n n u l a r r e a c t i o n o f t h e o t h e r e p o x i g r o u p ( t h e e x o - e p o x i d e a t C i r j ) w i t h t h e t r a n s - d o u b l e bond o f a h y p o t h e t i c a l i n t e r m e d i a t e at C , which would y i e l d p e r i p l a none-A. V i a another h y p o t h e t i c a l i n t e r m e d i a t e , the 5 - c i s isomer, p e r i p l a n o n e - A c o u l d t h e n be t r a n s f o r m e d i n t o a s t a b l e , b u t i n a c t i v e compound ( F i g u r e 1 5 ) . R e c e n t l y e l e c t r o p h y s i o l o g i c a l e x p e r i m e n t s by S a s z h a v e y i e l d e d r e s u l t s which would i n d i c a t e t h a t not o n l y p e r i p l a n o n e - B , b u t a l s o p e r i p l a n o n e - A c o u l d be a g e n u i n e pheromone. W i t h s i n g l e c e l l r e c o r d i n g t e c h n i q u e s he d e m o n s t r a t e d t h e p r e s e n c e o f many o l f a c t o r y h a i r s on t h e a n t e n n a e o f P e r i p l a n e t a a m e r i c a n a t h a t w e r e v e r y s e n s i t i v e t o p e r i p l a n o n e - A , whereas o t h e r s were s e n s i t i v e f o r periplanone-B (41). A l t h o u g h p e r i p l a n o n e - A i s a v e r y p o t e n t s e x pheromone, i t s p r a c t i c a l a p p l i c a t i o n m i g h t meet more d i f f i c u l t i e s t h a n t h a t o f p e r i p l a n o n e - B and w o u l d r e q u i r e a s o l u t i o n o f t h e p r o b l e m s r e l a t e d to i t s i n s t a b i l i t y . We w i s h t o a c k n o w l e d g e t h e c o n t r i b u t i o n o f W.J. N o o i j e n t o t h e i s o l a t i o n and o f P . E . J . V e r w i e l t o t h e NMR a n a l y s e s o f p e r i planone-A.


1 , 5

5

The

Sex Pheromone o f t h e B e e t Armyworm, S p o d o p t e r a

exigua

The b e e t armyworm, S p o d o p t e r a e x i g u a , i s a r a t h e r r e c e n t p e s t i n H o l l a n d , where i t i s now c a l l e d t h e F l o r i d a moth. A f t e r i t was a c c i d e n t a l l y i n t r o d u c e d i n t o t h e N e t h e r l a n d s i n 1976, i t i m m e d i a t e l y became a g r a v e p e s t i n g r e e n h o u s e s , m a i n l y f o r chrysanthemums and g e r b e r a e , b u t t o a l e s s e r e x t e n t a l s o f o r e g g - p l a n t s . As a p p l i c a t i o n o f i n s e c t i c i d e s s u c h as s y n t h e t i c p y r e t h r o i d s , i n i t i a l l y e f f e c t i v e , s o o n p r o v e d t o have become i n a d e q u a t e , o t h e r methods w e r e n e e d e d , and pheromones w e r e i n v e s t i g a t e d as p o s s i b l e alternatives.



Figure 15.

Hypothetical scheme for the transformation of periplanone-B into periplanone-A of periplanone-A into the stable, biologically inactive product.


and


126


TECHNOLOGY

B r a d y and G a n y a r d i d e n t i f i e d (Z,E)-9,12-tetradecadienyl a c e t a t e i n t h i s moth ( 4 2 ) , b u t t r a p p i n g e x p e r i m e n t s w i t h t h i s com pound, a l o n e o r i n c o m b i n a t i o n w i t h ( Z ) - 9 - t e t r a d e c e n y l a c e t a t e were u n s u c c e s s f u l (43,44). A j o i n t e f f o r t t o r e i n v e s t i g a t e t h i s p r o b l e m was t h e r e f o r e s t a r t e d by r e s e a r c h teams i n D e l f t and Wageningen. F r a c t i o n s obtained from abdominal t i p s , e x c i s e d glands or t r a p p e d a i r v o l a t i l e s o b t a i n e d f r o m v i r g i n f e m a l e s w e r e t e s t e d by electroantennography and a b e h a v i o u r a l b i o a s s a y (Van d e r K r a a n , p u b l i c a t i o n i n p r e p a r a t i o n ) . The l a t t e r u s e d w i n d t u n n e l s w i t h f u n n e l s u p w i n d and a common chamber downwind. T e s t s a m p l e s w e r e p l a c e d i n t h e f u n n e l s , and t h e b e h a v i o u r o f m a l e m o t h s , r e l e a s e d i n t h e m i d d l e o f t h e t u n n e l s , as w e l l as t h e numbers t r a p p e d i n the f u n n e l s , were examined. Of 150 m o d e l compounds s c r e e n e d by t h e EAG t e c h n i q u e , o n l y t h e ( Z , E ) - 9 , 1 2 - 1 4 : A c i s o l a t e d by B r a d y and G a n y a r d gave a c l e a r p o s i t i v e response. The b e h a v i o u r a l a s s a y o f f r a c t i o n s e l u t e d f r o m s i l i c a g e l w i t h , r e s p e c t i v e l y , h e x a n e , 10% e t h e r i n h e x a n e , 50% e t h e r i n h e x a n e and e t h e r , i n d i c a t e d t h a t t h e a c t i v i t y was c o n f i n e d t o t h e f r a c t i o n c o n t a i n i n g Cii+ a c e t a t e s ( t h e 10% e t h e r f r a c t i o n ) . Combi n a t i o n w i t h other f r a c t i o n s d i d not i n c r e a s e the a c t i v i t y . A n a l y s e s by mass s p e c t r o m e t r y , o z o n o l y s i s and r e t e n t i o n i n d i c e s r e v e a l e d t h e p r e s e n c e o f t h e ( Z , E ) - 9 , 1 2 and t h e ( Z ) - 9 com pounds m e n t i o n e d e a r l i e r , b u t , i n a d d i t i o n , a l s o o f t h e ( Z , Z ) 9,12 d i - u n s a t u r a t e d , t h e (Z)-11 m o n o u n s a t u r a t e d and t h e s a t u r a t e d Cii+ a c e t a t e s . The d e t a i l s o f t h e a n a l y s e s and t h e d i f f e r e n t r a t i o s f o u n d i n a b d o m i n a l t i p s , e x c i s e d g l a n d s and a i r v o l a t i l e s a r e given i n a recent communication (45). The b e h a v i o u r a l a s s a y i n d i c a t e d t h a t t h e ( Z , E ) - 9 , 1 2 and t h e ( Z ) - 9 components a p p e a r e d t o be n e c e s s a r y f o r u p w i n d s e a r c h b e h a v i o u r , w h i l e t h e ( Z , Z ) - 9 , 1 2 compound i s i m p o r t a n t f o r s h o r t r a n g e c o u r t s h i p b e h a v i o u r , l i k e h a i r p e n c i l d i s p l a y and c o p u l a t i o n attempts. F i e l d e x p e r i m e n t s w e r e c a r r i e d o u t by T.C. B a k e r n e a r R i v e r s i d e i n C a l i f o r n i a , who u s e d P h e r o c o n - 2 s t i c k y t r a p s and p o l y e t h y l e n e c a p s as d i s p e n s e r s . The p r e l i m i n a r y r e s u l t s show t h a t t r a p s w i t h m i x t u r e s o f t h e f o u r u n s a t u r a t e d a c e t a t e s c o l l e c t e d as an a v e r a g e s l i g h t l y more m a l e s t h a n t r a p s w i t h l i v e f e m a l e s d i d . R e m a r k a b l y , m i x t u r e s o f t h e two compounds f o u n d t o be i n e f f e c t i v e by M i t c h e l l and D o o l i t t l e (44) c a u g h t o n l y s l i g h t l y f e w e r m a l e s t h a n t h e s e two l i v e v i r g i n females d i d . W h i l e our most r e c e n t c o m m u n i c a t i o n was i n p r e s s (45) , a p u b l i c a t i o n on t h e same s u b j e c t by T u m l i n s o n , M i t c h e l l and S o n n e t a p p e a r e d ( 4 6 ) . They i s o l a t e d 11 compounds f r o m f e m a l e S p o d o p t e r a e x i g u a , but t h e i r f i e l d t e s t s demonstrated t h a t a m i x t u r e of o n l y two o f them, (Ζ,E)-9,12-tetradecadienyl a c e t a t e and t h e f r e e a l c o h o l , ( Z ) - 9 - t e t r a d e c e n o l , i n a r a t i o o f 5:4, w e r e needed f o r a t t r a c t a n c y . Such a m i x t u r e was e q u a l i n a c t i v i t y t o l i v e f e m a l e s ,



5.

RiTTER E T A L .

Pheromone

Research

on

Muskrats

and

Insects

127

t o t h e t o t a l h e x a n e wash o f f e m a l e s and t o a m i x t u r e o f a l l 11 compounds i n a t t r a c t i n g w i l d m a l e s i n t h e f i e l d . To o b t a i n a t t r a c t i o n c o m p a r a b l e t o t h a t o f l i v i n g f e m a l e s , the a u t h o r s u s e d about 4 5 mg o f t h e i r m i x t u r e i n p o l y e t h y l e n e v i a l s , w h i c h i n o u r o p i n i o n , i s an e x c e s s i v e l y h i g h d o s e . We a l w a y s u s e d d o s e s b e l o w t h e 1 mg l e v e l . T a b l e I I I compares t h e r e s u l t s o b t a i n e d by M i t c h e l l and D o o l i t t l e i n 1 9 7 6 ( 4 4 ) , by T u m l i n s o n e t a l . i n 1 9 8 1 ( 4 5 ) and by P e r s o o n s e t a l . ( 4 6 7 7 I t l i s t s t h e 1 1 compounds f o u n d by T u m l i n s o n e t a l . . Of t h e f i v e a c e t a t e s w h i c h we i n v e s t i g a t e d , f o u r w e r e a l s o f o u n d by t h e o t h e r g r o u p . The r e s u l t s o f t h e f i e l d t e s t s d i f f e r w i d e l y . Whereas M i t c h e l l and D o o l i t t l e f o u n d no a t t r a c t a n c y i n t h e two-component m i x t u r e , B a k e r s ' f i e l d t e s t s f o u n d a s i m i l a r m i x t u r e t o be a l m o s t as a t t r a c t i v e as l i v e females. V e r y r e c e n t l y , B a k e r compared t h e two-component m i x t u r e o f Tumlinson e t a l . w i t h our four-component m i x t u r e i n f i e l d t e s t s n e a r R i v e r s i d e . A l t h o u g h , due t o low p o p u l a t i o n d e n s i t i e s , t h e c a t c h e s were r a t h e r poor, t h e r e s u l t s are c l e a r l y i n f a v o u r o f the f o u r - c o m p o n e n t m i x t u r e ( T a b l e I V ) . We c o n s i d e r a l l t h e s e f i e l d t e s t s s t i l l t o be p r e l i m i n a r y . They w i l l h a v e t o be r e p e a t e d on a l a r g e r s c a l e and i n d i f f e r e n t l o c a t i o n s and c o u n t r i e s b e f o r e a c o m p l e t e p i c t u r e c a n be g a i n e d . The work on S p o d o p t e r a e x i g u a i n H o l l a n d was done i n c o l l a b o r a t i o n w i t h W.J. N o o i j e n o f t h e D e l f t group and C. v a n d e r K r a a n and S. V o e r m a n o f t h e I n s t i t u t e f o r P e s t i c i d e R e s e a r c h i n Wageningen. We a l s o g r a t e f u l l y a c k n o w l e d g e t h e f i e l d t e s t s by T.C. Baker- i n R i v e r s i d e and t h e s u g g e s t i o n s and c r i t i c a l comments by A.K. M i n k s of t h e I n s t i t u t e f o r P l a n t P r o t e c t i o n i n Wageningen.



128

TECHNOLOGY

Table I I I RESPONSE OF WILD SPODOPTERA EXIGUA MALES TO SYNTHETIC PHEROMONE IN POLYETHYLENE CAPS M i t c h e l l and D o o l i t t l e 1976


mg 14:Ac (Z)-7-14:Ac (E)-9-14:Ac (Z)-9-14:Ac (Z)-11-14:Ac (Z,E)-9,12-14:Ac (Z,Z)-9,12-14:Ac 14:OH (E)-9-14:OH (Z)-9-14:OH (Z,E)-9,12-14:OH (Z,Z)-9,12-14:OH χ no. c r / t r a p / n i g h t

mg

+

3

5

+

7

5

0.0

3 ?

Tumlinson e t a l . 1981

Persoons e t a l . 1981

μΐ

μg

μΐ

4?

+ + + + 12.5

19.7

(+) + 450 300 + 45 + 450 600 +22.5

26.7 12.0

8.5 7.8 8.0

+ compound d e t e c t e d

Table IV RESPONSE OF A LOW POPULATION OF WILD SPODOPTERA EXIGUA MALES TO SYNTHETIC PHEROMONE I N POLYETHYLENE CAPS. COMPARATIVE FIELD TEST OF "TUMLINSON AND "PERSOONS" MIXTURES, TESTED BY BAKER 11

Persoons

Tumlinson

(Z)-9-14:Ac (Z)-11-14:Ac (Z,E)-9,12-14:Ac (Z,Z)-9,12-14:Ac (Z)-9-14:0H

500

5000

400

4000

χ no. d'/trap/night

0.4

0.0

2?

+

+ 25 25 + + + + 20. 20 + 10 +

0.0 5.3

pg

μ8

μ8

600 60 400 20

200 20 200 10

1.8

2.0

1000 100 750 37.5 1 .5


5.

RITTER E T A L .


Literature

Pheromone

Research

on Muskrats and Insects

129

cited

1. Mykytowycz, R., in: Ritter, F.J., Ed.; "Chemical Ecology: Odour Communication in A n i m a l s " ; Elsevier: Amsterdam, 1979; pp. 105-115. 2. Beauchamp, G . K . ; Doty, R.L.; Moulton, D . G . ; Mugford, R . A . J. Chem. E c o l . 1979, 5, 301-305. 3. K a t z , R.A.; Shorey, H . H . J. Chem. E c o l . 1979, 5, 299-301. 4. Rutowski, R . L . J. Chem. E c o l . 1981, 7, 481-484. 5. C l a u s , R. "Pheromone b e i Säugetieren unter besonderer Berücks i c h t i g u n g des Ebergeruchstoffes und s e i n e r Beziehung zu anderen Hodensteroiden", Paul Parey: Hamburg, 1979. 6. M ü l l e r - S c h w a r z e , D.; M o z e l l , M . M . , E d s . ; "Chemical S i g n a l s in V e r t e b r a t e s " ; Plenum: New York, 1977. 7. M ü l l e r - S c h w a r z e , D.; Silverstein, R . M . , E d s . ; "Chemical S i g n a l s . Vertebrates and Aquatic I n v e r t e b r a t e s " ; Plenum: New Y o r k , 1980. 8. Ritter, F.J., Ed.; "Chemical Ecology: Odour Communication i n A n i m a l s " ; E l s e v i e r : Amsterdam, 1979. 9. Shorey, H . H . "Animal Communication by Pheromones"; Academic P r e s s : New Y o r k , 1978. 10. B i r c h , M . C . , Ed.; "Pheromones"; E l s e v i e r : New Y o r k , 1974. 11. C h r i s t i a n s e n , Ε . ; Døving, K., in: Hanson, L.; Nilson, B., E d s . ; " B i o c o n t r o l of Rodents"; Swed. N a t l . Res. C o u n c i l : Stockholm, 1975; 221-227. 12. Marsh, R.E.; Howard, W.E. Pest C o n t r o l 1979, 7, 22-23. 13. Stacewycz-Sapuntzakis, M . ; Gawienowski, A . M . J. Chem. E c o l . 1977, 3, 411-417. 14. Christian, J.J. Am. J. P h y s i o l . 1955, 182, 292-300. 15. D a v i s , D.E.; Christian, J.J. Nav. Med. Res. I n s t . Res. Rep. 1957, 15, p 311. 16. Bronson, F.H.; Caroom, D. J. Reprod. Fertil. 1971, 25, 279-282. 17. McKinney, T . D . ; Christian, J.J. P r o c . Soc. exp. Biol. Med. 1970, 134, 291-293. 18. M ü l l e r - S c h w a r z e , D., in: E i s e n b e r g , J.F.; K l e i m a n , D.G., E d s . ; "The Behavior of Mammals"; Amer. Soc. Mammalogists P u b l . ( i n press). 19. Hoffmann, M. Z. angew. Z o o l . 1976, 63, 187-239. 20. W i l l i a m s , R . M . J. Wildlife Management 1951, 15, 117-118. 21. Brüggemann, I.E.M.; Ritter, F.J.; Gut, J. "Onderzoek naar s i g n a a l s t o f f e n voor de muskusrat"; 1981; TNO report CL 81/24. 22. Akkermann, R. Z. angew. Z o o l . 1975, 62, 281-338. 23. M ü l l e r - S c h w a r z e , D.; Heckman, S. J. Chem. E c o l . 1980, 6, 81-95. 24. Stevens, P.G.; E r i c k s o n , J.L.E. J . Am. Chem. Soc. 1942, 64, 144-147. 25. Stevens, P . G . J. Am. Chem. Soc. 1945, 67, 907-909. 26. Van Dorp, D.A.; K l o k , R . ; Nugteren, D . H . Rec. Trav. Chim. Pays-Bas 1973, 915-928.


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27. 28. 29. 30. 31. 32.


33. 34. 35.

36.

37.

38. 39. 40. 41. 42. 43. 44. 45.

46.


TECHNOLOGY

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RECEIVED February 24,

1982.


Insect Pheromone Technology: Chemistry and ... - ACS Publications

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