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Chemical determinants of tree susceptibility to mountain pine beetle (Dendroctonus ponderosae Hopkins) Syed, Akbar 1972

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CHEMICAL DETERMINANTS OF TREE S U S C E P T I B I L I T Y TO MOUNTAIN PI N E BEETLE ( D e n d r o c t o n u s p o n d e r o s a e H o p k i n s ) b y AKBAR SYED B.Sc. ( A g r i . ) Osmania U n i v e r s i t y , 1964 M.Sc. ( A g r i . ) A n d h r a P r a d e s h A g r i c u l t u r a l U n i v e r s i t y , 1966 A THESIS SUBMITTED I N PAR T I A L FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF FORESTRY i n t h e F a c u l t y o f F o r e s t r y We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d s THE UNIVERSITY OF B R I T I S H COLUMBIA A p r i l , 1972 In present ing t h i s thes is in p a r t i a l f u l f i l m e n t o f the requirements fo r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y sha l l make i t f r e e l y a v a i l a b l e for reference and study. I fu r ther agree that permission for extensive copying of t h i s t h e s i s for s c h o l a r l y purposes may be granted by the Head of my Department or by h is representa t ives . It is understood that copying or p u b l i c a t i o n of th is thes is f o r f i n a n c i a l gain s h a l l not be allowed without my wr i t ten permiss ion . Department of The Un ive rs i t y o f B r i t i s h Columbia Vancouver 8, Canada i ABSTRACT V o l a t i l e c o n s t i t u e n t s o f b a r k f r o m t h e two s p e c i e s o f p i n e h o s t s o f t h e m o u n t a i n p i n e b e e t l e , ( D e n d r o c t o n u s p o n - d e r o s a e H o p k i n s ) w e r e i n v e s t i g a t e d a s p o s t u l a t e d f a c t o r s i n h o s t t r e e d i s c o v e r y a n d s e l e c t i v e a t t a c k by t h e i n s e c t . The a n e m o - o l f a c t o r y , k l i n o k i n e t i c a n d k l i n o t a c t i c r e s -p o n s e s o f p e d e s t r i a n a d u l t f l o w n f e m a l e s t o o d o r s o f d i f f e r e n t a g e d p i n e s o f t h e s p e c i e s P i n u s p o n d e r o s a Laws, ( p o n d e r o s a p i n e ) and P i n u s m o n t i c o l a D o u g l . ( w e s t e r n w h i t e p i n e ) w e r e u s e d a s c r i t e r i a t o t e s t f o r e v i d e n c e a s t o p o s s i b l e i n v o l v e -ment o f h o s t t r e e v o l a t i l e s i n h o s t s e l e c t i o n . V o l a t i l e s u b -s t a n c e s f r o m p i n e b a r k w e r e c o l l e c t e d t h r o u g h s u b l i m a t i o n u n d e r vacuum f r o m f r o z e n s t a t e . The v o l a t i l 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 - L i q u i d C h r o m a t o g r a p h y . T o t a l v o l a t i l e e x -t r a c t s a n d t h e i r e t h e r s o l u b l e f r a c t i o n f r o m m a t u r e p i n e t r e e s w e r e " a t t r a c t i v e " t o b e e t l e s , w h e r e a s t h o s e f r o m s a p -l i n g s w e r e " r e p e l l e n t . " No q u a l i t a t i v e d i f f e r e n c e s w e r e f o u n d i n t h e e t h e r - s o l u b l e f r a c t i o n o f m a t u r e t r e e s a n d s a p l i n g s r e s p e c t i v e l y , b u t p r o p o r t i o n s o f i n d i v i d u a l c o n s t i -t u e n t s i n t h e e x t r a c t s d i f f e r e d . E t h a n o l a t v a r i o u s c o n c e n -t r a t i o n s c a u s e d a r r e s t a n c e o f t h e a n e m o - o l f a c t o r y r e s p o n s e o f p e d e s t r i a n b e e t l e s . As a c o n s t i t u e n t o f t r e e s u n d e r s t r e s s e t h a n o l may t h u s p l a y a n i m p o r t a n t r o l e i n t h e p r o -gramme o f r e s p o n s e s w h i c h l e a d u l t i m a t e l y t o a t t a c k . P r o b l e m s e n c o u n t e r e d d u r i n g t h e s t o r a g e o f t r e e s a m p l e s a n d t h e i r e x t r a c t s h a v e b e e n d i s c u s s e d . i i TABLE OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS i i LIST OF TABLES i v LIST OF ILLUSTRATIONS v LIST OF APPENDICES »v£ii ACKNOWLEDGEMENTS i x INTRODUCTION 1 MATERIALS AND METHODS 6 The Experimental Insect 6 1. Treatment of the "beetles 7 2. Bioassay studies 8 3. Olfactometer observations . . . . . . . . . . 9 The A n a l y t i c a l Technique 12 1. Extraction of v o l a t i l e compounds and preparation of samples 14 2. Gas-Liquid Chromatography 17 EXPERIMENTAL RESULTS 20 1. Response of the beetles to l i v i n g and dead bark of mature ponderosa pine and western white pine . 2 0 2. Response of the beetles to l i v i n g and dead bark of ponderosa pine saplings . . . . . . . . 26 3. Response of the beetles to t o t a l v o l a t i l e extracts . 32 4. Response of the beetles to ether soluble fractions 38 5. Response of the beetles to ethanol . . . . . . 44 • • • 111 TABLE OF CONTENTS ( c o n t i n u e d ) Page 6. E f f e c t o f s t o r a g e on a t t r a c t a n c y o f l o g s a n d e x t r a c t e d s a m p l e s . * . . . . . . 50 7. G a s - L i q u i d C h r o m a t o g r a p h y A n a l y s e s . . . . . 50 DISCUSSION 5^ 1. A n e m o - o l f a c t o r y r e s p o n s e t o h o s t o d o r s . . . 54 2. A t t r a c t a n c y a n d r e p e l l e n c y o f d i f f e r e n t a g e d t r e e s 56 3. R o l e o f e t h a n o l 60 k. G e n e r a l r e m a r k s 61 SUMMARY AND CONCLUSIONS 63 LITERATURE CITED 66 APPENDICES ?k i v L I S T OF TABLES T a b l e No. Page I D a t e o f c u t t i n g , s t o r a g e and l o c a t i o n f r o m w h e r e t h e h o s t t r e e s a m p l e s o f D. p o n d e r o s a e w e r e o b t a i n e d 13 I I R e s p o n s e o f D. p o n d e r o s a e f e m a l e b e e t l e s t o l i v i n g a n d d e a d b a r k o f m a t u r e p o n d e r o s a p i n e a n d w e s t e r n w h i t e p i n e . • 21 I I I R e s p o n s e o f D. p o n d e r o s a e f e m a l e b e e t l e s t o l i v i n g a n d d e a d b a r k o f p o n d e r o s a p i n e s a p l i n g s . 27 I V R e s p o n s e o f D. p o n d e r o s a e t o t o t a l v o l a t i l e e x t r a c t s o f m a t u r e p o n d e r o s a p i n e a n d w e s t e r n w h i t e p i n e • 33 V R e s p o n s e o f D. p o n d e r o s a e f e m a l e b e e t l e s t o e t h e r s o l u b l e f r a c t i o n s o f m a t u r e p o n d e r o s a p i n e a n d w e s t e r n w h i t e p i n e 39 V I R e s p o n s e o f D. p o n d e r o s a e f e m a l e b e e t l e s t o v a r i o u s c o n c e n t r a t i o n s o f e t h a n o l 4-5 V I I E f f e c t : o f s t o r a g e on t h e a t t r a c t a n c y o f b a r k a n d e x t r a c t e d s a m p l e s 51 V I I I P e r c e n t a g e c o m p o s i t i o n o f i n d i v i d u a l v o l a t i l e compounds p r e s e n t i n e t h e r s o l u b l e f r a c t i o n s o f m a t u r e p o n d e r o s a p i n e a n d s a p l i n g s a n d m a t u r e w e s t e r n w h i t e p i n e . • 52 V LIST OF ILLUSTRATIONS Figure No. Page 1. Schematic diagram showing i n i t i a l points of start of beetles, source of odor and light 11 2. Schematic diagram of the apparatus for trapping the volatile compounds from bark samples 15 3. Scheme of programme of fractionation, analysis and bioassay studies 16 4a. Representative path of response to living and dead bark of mature western white pine by flight-experienced D. ponderosae female—Beetle No. 1 22 4b. Representative path of response to li v i n g and dead bark of mature western white, pine by flight-experienced D. ponderosae female—Beetle No. 2 23 4c. Representative path of response to l i v i n g and dead bark of mature ponderosa pine by flight-experienced D. ponderosae female—Beetle No. 1 24 4d. Representative path of response to l i v i n g and dead bark of mature ponderosa pine by flight-experienced D. ponderosae female—Beetle No. 2 25 5a. Representative path of response to l i v i n g and dead bark of ponderosa pine sapling by flight-experienced 2« ponderosae female—Beetle No. 1 28 5b. Representative path of response to li v i n g and dead bark of ponderosa pine sapling by flight-experienced D. ponderosae female—Beetle No. 2 29 5c. Representative path of response to ether soluble fraction of ponderosa pine, sapling by flight-experienced D. ponderosae female—Beetle No. 1 30 v i L I S T OF ILLUSTRATIONS ( c o n t i n u e d ) F i g u r e No. Page 5d. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o e t h e r s o l u b l e f r a c t i o n o f p o n d e r o s a p i n e s a p l i n g b y f l i g h t - e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 2 . . . . . 31 6a. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o t o t a l v o l a t i l e e x t r a c t o f m a t u r e w e s t e r n w h i t e p i n e by f l i g h t -e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 1 34 6b. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o t o t a l v o l a t i l e e x t r a c t o f m a t u r e w e s t e r n w h i t e p i n e . b y f l i g h t -e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 2 35 6c. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o t o t a l v o l a t i l e e x t r a c t o f m a t u r e p o n d e r o s a p i n e by f l i g h t - e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 1 . . . . . 36 6d. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o t o t a l v o l a t i l e e x t r a c t o f m a t u r e p o n d e r o s a p i n e b y f l i g h t - e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 2 37 ? a . R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o e t h e r s o l u b l e f r a c t i o n o f w e s t e r n w h i t e p i n e b y f l i g h t - e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 1 . . . . . 4-0 7b. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o e t h e r s o l u b l e f r a c t i o n o f w e s t e r n w h i t e p i n e by f l i g h t - e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 2 . . . . . 4-1 7c. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o e t h e r s o l u b l e f r a c t i o n o f p o n d e r o s a p i n e by f l i g h t - e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 1 . 42 7d. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o e t h e r s o l u b l e f r a c t i o n o f p o n d e r o s a p i n e b y f l i g h t - e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 2 43 L I S T OF ILLUSTRATIONS ( c o n t i n u e d ) F i g u r e No. 8. P e r c e n t a g e a r r e s t a n t r e s p o n s e t o v a r i o u s c o n c e n t r a t i o n s o f e t h a n o l by f l i g h t - e x p e r i e n c e d D. p o n d e r o s a e f e m a l e s 9a. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o 0.2 p e r c e n t e t h a n o l by f l i g h t -e x p e r i e n c e d D, p o n d e r o s a e f e m a l e . 9b. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o 1.0 p e r c e n t e t h a n o l by f l i g h t -e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 1 . . . . . . . . . . . 9c. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o 1.0 p e r c e n t e t h a n o l by f l i g h t -e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 2 . . . . v i i Page 46 47 48 49 v i i i L I S T OF APPENDICES Number Page I C h r o m a t o g r a m o f e t h e r s o l u b l e f r a c t i o n ( m a t u r e w e s t e r n w h i t e p i n e ) ?4 I I C h r o m a t o g r a m o f e t h e r s o l u b l e f r a c t i o n ( m a t u r e p o n d e r o s a p i n e ) 75 I I I C h r o m a t o g r a m o f e t h e r s o l u b l e f r a c t i o n ( p o n d e r o s a p i n e s a p l i n g ) 76 ACKNOWLEDGEMENTS i x I am d e e p l y i n d e b t e d t o my s u p e r v i s o r Dr. K e n n e t h Graham, P r o f e s s o r o f F o r e s t E n t o m o l o g y , U n i v e r s i t y o f B r i t i s h C o l u m b i a , f o r h i s i n v a l u a b l e a d v i c e , g r e a t e n c o u r a g e m e n t a n d k i n d n e s s t h r o u g h o u t t h e c o n d u c t o f t h e s e s t u d i e s a n d p r e p a r a t i o n o f t h i s t h e s i s . To D r . W. H a n c o c k , R e s e a r c h S c i e n t i s t , W e s t e r n F o r e s t P r o d u c t s L a b o r a t o r y , D e p a r t m e n t o f t h e E n v i r o n m e n t , V a n c o u v e r , f o r m a k i n g t r e e s a m p l e s a v a i l a b l e ; t o Mr. H e n r y A. Moeck, f o r m e r E n t o m o l o g i s t , W e s t e r n F o r e s t P r o d u c t s L a b o r a t o r y , V a n c o u v e r , f o r h i s s u g g e s t i o n s a n d p r o v i d i n g n e c e s s a r y e q u i p -ment a n d t o Dr. L. S a f r y a n i k , E n t o m o l o g i s t , C a n a d i a n F o r e s t r y S e r v i c e , D e p a r t m e n t o f t h e E n v i r o n m e n t , B a n f f , A l b e r t a , f o r s u p p l y i n g t h e s t o c k o f b e e t l e s , I am d e e p l y g r a t e f u l . S p e c i a l t h a n k s t o D r s . G. H. N. T o w e r s , P r o f e s s o r , D e p a r t -ment o f B o t a n y , U n i v e r s i t y o f B.C., a n d G i l l e s D u p r i e s , P o s t -D o c t o r a l f e l l o w , D e p a r t m e n t o f B o t a n y , U . B . C , f o r t h e i r a s s i s t a n c e a n d p e r m i s s i o n t o u s e t h e l a b o r a t o r y e q u i p m e n t . To members o f my a d v i s o r y c o m m i t t e e D r s . J . P. K i m m i n s , D. B. M u l l i c k , G. G. E. S c u d d e r a n d J . W o r r a l l f o r t h e i r i n t e r e s t i n t h e c o n d u c t o f t h i s w o r k a n d p r e p a r a t i o n o f t h e t h e s i s , g r a t e f u l a c k n o w l e d g e m e n t s a r e e x t e n d e d . To N a t i o n a l R e s e a r c h C o u n c i l o f C a n a d a f o r t h e i r f i n a n -c i a l a s s i s t a n c e , I d e e p l y e x t e n d my a c k n o w l e d g e m e n t s . L a s t l y I w i s h t o t h a n k my b r o t h e r Y o u s u f , whose m o r a l e n c o u r a g e m e n t f r o m a l o n g d i s t a n c e h a s a l w a y s g i v e n me a g r e a t i n s p i r a t i o n d u r i n g my s t u d i e s . INTRODUCTION The m e c h a n i s m s o f h o s t s e l e c t i o n b y i n s e c t s c o n s t i t u t e a s u b j e c t o f c o n s i d e r a b l e s c i e n t i f i c a n d p r a c t i c a l i m p o r t a n c e (Beck, 1963, 1965; B i r u k o w , 1966; D e t h i e r , 1947a, 1947b, 1953a, 1953b, 1957, 1963, 1966, 1970; P r a e n k e l , 1959, 1969; H a s k e l l , 1966; Kennedy 1958a, 1958b, 1966; P a i n t e r , 1963? T h o r s t e i n s o n , 1953a, 1953b, I960; W i g g l e s w o r t h , 1965? W r i g h t , 1966). The s c i e n t i f i c i n t e r e s t l i e s i n t h e f i e l d s o f b e h a v i o u r a n d o f i n s e c t h o s t p l a n t i n t e r a c t i o n s , c o - e v o l u t i o n a n d e c o l o g y . The p r a c t i c a l i n t e r e s t l i e s i n t h e f i e l d o f u s e a n d m a n i p u -l a t i o n o f t h e f a c t o r s o f h o s t s e l e c t i o n . I f t h e f a c t o r s a n d m e c h a n i s m s c a n be u n d e r s t o o d , t h e y may be u s e d d i a g n o s t i c a l l y and p r e d i c t i v e l y f o r e v a l u a t i n g t h e s u s c e p t i b i l i t y o f n a t u r a l o r g e n e t i c a l l y d e v e l o p e d h o s t p l a n t s o r a n i m a l s t o i n s e c t a t t a c k s . The f a c t o r s may a l s o b e m a n i p u l a t e d f o r a t t r a c t i n g s p e c i f i c i n s e c t s f o r t h e p u r p o s e o f e i t h e r d e t e c t i n g , t r a p p i n g o r c o n f u s i n g them. Amongst t h e v a r i o u s p r o b l e m s c o n c e r n i n g i n s e c t h o s t p l a n t r e l a t i o n s , one o f t h e most i m p o r t a n t w h i c h h a s r e c e i v e d a g r e a t amount o f a t t e n t i o n i s t h a t o f a r b o r i c o l u s b a r k b e e t l e s . B a r k b e e t l e s a r e n o t e w o r t h y f o r t h e i r a p p a r e n t s e l e c t i v i t y i n a t t a c k s on h o s t t r e e s . They a t t a c k d i f f e r e n t i a l l y a c c o r d i n g t o t r e e s p e c i e s , g e n u s o r o t h e r g r o u p r e l a t i o n s h i p s (Graham, 1963; Richmond, 1933 J R u d i n s k y , 1962; Swaine, 1917)» a s w e l l a s a c c o r d i n g t o age o f t r e e a n d d i a m e t e r c l a s s ( B a l c h , 1942; H o p p i n g a n d B e a l l . 1948; Keen, 1936, 1943)1 p o r t i o n o f t h e t r e e 2 s t e m ( J o h n s o n , 1967; T r a g a r d h , 1927) a n d a c c o r d i n g t o t h e p h y s i -o l o g i c a l s t a t e o f t h e t r e e ( Beal , 1 9 4 3 ) . F o r e x a m p l e , t h e D o u g l a s - f i r b e e t l e , D e n d r o c t o n u s p s e u d o t s u g a e Hopk., u s u a l l y c o n f i n e s i t s a t t a c k s t o D o u g l a s - f i r , a n d e s p e c i a l l y m a t u r e a n d o l d t r e e s ( W a l t e r s , 1956). W i n d t h r o w n , f r e s h l y f e l l e d o r w e a k e n e d t r e e s become t h e b r e e d i n g p l a c e s f o r t h i s b e e t l e . The m o u n t a i n p i n e b e e t l e , D e n d r o c t o n u s p o n d e r o s a e H o p k i n s , a t t a c k s s e v e r a l s p e c i e s o f p i n e s a n d " p r e f e r s " h e a l t h y s t a n d i n g t r e e s . When t h e b e e t l e i s n o t n u m e r o u s , i t b r e e d s i n p h y s i o -l o g i c a l l y w e a k e n e d t r e e s o r t h o s e i n j u r e d b y l i g h t n i n g ( B e a l , 1939)* C e r t a i n a u t h o r s i n d i c a t e t h a t t h i s b e e t l e e x h i b i t s a d e g r e e o f " p r e f e r e n c e " f o r c e r t a i n s p e c i e s among i t s p i n e h o s t s ( E v e n d e n e t §1 ,1943) . The a t t a c k s a r e common a t t h e b a s a l p o r t i o n o f t h e s t e m ( E v e n d e n e t §1,1943; S h e p h e r d , I965). I p s c o n f u s u s p r e f e r s y o u n g t r e e s o r t o p s o f o l d e r t r e e s ( A n d e r s o n , 1948, i 9 6 0 ) . F i e l d d a t a a r e a d v a n c e d b y many a u t h o r s t o s u p p o r t a n i n t e r p r e t a t i o n t h a t d i f f e r e n t i a l a t t a c k s s i g n i f y p r e - s e l e c t i v e c a p a b i l i t i e s o f t h e b a r k b e e t l e s (Chapman,1961; D y e r a n d Chap-man, 1965? P r e b b l e a n d Graham, 1957)• A d d i t i o n a l e v i d e n c e i n d i -c a t e s t h a t t h e b e e t l e s f i n d t h e i r h o s t s b y a i r - b o r n e o d o r s f r o m t h e t r e e (Chapman, 1967; M c M u l l e n a n d A t k i n s , 1962; C o s t e r a n d G a r a , 1968; V i t e , 1969; V i t e a n d Gara,1962) . The m e c h a n i s m o f a t t r a c t i o n i s c o n s i d e r e d t o be a n a n e m o - o l f a c t o r y r e s p o n s e i n w h i c h t h e a p p r o p r i a t e o d o r i n d u c e s t h e i n s e c t t o f l y u p -w i n d . 3 O t h e r a u t h o r s a d v a n c e d a t a w h i c h t h e y b e l i e v e d e m o n s t r a t e n o n - d i r e c t i o n a l p r o c e s s e s ? t h e i r e v i d e n c e d e n i e s t h e e x i s t e n c e o f p r i m a r y a t t r a c t a n t s d e r i v e d f r o m t h e t r e e ( B e r r y m a n , 1968} N o r r i s , 1965; V i t e a n d Wood, 1961; Wood, 1963). They a r g u e t h e i r c l a i m o n t h e e v i d e n c e t h a t a t t a c k s a r e random a n d t h a t some b e e t l e s i n t h e v i c i n i t y o f s u s c e p t i b l e t r e e s l a n d on t h e "wrong o b j e c t s " , s u c h a s d e a d t r e e s o r n o n - h o s t s p e c i e s . The e v i d e n c e f o r random a t t a c k s d o e s n o t c l a r i f y w h e t h e r s u s c e p -t i b l e t r e e s a r e d i s t r i b u t e d a t random o r w h e t h e r a l l t r e e s i n t h e i r s t u d i e s a r e u n i f o r m l y s u s c e p t i b l e . N e i t h e r d o e s i t t a k e i n t o a c c o u n t t h e p o s s i b i l i t y t h a t w r o n g l a n d i n g s may be t h e r e s u l t o f a c h e m i c a l l y o r v i s u a l l y - i n d u c e d a r r e s t a n c e o f f l i g h t . S u c h a r r e s t a n c e m i g h t c o n c e i v a b l y be s p o n s o r e d e i t h e r by c o n c e n t r a t i o n s o f c e r t a i n o d o r s g e n e r a t e d b y s u s c e p t i b l e t r e e s i n t h e v i c i n i t y , o r b y v i s u a l i m a g e s . B e n n e t t a n d B o r d e n (1971) d e m o n s t r a t e d t h e a r r e s t a n t i n f l u e n c e o f h o s t t r e e o d o r s a n d pheromones on s c o l y t i d s i n t e t h e r e d f l i g h t . The e v i d e n c e w h i c h i n d i c a t e s a random p a t t e r n o f a t t a c k d o e s n o t t h e r e f o r e n e c e s s a r i l y p r o v e t h e a b s e n c e o f a t t r a c t a n c y . E i t h e r r andom o r "wrong" l a n d i n g s c o u l d be t h e r e s u l t o f a r r e s t a n c e c a u s e d b y t h e h o s t o d o r s p e r m e a t i n g t h e a i r . Then v i s u a l c u e s may come i n t o o p e r a t i o n . I t i s a l s o c o n c e i v a b l e t h a t a n y s e l e c t i v i t y b y a s m a l l p r o p o r t i o n o f i n d i v i d u a l s may be masked b y t h e n o n - g u i d e d b e h a v i o r o f t h e m a j o r i t y . The c o n f l i c t i n g v i e w s a r e c o n c e i v a b l y t h e r e s u l t o f e x p e r i m e n t e r s e x t r a p o l a t i n g f r o m t h e i r own e x p e r i e n c e s . T hose 4 who w o r k w i t h D o u g l a s - f i r " b e e t l e h a v e s t r o n g p r e s u m p t i v e e v i d e n c e t o b e l i e v e t h a t a p r i m a r y h o s t - g e n e r a t e d a t t r a c t a n t e x i s t s . T h o s e who w o r k w i t h w e s t e r n p i n e b e e t l e i n p o n d e r o s a p i n e a r e c o n v i n c e d t h a t no e v i d e n c e e x i s t s f o r a h o s t - g e n e r a t e d a t t r a c t a n t . P e r h a p s t h e s t r o n g e s t p r e s u m p t i v e e v i d e n c e f o r d i f f e r e n c e s i n modus o p e r a n d i o f t h e s e two s p e c i e s o f i n s e c t i s t h e f a c t t h a t f r e s h l y f e l l e d d o u g l a s - f i r become a t t a c k e d r e a d i l y , w h e r e a s f e l l e d p o n d e r o s a p i n e i s i n e f f e c t i v e b a i t f o r b e e t l e s . I t i s t h u s c o n c e i v a b l e t h a t w e s t e r n p i n e b e e t l e s a r e more i n f l u e n c e d b y v i s u a l c u e s f r o m t h e s i l h o u e t t e s o f s t a n d i n g t r e e s . The e x i s t e n c e o f a n e m o - o l f a c t o r y r e s p o n s e s i n i n s e c t s i s w e l l known. I n t h i s c a s e t h e a n e m o t a c t i c u p - s t r e a m o r i e n t a -t i o n i n p e d e s t r i a n b e e t l e s i s i n d u c e d by " a t t r a c t i v e ' 1 o d o r , b o t h i n t h e d a r k a n d u n d e r e x p o s u r e t o p o t e n t i a l d i s t r a c t i o n o f a u n i d i r e c t i o n a l s o u r c e o f l i g h t . No a n e m o t a c t i c r e s p o n s e o c c u r s i n t h e a b s e n c e o f a p p r o p r i a t e o d o r . R e s p o n s e o f t h e b e e t l e s t o a s t e e p o d o r g r a d i e n t o v e r a v e r y s h o r t d i s t a n c e o f a f e w c e n t i m e t e r s w i t h o u t a i r c u r r e n t s c o u l d be t h r o u g h k l i n o -k i n e s i s a n d k l i n o t a x i s m e c h a n i s m s . A t t r a c t i v e o d o r w i l l h a v e a n i n f l u e n c e on t h e r a t e and s p e e d o f s u c h r e a c t i o n s . The e x i s t e n c e o f b e h a v i o r i s t i c a l l y i n f l u e n t i a l v o l a t i l e s u b s t a n c e s h a s b e e n d e m o n s t r a t e d t o o c c u r f o r c e r t a i n s c o l y t i d b e e t l e s (Chapman, 1962; F r a n c i a a n d Graham, 1967)• However l i t t l e i s known a b o u t t h e c h e m i s t r y o f a t t r a c t i v e o d o r s p r e s e n t i n a h o s t t r e e w h i c h g o v e r n t h e b e h a v i o r o f b a r k b e e t l e s i n i n i t i a l a t t a c k s e q u e n c e . 5 The p u r p o s e o f t h e p r e s e n t s t u d y i s t o e x a m i n e e v i d e n c e as t o t h e p o s s i b l e i n f l u e n c e o f h o s t t r e e v o l a t i l e s i n h o s t s e l e c t i o n b y t h e m o u n t a i n p i n e b e e t l e . I t i s p o s t u l a t e d t h a t p r i m a r y a i r - b o r n e o d o r s o r i g i n a t i n g f r o m t h e h o s t t r e e p l a y a n i m p o r t a n t r o l e i n h o s t t r e e s e l e c t i o n b y t h i s b a r k b e e t l e . T h i s d o e s n o t d e n y t h e a g g r e g a t i n g e f f e c t o f s e x a t t r a c t a n t s ; i . e . p h e r o m o n e s w h i c h b e e t l e s r e l e a s e o n c e t h e y b e g i n t o e s t a b l i s h t h e m s e l v e s . S e c o n d l y i t i s p o s t u l a t e d t h a t t h r e e f u n c t i o n a l l y d i f f e r e n t c h e m i c a l i n f l u e n c e s a r e i n v o l v e d i n o l f a c t o r y c u e i n g i n l a n d i n g ( a p a r t f r o m p o s s i b l e v i s u a l c u e s ) on a s u i t a b l e h o s t b e f o r e t h e y c o u l d e s t a b l i s h i n i t . T h e s e may c o n s i s t o f s e p a r a t e c h e m i c a l e n t i t i e s f o r h o s t s p e c i f i c i t y a n d p h y s i o l o g i c a l m a t u r i t y a n d p h y s i o l o g i c a l " s t r e s s " , o r s i n g l e m o l e c u l a r e n t i t i e s may f o r m o n l y i n c e r t a i n t r e e s p e c i e s u n d e r c e r t a i n c o n d i t i o n s o f age a n d s t r e s s . MATERIALS AND METHODS The E x p e r i m e n t a l I n s e c t The m o u n t a i n p i n e b e e t l e , D e n d r o c t o n u s p o n d e r o s a e 1 H o p k i n s , was c h o s e n a s t h e t e s t i n s e c t , t o s e r v e a s a r e p r e s e n t a t i v e s p e c i e s o f b a r k b e e t l e o f c o n s i d e r a b l e i m p o r t a n c e e c o l o g i c a l l y a n d e c o n o m i c a l l y ( B e a l , 1939; E v e n d e n e t a l , 19^3; S t r u b l e a n d J o h n s o n , 1955)* I t a l s o h a s much b a c k g r o u n d l i t e r a t u r e a v a i l a b l e on i t . I t h a s t h e d e s i r a b l e f e a t u r e o f p r o d u c i n g s u c c e s s i v e g e n e r a t i o n s i n l a b o r a t o r y r e a r i n g s w i t h o u t i n t e r r u p -t i o n b y d i a p a u s e ( R e i d , 1958, I 9 6 0 ) . T h i s i s a n a d v a n t a g e o v e r c e r t a i n o t h e r b a r k b e e t l e s w h i c h h a v e g e n e r a t i o n s s e p a -r a t e d b y p e r i o d s o f d i a p a u s e . T h i s s p e c i e s a c c e p t s p o n d e r o s a p i n e among i t s h o s t s a n d i s t h u s a c o n v e n i e n t i n s e c t t o r e a r , b e c a u s e t h e t h i c k b a r k o f p o n d e r o s a p i n e p r o v i d e s a n a d v a n -t a g e o u s r e a r i n g medium. A f u r t h e r a d v a n t a g e i n t h e u s e o f m o u n t a i n p i n e b e e t l e was i t s r a n g e o f p i n e s p e c i e s w h i c h i t a t t a c k s ( F u r n i s s a n d S c h e n k , 1 9 6 9 ) . T h i s f a c t makes i t p o s s i b l e t o s t u d y c o m p a r a t i v e a n a l y s e s o f e x t r a c t i v e s . I n i t i a l l y , a d u l t b e e t l e s w e r e o b t a i n e d f r o m E i s e n h o w e r R e s e a r c h S t a t i o n , C a n a d i a n F o r e s t r y S e r v i c e , D e p a r t m e n t o f t h e E n v i r o n m e n t , B a n f f , A l b e r t a . L a t e r a c o n s t a n t s u p p l y o f t h e s e D e n d r o c t o n u s m o n t i c o l a e H o p k i n s a n d D e n d r o c t o n u s p o n d e r o s a e H o p k i n s a r e t h e same s p e c i e s a n d l a t e r i s t h e a c c e p t e d s c i e n -t i f i c name f o r B l a c k H i l l b e e t l e s a n d m o u n t a i n p i n e b e e t l e (Wood, L.S., 1963t The G r e a t B a s i n N a t u r a l i s t V o l . 23 (1-2) » 1 - 1 1 7 ) . 7 b e e t l e s was o b t a i n e d b y r e a r i n g t h e m on p o n d e r o s a p i n e l o g s i n t h e l a b o r a t o r y a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a . The r e a r i n g p e r i o d a t t h e p r e v a i l i n g room c o n d i t i o n s d i d n o t e x c e e d more t h a n e i g h t w e e k s . 1) T r e a t m e n t o f t h e B e e t l e s t A d u l t b e e t l e s w h i c h emerged f r o m t h e l o g s w e r e c o l l e c t e d a n d s e g r e g a t e d i n t o m a l e s a n d f e m a l e s . O n l y f e m a l e b e e t l e s w e r e s e l e c t e d f o r b i o a s s a y s t u d i e s , s i n c e i t i s t h e f e m a l e o f D. p o n d e r o s a e w h i c h f i r s t e s t a b l i s h e s the g a l l e r y , l a t e r j o i n e d b y t h e m a l e ( R e i d , 1962). E a c h t e s t c o m p r i s e d 10 t o 15 f e m a l e a d u l t s . B e e t l e s w i t h b r o k e n t a r s i , l e g s , a n t e n n a e o r a n y o t h e r p a r t o f t h e b o d y w e r e d i s c a r d e d a s b e i n g u n s u i t a b l e f o r t h e t e s t s . The s e l e c -t e d b e e t l e s w e r e f i r s t p r e - c o n d i t i o n e d f o r one t o two h o u r s a t t h e d e s i r e d t e s t c o n d i t i o n s o f t e m p e r a t u r e a n d r e l a t i v e h u m i d i t y . F o l l o w i n g t h i s , b e e t l e s w e r e s e t up f o r a n c h o r e d f l i g h t b y g l u e i n g t h e i r p r o t h o r a x t o one end o f a f i n e c o p p e r w i r e . The o t h e r e nd o f t h i s w i r e was f o r m e d i n t o a l o o s e l o o p a t t a c h e d t o a n e n t o m o l o g i c a l p i n p l a c e d h o r i z o n t a l l y on t h e edge o f a s t y r o f o a m b l o c k . B e e t l e s w e r e i n d u c e d t o f l y f o r a t l e a s t one h o u r b y t o s s i n g t h e c o p p e r w i r e g e n t l y . T h e i r f l i g h t was c o n t i n u o u s l y o b s e r v e d a n d t h o s e b e e t l e s w h i c h s t o p p e d f l y i n g d u r i n g t h i s p e r i o d w e r e i n d u c e d t o f l y a g a i n b y r e - t o s s i n g . F l y i n g o f t h e b e e t l e s i s e s s e n t i a l b e -c a u s e u n t i l t h e r e h a s b e e n f l i g h t e x e r c i s e a f l i g h t p o s i t i v e r e s p o n s e t e n d s t o d o m i n a t e o v e r o l f a c t o r y r e s p o n s e s (Graham, 1959, I960). 8 2) B i o a s s a y S t u d i e s t Many t e c h n i q u e s s u c h a s c h o i c e c h a m b e r , "Y" t u b e o l f a c t o m e t e r a n d o p e n o d o r f i e l d h a v e b e e n u s e d i n t h e p a s t f o r t h e s t u d y o f o l f a c t o r y b e h a v i o u r o f i n s e c t s t o v a r i o u s c h e m i c a l a n d p h y s i c a l s t i m u l i . However t h e y h a v e c e r t a i n d i s a d v a n t a g e s a s w e l l a s a d v a n t a g e s , a n d do n o t p r o -v i d e a n a c c u r a t e m e a s u r e f o r o l f a c t o r y r e s p o n s e s . Hughes a n d P i t m a n (1970) h a v e d e s c r i b e d a s y s t e m w h e r e b y o l f a c t o r y s t u d i e s c a n be c o n d u c t e d on f l y i n g b e e t l e s u n d e r l a b o r a t o r y c o n d i t i o n s . I n t h e p r e s e n t s t u d i e s , t h e o l f a c t o m e t e r d e s i g n e d b y Moeck (1970a) t o s t u d y t h e r e s p o n s e s o f p e d e s t r i a n b e e t l e s t o v a r i o u s s t i m u l i was u s e d . I t h a s one c e r t a i n advantage« t h e i n s e c t u n d e r o b s e r v a t i o n h a s a f r e e f i e l d t o move i n t h e o l f a c t o m e t e r . The c h e m i c a l s t i m u l u s i s p r e s e n t e d i n t h e f o r m o f a n o d o r -s t r e a m w h i c h d o e s n o t d i f f u s e o r m i x i n t h e o l f a c t o m e t e r f i e l d . T h i s was a c h i e v e d by d r a w i n g t h e a i r f r o m i t by a s u c t i o n pump a n d e x h a u s t i n g t h e o d o r o u s s u b s t a n c e s t o t h e o u t s i d e . L i g h t c o u l d be p r o v i d e d a t r i g h t a n g l e s t o t h e o d o r -s t r e a m a s a c o u n t e r - s t i m u l u s . The s o u r c e o f w h i t e l i g h t was a R e i c h e r t m i c r o s c o p e i l l u m i n a t o r w i t h a h e a t f i l t e r . A r h e o s t a t was a d o p t e d t o k e e p t h e w a v e l e n g t h b a l a n c e . The i n c i d e n t l i g h t r a n g e d f r o m 20 t o 30 f o o t c a n d l e s . D u r i n g t h e t e s t s , c o n s t a n t t e m p e r a t u r e a n d r e l a t i v e h u m i -d i t y w e r e m a i n t a i n e d b y k e e p i n g t h e o l f a c t o m e t e r i n a c o n -t r o l l e d e n v i r o n m e n t room. The t e m p e r a t u r e a n d r e l a t i v e h u m i -d i t y a t w h i c h b i o a s s a y t e s t s w e r e c o n d u c t e d w e r e 21°c a n d 55 t o 65$ r e s p e c t i v e l y . D. p o n d e r o s a e a d u l t s p e r f o r m many o f 9 t h e i r v i t a l a c t i v i t i e s u n d e r t h e s e c o n d i t i o n s ( R e i d , I 9 6 0 ; S h e p h e r d , 1 9 6 6 ) . 3 ) O l f a c t o m e t e r O b s e r v a t i o n s i B e e t l e s , s e r v i n g a s i n s t r u -m e n t s f o r b i o a s s a y , w e r e t e s t e d one a t a t i m e . F o r e a c h t r i a l a f l o w n b e e t l e was p l a c e d i n t h e o l f a c t o m e t e r . I t s r e s p o n s e t o a n a i r - s t r e a m ; a n d l i g h t was r e c o r d e d b y t r a c i n g i t s p a t h on t h e t r a n s p a r e n t t o p o f t h e o l f a c t o m e t e r . E a c h b e e t l e was ob-s e r v e d f i v e t i m e s b y p l a c i n g i t i n d i f f e r e n t d i r e c t i o n s ( F i g . 1 ) r e l a t i v e t o t h e l i g h t a n d o d o r s o u r c e . The r e s p o n s e s o f t h e b e e t l e s t o l i g h t a n d a i r - s t r e a m w e r e f i r s t r e c o r d e d a s c h e c k s o r c o n t r o l s a g a i n s t s u b s e q u e n t t e s t i n g w i t h c h e m i c a l s t i m u l i . Then t h e s a m p l e t o be t e s t e d f o r i t s o l f a c t o r y i n f l u e n c e was p l a c e d i n t h e o d o r s o u r c e t u b e . L i q u i d s a m p l e s w e r e a p p l i e d on a f i l t e r p a p e r . A l l t h e s a m p l e s , e i t h e r l i v i n g a n d d e a d b a r k o r e x t r a c t s on f i l t e r p a p e r s , w e r e p l a c e d e a c h t i m e on s h e e t s o f a l u m i n u m f o i l w h e n e v e r u s e d . T h i s a v o i d e d a n y p o s s i b l e c o n t a m i n a t i o n i n t h e o d o r s o u r c e t u b e w h i c h m i g h t i n t e r f e r e w i t h s u b s e q u e n t t e s t s . R e s p o n s e s t o o d o r o u s s t i m u l i w e r e r e c o r d e d i n t h e same manner a s d e s c r i b e d a b o v e . The f o l l o w i n g c r i t e r i a w e r e u s e d f o r d e s i g n a t i n g t h e o l f a c t o r y r e s p o n s e o f t h e b e e t l e s t o a i r - s t r e a m a n d c h e m i c a l s t i m u l i 1 Term d e s i g n a t e d f o r P a t t e r n o f R e s p o n s e t h e s t i m u l u s R e s p o n s e t o C h e m i c a l S t i m u l i a ) B e e t l e p a u s e d a n d showed k l i n o k i n e t i c r e s p o n s e i n t h e o d o r - s t r e a m , b u t d i d n o t move u p - s t r e a m o r d o w n - s t r e a m . . . . A r r e s t a n t 10 b) Beetle walked i n the odor either up-stream or down-stream Attractant c) Beetle avoided or attempted to avoid the odor-stream a f t e r intercepting i t or walked along the boundary of odor-stream • • Repellent Beetle response to any one of the above categories was designated as p o s i t i v e anemo-olfactory. Absence of response to the category mentioned above was designated as negative anemo-olfactory. Sim i l a r l y the response of the beetles to air-stream without odor could be designated as p o s i t i v e anemotactic and absence of response to such a stimulus as negative anemotactic. The following i s the l i s t of chemical s t i m u l i against which the response of adult females of D. ponderosae was observed. 1) Pieces of l i v i n g and dead bark; mature ponderosa pine and saplings and mature western white pine. 2) Total v o l a t i l e extracts from l i v i n g and dead bark of mature ponderosa pine and western white pine (2 to 3 ml. of the trapped material). 3) Ether soluble fractions (fractionation of t o t a l v o l a t i l e extracts) of mature ponderosa pine and saplings and mature western white pine (80 to 100 u l . of the con-centrate). 4) Ethanol at concentrations of 0.1, 0.2, 0.5, 1.0 and 10 percent. F i g . 1. S c h e m a t i c d i a g r a m s h o w i n g i n i t i a l p o i n t s o f s t a r t o f b e e t l e s , s o u r c e o f o d o r a n d l i g h t . HI j l • Dovn-ktream Diri Source of l i g h t ection Source of odor Up-i>tream Direction N.B. 1, 2, 3, k and 5 P o s i t i o n of the beetles and the d i r e c t i o n i n which they were pointing. 2^ R I 5 12 The A n a l y t i c a l Technique The "bioassay of an attractant, repellent or any other chemical stimulant depends on the i s o l a t i o n and i d e n t i f i c a -t i o n of the chemical components, and the confirmation of active chemical depends on bioassay r e s u l t s . These two f i e l d s of study are inter-dependent f o r the f i n a l answer, but f o r convenience are dealt herewith separately under the headings i A n a l y t i c a l Techniques and Bioassay Studies. The techniques were devised on the hypothesis that o l -factory responses of the insects are involved i n host selec-t i o n . Odorous compounds must be v o l a t i l e . The techniques of i s o l a t i o n were employed so as to cause a minimum possible a l t e r a t i o n of the natural state of l i v i n g samples and chemi-cals i n them. This was accomplished by obtaining the samples of trees from forests and storing them at low temperatures (0 to - 1 5°C) i n the laboratory. Storage of logs at these temperatures minimized the losses of v o l a t i l e compounds, and further prolonged the l i v i n g period of tissues i n a log. I n i t i a l l y studies were directed to f i n d the response of beetles to t o t a l v o l a t i l e extracts of l i v i n g and dead bark of mature western white pine trees. Later samples of mature ponderosa pine trees and saplings were obtained and used f o r extraction purposes, analysis and bioassay studies. Samples of ponderosa pine (Pinus ponderosa Laws.) mature trees and saplings and western white pine (Pinus monticola Table 1. Date of cutting, storage and loc a t i o n from where the host tree species of D. ponderosae were obtained. Sample No. Host Species Location Date of Cutting Date of Storage Approx. Age Physical Characteristics of Samples 1 western white pine Haney, U.B.C. Forest Res. Station 15-7-69 17-7-69 100 yrs. 3 pieces of logs, 2' long and l i l ' width, phloem and outer bark thick. Selec-ted from breast height to 20' high. Healthy & mature. 2 western white pine Merritt Lake (B.C. ) 22-11-70 25-11-70 80 yrs. 2 pieces of logs 2j-' long and 1'8" i n width, bark and phloem thick. Selected from breast height to 20' high. Healthy & mature. 3 ponderosa pine Merritt Lake (B.C.) 25-5-70 27-5-70 40 y r s . 3 pieces of logs 2 f long and 1' i n width, bark and phloem 1" thick. Selected from breast height to 20* high. Healthy & mature. ponderosa pine Merritt Lake (B.C. ) 22-11-70 25-11-70 50 yrs. 2 pieces of logs 2' long and 1' i n width, bark and phloem l£ M thick, selected from breast height to 20' high. Healthy & mature. 5 western white pine Haney, U.B.C. Forest.Res. Station 15-5-71 15-5-71 80 yrs. 2 pieces of logs 2' long and 1^' i n width. Bark and phloem thick. Selected from breast height to 20* high. Healthy & mature. 6 ponderosa pine Kelowna (B.C.) 25-5-71 27-5-71 80 y r s . 2 pieces of logs 2' long and 2' i n width. Bark and phloem l i " t hick. Selected from breast height to 20'. Healthy & mature. 7 ponderosa pine (sapling) Kelowna (B.C.) 25-5-71 27-5-71 8 y r s . 2 saplings 5' i n height, 4" & 6" i n width. Bark and phloem 1/8". Healthy saplings. 14 Dougl*) mature trees were obtained from various sources. The history of samples was as shown i n Table I. 1) Extraction of. V o l a t i l e Compounds and Preparation of Samples i  Conventional techniques f o r extracting v o l a t i l e compounds from l i v i n g tissues of plants consist of grinding and squeezing the samples, steam d i s t i l l a t i o n , extraction with d i f f e r e n t organic solvents of dried or l i v i n g tissues and vacuum d i s -t i l l a t i o n etc. The method adopted was that of Moeck (19?0b), whereby frozen samples of phloem and bark could be extracted through a sublimation process. The technique consists of placing frozen samples of l i v i n g and dead bark i n a plexiglass chamber (6.25 cm. diameter), closed above and below by two thick glass plates. The rims of the chamber were sealed with high vacuum s i l i c o n grease. The outlet from the chamber was connected to a "U" tube (drying tube) which served to trap the v o l a t i l e s . The U-tube was immersed i n a Dewar f l a s k containing l i q u i d nitrogen. The other arm of the drying tube was connected to a vacuum pump (Fig. 2). The extracts then obtained were used f o r bioassay and f o r f r a c t i o n a t i n g the active materials. The flow diagram (3) explains i n d e t a i l the steps followed during experimental work. Each sample of 5 to 10 square centimeters of l i v i n g and dead bark yielded 2 to 3 ml. of the trapped material i n the U-tube. A t o t a l amount of 30 ml. of the trapped material was F i g . 2. S c h e m a t i c d i a g r a m o f t h e a p p a r a t u s f o r t r a p p i n g t h e v o l a t i l e compounds f r o m b a r k s a m p l e s . Connecting tube Glass covering (top) Plexiglass chamber J I Bark sample Glass covering (bottom) F i g . 3« Scheme o f programme o f f r a c t i o n a t i o n , a n a l y s i s a n d h i o a s s a y s t u d i e s . 16 Fig. 3» Samples of Living and Dead Bark Bioassay (Host Tree) l Chemical Analysis Extraction of Total V o l a t i l e Compounds by Sublimation Method Bioassay [ Fractionation Ether Soluble Fraction Bioassay Gas-Liquid Chromatography Analysis accumulated. To t h i s was added an equal amount of ether, and a f t e r vigorous shaking i n a separatory funnel, the ether and water layers were co l l e c t e d i n separate f l a s k s . The water layer was re-extracted with equal amount of ether and the pro-cedure was repeated twice. The combined ether soluble f r a c -t i o n was then treated with anhydrous sodium sulphate to remove water present in, the f r a c t i o n . The mixture was f i l t e r e d and the ether soluble f r a c t i o n c o l l e c t e d i n a separate f l a s k . The combined f r a c t i o n (60-70 ml.) was f i n a l l y concentrated to 1 ml. by passing a stream of nitrogen through the fl a s k by heating gently on a heating pan (temperature between 40°G to 45°C). The concentrated ether soluble f r a c t i o n was used f o r a n a l y t i c a l and bioassay studies. 2) Gas-Liquid Chromatography•(GLC)t Since the analysis was to be done f o r the unknown com-pounds of the trapped v o l a t i l e mixture, the following points were noted and give guidance f o r the Gas-Liquid Chromatography. Sample history gives some idea of the nature of compounds present. (a) Pine trees contain large amounts of terpenes and other hydrocarbons (Mirov, 1967). These are the major v o l a t i l e compounds present i n these trees. (b) Samples might also have v o l a t i l e f a t t y acids, primary and secondary alcohols and esters etc. (c) Resin acids are present i n large amounts i n many species of Pines. 18 In the l i g h t of the aforementioned considerations, i t was f i r s t necessary to f i n d out whether any polar or non-polar types of compounds were present i n the v o l a t i l e mixtures of l i v i n g and dead hark. The s e l e c t i o n of the column with a p a r t i c u l a r l i q u i d phase depends on the nature of compounds searched for i n the sample mixture. Much progress has been made during recent years i n the Gas-Liquid Chromatography and separation of essential o i l components (Guenther et a l , 1959)» in ascertaining the Gas-Liquid Chromatographic behavior of terpenoid compounds and i n the se l e c t i o n of the most suitable column materials and operating conditions (von Rudloff, 1970). The resolution of a v o l a t i l e mixture into i t s i n d i v i d u a l com-ponents i s affected by three main parameters: (1) column siz e (length and diameter), (2) column temperature, and (3) nature of l i q u i d phase and s o l i d support (von Rudloff, 1970). Apizeon L was selected as one of the l i q u i d phase f o r determining the presence of a c y c l i c hydrocarbons and also monoterpene hydrocarbons and other non-polar compounds. L i -quid phases such as b, b d i o x y p r o p i o n i t r i l e acetophenyl and Carbowax 20 M were selected f o r separating compounds which have functional groups on them. Abundant l i t e r a t u r e i s a v a i l -able f o r the s e l e c t i o n of l i q u i d phases and s o l i d supports suitable f o r the analysis of d i f f e r e n t compounds. The following conditions prevailed i n the Gas-Liquid Chromatography of v o l a t i l e compounds i n the present studies. 19 Gas-Liquid Chromatograph Model PYE 105 preparative series Single Flame Ionization Detector. Columns Copper columns f inch O.D. 8 feet long, packed witht 1) Apizeon L 5% on chromo-sorb P acid washed 60/80 mesh. 2) b»b d i o x y p r o p i o n i t r i l e acetophenyl 10% on chromosorb p acid washed 60/80 mesh. Carrier gas Nitrogen @ 30 cc/min. Operating conditions Isothermal temperature 1) Oven temperature 55° and 100° C 2) Injecting temperature 90° and 135°C. Solvent Ethyl ether chromato-graphic grade. Syringe Precision Sampling Co. Size 10 micro l i t r e . Recorder Perkin Elmer No. I 6 5 1 MV, chart speed 10 mm/ minute. The column packing was done i n the laboratory. Packed columns were pre-heated for 24 hours at temperatures 180°C (for Apizeon L) and 120°C (for b, b d i o x y p r o p i o n i t r i l e acetophenyl) and were checked f o r possible bleeding. EXPERIMENTAL RESULTS 1) Response of the Beetles to Living and Dead Bark .of Mature Ponderosa Pine and Western White Pine i  Altogether 92 female "beetles (comprising 460 observations) were tested to evaluate t h e i r response to combined odors of l i v i n g and dead bark pieces of mature ponderosa pine and western white pine (Table I I ) . Forty-six to ?0 percent of the beetles were attracted to odors of ponderosa pine and a si m i l a r percentage (42 to 70 percent) were attracted to those of western white pine. The average response to odors of these two host species varied between 52 percent to 55 percent res-p e c t i v e l y . The representative trackings of beetles i n the presence of host odors are i l l u s t r a t e d i n Figs. 4a to d. In many instances response varied from i n d i v i d u a l to i n d i v i d u a l . Beetles when released i n the olfactometer moved either towards the source of l i g h t or at random. On intercepting the odor-stream, most turned up-stream, maintaining t h e i r path straight towards the source of odor, while others a f t e r crossing the odor-stream made a few turnings, returned to the odor-stream (probably through k l i n o k i n e t i c and k l i n o t a c t i c reaction), and then maintained t h e i r path towards the source of odor u n t i l they reached the odor delivery tube (Figs. 4a, b, c and d). In some instances the beetles which had crossed the odor-stream, a f t e r a few turnings as stated above, could not suc-c e s s f u l l y return to the odor-stream and walked aimlessly (Figs. 4b and d). Some of the beetles exhibited down-stream Table II. Response of D. ponderosae female beetles to l i v i n g and dead bark of mature ponderosa pine and western white pine WESTERN WHITE PINE PONDEROSA PINE Number o f 1 N i Number o f l Beetles Percent Number of-1- Beetles Percent Number Number of responded response Number Beetles responded response of Beetles to l i v i n g to dead of responded to l i v i n g to dead Test Beetles responded and dead & l i v i n g Test Beetles to A i r and dead. & l i v i n g No. Tested to A i r bark bark No. Tested Stream bark bark 1 10 0 7 70 11 10 0 7 70 2 14 0 7 50 12 10 0 5 50 3 19 0 8 42 13 15 0 6 46 5 14 0 7 50 ..Average 52 55 Total Observations 285 175 Each figure based on 5 observations. F i g . 4a. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o l i v i n g a n d d e a d h a r k o f m a t u r e w e s t e r n w h i t e p i n e b y f l i g h t - e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 1. ( V ) / V ) F i g . 4b. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o l i v i n g a n d d e a d b a r k o f m a t u r e w e s t e r n w h i t e p i n e b y f l i g h t - e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 2. F i g . 4-c. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o l i v i n g a n d d e a d "bark o f m a t u r e p o n d e r o s a p i n e "by f l i g h t - e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 1. F i g . 4d. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o l i v i n g a n d d e a d h a r k o f m a t u r e p o n d e r o s a p i n e b y f l i g h t - e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 2. 26 response to odors and t h i s was also common i n those beetles which moved up-stream (Fig. 4a). In a l l the above observations, i t was noted that beetles responding to the up or down odor-streams, oriented k l i n o k i -n e t i c a l l y by making a few turnings between 90° and 36O0. Beetles moving down the odor-stream made more turnings within and around the stream as compared to those which moved up-stream (Fig. 4a). Thus, according to the previously defined parameters (pages 9 and 10), the above responses of the beetles are due to attractancy to combined odors of l i v i n g and dead bark from the two host species. 2) Response of the Beetles to Living and Dead Bark of Ponderosa Pine Saplings« Altogether 37 female beetles (comprising 185 observa-tions ) were used to determine the e f f e c t of l i v i n g and dead bark odors of ponderosa pine saplings. The r e s u l t s are sum-marized i n Table I I I , and the representative paths of beetles are i l l u s t r a t e d i n Figs. 5a and b. It i s evident from the trackings that i n i t i a l l y the beetles following release i n the olfactometer, moved either towards the source of l i g h t , or at random. On intercepting the boundary of the odor-stream, they frequently followed a short distance down-stream s t r i c t l y along the margins of the flowing a i r , but neither up, nor within the stream. They then turned away and walked i n the odorless f i e l d at random. Less frequently, they either turned back on reaching the boundary of the odor-stream, or Table III. Response of D. ponderosae female beetles to living and dead bark and ether soluble fractions of ponderosa pine saplings.  LIVING AND DEAD BARK ETHER SOLUBLE FRACTION. Number ofJ- Number o f l Number o f l Beetles Percent Number o f l Beetles Percent Number Beetles responded response Number Beetles responded response of responded to living to l i v i n g of responded to EtpO to Et20 Test Beetles to Air & dead & dead Test Beetles to Air soluble soluble No. Tested Stream bark bark No. Tested Stream Frac. Frac. 24 12 0 8 66 28 14 0 7 51 25 10 0 7 70 29 10 0 7 70 : 26 . 15 0 12 80 30 10 0 8 80 Average 72 67 Total observations 185 170 Each figure based on 5 observations. ^ 3 F i g . 5a. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o l i v i n g a n d d e a d b a r k o f p o n d e r o s a p i n e s a p l i n g s b y f l i g h t - e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 1. F i g . 5b. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o l i v i n g a n d d e a d b a r k o f p o n d e r o s a p i n e s a p l i n g s b y f l i g h t - e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 2. Source of Light Source of Odor ^_ to \0 F i g . 5c. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o e t h e r s o l u b l e f r a c t i o n o f p o n d e r o s a p i n e s a p l i n g s b y f l i g h t - e x p e r i e n c e d B. p o n d e r o s a e f e m a l e — B e e t l e No. 1. F i g . 5d. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o e t h e r s o l u b l e f r a c t i o n o f p o n d e r o s a p i n e s a p l i n g s b y f l i g h t - e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 2. 32 attempted to cross i t continuing towards the source of l i g h t . In no case, did they pause or remain i n the odor-stream. Frequently on intercepting the odor-stream the beetles rubbed t h e i r antennae vigorously with the front legs. The above responses of the beetles c l e a r l y indicate avoidance of the odor-stream and hence the stimulus can be c l a s s i f i e d as rep e l l e n t . In a l l the t e s t s , beetles showed negative anemo-taxis to the air-stream alone. 3) Responseof the Beetles to Total V o l a t i l e Extracts i The percentage response of 140 female beetles based on 700 observations (280 observations f o r mature ponderosa pine and 420 f o r mature western white pine) i s presented i n Table IV. The data show that the a t t r a c t i v e response of the beetles to the two host odors varied between 50 percent to 90 percent. The average percentage response to ponderosa pine was 77 per-cent and f o r western white pine 66 percent. The representa-t i v e trackings of the beetles responding to t o t a l v o l a t i l e extracts of the two species are shown i n Figs. 6a to d. The t o t a l v o l a t i l e extracts from l i v i n g and dead bark of the two host species were a t t r a c t i v e to beetles. A majority of the beetles moved str a i g h t towards the odor source on intercepting the odor-stream. Moreover they also showed k l i n o k i n e t i c and k l i n o t a c t i c behavior. A l l of the above mentioned responses were i n d i c a t i v e of the attractancy of the extracts. The attractancy observed under these tests was s i m i l a r to that observed f o r the odors of l i v i n g and dead bark pieces of the Table IV. Response of D. ponderosae female beetles to t o t a l - v o l a t i l e extracts of mature - ponderosa pine-and western white pine. WESTERN WHITE PINE PONDEROSA PINE Number o f l 1 Number o f i Number o f 1 Beetles Percent Number o f x Beetles Percent Number Beetles responded response Number Beetles responded response of responded to Total to Total of responded to Total to Total Test Beetles to A i r . . Volatile- . V o l a t i l e Test Beetles -to Air- Volatile. V o l a t i l e No. Tested Stream Ext. Ext. No. Tested Stream Ext. Ext. 4 12 0 6 50 14 14 0 12 86 6 10 0 9 90 15 12 0 8 67 7 14 0 13 90 16 10 0 9 90 8 19 0 14 70 17 10 0 6 60 9 14 0 11 51 17a 10 0 8 80 10 15 0 9 60 .. Average 68 78 • - Total observations 420 280 Each figure based on 5 observations. F i g . 6 a . R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o t o t a l v o l a t i l e e x t r a c t s o f m a t u r e w e s t e r n w h i t e p i n e "by f l i g h t -e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 1. F i g . 6b. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o t o t a l v o l a t i l e e x t r a c t o f m a t u r e w e s t e r n w h i t e p i n e by f l i g h t -e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 2. F i g . 6 c . R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o t o t a l v o l a t i l e e x t r a c t o f m a t u r e p o n d e r o s a p i n e "by f l i g h t - e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 1. F i g . 6d. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o t o t a l v o l a t i l e e x t r a c t o f m a t u r e p o n d e r o s a p i n e b y f l i g h t - e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 2. 'I •i I Source of.Light 38 mature ponderosa pine and western white pine. This establishes the v a l i d i t y of the extraction technique of host tree odors i n bioassay studies. 4) Response of the Beetles to Ether Soluble Fractions i In t o t a l , 11 tests comprising 118 female beetles, were conducted to evaluate the responses of beetles to ether soluble f r a c t i o n s from mature ponderosa pine and saplings and mature western white pine. There were 210, 1?0 and 210 obser-vations i n each case, respectively (Table V). The represen-t a t i v e trackings are i l l u s t r a t e d i n Figs. ?a to d. The ether soluble f r a c t i o n s of mature ponderosa pine and western white pine e l i c i t e d attractant responses i n pedestrian beetles s i m i l a r to those obtained with t o t a l v o l a t i l e extracts of the two host species. In general, the response was up-stream towards the source of odor. However, i n a few i n -stances beetles showed k l i n o k i n e t i c and k l i n o t a c t i c reactions also. After reaching the tube which delivered the odor i n the olfactometer some of the beetles entered the tube, thereby i n d i c a t i n g the attractancy of the f r a c t i o n s . It i s stated here that these types of observations were not unique to ether soluble f r a c t i o n s , but were also noticed with other attractant odors described e a r l i e r . The ether soluble f r a c t i o n s of ponderosa pine saplings were found to be repellent (Table I I I ) . It i s evident from t h e i r representative trackings (Figs. 5c and d) that beetles Table V. Response of D. ponderosae female beetles to ether soluble fractions of mature ponderosa pine and western white pine. .WESTERN WHITE PINE PONDEROSA PINE Number o f l Number o f l Number o f 1 Beetles Percent Number o f x Beetles Percent Number Beetles responded response Number Beetles responded response of responded to Et20 to Et20 of responded to Et£0 to Et£0 Test Beetles to Ether soluble . soluble Test Beetles to Ether soluble . soluble No. Tested alone Frac. Frac. No. Tested alone Frac. Frac. 18 10 0 8 80 21 10 0 8 80 19 10 0 6 60 22 12 0 8 67 20 12 0 6 50 23 10 0 9 90 20a 10 . 0 7 70 24 10 0 6 60 . Average . 6.6 . 74 ... .... Total observations 210 210 Each figure based on 5 observations. F i g . 7a. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o e t h e r s o l u b l e f r a c t i o n s o f w e s t e r n w h i t e p i n e by f l i g h t - e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 1. '1 o F i g . 7b. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o e t h e r s o l u b l e f r a c t i o n s o f w e s t e r n w h i t e p i n e by f l i g h t - e x p e r i e n c e d D. p o n d e r o s a e f e m a l e - - B e e t l e No. 2. F i g . 7c. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o e t h e r s o l u b l e f r a c t i o n s o f p o n d e r o s a p i n e by f l i g h t - e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 1. 'I F i g . ? d . R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o e t h e r s o l u b l e f r a c t i o n s o f p o n d e r o s a p i n e by f l i g h t - e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 2. Source of ! Lifeht 44 on intercepting the odor-stream t r i e d to avoid i t by changing t h e i r course of movement. In a l l of these observations the response pattern was s i m i l a r to that noticed with l i v i n g and dead bark pieces of the saplings (Figs. 5a and b). Beetles showed no response to ether alone. 5) Response of the Beetles to Ethanolt In t o t a l , 157 beetles were used i n 12 tests to study response to several concentrations of ethanol. The res u l t s of 885 observations are summarized i n Table VI. The percent-age response to each concentration of ethanol i s presented as histograms i n Fig. 8. Thirty-eight percent of the beetles responses to air-borne EtOH evaporating from f i l t e r paper moistened with 0.1 percent EtOH. The response increased with increasing concentrations of EtOH (0.2 percent, 0.5 percent and 1.0 percent). F i f t y - e i g h t percent of the beetles responded to 1.0 percent EtOH. However the response decreased to 28 percent with 10.0 percent EtOH applied to the f i l t e r paper surface. From the trackings of the pedestrian beetles (Figs. 9a to c ) , i t i s evident that a majority of beetles paused for a period of 5 to 50 seconds on intercepting the odor-stream. After the period of pause, the majority of beetles did not move up or down the odor-stream but moved either at random or towards the source of l i g h t . A few beetles also showed a very b r i e f k l i n o k i n e t i c response. A l l of these observations were a c l e a r i n d i c a t i o n of the arrest-ant e f f e c t of ethanol on D. ponderosae female beetles. Table VI. Response of D. ponderosae female beetles to various concentrations of ethanol Number o f l Test No. Number of Beetles Tested Beetles responded to Air Stream Number ofl Beetles responded to 0.156 EtOH Number ofl Beetles responded to 0.2% EtOH Number o f l Beetles responded to 0.5% EtOH Number o f l Beetles responded to 1.0% EtOH Number ofl Beetles responded to 10% EtOH IE 14 0 5 - - - -2E 14 0 5 - - - -3E 12 0 - 5 - - -4E 14 0 - - 6 - -5E 15 0 - - 6 - -6E 10 0 - - - 7 -7E 14 0 - - - 6 -8E 19 0 - - - 11 -9E 10 0 - - - 6 -10E 10 0 - - - 7 -H E 10 0 - - - - 3 12E 15 0 - - 4 Responded /Total °/l57 10/28 Viz- 1 2 / 2 9 ? / 2 5 Total observations 885* Figures based on 5 runs of each beetle. F i g . 8. P e r c e n t a g e a r r e s t a n t r e s p o n s e t o v a r i o u s c o n c e n t r a t i o n s o f e t h a n o l b y f l i g h t - e x p e r i e n c e d D. p o n d e r o s a e f e m a l e s . 'I w w hi e-< w w w < £ w PK o •z. w < c/5 E H 2: W o W « w < a: < W o W O H 100 80 60 kO -20 0.1 0.2 0.5 1.0 1 0 ' ° Concentration of Ethanol (Percent) F i g . 9 a . R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o 0.2 p e r c e n t e t h a n o l "by f l i g h t -e x p e r i e n c e d D. p o n d e r o s a e f e m a l e . Source of Light I Beetle paused ( t u n ) F i g . 9b. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o 1.0 p e r c e n t e t h a n o l by f l i g h t -e x p e r i e n c e d D. p o n d e r o s a e f e m a l e — B e e t l e No. 1. 00 F i g . 9c. R e p r e s e n t a t i v e p a t h o f r e s p o n s e t o 1.0 p e r c e n t e t h a n o l b y f l i g h t e x p e r i e n c e d D. p o n d e r o s a f e m a l e -B e e t l e No. 2. 50 6) Effect of Storage on the Attractancy of Logs and Extracted Samples t  The log samples which were stored f o r more than 2 months below freezing temperatures (-15 to -18°C) were l e s s a t t r a c -t i v e as compared with freshly c o l l e c t e d material. In order to f i n d the e f f e c t of storage on the attractancy of logs to beetles, samples were tested at i n t e r v a l s of 2 and 6 weeks of storage. Observations revealed that the attractancy of stored logs was less a f t e r storage for a 6 week period. Similar e v i -dence was obtained with t o t a l v o l a t i l e extracts of the ponderosa pine tree. The GLC analysis of ether soluble fractions from logs stored for more than 2 months d i f f e r e d i n retention time and broadening of the i n d i v i d u a l peaks which remained o f f the chart scale. A summary of these observations i s presented i n Table VII. It i s interpreted here that the polymerisation and auto-oxidation of v o l a t i l e compounds slowly takes place. 7) Gas-Liquid Chromatographic Analysist The GLC analysis of ether soluble fractions of ponderosa pine, western white pine mature trees and ponderosa pine saplings resulted i n i d e n t i c a l peaks which could be compared with t h e i r retention times. Columns such as Apizeon L and b, b d i o x y p r o p i o n i t r i l e acetophenyl resolved s i m i l a r peaks. There were 5 major peaks and 2 minor peaks i n the chromato-grams of a l l samples. When the retention times of these peaks were compared with standards of monoterpenes avai l a b l e , two peaks i n the samples could be i d e n t i f i e d with certainty. These peaks were of 3-carene and limonene. The two peaks, T a b l e V I I . E f f e c t o f S t o r a g e on A t t r a c t a n c y o f L o g s a n d E x t r a c t e d S a m p l e s . ! L i v i n g a n d Dead B a r k T o t a l V o l a t i l e E x t r a c t s ( p o n d e r o s a p i n e ) ( p o n d e r o s a p i n e ) P e r i o d of-.. P e r c e n t R e s p o n s e P e r i o d , of. P e r c e n t .Response S t o r a g e b y B e e t l e s S t o r a g e b y B e e t l e s 2 weeks 70 2 weeks 83 6 weeks J6 6 weeks 50 F i g u r e s b a s e d on 3 t e s t s . E a c h b e e t l e o b s e r v e d 5 t i m e s . ponderosa pine I (sapling) j (8 yrs.) western white pine (80 yrs.) ponderosa pine (80 yrs.) Tree ; Sample • ON • H 00 o • NO H • r - 1 O • 00 Peak No. 1 J CD P 1-3 >-3 c3 c3 CJ o CJ cj cj JB P • p P P 3 O o o o o o CD CD CD CD CD • r o CD p i H 1—1 H H rV O ON NO r o H • m • • • • 3 o o M O 00 . CD ON NjJ ON r o £• • • • • • • i °° NO 00 c o ON • -i c d CD CO ON ON r o r o £ ON O r o NO 00 • • • • • • 3 ON o NO Q • ! W CD M H H : r o r o NO ON r o • • • # o H o H o • • • o o t o • r o 18.9 20.0 H NO • Peak No. 7 C3 P cr CD < CH M set* CO <! crj 3* H- O O CD H* 3 H H cj c f CD £ P O CD cr c+ CD H H - 3 3 CD M c f H" p< CD P 3 : Hi m CD CO CJ O CD • p p O 0 3 0 H O H* W O 3 3 0 d ' d TO 3 3 0 CO CO PJ CQ CO H* e O c+ 3 H)U H* P- cj O 3 CD 3 3 p co P c f CD O c f C 3 H , cj c f cj CD H* CD P« 3 is O H« CD 3 CD <! CO P . c f H' c f CD 3* P " CD CJ CD C CJ O CJ fD 3 CQ 1—1 p 53 No. 1 a n d 3 w e r e o f p i n e n e . However i t r e m a i n e d u n i d e n t i f i e d a s t o w h i c h one o f them was a l p h a - p i n e n e a n d b e t a - p i n e n e ( A p p e n d i x I , I I a n d I I I ) . A c o n s i d e r a b l e d i f f e r e n c e a p p e a r e d i n t h e q u a n t i t a t i v e c o m p o s i t i o n o f r e s o l v e d p e a k s o f t h e a b o v e t r e e s a m p l e s . The q u a n t i t a t i v e d i f f e r e n c e s w e r e c a l c u l a t e d f r o m t h e p e a k a r e a s a n d r e p r e s e n t e d a s p e r c e n t a g e s . T a b l e V I I I shows t h e p e r c e n t a g e c o m p o s i t i o n o f i n d i v i d u a l p e a k s o f p o n d e r o s a p i n e a n d w e s t e r n w h i t e p i n e . DISCUSSION 1) A n e m o - o l f a c t o r y R e s p o n s e t o H o s t O d o r s j The r e s p o n s e o f D. p o n d e r o s a e f l o w n a d u l t s t o a n o d o r b e a r i n g a i r - s t r e a m i n t h e o l f a c t o m e t e r was p o s i t i v e l y anemo-o l f a c t o r y . B e e t l e s when r e l e a s e d i n t h e o l f a c t o m e t e r , i n i -t i a l l y e i t h e r o r i e n t a t e d a n d p r o c e e d e d t o w a r d t h e s o u r c e o f l i g h t o r w a l k e d i n t h e d i r e c t i o n i n w h i c h t h e y w e r e a l r e a d y p o i n t i n g . As t h e y a r r i v e d i n t h e o d o r - s t r e a m , t h e y c h a n g e d t h e i r c o u r s e . They r e a c h e d t h e s o u r c e o f o d o r e i t h e r b y m a k i n g a few t u r n s o f 90° t o 360 0 a n d r e t r i e v i n g t h e i r p a t h i n t h e o d o r - s t r e a m o r by a l i g n i n g t h e i r c o u r s e w i t h i t . The f i n d i n g s on a n e m o - o l f a c t o r y r e s p o n s e s i n t h e p r e s e n t s t u d y w i t h m o u n t a i n p i n e b e e t l e a r e i n a g r e e m e n t w i t h t h o s e o f F r a n c i a a n d Graham (1967) f o r t h e t w o - l i n e d a m b r o s i a b e e t l e . The n e e d f o r s u b j e c t i n g t h e b e e t l e s t o f l i g h t " e x e r c i s e " i n o r d e r t o l o w e r t h e t h r e s h o l d o f r e s p o n s e t o o d o r s i n t h e p r e s e n c e o f l i g h t , c o n f o r m s w i t h t h e f i n d i n g s o f Graham (1959)» F r a n c i a a n d Graham (1967), a n d B e n n e t t and B o r d e n (1971). F l o w n b e e t l e s when t e s t e d a g a i n s t a i r - s t r e a m a n d l i g h t d i d n o t show any a n e m o t a x i s . The u p - s t r e a m o r i e n t a t i o n o f t h e m a j o r i t y o f b e e t l e s t o a n o d o r - s t r e a m was i n t e r p r e t e d a s s i g n i f y i n g t h a t t h e o d o r s w e r e " a t t r a c t i v e . " S u c h r e s p o n s e t o a n o d o r - s t r e a m i n t h e o l f a c t o m e t e r s u g g e s t s t h a t t h e b e e t l e s o r i e n t t o w a r d s t h e s o u r c e o f o d o r b y a n a n e m o t a c t i c m e c h a n i s m w h i c h i s s w i t c h e d on i n t h e p r e s e n c e o f o d o r . O b s e r v a t i o n s a r e a l s o a v a i l a b l e 55 t o show t h a t t h e b e e t l e s may r e s p o n d t o a t t r a c t i v e o d o r s b y p r o c e e d i n g d o w n - s t r e a m ( n e g a t i v e a n e m o t a x i s a c c o r d i n g t o Kennedy a n d M o o r h o u s e , 1969t i n c a s e o f a r e p e l l e n t ) a n d l a t e r c h a n g e d t o p o s i t i v e a n e m o t a x i s . I t i s n o t c l e a r a t t h i s s t a g e w h a t s w i t c h e s o n t h e i r u p -s t r e a m o r d o w n - s t r e a m movements i n t h e o l f a c t o m e t e r . How-e v e r i t i s p o s s i b l e t h a t i f a t u r n o f 90° t o 180° i n t h e i n i t i a l s t a g e s when b e e t l e s r e s p o n d t o o d o r , d o e s n o t make them move u p - s t r e a m , t h e y c o n t i n u e t h e i r p a t h d o w n - s t r e a m a n d r e s p o n d t o o d o r k l i n o k i n e t i c a l l y . I t c o u l d a l s o be p o s s i b l e t h a t t h e c o n c e n t r a t i o n o f t h e v o l a t i l e compounds was n o t c o n -s t a n t i n t h e o d o r - s t r e a m when t h e b e e t l e was u n d e r t e s t f o r a c e r t a i n p e r i o d , t h u s s u g g e s t i n g t h e n e e d f o r q u a n t i f i c a t i o n o f t h e c h e m i c a l s t i m u l u s . The r e s p o n s e o f b e e t l e s t o l i v i n g a n d d e a d b a r k a n d e x t r a c t s o f p o n d e r o s a p i n e s a p l i n g s r e s u l t e d i n d o w n - s t r e a m a n e m o t a x i s . B e e t l e s d i d c r o s s o r i n t e r c e p t , b u t d i d n o t r e m a i n i n t h e o d o r - s t r e a m a n d moved p a r a l l e l t o t h e b o u n d a r i e s o f t h e o d o r . T h i s a p p a r e n t a v o i d a n c e r e s p o n s e i s t a k e n t o s i g n i f y r e p e l l e n c y o f t h e c h e m i c a l s . Kennedy a n d Mo o r h o u s e (1969) d e s c r i b e d a s i m i l a r r e s p o n s e b y g r a s s h o p p e r s t o r e -p e l l e n t g r a s s - o d o r s . Moeck (1970a) o b s e r v e d p o s i t i v e anemo-t a x i s o f T. l i n e a t u r n a l o n g t h e b o u n d a r y o f a n o d o r - s t r e a m o f a l p h a - p i n e n e . B e c a u s e t h e o d o r - s t r e a m a c t e d 4 i n t h e manner o f a n i n v i s i b l e b a r r i e r , he i n t e r p r e t e d t h e r e s p o n s e a s s i g n i f y i n g t h e r e p e l l e n c y o f t h e c h e m i c a l t o t h e i n s e c t . The p r e s e n t s t u d i e s on t h e a n e m o - o l f a c t o r y r e s p o n s e "by D. p o n d e r o s a e t o o d o r s show t h a t b e e t l e s a r e a t t r a c t e d t o m a t u r e h o s t t r e e s . T h i s e s t a b l i s h e s t h e p r e s e n c e o f p r i m a r y a t t r a c t a n t s i n p i n e l o g s . S i n c e t h e h o s t t r e e s i n t h e p r e s e n t s t u d y d i d n o t d i f f e r v e r y much i n t h e i r age c l a s s , l i t t l e d i f f e r e n c e was f o u n d i n t h e a t t r a c t a n c y o f p o n d e r o s a p i n e a n d w e s t e r n w h i t e p i n e l i v i n g a n d d e a d b a r k . T h i s s u g g e s t s t h a t t h e r e s h o u l d n o t be a n y q u a l i t a t i v e d i f f e r e n c e s i n t h e o d o r o u s compounds p r e s e n t i n t h e t w o h o s t s p e c i e s . T h i s i s c o n f i r m e d f r o m t h e d a t a on GLC a n a l y s i s and w i l l be d i s -c u s s e d l a t e r . 2) A t t r a c t a n c y a n d R e p e l l e n c y o f D i f f e r e n t Aged T r e e s i The r e s p o n s e o f t h e b e e t l e s t o h o s t o d o r s o f d i f f e r e n t age r e s u l t e d i n a r e m a r k a b l e c h a n g e . P o n d e r o s a p i n e a n d w h i t e p i n e t r e e s a b o v e 50 y e a r s o l d w e r e f o u n d t o be a t t r a c -t i v e . I n c o n t r a s t t h e p o n d e r o s a p i n e s a p l i n g s o f 8 y e a r s o l d w e r e r e p e l l e n t . T h e s e d i f f e r e n c e s i n t h e r e s p o n s e o f t h e b e e t l e s t o t h e i r h o s t t r e e s o f v a r i e d age p o s s i b l y e x p l a i n s t h e d i f f e r e n t i a l s e l e c t i o n b y b a r k - b e e t l e s o f c e r t a i n age g r o u p s w i t h i n a h o s t s p e c i e s . T h i s d i f f e r e n t i a l s e l e c t i o n o f h o s t t r e e s may be due t o q u a n t i t a t i v e d i f f e r e n c e s o f t h e o d o r o u s compounds w h i c h a c t a s a t t r a c t a n t s o r r e p e l l e n t s a t v a r i o u s c o n c e n t r a t i o n s when p r e s e n t i n t h e h o s t . D e t h i e r (1947b) a n d F r a n k e - G r o s s m a n ( 1 9 6 3 ) h a v e d e s c r i b e d a s i m i l a r phenomenon when o d o r o u s c h e m i c a l s a r e t e s t e d i n d i f f e r e n t c o n c e n t r a t i o n s . From t h e GLC a n a l y s i s a n d b i o a s s a y s t u d i e s o f t h e v o l a t i l e e x t r a c t s , i t became e v i d e n t t h a t t h e r e w e r e 57 no q u a l i t a t i v e d i f f e r e n c e s i n t h e c h e m i c a l c o m p o s i t i o n o f t h e two h o s t s p e c i e s . On t h e o t h e r h a n d , t h e r e w e r e d i f f e r -e n c e s i n t h e amounts o f e a c h compound p r e s e n t i n t h e e x t r a c t s . From T a b l e V I I I , i t i s e v i d e n t t h a t t h e 3 - c a r e n e p e r c e n t a g e was h i g h e r i n p o n d e r o s a p i n e s a p l i n g s a s c o m p a r e d t o m a t u r e t r e e s o f p o n d e r o s a p i n e a n d w e s t e r n w h i t e p i n e . The p e r c e n t -age o f l i m o n e n e was l o w i n p o n d e r o s a p i n e s a p l i n g s a s com-p a r e d t o m a t u r e t r e e s . H o wever, t h e r e w e r e some v a r i a t i o n s i n t h e amounts o f p e a k No. 1 a n d 3 ( p i n e n e i s o m e r s ) . P r e -s u m a b l y t h e c o n c e n t r a t i o n o f 3 - c a r e n e when p r e s e n t i n h i g h p e r c e n t a g e m i g h t h a v e a c t e d a s a r e p e l l e n t i n t h e c a s e o f p o n d e r o s a p i n e s a p l i n g s ; a n d when i t s amount d e c r e a s e d w i t h t h e i n c r e a s e i n t h e age o f t h e h o s t t r e e s , i t a c t e d a s a n a t t r a c t a n t . A n d e r s o n e t a l (1969a, 1969b) h a v e a n a l y z e d t h e v o l a t i l e c o n s t i t u e n t s o f v a r i o u s p i n e s . The p r i n c i p l e q u a l i t a t i v e d i f f e r e n c e s b e t w e e n t h e t e r p e n e c o m p o s i t i o n w e r e t h e p r e s e n c e o f 3 - c a r e n e i n s i g n i f i c a n t a m o u nts i n s u g a r p i n e a n d i t s " a p p a r e n t " a b s e n c e i n w e s t e r n w h i t e p i n e . The GLC a n a l y s i s was c a r r i e d o u t on 10 p e r c e n t b , b d i o x y p r o p i o n i t r i l e c o l u m n a n d e x t r a c t s w e r e o b t a i n e d b y e x h a u s t i v e s t e a m d i s t i l l a t i o n . D u r i n g t h e p r e s e n t s t u d y i d e n t i f i c a t i o n o f p i n e n e (peak No. 1 a n d 3), 3 - c a r e n e a n d l i m o n e n e p e r m i t s c o m p a r i s o n o f t h e r e -s u l t s w i t h t h o s e o b t a i n e d b y A n d e r s o n e t a l . I t i s e v i d e n t f r o m T a b l e V I I I t h a t 3 - c a r e n e was p r e s e n t i n w e s t e r n w h i t e p i n e b u t was i n l o w p r o p o r t i o n s o f 2.5 t o 3 p e r c e n t . S m i t h 58 (1964) s t u d i e d t h e v a r i a t i o n o f m o n o t e r p e n e s o f P i n u s p o n - d e r o s a * He c o n c l u d e d t h a t t h e d i f f e r e n c e s t h a t w e r e a t t r i b u -t a b l e t o v a r i a t i o n w i t h i n t h e t r e e , t o s e a s o n o f s a m p l i n g , t o t h e y e a r o f s a m p l i n g a n d method s o f a n a l y s i s by GLC w e r e o n l y s l i g h t . The s a m p l e s u s e d d u r i n g t h e p r e s e n t s t u d y w e r e ob-t a i n e d d u r i n g summer months a n d when a n a l y z e d d i d n o t i n d i c a t e much v a r i a t i o n . ( T a b l e V I I I ) T h e s e f i n d i n g s a r e i n a g r e e m e n t f o r t h e q u a n t i t a t i v e v a r i a t i o n o f v o l a t i l e compounds o f p o n -d e r o s a p i n e m a t u r e t r e e s w i t h t h o s e o b t a i n e d b y S m i t h . How-e v e r , R o b e r t s (1970) r e p o r t s on t h e v a r i a t i o n w i t h i n t r e e o f m o n o t e r p e n e c o m p o s i t i o n o f s l a s h p i n e . O l e o r e s i n f r o m n e e d l e s , b r a n c h - x y l e m , t r u n k - x y l e m a n d r o o t - x y l e m a l l d i f f e r e d f r o m one a n o t h e r i n c o m p o s i t i o n . W h e t h e r he r e f e r s t o v a r i a t i o n i n q u a l i t a t i v e o r q u a n t i t a t i v e c o m p o s i t i o n i s n o t c l e a r . V a r i o u s s t u d i e s on t h e a t t r a c t a n c y o f b a r k b e e t l e s h a v e r e s u l t e d i n t e s t i n g t h e v o l a t i l e compounds p r e s e n t i n h o s t t r e e s . C h a r a r a s (1959) s t a t e s t h a t t h e a t t r a c t a n c y o f b a r k b e e t l e s t o s u s c e p t i b l e h o s t t r e e s i s g o v e r n e d b y c h e m o t a c t i c r e s p o n s e s o f t h e b e e t l e s t o s u c h t u r p e n t i n e c o m p o n e n t s a s a l p h a - p i n e n e , b e t a - p i n e n e a n d l i m o n e n e . R u d i n s k y (1966) s u b s t a n t i a t e d t h e i n f o r m a t i o n f o r D o u g l a s - f i r b e e t l e a n d o t h e r b a r k b e e t l e s w h i c h showed t h a t t h e y w e r e a t t r a c t e d t o a l p h a - p i n e n e , camphene, l i m o n e n e , g e r a n i o l a n d a l p h a -t e r p i n e o l . Kangas ejt a l (1965t 1967) a n d Y a s u n a g a ejt a l (1962, I963) f o u n d compounds l i k e a l p h a - t e r p i n e o l a n d b e n z o i c a c i d a s a t t r a c t a n t s t o b a r k b e e t l e s r e s p e c t i v e l y . R e n w i c k 59 (1970) s t a t e s t h a t a l p h a - p i n e n e a n d t r a n s - v e r b e n o l ( b e e t l e p h e r o m o n e ) a r e a t t r a c t a n t t o D. p o n d e r o s a e when o f f e r e d t o g e t h e r a n d n e i t h e r compound i s a t t r a c t i v e on i t s own. From t h e s e d a t a a n d r e s u l t s f r o m t h e p r e s e n t s t u d y i t b e -comes e v i d e n t t h a t h o s t v o l a t i l e s do p l a y a m a j o r r o l e i n a t t r a c t i n g t h e b a r k b e e t l e s , a n d e m p h a s i z e t h e n e e d t o i n v e s t i g a t e t h e r o l e o f e a c h compound a l o n e a n d i n t h e p r e s e n c e o f o t h e r o d o r o u s compounds p r e s e n t . However t h e i r n o n - a t t r a c t a n c y o r r e p e l l e n c y when t e s t e d a l o n e c o u l d be o f d i f f e r e n t s i g n i f i c a n c e . I t m i g h t a l s o be p o s s i b l e t h a t t h e h o s t r a n g e i s d e t e r m i n e d on t h e one h a n d b y more o r l e s s s p e c i f i c r e s p o n s e s o f some c h e m o r e c e p t o r s t o a t t r a c t i v e s t i m u l u s a n d on t h e o t h e r h a n d b y t h e s e n s i -t i v i t y o f o t h e r r e c e p t o r s t o r e p e l l e n t compounds. Jermy (I965) h a s r e p o r t e d o n t h e f e e d i n g s t i m u l a n t s a n d i n h i b i -t o r s a n d t h e r o l e o f r e j e c t i v e s t i m u l i i n h o s t s e l e c t i o n o f p h y t o p h a g o u s i n s e c t s . P l a n t o d o r s a r e u s u a l l y m i x t u r e s o f f a i r l y c o m p l e x com-p o u n d s . Many o d o r o u s f a c t o r s o f t e n i n s p e c i f i c c o m b i n a t i o n s a r e t y p i c a l f o r a s i n g l e p l a n t s p e c i e s o r h i g h e r t a x o n ( S c h o o n h o v e n , 1968). D. p o n d e r o s a e i s i n i t i a l l y a t t r a c t e d b y v o l a t i l e compounds o f t h e t r e e s w h i c h a r e common t o t h e two h o s t s s t u d i e d h e r e . A t l e a s t t h i s i s s u g g e s t e d by t h e GLC a n a l y s i s o f t h e two h o s t s p e c i e s i n t h e p r e s e n t s t u d i e s . 6o However t h i s a s p e c t must be e x p l o r e d i n d e t a i l . The g r e a t e r a t t r a c t a n t e f f e c t o f t h e v o l a t i l e e x t r a c t s may b e t h e r e s u l t o f a p a r t i c u l a r c o n c e n t r a t i o n o f v o l a t i l e c o n s t i t u e n t s w h i c h c o u l d be o b t a i n e d b y t h e m e t h o d d e s c r i b e d i n t h e p r e s e n t s t u d y . H owever, a c o m p a r i s o n o f t h e e f f e c t o f o d o r o u s compounds o f h o s t a n d n o n - h o s t t r e e s on a s i n g l e s p e c i e s o f b a r k - b e e t l e m i g h t c o n t r i b u t e t o a n u n d e r s t a n d i n g o f t h e " c h e m i c a l s p e c i -f i c i t y " a n d h o s t s e l e c t i o n b y t h e s e i n s e c t s (Graham, 1971a p e r s o n a l c o m m u n i c a t i o n ) . 3) R o l e o f E t h a n o l i Moeck (1970b) d e s c r i b e d t h e a t t r a c t a n c y o f e t h a n o l f o r T. l i n e a t u m a n d o t h e r s c o l y t i d s . T h i s was c o n f i r m e d by Cade e t a l (1970). From t h e r e s u l t s o f t h e p r e s e n t s t u d y , i t i s e v i d e n t t h a t , t o t h e m o u n t a i n p i n e b e e t l e s , v a r i o u s c o n c e n t r a -t i o n s o f e t h a n o l a c t e d a s a n " a r r e s t a n t . " The c e s s a t i o n o f l o c o m o t i o n i n t h e o d o r s t r e a m c o m b i n e d w i t h k l i n o k i n e t i c r e s p o n s e a n d s l o w o r t h o k i n e s i s ( D e t h i e r e t a l , I 9 6 0 ; B e c k , 1965) was a c l e a r i n d i c a t i o n o f a n a r r e s t a n t e f f e c t . T. l i n e a t u m d o e s n o t a t t a c k l i v i n g , f r e s h l y f e l l e d , c u t o r w i n d -t h r o w n t r e e s (Chapman, 1961; Graham, 1963; R u d i n s k y , 1 9 6 2 ) . I t i s p r o b a b l e t h a t t h i s b e e t l e i s a t t r a c t e d b y t h e p r o d u c t s o f d e t e r i o r a t i o n a n d f e r m e n t a t i o n (Graham, 1968; Graham a n d W e r n e r , 1956; R u d i n s k y , 1 9 6 6 a ) . M o r e o v e r t h i s b e e t l e i n v a d e s a w i d e r a n g e o f c o n i f e r o u s h o s t s a n d i t s s e l e c t i o n f o r c e r t a i n h o s t s p e c i e s b a s e d on t h e i r " t y p i c a l " o r s p e c i f i c o d o r s w o u l d be a n u n e x p e c t e d phenomenon. Thus i t i s t o be e x p e c t e d t h a t 61 t h e c h e m i c a l common t o a l l h o s t s w i l l be t h e one s e r v i n g t o f a c i l i t a t e t h e b e e t l e i n s e l e c t i o n o f s u i t a b l e l o g s . O l f a c -t o r y r e s p o n s e t o e t h a n o l i s t h e s t i m u l u s w h i c h s e r v e s a s a " p r i m a r y a t t r a c t a n t . " E t h a n o l a c t s a s a t u n n e l i n g ( f e e d i n g ) s t i m u l a n t i n t h e d i e t s f o r X y l e b o r u s f e r r u g i n e u s ( N o r r i s a n d B a k e r , 1969)» I t s i n f l u e n c e on D. p o n d e r o s a e a s a n a r r e s t a n t may be i n v o l v e d i n s t o p p i n g o r s l o w i n g t h e f l i g h t o f b e e t l e s when p r e s e n t i n s u s c e p t i b l e h o s t t r e e s . The p r e s e n c e o f e t h a n o l i n l i v i n g t r e e s w h i c h a r e s u s c e p t i b l e t o b a r k b e e t l e s h a s n o t y e t b e e n shown. 4) G e n e r a l Remarks i I n many b i o a s s a y t e s t s , more t h a n 50 p e r c e n t o f t h e b e e t l e s r e s p o n d e d t o a t t r a c t i v e o r r e p e l l e n t h o s t o d o r s . I t i s d i f f i c u l t a t t h i s s t a g e t o e x p l a i n t h e n e g a t i v e o l f a c -t o r y b e h a v i o r o f t h e r e m a i n i n g b e e t l e s . E v e n t h o s e b e e t l e s w h i c h r e s p o n d e d t o c h e m i c a l s t i m u l u s , when t e s t e d a g a i n w i t h t h e same s t i m u l u s , d i d n o t a l w a y s r e p e a t t h e i r i n i t i a l r e s p o n s e . T h e s e o b s e r v a t i o n s i n d i c a t e t h a t t h e b e h a v i o r o f i n d i v i d u a l b e e t l e s i n a t e s t v a r i e d c o n s i d e r a b l y f r o m t h e a v e r a g e a n d was n o t p r e d i c t a b l e . However t h e r e was some c o n s i s t e n c y i n t h e r e s p o n s e o f f l i g h t - e x p e r i e n c e d b e e t l e s t o s u c h s t i m u l i i n a l l t e s t s . A t k i n s (1966a) d e s c r i b e d s i m i l a r v a r i a t i o n s i n t h e r e s p o n s e o f i n d i v i d u a l s o f D, p s e u d o t s u g a e when t e s t e d a g a i n s t v a r i o u s s t i m u l i u n d e r l a b o r a t o r y c o n d i t i o n s . The e c o l o g i c a l s i g n i f i c a n c e o f b e h a v i o r a l v a r i a t i o n among s c o l y -t i d s i n r e l a t i o n t o t h e i r h a b i t a t h a s b e e n d i s c u s s e d ( A t k i n s , 62 1966b; B e d a r d , 1966). I t i s n e c e s s a r y t o s t u d y t h e p a r a -m e t e r s o f t h e s e v a r i a t i o n s i n t h e r e s p o n s e o f s c o l y t i d s . T h e s e v a r i a t i o n s c o u l d be due t o v a r i a t i o n i n t h e p h y s i o -l o g i c a l c o n d i t i o n s o r g e n e t i c n a t u r e o f b e e t l e s i n a p o p u l a -t i o n ( A t k i n s , 1966b; Graham, 1971b; p e r s o n a l c o m m u n i c a t i o n ) . SUMMARY AND CONCLUSIONS The p r e s e n t s t u d i e s d e m o n s t r a t e t h e e x i s t e n c e o f e x t r a c t a b l e v o l a t i l e o r g a n i c s u b s t a n c e s i n t h e l i v i n g a n d d e a d b a r k o f p o n d e r o s a p i n e a n d w e s t e r n w h i t e p i n e w h i c h e l i c i t p o s i t i v e o r i e n t a t i o n r e s p o n s e s i n t h e m o u n t a i n p i n e b e e t l e . Q u a n t i t a t i v e d i f f e r e n c e s i n t h e p r o p o r t i o n s o f d i f f e r e n t c o n s t i t u e n t s a r e a s s o c i a t e d w i t h d i f f e r e n c e s i n r e l a t i v e s u s c e p t i b i l i t y o f y o u n g a n d o l d t r e e s , r e s p e c -t i v e l y . I t i s i n f e r r e d h e r e t h a t c e r t a i n o f t h e c o n s t i t u -e n t s c a n p r o v i d e g u i d i n g c u e s f o r t h e b e e t l e i n n a t u r e , a n d c o n s t i t u t e w h a t may b e d e s i g n a t e d a s " p r i m a r y a t t r a c t a n t s . " The f o l l o w i n g c o n c l u s i o n s a r e b a s e d o n o b s e r v a t i o n s o f p e d e s t r i a n f l i g h t " e x e r c i s e d " f e m a l e b e e t l e s . 1) P o s i t i v e ( u p - s t r e a m o r d o w n - s t r e a m ) a n e m o t a x i s i n a n a i r - s t r e a m c o n t a i n i n g v o l a t i l e e x t r a c t s o f l i v i n g a n d d e a d b a r k was i n t e r p r e t e d a s a p o s i t i v e r e s p o n s e t o a t t r a c t i v e o d o r s , w h e r e a s a c o u r s e t a k e n a l o n g t h e m a r g i n o f t h e a i r - s t r e a m was i n t e r p r e t e d a s a n a v o i d a n c e r e s p o n s e t o r e p e l l e n t o d o r s . 2) The b e e t l e s showed a p o s i t i v e a t t r a c t a n t r e s p o n s e t o o d o r s p r e s e n t i n t h e l i v i n g a n d d e a d b a r k o f p o n d e r o s a p i n e a n d w e s t e r n w h i t e p i n e m a t u r e t r e e s . 3) The o d o r s p r e s e n t i n t h e l i v i n g a n d d e a d b a r k o f p o n d e r o s a p i n e s a p l i n g s (8 y e a r s o l d ) w e r e r e p e l l e n t t o a d u l t f l o w n b e e t l e s . 64 The v o l a t i l e e x t r a c t s a n d t h e i r e t h e r s o l u b l e f r a c t i o n s o f p o n d e r o s a p i n e and w e s t e r n w h i t e p i n e m a t u r e t r e e s o b t a i n e d t h r o u g h s u b l i m a t i o n p r o c e s s w e r e a t t r a c t a n t and t h a t o f p o n d e r o s a p i n e s a p l i n g s r e p e l l e n t . The s u b l i m a t i o n t e c h n i q u e h a s c e r t a i n a d v a n t a g e s o v e r c o n v e n t i o n a l m e t h o d s . The GLC a n a l y s i s o f v o l a t i l e e x t r a c t s o f t h e a b o v e s a m p l e s showed no q u a l i t a t i v e d i f f e r e n c e s i n c h e m i c a l c o n s t i t u -e n t s . D i f f e r e n t c o l u m n s c o u l d r e s o l v e s e v e n p e a k s . F o u r o f t h e m w e r e i d e n t i f i e d a s p i n e n e ( i d e n t i t y o f i s o m e r s n o t k n o w n ) , 3 - c a r e n e a n d l i m o n e n e . The r e s t o f t h e t h r e e p e a k s r e m a i n unknown. T h e r e w e r e q u a n t i t a t i v e d i f f e r e n c e s i n t h e p r o p o r t i o n a t e a mounts o f e a c h c h e m i c a l p r e s e n t i n v o l a t i l e e x t r a c t s o f p o n d e r o s a p i n e , w e s t e r n w h i t e p i n e m a t u r e t r e e s a n d a l s o p o n d e r o s a p i n e s a p l i n g s . From t h e GLC a n a l y s i s a n d b i o a s s a y s t u d i e s , i t i s c o n -c l u d e d t h a t p r i m a r i l y q u a n t i t a t i v e d i f f e r e n c e s i n t h e v o l a t i l e c o n s t i t u e n t s may be i n v o l v e d i n a t t r a c t i n g o r r e p e l l i n g t h e b e e t l e s . E t h a n o l a t d i f f e r e n t c o n c e n t r a t i o n s e x e r t e d a n a r r e s t a n t i n f l u e n c e . However i t i s c o n c e i v a b l e t h a t e t h a n o l a c t i n g a s a n a r r e s t a n t may p l a y a r o l e i n t h e s e t t l i n g o f b e e t l e s on " t a r g e t " m a t e r i a l . S t o r a g e o f l o g s a t -15°C o r a t 0°C a n d v o l a t i l e e x t r a c t s a t 0°C f o r a p e r i o d o f two t o s i x w e e k s , w h i c h w e r e 65 o r i g i n a l l y a t t r a c t i v e , i s a c c o m p a n i e d b y d e c r e a s e o f a t t r a c t a n c y . 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Can. Entomol. 98 »1083-1093. Yasunaga, K., Y. Oshima, and Y. Kuwatsuka. 1962. Attractants of the pine hark beetles. I. Isolation of an attractant Benzoic Acid from red pine bark. J. Agric. Chem. Soc. Japan 3_6« 802-804. t 1963. Studies on attractants of the pine bark beetles. I II. Screening tests of attractancy and synergism of benzoic acid derivatives, higher f a t t y acids and t h e i r esters and terpineols. J. Agric. Chem. Soc. Japan 37t642-644. APPENDICES Appendix I Chromatogram of ether soluble f r a c t i o n (mature western white pine) Column - Apizeon L 5% on chromosorb P acid washed 60/80 mesh. Temperature - oven 90°C injector 135°C Nitrogen 30 cc/min. Peaks 1 & 3» pinene (isomers unknown) 2, unknown 4. unknown 5. 3-carene 6. unknown ?. limonene Appendix II Chromatogram of ether soluble f r a c t i o n (mature ponderosa pine) Column - Apizeon L 5#> on chromosorb P acid washed 60/80 mesh. Temperature - oven 90°C injector 135 C Nitrogen 30 cc/min. Peaks 1 & 3. 2. k. 5. 6. 7. pinene (isomers unknown) unknown unknown 3-carene unknown limonene o • H +» O B cd 18 15 1 2 9 6 Retention time (in minutes) Appendix III Chromatogram of ether soluble f r a c t i o n (ponderosa pine sapling) Column - Apizeon L 5% on Peaks chromosorb P acid washed 60/80 mesh 1 & Temperature Nitrogen 3 0 cc/min. oven 90°G injecxor 135°C 3. 2. k. o . ? . pinene (isomers unknown) unknown }. unknown 5 3-carene unknown limonene 18 15 12 9 6 Retention time (in minutes, 

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