Patterns in Defensive Natural Product Chemistry: Douglas Fir and

1 Patterns in Defensive Natural Product Chemistry: Douglas Fir and Western Spruce Budworm Interactions REX G. C A T E S , R I C H A R D A . R E D A K , and C O L I N B . H E N D E R S O N

Downloaded by 80.82.77.83 on May 29, 2018 | https://pubs.acs.org Publication Date: January 20, 1983 | doi: 10.1021/bk-1983-0208.ch001

University of New Mexico, Chemical Ecology Laboratory, Department of Biology, Albuquerque, NM 87131

Studies were undertaken to t e s t various aspects of current p l a n t - h e r b i v o r e theory. P a r t i c u l a r emphasis was given to i n v e s t i g a t i n g the e f f e c t s of D o u g l a s - f i r f o l i a g e q u a l i t y on the success of the western spruce budworm. Results i n d i c a t e d that the most important v a r i a b l e s i n f l u e n c i n g budworm success were the concentrations of s e v e r a l s p e c i f i c terpenes and changes i n the distribution of foliar terpenes. A d d i t i o n a l l y , the p r o d u c t i v i t y of trees was i n v e r s e l y c o r r e l a t e d with budworm success. The a v a i l a b l e foliar n i t r o g e n did not appear to be an important f a c t o r determining i n s e c t success. F i n a l l y , escape i n time appears to be another f a c t o r limiting i n s e c t utilization of newly emerged D o u g l a s - f i r t i s s u e .

Some of the major o b j e c t i v e s i n coevolutionary b i o l o g y are to e x p l a i n the patterns of interaction between plants and h e r b i v o r e s , the s e l e c t i o n pressures maintaining these p a t t e r n s , the ways in which these patterns differ among plant communities, and how they might change over e c o l o g i c a l and e v o l u t i o n a r y time ( 1 ) . The r e c e n t l y developed theory of plant phytophagous i n s e c t i n t e r a c t i o n s suggests that the population biology of phytophagous insects i s influenced significantly by the diversity of plant species w i t h i n a community, the i n t e r t w i n i n g r e l a t i o n s h i p s among invidual p l a n t s , the predictability of the plant resources i n space and time to h e r b i v o r e s , the nutritional l e v e l s of plant t i s s u e s of various growth forms, and the d i v e r s i t y of mechanical and chemical defense mechanisms of plant t i s s u e s (1-6). This theory has sparked considerable research e f f o r t w i t h i n s e v e r a l d i f f e r e n t areas. Appropriate i n v e s t i g a t i o n s , however, of the ways i n which the above f a c t o r s i n t e r a c t i n molding plant and h e r b i v o r e l i f e h i s t o r i e s and community s t r u c t u r e are only beginning to surface i n the l i t e r a t u r e ( 3 _ 7 - 9 ) . Since we wish to address various aspects of current p l a n t - h e r b i v o r e theory, a b r i e f d i s c u s s i o n of some of the b a s i c premises f o l l o w s , emphasizing those that p e r t a i n to woody

0097-6156/83/0208-0003$06.00/0 © 1983 American Chemical Society

Hedin; Plant Resistance to Insects ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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PLANT RESISTANCE TO INSECTS

p e r e n n i a l s and t h e i r associated h e r b i v o r e s . Rhoades and Cates (6) suggest that plant resources f o r herbivores vary along a continuum of i n c r e a s i n g p r e d i c t a b i l i t y . Resources such as l o n g - l i v e d woody p e r e n n i a l s , or mature, evergreen l e a f t i s s u e s , are suggested to be p r e d i c t a b l e , or i n Feeny's (5) terminology, apparent, resources to h e r b i v o r e s . Examples, on the other hand, of the unpredictable or unapparent resources that may vary g r e a t l y i n t h e i r occurrence and a v a i l a b i l i t y to herbivores are the ephemeral annuals or the q u i c k l y developing young l e a f t i s s u e s of perennials ( 6 ) . These d i f f e r e n c e s i n plant and t i s s u e p r e d i c t a b i l i t y and a v a i l a b i l i t y as a food resource to h e r b i v o r e s , along with various t i s s u e developmental c o n s t r a i n t s , are suggested to be important determinants of the defensive system evolved by a plant (_1 6 ) . Although p l a n t s produce many secondary products, chemical defensive compounds can be categorized on the b a s i s of f u n c t i o n as t o x i n s or d i g e s t i b i l i t y - r e d u c i n g substances. Toxins are t r e a t e d u s u a l l y as small molecular weight compounds that a f f e c t the metabolic processes w i t h i n herbivores and include cyanogenic g l y c o s i d e s , c a r d i a c g l y c o s i d e s , terpenes, a l k a l o i d s , and numerous o t h e r s . D i g e s t i b i l i t y - r e d u c i n g substances, on the other hand, are o f t e n l a r g e molecular weight substances capable of bonding w i t h p r o t e i n s and polysaccharides r e s u l t i n g i n a complex that may be d i f f i c u l t to d i g e s t . These two c l a s s e s are not a b s o l u t e l y d i s t i n c t but represent a continuum, and i n some cases, compounds may f u n c t i o n both as a t o x i n as w e l l as a p r o t e i n complexing substance ( 6 ) . The a v a i l a b l e data suggest that t o x i n s , or q u a l i t a t i v e defenses, are c h a r a c t e r i s t i c of ephemeral p l a n t s and plant t i s s u e s , and that d i g e s t i b i l i t y - r e d u c i n g substances, or q u a n t i t a t i v e defenses, appear to be c h a r a c t e r i s t i c of p r e d i c t a b l e p l a n t s and plant t i s s u e s (_5,6). Furthermore, unpredictable plant resources are thought to escape i n space and/or time from t h e i r herbivores to a l a r g e r extent than do p r e d i c t a b l e resources ( 6 ) . This escape i n space and/or time may be more e f f e c t i v e against s p e c i a l i z e d or monophagous-oligophagous herbivores than against the g e n e r a l i z e d or polyphagous herbivores since the former l a c k a l t e r n a t e food resources. Our o b j e c t i v e i s to examine some aspects of current plant h e r b i v o r e theory using D o u g l a s - f i r (Pseudotsuga m e n z i e s i i ) and western spruce budworm (Choristoneura o c c i d e n t a l i s ) . Both plant and herbivore are widespread i n western North America. N a t u r a l hosts of the budworm i n c l u d e D o u g l a s - f i r , species of A b i e s , and, on occasion, other c o n i f e r s ( 9 ) . V a r i a t i o n i n budworm d e n s i t y occurs on both a geographic and l o c a l s c a l e . We have f r e q u e n t l y observed d i f f e r e n t i a l d e f o l i a t i o n ' i n trees having overlapping crowns at s i t e s i n Montana, Idaho, and New Mexico. I n t e r e s t i n g l y , considerable geographic v a r i a t i o n i s known to e x i s t i n the terpene chemistry of D o u g l a s - f i r (10,11). Our


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p r e l i m i n a r y data show that t h i s v a r i a t i o n a l s o e x i s t s at a l o c a l l e v e l (Table I ) . Table I . V a r i a t i o n i n some of the chemical c o n s t i t u e n t s i n the young needles of D o u g l a s - f i r from s i t e s a t Boulder, Montana (MT) and Taos, New Mexico (NM). Value


Constituent

Site

X

SD

Min

Max

1.8 1.3

0.5 0.1

1.3 1.1

3.5 1.4

T o t a l Nitrogen

MT NM

a-pinene

MT NM

176.5 105.1

223.6 48.4

0.0 34.0

1001.0 221.0

Camphene

MT NM

62.9 190.8

63.3 84.4

0.0 67.0

211.0 351.0

0-pinene

MT NM

134.8 75.9

93.7 52.5

0.0 3.0

429.0 221.0

Limonene

MT NM

84.7 81.5

69.2 38.0

0.0 24.0

403.0 193.0

B o r n y l Acetate

MT NM

63.1 92.6

38.4 42.1

6.0 26.0

189.0 190.0

Cadinene Isomer

MT NM

6.9 4.4

5.4 8.1

0.0 0.0

33.2 32.0

T o t a l Terpenes

MT NM

502.1 270.2

147.8 246.0

2442.5 1119.0

Polyphenols

MT NM

0.2 9.1

0.1 0.0

1.0 40.0

713.6 616.6 0.4 16.6

Polyphenols = % f r e s h wt; n i t r o g e n =» % dw; terpenes counts/20 mg t i s s u e Objectives Our focus was upon n a t u r a l and experimental s t u d i e s that were designed to I n v e s t i g a t e v a r i o u s aspects of the current p l a n t - h e r b i v o r e theory using the D o u g l a s - f i r / w e s t e r n spruce budworm system. S p e c i f i c a l l y , we wished to t e s t whether there are chemical c h a r a c t e r i s t i c s l a the young needle t i s s u e of D o u g l a s - f i r that reduce the growth, s u r v i v a l , and adult f e c u n d i t y of the western spruce budworm. Secondly, we were i n t e r e s t e d i n



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e l u c i d a t i n g the r e l a t i o n s h i p between budworm success and p h y s i c a l a t t r i b u t e s of D o u g l a s - f i r . F i n a l l y , we desired to determine whether there are any changes i n f o l i a g e q u a l i t y due to increased water s t r e s s that could account f o r v a r i a b i l i t y i n budworm success. Resistance i s defined as the s u i t e of " h e r i t a b l e c h a r a c t e r i s t i c s by which a plant species, race, clone or i n d i v i d u a l may reduce the p r o b a b i l i t y of s u c c e s s f u l u t i l i z a t i o n of that plant as a host by an i n s e c t species, race, biotype, or i n d i v i d u a l " ( 2 ) . The important aspect of t h i s concept i s that r e s i s t a n c e c h a r a c t e r i s t i c s are h e r i t a b l e , even though they may be modified by the p h y s i c a l environment. With regard to the above c o n s i d e r a t i o n s and questions, emphasis was placed upon determining the e f f e c t s of v a r i a t i o n i n n u t r i t i o n a l (water, n i t r o g e n sources) as w e l l as secondary chemical c h a r a c t e r i s t i c s (terpenes, r e s i n a c i d s , polyphenols, d i g e s t i b i l i t y - r e d u c i n g c a p a c i t y ) i n the f o l i a g e of D o u g l a s - f i r trees upon budworm l a r v a l growth, s u r v i v a l , d e n s i t y , l e v e l of d e f o l i a t i o n , a d u l t dry weight, and f e c u n d i t y . Several p h y s i c a l and phenological measurements of the trees were made at each s i t e i n c l u d i n g age, h e i g h t , dbh, crown diameter, crown r a t i o , bole r a d i u s , f i v e year growth increment, average internode l e n g t h , xylem pressure p o t e n t i a l , and time of budburst. S i t e S e l e c t i o n and General Methodology Resistance-Susceptibility

Studies

Studies to determine the l e v e l of r e s i s t a n c e or s u s c e p t i b i l i t y due to f o l i a g e q u a l i t y and tree p h y s i c a l parameters of D o u g l a s - f i r to the budworm were conducted at the Boulder, Montana and Barley Canyon, New Mexico s i t e s . The study a t the Montana s i t e involved trees exposed to moderate budworm d e n s i t i e s , while the study at Barley Canyon was done using budworm that were placed on trees that had very low n a t u r a l d e n s i t i e s of budworm. Near Boulder, Montana i n the Deer Lodge N a t i o n a l Forest a s i t e was s e l e c t e d that c o n s i s t e d of a near monoculture of D o u g l a s - f i r i n which were s c a t t e r e d ponderosa pine, lodgepole p i n e , and j u n i p e r s . During May and e a r l y June, 1979, 147 trees were located and tagged at the s i t e which was a t an e l e v a t i o n of 1620 m. The i n i t i a l s e l e c t i o n of these trees was done such that a l l were of s i m i l a r age, h e i g h t , dbh, and crown diameter. Trees were selected that were growing i n s i m i l a r microenvironments ( p r o x i m i t y to drainage, angle and a t t i t u d e of slope, and distance t o neighboring t r e e s ) . A f t e r the i n i t i a l s e l e c t i o n a more exact determination of the above p h y s i c a l parameters was made, and from the i n i t i a l 147 t r e e s , 80 were s e l e c t e d f o r t h i s r e s i s t a n c e - s u s c e p t i b i l i t y study.



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N a t u r a l budworm d e n s i t i e s were determined by sampling 6 sprays, each 40 cm long, i n the same quarter of the tree used to c o l l e c t t i s s u e f o r chemical a n a l y s i s and to c o l l e c t d e f o l i a t i o n data. D e n s i t i e s were expressed as the average number of budworm l a r v a e per 100 buds per t r e e . A v i s u a l estimate of the amount of d e f o l i a t i o n also was made i n the same a r e a of the crown where the d e n s i t i e s and needle t i s s u e were c o l l e c t e d . Since budworm may d i s p e r s e from h e a v i l y d e f o l i a t e d t r e e s , (Greenbank, 1963) budworm d e n s i t i e s from each tree were weighted by the l e v e l of d e f o l i a t i o n that each tree sustained. This r e s u l t e d i n an i n f e s t a t i o n i n t e n s i t y measurement (dependent v a r i a b l e ) which was subjected to m u l t i p l e stepwise c o r r e l a t i o n a n a l y s i s using v a r i o u s f o l i a g e q u a l i t y and p h y s i c a l tree parameters as the independent v a r i a b l e s . Thirty-one parameters were used as independent variables i n this analysis. F i f t e e n to 20 g of the young or current year's f o l i a g e were c o l l e c t e d during the time period that corresponded to the 4 t h and 5th l a r v a l i n s t a r . The t i s s u e was f r o z e n , and returned to the l a b o r a t o r y a t the U n i v e r s i t y of New Mexico f o r a n a l y s i s . Polyphenols and p r o t e i n complexing c a p a c i t y were determined by p r e c i p i t a t i n g the polyphenols, weighing t h i s f r a c t i o n , r e d i s s o l v i n g i n 20% acetone, and f i n a l l y , measuring the p r o t e i n complexing c a p a c i t y of the e x t r a c t using a 1.5% buffered g e l a t i n s o l u t i o n . This method, w i t h s l i g h t m o d i f i c a t i o n , i s taken from Feeny and Bostock (13) and Feeny ( 1 4 ) . T o t a l n i t r o g e n was determined using standard m i c r o k j e l d a h l d i g e s t i o n , and terpenes were analyzed using a P e r k i n Elmer 3920 gas chromatograph equipped w i t h a P e r k i n Elmer 0.10 i n x 150 f t c a p i l l a r y column packed with 85% 0S-138, 14% CO-880, and 1% V-930. The method foe terpene a n a l y s i s followed Redak (1982) w i t h only minor m o d i f i c a t i o n s i n column loading time. Dr. D. F. Z i n k e l (Forest Products Laboratory, USDA, Madison, Wisconsin) k i n d l y analyzed the young f o l i a g e f o r r e s i n a c i d s . The r e s i n a c i d s were not detected and no d e t a i l e d a n a l y s i s of the needles f o r these chemicals was done i n any of our s t u d i e s . The r e s i s t a n c e - s u s c e p t i b i l i t y study, where budworm larvae were placed on trees that n a t u r a l l y had few, i f any, budworm, was conducted In 1980 a t the Barley Canyon s i t e i n the Santa Fe N a t i o n a l F o r e s t . This s i t e was approximately 3.2 km long and 0.5 km wide at an average e l e v a t i o n of 2440 m. The dominant v e g e t a t i o n included D o u g l a s - f i r , white f i r , ponderosa pine, and aspen. Tree s e l e c t i o n was i d e n t i c a l to that described f o r the Montana s i t e except that i n i t i a l l y 200 t r e e s were s e l e c t e d . From t h i s group 105 t r e e s e v e n t u a l l y were used f o r the study reported here. T h i r t y - f o u r of the 105 were trenched i n 1980 to induce water s t r e s s i n 1981. One-way m u l t i p l e a n a l y s i s of variance showed no s i g n i f i c a n t d i f f e r e n c e i n the measurements of 34 f o l i a g e q u a l i t y and tree p h y s i c a l c h a r a c t e r i s t i c s between the 34 t r e e s that were trenched i n 1980 and the remaining 71 trees ( 1 5 ) .



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Consequently a l l 105 trees were used i n the a n a l y s i s reported here. Larvae were c o l l e c t e d from a nearby i n f e s t e d area and transported i n the cool of the evening to the U n i v e r s i t y of New Mexico l a b o r a t o r y (15). Approximately 3000, 4th i n s t a r larvae were c o l l e c t e d from the i n f e s t e d s i t e and placed i n v i a l s c o n t a i n i n g a small amount of young D o u g l a s - f i r t i s s u e . Each v i a l contained 25 l a r v a e . V i a l s were then returned to the environmental chamber to minimize l a r v a l s t r e s s . The f o l l o w i n g day the experimental larvae were placed on the s e l e c t e d trees at the Barley Canyon s i t e . Five l a r v a e were placed on each branch t h a t had at l e a s t 10 new, expanding f o l i a g e buds, and were contained on the branches using screen s l e e v e s . Seventy percent of f u l l s u n l i g h t penetrated these s l e e v e s . Five sleeves, each w i t h f i v e l a r v a e , were placed on each tree to y i e l d a t o t a l of 25 l a r v a e per t r e e . The l a r v a e were allowed to pupate before t r a n s f e r r i n g them to the l a b o r a t o r y where a d u l t s emerged and were sexed. L a r v a l s u r v i v o r s h i p and adult dry weight could then be determined. In a d d i t i o n , f o r 81 randomly s e l e c t e d females, egg mass dry weight and numbers of eggs per female were determined to examine the r e l a t i o n s h i p between f e c u n d i t y and female dry weight (15) . During the 5th and 6th i n s t a r , approximately 30 g of f o l i a g e were c o l l e c t e d from the same side and i n the same midcrown l e v e l where the larvae were placed. The f o l i a g e samples were put i n z i p - l o c bags, transported on Ice to the l a b o r a t o r y , and frozen u n t i l analyzed f o r n i t r o g e n content and p r o t e i n complexing c a p a c i t y . Nitrogen was analyzed as above. P r o t e i n complexing c a p a c i t y was determined using the method described by Bate-Smith (16) w i t h minor m o d i f i c a t i o n s ( 1 5 ) . For the a n a l y s i s of f o l i a g e terpene content, 30 mg of young f o l i a g e were c o l l e c t e d and weighed i n the f i e l d , encapsulated i n indium tubing, placed on i c e , transported to the l a b o r a t o r y , and frozen u n t i l i t was analyzed f o r terpene content. Terpenes were analyzed as described by (15). A t o t a l of 34 v a r i a b l e s were used i n the m u l t i p l e stepwise c o r r e l a t i o n a n a l y s i s . Twenty-three were used to determine f o l i a g e q u a l i t y , while 11 v a r i a b l e s were used to define the p h y s i c a l and phenological a t t r i b u t e s of the sample t r e e s . The dependent v a r i a b l e s used were average adult female budworm dry weight and average adult male budworm dry weight f o r each t r e e . D e t a i l s are found i n Redak ( 1 5 ) . Water S t r e s s

Studies

Studies to determine the e f f e c t of water s t r e s s on f o l i a g e q u a l i t y and budworm success were conducted at 2 s i t e s west of Jemez s p r i n g s , New Mexico, Santa Fe N a t i o n a l F o r e s t . These s i t e s were chosen such that d i f f e r e n c e s i n water a v a i l a b i l i t y to D o u g l a s - f i r trees would be maximized. T h i r t y trees were s e l e c t e d



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that were growing on a north f a c i n g slope ( n o n - s t r e s s ) , while another 30 were selected from a south f a c i n g slope ( s t r e s s s i t e ) . A n a l y s i s of p r e l i m i n a r y data suggested that these trees d i d not d i f f e r i n i t i a l l y i n foliage quality characteristics. A d d i t i o n a l l y , a t the s t r e s s s i t e , about a t h i r d of the area beneath each tree was trenched to cut roots i n an e f f o r t to f u r t h e r maximize water s t r e s s . Xylem water p o t e n t i a l s were measured using a Scholander pressure bomb when the larvae were i n t h e i r 5th i n s t a r . Budworm larvae were placed on each of the t r e e s at the s i t e s as described above f o r the Barley Canyon study. Terpenes and n i t r o g e n were analyzed as described above. Polyphenols and p r o t e i n completing c a p a c i t y were measured as described f o r the Montana s i t e . Female adult budworm dry weights and the number of s u r v i v o r s of budworm were analyzed by m u l t i v a r i a t e a n a l y s i s of v a r i a n c e to t e s t f o r the e f f e c t s of s i t e and sex. Stepwise d i s c r i m i n a n t a n a l y s i s was used to determine i f tree chemical and p h y s i c a l parameters d i f f e r e d between s i t e s ( 1 7 ) . Results Resistance - S u s c e p t i b i l i t y Studies Using N a t u r a l Budworm D e n s i t i e s and " D e f o l i a t i o n

R e s u l t s of the data gathered a t the Montana s i t e i n d i c a t e that 50% of the v a r i a t i o n i n the n a t u r a l budworm i n f e s t a t i o n i n t e n s i t y v a r i a b l e was explained by 9 v a r i a b l e s (Table I I ) . The acetate f r a c t i o n of the terpenes, myrcene, an u n i d e n t i f i e d terpene, time of budburst, and bole radius were i n v e r s e l y c o r r e l a t e d with budworm i n f e s t a t i o n i n t e n s i t y , i n d i c a t i n g that some aspects of f o l i a g e q u a l i t y may confer r e s i s t a n c e against the budworm. The evenness i n the q u a n t i t a t i v e d i s t r i b u t i o n of the terpenes i n the f o l i a g e among the t r e e s , beta-pinene, t o t a l f o l i a r n i t r o g e n , and tree age were a l l c o r r e l a t e d p o s i t i v e l y with budworm i n f e s t a t i o n i n t e n s i t y . Examination of the standardized c o r r e l a t i o n c o e f f i c i e n t s i n d i c a t e d that the acetate f r a c t i o n of the terpenes, the q u a n t i t a t i v e d i s t r i b u t i o n of the terpenes i n the f o l i a g e among t r e e s , age, and time of budburst were the most important of the included v a r i a b l e s i n e x p l a i n i n g the v a r i a t i o n in infestation intensity. The evenness measurement, c a l c u l a t e d from the ShannonWiener formula, suggests that trees which have an uneven d i s t r i b u t i o n of terpenes are more r e s i s t a n t to the budworm. I t i s l i k e l y that t h i s imbalance i n the terpene d i s t r i b u t i o n i s represented by the s p e c i f i c terpenes (acetate f r a c t i o n , myrcene, and the u n i d e n t i f i e d terpene) that were found to be important i n the a n a l y s i s . The a n a l y s i s a l s o i n d i c a t e d that the polyphenol and p r o t e i n complexing c a p a c i t y of the e x t r a c t s from the f o l i a g e


10


were not important i n determining the budworm d e n s i t i e s and l e v e l s of d e f o l i a t i o n * Studies Using Experimental Female Budworm L e v e l s on Non-Infested Trees R e s u l t s of the data gathered at the Barley Canyon s i t e , where budworm were placed on trees that n a t u r a l l y had few l a r v a e , i n d i c a t e d that terpenes again were important i n determining


Table I I . M u l t i p l e c o r r e l a t i o n a n a l y s i s using i n f e s t a t i o n i n t e n s i t y as the dependent v a r i a b l e R^ = 0.50, p < 0.001). Boulder, Montana s i t e , 1979.

Independent Variable Acetate F r a c t i o n Evenness i n Terpene Distribution Tree Age Budburst Bole Radius 3-pinene T o t a l Nitrogen Myrcene Unidentified Terpene No. 10

Coefficient

Standard Error

Standardized Regression Coefficient

-0.17828

0.0432

-0.547

45.85419 0.38511 -3.05608 -1.74379 0.02624 5.17278 -0.03534

15.7133 0.1327 1.00207 0.6490 0.0138 2.3350 0.0162

0.389 0.339 -0.331 -0.302 0.246 0.239 -0.227

-0.22199

0.1247

-0.193

N i t r o g e n = % dw; Terpenes • area counts/20 mg dw budworm success (Table I I I ) . When the u n i d e n t i f i e d terpene, t o t a l n i t r o g e n , beta-pinene, and myrcene were i n high c o n c e n t r a t i o n i n the needles of D o u g l a s - f i r t r e e s , the adult female d r y weights were reduced s i g n i f i c a n t l y . The f i v e year growth increment a l s o was i n v e r s e l y r e l a t e d with female dry weight. When b o r n y l a c e t a t e , t e r p i n o l e n e , and geranyl acetate were higher i n the f o l i a g e , budworm success increased. A l s o , budburst, tree age, and twig internode l e n g t h f o r 1980 were a s s o c i a t e d p o s i t i v e l y with budworm success. Examination of the standardized c o r r e l a t i o n c o e f f i c i e n t s i n d i c a t e d that b o r n y l a c e t a t e , the u n i d e n t i f i e d terpene, t o t a l n i t r o g e n content, and beta-pinene were the most important v a r i a b l e s i n determining female adult dry weight. I t i s i n t e r e s t i n g to note that once more p r o t e i n complexing c a p a c i t y of the e x t r a c t was not important i n determining female dry weight. I n t e r e s t i n g l y , s e v e r a l more v a r i a b l e s were included i n the model d e a l i n g w i t h male budworm success on the trees at the B a r l e y Canyon s i t e (Table I V ) . Terpinolene, c i t r o n e l l y l a c e t a t e ,


1.

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11

Chemistry


alpha-pinene, bornyl acetate, myrcene, an u n i d e n t i f i e d terpene, and crown r a t i o were i n v e r s e l y r e l a t e d with average adult male dry weight production per t r e e . P o s i t i v e l y c o r r e l a t e d with male dry weight were limonene, young needle water content, gammat e r p i n e n e , 3 u n i d e n t i f i e d terpenes, the r a t i o of s o l u b l e to I n s o l u b l e n i t r o g e n , and the amount of twig internode growth i n 1980. The most important of these 15 v a r i a b l e s i n i n f l u e n c i n g male adult dry weight production were limonene, t e r p i n o l e n e , c i t r o n e l l y l acetate, alpha-pinene, and bornyl acetate. Once a g a i n , p r o t e i n complexing c a p a c i t y explained none of the v a r i a t i o n i n average male adult success. Table I I I . M u l t i p l e c o r r e l a t i o n a n a l y s i s using adult female dry weight as the dependent v a r i a b l e . (R2 = 0.35; F - 3.31; p < 0.001). Barley Canyon, New Mexico (Redak, 1982).

Independent Variable B o r n y l Acetate Unidentified Terpene No. 5 T o t a l Nitrogen 3-pinene Budburst Terpinolene Myrcene Tree Age Geranyl Acetate F i v e Yr Growth Increment 1980 Internode Length

Coefficient 0.00334

Standard Error 0.00122

Standardized Regression Coefficient 0.354

0.01862 51.62314 0.00290 0.22878 0.02703 0.000948 0.12241 0.00376

-0.262 -0.232 -0.229 0.193 0.172 -0.165 0.145 0.137

-1.59537

1.31204

-0.133

0.48272

0.37711

0.133

-0.04165 -114.75060 -0.00541 0.41324 0.04044 -0.01387 0.16913 0.00436

Nitrogen = % dw; Terpenes » Area counts/20 mg dw Water S t r e s s and Budworm Success Hypotheses concerning drought s t r e s s and i t s i n f l u e n c e on budworm success and changes i n f o l i a g e q u a l i t y were tested using D o u g l a s - f i r trees growing on south f a c i n g ( s t r e s s ) and north f a c i n g slopes ( n o n - s t r e s s ) . We f i r s t demonstrated that the degree of water s t r e s s was d i f f e r e n t between the 2 groups of t r e e s . Xylem pressure p o t e n t i a l s averaged 23% higher f o r the t r e e s a t the s t r e s s s i t e when compared to the non-stress s i t e (p < 0.001).



12


Next, the male and female adult dry weights and the number of budworm s u r v i v i n g per tree between the 2 s i t e s were compared. Table V i n d i c a t e s the means f o r each of these v a r i a b l e s subdivided by s i t e and sex. These data were used i n the m u l t i v a r i a t e a n a l y s i s of v a r i a n c e , and are presented i n t h i s t a b l e as a point of reference. Since no dependency was found between weight and number of s u r v i v o r s ( r = 0.158; p • 0 . 1 2 ) , these v a r i a b l e s were subjected to m u l t i v a r i a t e a n a l y s i s of v a r i a n c e to t e s t f o r the e f f e c t s of s i t e and sex (Table V I ) . Budworm success, as measured by a d u l t dry weight and number of s u r v i v o r s , was found to be s i g n i f i c a n t f o r both s i t e and sex, but there was no i n t e r a c t i o n between these f a c t o r s . The r e l a t i v e c o n t r i b u t i o n s of weight and numbers of s u r v i v o r s to the d i f f e r e n c e s between s i t e s and sexes are i l l u s t r a t e d by the c h a r a c t e r i s t i c vector c o e f f i c i e n t s . Not s u r p r i s i n g l y , they i n d i c a t e that d i f f e r e n c e s between sexes were due to d i f f e r e n c e s i n weight between males and females. The s i g n i f i c a n t r e s u l t was that d i f f e r e n c e s between s i t e s were due to d i f f e r e n c e s i n weight and the number of s u r v i v o r s per t r e e . Stepwise d i s c r i m i n a n t a n a l y s i s was used to determine how t r e e chemical, p h e n o l o g i c a l , and p h y s i c a l parameters d i f f e r e d between s i t e s (Table V I I ) . Only seven of the 18 v a r i a b l e s used were needed to completely d i f f e r e n t i a t e the trees at the 2 s i t e s (F(7 193} = 210.36; p < 0 . 0 0 1 ) . The magnitudes of the standardized d i s c r i m i n a n t f u n c t i o n c o e f f i c i e n t s f o r the i n c l u d e d v a r i a b l e s i n d i c a t e d that the d i f f e r e n c e s between s i t e s were l a r g e l y due to terpene chemistry (Table V I I I ) . The d i s c r i m i n a n t f u n c t i o n c o n t r a s t s p r i m a r i l y the r e l a t i v e c o n c e n t r a t i o n of alpha-pinene versus the c o n c e n t r a t i o n of s e v e r a l terpenes, p a r t i c u l a r l y bornyl acetate and beta-pinene. Examination of the d i s c r i m i n a n t scores showed that the stressed t r e e s loaded n e g a t i v e l y on the f u n c t i o n (x d i s c r i m i n a n t score = -2.23), w h i l e the non-stressed trees loaded p o s i t i v e l y (x d i s c r i m i n a n t score = 3.38). In other words, t r e e s from the s t r e s s e d s i t e were higher i n alpha-pinene while the non-stressed trees contained more b o r n y l a c e t a t e , beta-pinene, and other terpenes i n t h e i r young needles. Discussion D o u g l a s - f i r F o l i a g e Q u a l i t y and Resistance

t o Budworm

Data from a l l three s t u d i e s show t h a t , i n a l l cases, the terpene chemistry of young f o l i a g e , or q u a l i t a t i v e defenses, was the most important f a c t o r i n reducing budworm success. The p r o t e i n complexing c a p a c i t y , or q u a n t i t a t i v e defenses, of t h i s t i s s u e was not important i n reducing budworm success i n any of the s t u d i e s .


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Chemistry

13

Table IV. M u l t i p l e c o r r e l a t i o n a n a l y s i s using adult male dry weight as the dependent v a r i a b l e . ( R - 0.35; B a r l e y Canyon, New Mexico F * 2.49; p < 0.005) (Redak, 1982). 2


Independent Variable Limonene Terpinolene Citronellyl Acetate a-Pinene B o r n y l Acetate Myrcene Water Content y-Terpinene Unidentified Terpene No. 8 Unidentified Terpene No. 5 Crown Ratio Unidentified Terpene No. 9 Soluble/ Insoluble Nitrogen Unidentified Terpene No. 4 1980 Internode Length

Standard Error

Coefficient

Standardized Regression Coefficient

0.00512 -0.02296

0.00200 0.00999

0.461 -0.300

-0.00335 -0.00079 -0.00062 -0.00526 6.74118 0.00825

0.00144 0.00055 0.00040 0.00315 4.43527 0.00543

-0.256 -0.234 -0.202 -0.192 0.177 0.167

-0.02614

0.01863

-0.159

0.00770 -5.52496

0.00598 4.97238

0.149 -0.124

0.00946

0.00831

0.121

0.71473

0.68189

0.119

0.00658

0.00686

0.104

0.11978

0.12284

0.102

N i t r o g e n * % dw; Terpenes = Area Counts/20 mg dw Table V. Mean adult dry weight and number of budworm s u r v i v i n g per tree on s t r e s s e d and non-stressed s i t e s . These data were used i n the m u l t i v a r i a t e a n a l y s i s , the r e s u l t s of which are given i n Table VI (Cates et a l . , 1982).

Sex

N

Weight (mg)

Number Survived

Non-stressed

Male Female

22 23

8.95 18.18

3.73 3.04

Stressed

Male Female

27 26

10.73 23.64

4.63 5.15

Site


14

PLANT RESISTANCE

TO INSECTS

Table V I . Results of m u l t i v a r i a t e a n a l y s i s of variance f o r the e f f e c t s of s i t e and sex on adult dry weight and number of s u r v i v o r s . C h a r a c t e r i s t i c vector coe f f i c i e n t s i n d i c a t e the r e l a t i v e c o n t r i b u t i o n of the dependent v a r i a b l e s to a p a r t i c u l a r e f f e c t (Cates et a l . , 1982). C h a r a c t e r i s t i c Vector Coefficients F


Source

?

10.78 73.97 2^42

0.0001 0.0001 0.0941

t

Site Sex S i t e x Sex Table V I I .

(2 93)

Weight

0.022 -0.007 0.018

0.018 0.023 0.019 _

Chemical, p h e n o l o g l c a l , and p h y s i c a l c h a r a c t e r i s t i c s of D o u g l a s - f i r trees that were subjected to d i s c r i m i n a n t a n a l y s i s .

Nitrogen Total Soluble P r o t e i n Complexing Capacity Table V I I I .

Number

Terpenes 13 I n d i v i d u a l Compounds Total Tree Age

Standardized d i s c r i m i n a n t f u n c t i o n c o e f f i c i e n t s f o r the 7 v a r i a b l e s r e s u l t i n g from the a n a l y s i s of chemical and p h y s i c a l parameters among trees growing on the stressed and non-stressed s i t e s (Cates et a l . , 1982).

Variable a-pinene S o l u b l e Nitrogen Age U n i d e n t i f i e d terpene 1 U n i d e n t i f i e d terpene 2 3-pinene B o r n y l Acetate

Standardized D i s c r i m i n a n t Function C o e f f i c i e n t -5.84 -0.71 0.34 1.22 1.66 2.57 2.81

N i t r o g e n was found to be of l i t t l e importance except f o r the female model generated from the Barley Canyon Study. In t h i s case, female success was i n v e r s e l y c o r r e l a t e d with f o l i a r n i t r o g e n l e v e l s . However, these l e v e l s , based on published data (18,19), do not appear high enough to be t o x i c to the budworm. This observation, i n view of the inverse c o r r e l a t i o n between t o t a l n i t r o g e n and female budworm success, leads us to



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Chemistry

suggest that t h i s r e l a t i o n s h i p i s more a r e f l e c t i o n of tree p r o d u c t i v i t y than i t s r o l e as a primary n u t r i e n t . In other words, higher l e v e l s of f o l i a r n i t r o g e n i n non-stressed trees t h a t were growing on good q u a l i t y s i t e s may have been i n d i c a t i v e of o v e r a l l tree p r o d u c t i v i t y and v i g o r (19,20,21). These trees would be b e t t e r able, p r o v i d i n g they were g e n e t i c a l l y predisposed, t o produce e f f e c t i v e defensive chemistry against the budworm. The inverse r e l a t i o n s h i p between female budworm success and the 5 year growth increment, as w e l l as the p o s i t i v e r e l a t i o n s h i p between tree age and female budworm success, are c o n s i s t e n t with t h i s hypothesis. Productive trees should be more vigorous, grow b e t t e r , and hence, possess wider annual r i n g s . The smaller width of annual r i n g s i n d i c a t e s that older trees may be l e s s productive, suggesting a reduced a b i l i t y to produce e f f e c t i v e defensive chemistry. A l l of our r e s u l t s i n d i c a t e that increased p r o d u c t i v i t y i s a s s o c i a t e d with reduced budworm success. In the Montana study t h i s was evidenced by the r e l a t i o n s h i p between budworm i n f e s t a t i o n i n t e n s i t y and bole r a d i u s . In the Barley Canyon study, the r e l a t i o n s h i p between budworm growth and crown r a t i o , the 5 year growth increment, and t o t a l n i t r o g e n support t h i s c o n c l u s i o n as w e l l . Assuming that p r o d u c t i v i t y d e c l i n e s with age, the p o s i t i v e c o r r e l a t i o n between budworm success and age a l s o i m p l i e s that a d e c l i n e i n v i g o r increases tree s u s c e p t i b i l i t y to budworm. A d d i t i o n a l l y , our data suggest that D o u g l a s - f i r may reduce the r i s k of damage to new t i s s u e through escape i n time. In Montana, trees which burst bud l a t e r i n the growing season s u f f e r e d l e s s damage from budworm. This i s c o n s i s t e n t with theory (1,5^6) which suggests that u n p r e d i c t a b i l i t y i n space and time may be an e f f e c t i v e mechanism f o r reducing the adverse e f f e c t s of monophagous-oligophagous herbivores on young ephemeral t i s s u e s . On the other hand, trees a t Barley Canyon show a p o s i t i v e r e l a t i o n s h i p between budburst and budworm s i z e . This appears to be i n d i r e c t c o n t r a d i c t i o n with the Montana data and theory, u n t i l the experimental design Is considered. At Barley Canyon, budworm were placed on trees a f t e r a l l had b u r s t bud. Consequently, most budworm were subjected to t i s s u e s that had developed f o r up to 10 days and which possessed more complete defensive systems. Therefore, the c o r r e l a t i o n we observed with budburst r e f l e c t e d the length of time the f o l i a g e was allowed to mature d e f e n s i v e l y before the budworm feeding occurred. I t d i d not r e f l e c t an escape i n time component as the Montana study d i d where n a t u r a l populations were s t u d i e d .


15

16



Water Stress and Budworm Success The study i n v o l v i n g water s t r e s s i n the Jemez mountains suggests that changes i n f o l i a g e q u a l i t y due to s t r e s s p o s i t i v e l y i n f l u e n c e s budworm success (17). Female adult budworm from larvae reared on stressed trees were 30% heavier, w h i l e the male a d u l t s were 20% h e a v i e r . In a d d i t i o n , a higher number of both sexes survived on stressed trees (Table V I ) . The water s t r e s s i n c u r r e d by trees growing on the south s i t e i s hypothesized to have modified f o l i a g e q u a l i t y such that trees became h i g h l y s u s c e p t i b l e to the budworm. This appears to have been p r i m a r i l y due to the decrease i n the r e s i s t a n c e f a c t o r s beta-pinene and bornyl acetate accompanied by an increase i n alpha-pinene. I f alpha-pinene c o n s i s t e n t l y Increases i n s t r e s s e d D o u g l a s - f i r t r e e s , i t i s then p o s s i b l e that budworm may use i t as an a t t r a c t a n t or cue i n l o c a t i n g s u i t a b l e hosts. The primary emphasis i n the l i t e r a t u r e has been to show the importance of n i t r o g e n i n f a c i l i t a t i n g outbreaks of phytophagous i n s e c t s (22). More r e c e n t l y , i t has been suggested that changes i n defensive chemistry are as important as the changes i n primary n u t r i e n t s (23,24). In our s t u d i e s , n i t r o g e n was not n e a r l y as important as were s e v e r a l terpenes. Budworm success was most s t r o n g l y a s s o c i a t e d with changes i n terpene defensive chemistry, p a r t i c u l a r l y i n the r e d u c t i o n of r e s i s t a n c e f a c t o r s , rather than with changes i n the primary n u t r i t i o n of the herbivore. I m p l i c a t i o n s f o r Current Plant-Herbivore

Theory

Ephemeral t i s s u e s , such as the young needles of D o u g l a s - f i r , are suggested to be defended by t o x i n s or q u a l i t a t i v e defenses (5,6). Because these t i s s u e s are under strong s e l e c t i o n f o r r a p i d development and growth to a l e v e l where they are c o n t r i b u t i n g to the net primary p r o d u c t i v i t y of the p l a n t , s e l e c t i o n i s postulated to favor a defensive system t h a t does not place a f u r t h e r burden on an already s t r a i n e d energy budget (1,6). Hence, q u a n t i t a t i v e defenses that r e q u i r e c o n s i d e r a b l e energy to produce, and must be compartmentalized so that they do not i n t e r f e r e with metabolic processes, are p o s t u l a t e d to be a minor or missing defensive system i n ephemeral t i s s u e s . Q u a l i t a t i v e , or t o x i n defensive systems are p r e d i c t e d i n these t i s s u e s i n s t e a d . Exceptions to t h i s general p r e d i c t i o n may p o s s i b l y i n c l u d e the young leaves of evergreen t r e e s or shrubs which should show slower growth when compared to s h o r t - l i v e d t i s s u e s of non-evergreen p l a n t s . While q u a n t i t a t i v e defenses or d i g e s t i b i l i t y reducing substances are also present i n small q u a n t i t i e s i n the .young needles, they do not seem to be e f f e c t i v e i n reducing female dry weight, l a r v a l d e n s i t y , or l e v e l of d e f o l i a t i o n . This i s c o n s i s t e n t with the reasoning that young t i s s u e development



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Natural Product

Chemistry

c o n s t r a i n t s render d i f f i c u l t the production of a w e l l developed d i g e s t i b i l i t y reducing system. As discussed i n the previous s e c t i o n , we have observed that escape i n time, another p r e d i c t i o n of current p l a n t - h e r b i v o r e theory, e x i s t s making newly emerged f o l i a g e l e s s p r e d i c t a b l e to the budworm. Some trees may burst bud as l a t e as 10-14 days a f t e r the f i r s t trees w i t h i n a stand, and as our data show, t h i s delay i s a s s o c i a t e d w i t h reduced damage by the budworm. Slansky and Feeny (25) and Feeny (_5) suggest that once q u a l i t a t i v e defenses are overcome they " a f f e c t l a r v a l growth to a much l e s s e r extent than do the n u t r i e n t c h a r a c t e r i s t i c s of food p l a n t s , " and that "once overcome by s p e c i f i c adaptation, they may have l i t t l e a f f e c t on growth o r f i t n e s s . " Our data do not corroborate these p r e d i c t i o n s . Budworm success was reduced s i g n i f i c a n t l y by the presence of higher q u a n t i t i e s of terpenes i n the young needles of some t r e e s . A d d i t i o n a l l y , i n a l l 3 s t u d i e s , n i t r o g e n content was not as important as were the terpene contents of the f o l i a g e i n i n f l u e n c i n g budworm success. I n a l l three s t u d i e s , but p a r t i c u l a r l y I n the 2 d e a l i n g w i t h r e s i s t a n c e - s u s c e p t i b i l i t y c h a r a c t e r i s t i c s of f o l i a g e q u a l i t y , s p e c i f i c a l l y the higher q u a n t i t i e s of terpenes were c o r r e l a t e d with reduced budworm success. At any p a r t i c u l a r s i t e a l l of the trees sampled contained the same b a s i c complement of terpenes, a l b e i t , some were present i n low q u a n t i t i e s . The e f f e c t , which we assume i s one of t o x i c i t y , of the terpenes i n reducing budworm success, then, was due to an Increase i n t h e i r q u a n t i t a t i v e amounts. I n other words, the terpenes were a c t i n g i n a q u a n t i t a t i v e o r dosage dependent fashion. Q u a l i t a t i v e defenses i n D o u g l a s - f i r a l s o may be s u c c e s s f u l a g a i n s t the r e l a t i v e l y s p e c i a l i z e d budworm because o f the d i v e r s i t y of terpenes that are present. I n other words, a l a r g e number of q u a l i t a t i v e t o x i c combinations are a v a i l a b l e f o r s e l e c t i o n to a c t upon i n D o u g l a s - f i r . I n t e r e s t i n g l y , we found that i n the Montana study the greater the Imbalance In the terpene d i s t r i b u t i o n i n the f o l i a g e , the l e s s w e l l the budworm performed. This suggests that the more a t r e e ' s d e f e n s i v e chemistry deviates from a balanced, o r "average" p a t t e r n , the l e s s l i k e l y i t i s t o be attacked by the c h e m i c a l l y w e l l adapted budworm. A d d i t i o n a l l y , adaptation by budworm to the p a r t i c u l a r imbalanced terpene p a t t e r n of one host tree would confer an advantage to any tree whose terpene p a t t e r n was predominated by other terpenes. Thus we f e e l that the e f f e c t of s e l e c t i o n by the budworm f o r q u a l i t a t i v e terpene defenses would be to produce a number of "chemical p a t t e r n phenotypes" among I n d i v i d u a l s a t a l o c a l or i n t r a p o p u l a t i o n a l l e v e l . Chemical d i v e r s i t y should e x i s t among populations as w e l l , and t h i s has been suggested i n our work as w e l l as by others (10,11). Such s e l e c t i o n f o r



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d i v e r s i t y i n the host plant chemistry should produce races of budworm that are g e n e r a l l y adapted to the average defensive chemistry of t h e i r host plants but that may not be w e l l adapted t o the chemistry of D o u g l a s - f i r from other populations. I f t h i s scenario i s c o r r e c t , then some very i n t e r e s t i n g s i l v i c u l t u r a l p r a c t i c e s i n managing f o r e s t s could r e s u l t using defensive n a t u r a l product chemistry along with other c o n t r o l measures i n an i n t e g r a t e d pest management system. I n a sense, many of the data presented from our work are p r e l i m i n a r y since the above d i s c u s s i o n i s based on c o r r e l a t i o n a n a l y s i s . Synthetic d i e t s i n c o r p o r a t i n g compounds that are suggested by these studies to adversely i n f l u e n c e budworm success, as w e l l as other experimental work with n a t u r a l populations of D o u g l a s - f i r and budworm, are needed to determine cause and e f f e c t . A thorough examination of the n a t u r a l product chemistry i s needed as some other t o x i c compounds may be i n f l u e n c i n g s i g n i f i c a n t l y the patterns being observed. Considerable e f f o r t needs to be given to various aspects of budworm b i o l o g y . But i n the f i n a l a n a l y s i s , these data suggest t h a t the Douglas-fir-budworm system may be very u s e f u l i n u n r a v e l i n g various aspects of p l a n t - h e r b i v o r e i n t e r a c t i o n s , and i n understanding ways i n which we might b e t t e r manage our f o r e s t s against phytophagous i n s e c t s . Acknowledgements We should l i k e to thank T. I. McMurray, H. J . Alexander, M. Alexander, J . Horner, M. F r e e h l i n g , and numerous undergraduate students f o r t h e i r help and f o r t h e i r constant q u e s t i o n i n g and d i s c u s s i o n of various aspects of the p r o j e c t s . We are g r a t e f u l to C l i f f o r d S. Crawford and F r i t z Taylor f o r p r o v i d i n g information and d i s c u s s i o n of budworm population b i o l o g y . Doug Parker and Mike Chavez (USDA FS, Region 3, Albuquerque), and Jed Dewey, John Hard, and Larry Stipe (USDA FS, Region 6, Missoula, MT), were h e l p f u l In numerous ways but p a r t i c u l a r l y i n helping us l o c a t e appropriate s i t e s . We are extremely g r a t e f u l to Linda L. DeVries f o r typing the manuscript s e v e r a l times. This research was supported i n part by NSF grants DEB 7619950 and DEB 7927067 to RGC f o r which we are g r a t e f u l . Work leading to t h i s p u b l i c a t i o n was funded by the Canada/United States Spruce Budworms Program, and A c c e l e r a t e d Research, Development and A p p l i c a t i o n Program, sponsored by the USDA Forest S e r v i c e .


1.

CATES ET AL.

Defensive

Natural Product

Chemistry

Literature Cited


1.

Cates, Rex G.; Rhoades, D. Biochem. S y s t . E c o l . 1977, 5, 185-93. 2. Cates, Rex G.; Alexander, H. J. In "Bark Beetles in North American C o n i f e r s : Ecology and E v o l u t i o n " : J . M i t t o n and K. Sturgeon, (Eds.); U n i v e r s i t y of Texas Press; A u s t i n , Texas, in press. 3. Cates, Rex G. Oecologia 1981, 48, 319-26. 4. Cates, Rex G. Oecologia 1980, 46, 22-31. 5. Feeny, P. P. In "Biochemical I n t e r a c t i o n s Between P l a n t s and I n s e c t s " ; Wallace, J.; M a n s e l l , R., Eds.; Plenum P r e s s : New York, NY, 1976; pp 1-40. 7. Futuyma, D.; Gould, F. Ecol. Monogr. 1979, 49, 33-50. 8. Rosenthal, G.; Janzen, D., Eds.; "Herbivores: Their I n t e r a c t i o n w i t h Secondary Plant M e t a b o l i t e s " ; Academic P r e s s : New York, NY, 1979. 9. Johnson, P.; Denton, R. USDA F o r . Ser. Gen. Tech. Rep. 1975, INT-20, 1-144. 10. von R u d l o f f , E. Pure A p p l . Chem. 1973, 34, 401-10. 11. von R u d l o f f , E. Can. J. Bot. 1972, 50, 1025-40. 12. Greenbank, D. Entomol. Soc. Can. Mem. 1963, 31, 174-80. 13. Feeny, P.; Bostock, H. Phytochemistry, 1968, 7, 871-80. 14. Feeny, P. Phytochemistry, 1969, 8, 2119-26. 15. Redak, R. "A Determination of the ResistanceSusceptibility C h a r a c t e r i s t i c s in D o u g l a s - f i r to the Western Spruce Budworm"; MS Thesis, Univ. of New Mexico, Albuquerque, NM, 1982, pp 1-74. 16. Bate-Smith, E. Phytochemistry, 1973, 12, 907-12. 17. Cates, Rex G.; McMurray, T.; Redak, R.; Henderson, C. Nature, (submitted). 18. Mattson, W. Ann. Rev. E c o l . Syst. 1980, 11, 119-61. 19. Gosz, J . Ecol. Bull, 1981,33,405-26. 20. Miller, H.; Cooper, J.; Miller, J . ; P a u l i n e , O. Can. J. For. Res. 1979, 9, 19-26. 21. Van den Driessche, R.; D a n g e r f i e l d , J . P l a n t and Soil 1975, 42, 685-02. 22. White, T. Ecology, 1969, 50, 905-9. 23. Rhoades, D. I n ( 8 ) . 24. Mattson, W.; Addy, N. Science, 1975, 190, 515-22. 25. Slansky, F.; Feeny, P. Ecol. Monogr. 1977, 47, 209-28. RECEIVED September 13, 1982


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Patterns in Defensive Natural Product Chemistry: Douglas Fir and

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