Journal of Chemical Ecology, Vol. 10, No. 10, 1984
VARIABILITY IN A C C U M U L A T I O N OF P R O A N T H O C Y A N I D I N S ( C O N D E N S E D TANNINS) IN N E E D L E S OF D O U G L A S - F I R (Pseudotsuga menziesii) FOLLOWING LONG-TERM B U D W O R M D E F O L I A T I O N
T O M W A L T E R S and H E L E N A. S T A F F O R D Biology Department, Reed College Portland, Oregon 97202
(Received October 26, 1983; revised February 16, 1984) Abstract--Long-term defoliation by budworms was associated with higher levels of soluble proanthocyanidins in the current year needles of Douglasfir trees. The proanthocyanidin contents of needles from defoliated Douglas-fir trees were considerably more variable than those levels of undefoliated ones. The increased mean and variability of proanthocyanidin levels following defoliation may have interesting ecological consequences for Douglas-fir and its defoliators. Key words--Proanthocyanidins, condensed tannins, plant-herbivore interactions, Douglas-fir, Pseudotsuga menziesii, western spruce budworm, Choristoneura occidentalis, Lepidoptera, Tortricidae, nested ANOVA. INTRODUCTION P r o a n t h o c y a n i d i n s (condensed tannins) are oligomers and polymers of hydroxy-flavan units, typically having molecular weights of 300-6000 daltons. They f o r m complexes with and precipitate proteins, a property which p r o b a b l y has significant ecological consequences for plants producing them (Swain, 1979; Haslam, 1981). P r o a n t h o c y a n i d i n s may lower the nutritive value of foliage, or they can act as feeding deterrents to insect herbivores (Reese et al., 1982; Klocke and Chan, 1982; Bernays, 1981; Dethier, 1982.) Variability in plant defense c o m p o u n d s is now considered to be an i m p o r t a n t obstacle for herbivores to overcome (Denno and McClure, 1983; see other articles in the same volume). Schultz (1983) and Baldwin and Schultz (1983) found sugar maple leaves to be highly variable in foliage feeding characteristics and argued that variability in plant defensive characteristics 1469 0098-0331/84/1000-1469503.50/09
1984 Plenum Publishing Corporation
1470
WALTERS AND STAFFORD
can be adaptive for trees defoliated by insects. On the basis of single-needle assays for proanthocyandin content of Douglas-fir Pseudtsuga mengiesii (Mirb.) Franco needles and a more detailed statistical analysis, we report not only an increase in the mean level of proanthocyanidins, but an increase in the variability of proanthocyanidin levels in budworm-defoliated trees. Therefore, since trees-may respond to herbivory by increasing proanthocyanidin synthesis in a highly variable fashion, western spruce budworms continuing to feed upon previously exploited Douglas-fir foliage may have to contend with higher and variable amounts of these feeding deterrents. M E T H O D S AND MATERIALS
Trees Defoliated by Budworms. Douglas-fir (Pseudotsuga menziesii) samples were taken from a mixed Douglas-fir-white fir stand (elevation 1500 m) near Heppner, Oregon, in the Umatilla National Forest on February 1, 1983. Trees at this site were defoliated by western spruce budworms the previous spring and for the two springs prior to that (private communication, Bill Helfenstein, USDA Forest Service, Heppner, Oregon, and to whom we are greatly indebted for information and guidance). Sample trees were not more than 100 m from each other and were at least 190 m from the road. Five budworm-defoliated (40-75% of the past several years' growth missing as estimated by visual inspection) and five undefoliated (less than 15% of the past three years' growth missing) trees were selected. The outer part of a branch was removed from each tree, sealed in a plastic bag, put on ice for 8 hr transit time, and stored in a freezer at - 2 0 ~C. Six undamaged needles were later removed from each branch; three were from the outside of the branch (the apical region of the current year's growth), and three were from just outside the previous year's terminal bud scar (the basal region of the current growth) (Table 1). (Fresh weight approximately 15 mg/needle, dry weight/ fresh weight 45%). Extraction Procedure. Single needles were cut into approximately 3-mm sections and homogenized for I0 sec at room temperature with a Tekmar Tissuemizer in 2 • 0.5 ml methanol-H20 (70: 30, v/v). After centrifugation, both supernatant (the "methanol-soluble" fraction) and pellet (the "methanolinsoluble" fraction) were analyzed. Proanthocyanidin Assay. Extracts and pellets were assayed for proanthocyanidins (Stafford and Lester, 1980) with modifications described below. We mixed 1.2 ml n-butanol-HC1 (95:5, v/v) into extract aliquots (0.050.2 ml) and water, for a total volume of 1.4 ml. Aliquots were chosen to give absorbances less than 0.2, since a biphasic curve has been reported (Stafford
1471
PROANTHOCYANIDINS IN DOUGLAS-FIR TABLE 1. MEAN METHANOL-SOLUBLE PROANTHOCYANIDINS
(&As50/100 m g FRESH WEIGHT) OF NEEDLES FROM RELATIVELY UNDEFOLIATEDTREES AND TREES DEFOLIATEDBY BUDWORMS(X -+ SE) Neighboring needles Relatively Undefoliated Ula a Ulb U2a U2b U3a U3b U4b U5a U5b Defoliated Dla Dlb D2a D2b D3a D3b D4a D4b D5a D5b
Trees
43.4-+3.6 }j 37.9 -+ 1.4 44.4 -+ 1.3 38.4 -+ 2.1 83.6 + 3.4 69.6 __+6.0 jr 42.7 • 2.8 63.7-+ 3.6 ~ 51.1 -+ 10.5 64.4-+ 2.9 59.4-+ 7.3 62.3 -+ 5.0 87~2 • 23.7 75.3 -+ 3.8 75.9 -+ 10.5 115.2• 10.2 73.4 -+ 7.7 69.1 -+ 2.5 75.9-+ 6.9
| I[ | [ ~ ~" ~ ~" / ~
] 40.7 _+ 2.1 41.4 • 1.7 76.6 -+ 4.4
53.1 • 2.91
49.5 -+ 3.4 57.4 _+ 5.8
61.9• 3.7 74.9 -+ 5.2 76.6 -+ 5.0
75.8~' _+ 3.85
94.3 -+ 11.1 72.5 _+ 3.6
~Groups: U, D (undefoliated, defoliated); trees: 1-5 (5 trees/group); Locations: a, b (apical, basal). ~43% greater than undefoliated trees.
and Lester, 1980). Pellets were s u s p e n d e d in 1.0 ml n - b u t a n o l - H C 1 (95: 5, v / v). The tubes were p l a c e d in a 95 ~ C b a t h for 60 rain. A f t e r c o o l i n g a n d r e m i x i n g , the a b s o r b a n c e s o f the m e t h a n o l - s o l u b l e p r o a n t h o c y a n i d i n s were r e c o r d e d . To m e a s u r e the p r o a n t h o c y a n i d i n c o n t e n t of the insoluble fraction, tubes Containing pellets were recentrifuged, a n d an a p p r o p r i a t e a l i q u o t of the s u p e r n a t a n t was diluted with n - b u t a n o l - H C 1 (95:5), v / v ) to a final v o l u m e of 1.2 ml. A f t e r mixing, the a b s o r b a n c e was r e c o r d e d . P r o c y a n i d i n values were e x p r e s s e d as A at 550 n m p r o d u c e d b y an e x t r a c t e q u i v a l e n t to 100 mg fresh weight of tissue per milliliter (AA55o/100 mg fresh weight). Statistical Analyses. M e a s u r e m e n t s of p r o a n t h o c y a n i d i n c o n t e n t of single needles were a n a l y z e d with a nested f a c t o r i a l analysis of v a r i a n c e c o m p u t e d b y an original F o r t r a n 1V plus p r o g r a m .
1472
WALTERS AND STAFFORD RESULTS
Long-Term Defoliation by Western Spruce Budworms. Soluble pro, anthocyanidin levels in the current year's foliage of budworm-defoliated trees were 45% higher than those in relatively undefoliated trees from the same site (Table 2). This difference was significant when the measurements were recorded on a fresh weight basis (P =
VARIABILITY IN A C C U M U L A T I O N OF P R O A N T H O C Y A N I D I N S ( C O N D E N S E D TANNINS) IN N E E D L E S OF D O U G L A S - F I R (Pseudotsuga menziesii) FOLLOWING LONG-TERM B U D W O R M D E F O L I A T I O N
T O M W A L T E R S and H E L E N A. S T A F F O R D Biology Department, Reed College Portland, Oregon 97202
(Received October 26, 1983; revised February 16, 1984) Abstract--Long-term defoliation by budworms was associated with higher levels of soluble proanthocyanidins in the current year needles of Douglasfir trees. The proanthocyanidin contents of needles from defoliated Douglas-fir trees were considerably more variable than those levels of undefoliated ones. The increased mean and variability of proanthocyanidin levels following defoliation may have interesting ecological consequences for Douglas-fir and its defoliators. Key words--Proanthocyanidins, condensed tannins, plant-herbivore interactions, Douglas-fir, Pseudotsuga menziesii, western spruce budworm, Choristoneura occidentalis, Lepidoptera, Tortricidae, nested ANOVA. INTRODUCTION P r o a n t h o c y a n i d i n s (condensed tannins) are oligomers and polymers of hydroxy-flavan units, typically having molecular weights of 300-6000 daltons. They f o r m complexes with and precipitate proteins, a property which p r o b a b l y has significant ecological consequences for plants producing them (Swain, 1979; Haslam, 1981). P r o a n t h o c y a n i d i n s may lower the nutritive value of foliage, or they can act as feeding deterrents to insect herbivores (Reese et al., 1982; Klocke and Chan, 1982; Bernays, 1981; Dethier, 1982.) Variability in plant defense c o m p o u n d s is now considered to be an i m p o r t a n t obstacle for herbivores to overcome (Denno and McClure, 1983; see other articles in the same volume). Schultz (1983) and Baldwin and Schultz (1983) found sugar maple leaves to be highly variable in foliage feeding characteristics and argued that variability in plant defensive characteristics 1469 0098-0331/84/1000-1469503.50/09
1984 Plenum Publishing Corporation
1470
WALTERS AND STAFFORD
can be adaptive for trees defoliated by insects. On the basis of single-needle assays for proanthocyandin content of Douglas-fir Pseudtsuga mengiesii (Mirb.) Franco needles and a more detailed statistical analysis, we report not only an increase in the mean level of proanthocyanidins, but an increase in the variability of proanthocyanidin levels in budworm-defoliated trees. Therefore, since trees-may respond to herbivory by increasing proanthocyanidin synthesis in a highly variable fashion, western spruce budworms continuing to feed upon previously exploited Douglas-fir foliage may have to contend with higher and variable amounts of these feeding deterrents. M E T H O D S AND MATERIALS
Trees Defoliated by Budworms. Douglas-fir (Pseudotsuga menziesii) samples were taken from a mixed Douglas-fir-white fir stand (elevation 1500 m) near Heppner, Oregon, in the Umatilla National Forest on February 1, 1983. Trees at this site were defoliated by western spruce budworms the previous spring and for the two springs prior to that (private communication, Bill Helfenstein, USDA Forest Service, Heppner, Oregon, and to whom we are greatly indebted for information and guidance). Sample trees were not more than 100 m from each other and were at least 190 m from the road. Five budworm-defoliated (40-75% of the past several years' growth missing as estimated by visual inspection) and five undefoliated (less than 15% of the past three years' growth missing) trees were selected. The outer part of a branch was removed from each tree, sealed in a plastic bag, put on ice for 8 hr transit time, and stored in a freezer at - 2 0 ~C. Six undamaged needles were later removed from each branch; three were from the outside of the branch (the apical region of the current year's growth), and three were from just outside the previous year's terminal bud scar (the basal region of the current growth) (Table 1). (Fresh weight approximately 15 mg/needle, dry weight/ fresh weight 45%). Extraction Procedure. Single needles were cut into approximately 3-mm sections and homogenized for I0 sec at room temperature with a Tekmar Tissuemizer in 2 • 0.5 ml methanol-H20 (70: 30, v/v). After centrifugation, both supernatant (the "methanol-soluble" fraction) and pellet (the "methanolinsoluble" fraction) were analyzed. Proanthocyanidin Assay. Extracts and pellets were assayed for proanthocyanidins (Stafford and Lester, 1980) with modifications described below. We mixed 1.2 ml n-butanol-HC1 (95:5, v/v) into extract aliquots (0.050.2 ml) and water, for a total volume of 1.4 ml. Aliquots were chosen to give absorbances less than 0.2, since a biphasic curve has been reported (Stafford
1471
PROANTHOCYANIDINS IN DOUGLAS-FIR TABLE 1. MEAN METHANOL-SOLUBLE PROANTHOCYANIDINS
(&As50/100 m g FRESH WEIGHT) OF NEEDLES FROM RELATIVELY UNDEFOLIATEDTREES AND TREES DEFOLIATEDBY BUDWORMS(X -+ SE) Neighboring needles Relatively Undefoliated Ula a Ulb U2a U2b U3a U3b U4b U5a U5b Defoliated Dla Dlb D2a D2b D3a D3b D4a D4b D5a D5b
Trees
43.4-+3.6 }j 37.9 -+ 1.4 44.4 -+ 1.3 38.4 -+ 2.1 83.6 + 3.4 69.6 __+6.0 jr 42.7 • 2.8 63.7-+ 3.6 ~ 51.1 -+ 10.5 64.4-+ 2.9 59.4-+ 7.3 62.3 -+ 5.0 87~2 • 23.7 75.3 -+ 3.8 75.9 -+ 10.5 115.2• 10.2 73.4 -+ 7.7 69.1 -+ 2.5 75.9-+ 6.9
| I[ | [ ~ ~" ~ ~" / ~
] 40.7 _+ 2.1 41.4 • 1.7 76.6 -+ 4.4
53.1 • 2.91
49.5 -+ 3.4 57.4 _+ 5.8
61.9• 3.7 74.9 -+ 5.2 76.6 -+ 5.0
75.8~' _+ 3.85
94.3 -+ 11.1 72.5 _+ 3.6
~Groups: U, D (undefoliated, defoliated); trees: 1-5 (5 trees/group); Locations: a, b (apical, basal). ~43% greater than undefoliated trees.
and Lester, 1980). Pellets were s u s p e n d e d in 1.0 ml n - b u t a n o l - H C 1 (95: 5, v / v). The tubes were p l a c e d in a 95 ~ C b a t h for 60 rain. A f t e r c o o l i n g a n d r e m i x i n g , the a b s o r b a n c e s o f the m e t h a n o l - s o l u b l e p r o a n t h o c y a n i d i n s were r e c o r d e d . To m e a s u r e the p r o a n t h o c y a n i d i n c o n t e n t of the insoluble fraction, tubes Containing pellets were recentrifuged, a n d an a p p r o p r i a t e a l i q u o t of the s u p e r n a t a n t was diluted with n - b u t a n o l - H C 1 (95:5), v / v ) to a final v o l u m e of 1.2 ml. A f t e r mixing, the a b s o r b a n c e was r e c o r d e d . P r o c y a n i d i n values were e x p r e s s e d as A at 550 n m p r o d u c e d b y an e x t r a c t e q u i v a l e n t to 100 mg fresh weight of tissue per milliliter (AA55o/100 mg fresh weight). Statistical Analyses. M e a s u r e m e n t s of p r o a n t h o c y a n i d i n c o n t e n t of single needles were a n a l y z e d with a nested f a c t o r i a l analysis of v a r i a n c e c o m p u t e d b y an original F o r t r a n 1V plus p r o g r a m .
1472
WALTERS AND STAFFORD RESULTS
Long-Term Defoliation by Western Spruce Budworms. Soluble pro, anthocyanidin levels in the current year's foliage of budworm-defoliated trees were 45% higher than those in relatively undefoliated trees from the same site (Table 2). This difference was significant when the measurements were recorded on a fresh weight basis (P =
