Journal of Chemical Ecology, Vol. 17, No. 6, 1991
STABILITY OF PHENOLIC A N D PROTEIN MEASURES IN EXCISED OAK FOLIAGE
K A R L W. K L E I N E R Department of Entomology, Pesticide Research Lab The Pennsylvania State University University Park, Pennsylvania 16802
(Received December 12, 1990; accepted February 19, 1991) Abstract--The stability of protein and phenolic measures in excised foliage from two oak species was measured under conditions that simulated the handling and treatment of foliage during insect rearing trials. Excised foliage kept hydrated under refrigeration or insect-rearing conditions maintained stable levels of protein content, proanthocyanidins, gallotannins, total phenolics, and protein-binding capacity for up to 48 hr following field sampling. Measures of protein content, total phenolics, protein-binding capacity, and proanthocyanidins were significantly greater 48-72 hr after field sampling, followed by declines to near field levels within 120 hr. Key Words--Quercus rubra, Quercus prinus, phenolics, tannins, foliage quality, insect-rearing trials. INTRODUCTION Investigations o f the suitability of food plants for herbivorous insects usually require rearing trials with foliage. Ideally, such trials should be performed in situ, but logistical constraints (access to foliage on mature trees, location o f food plants) and environmental variability (temperature, humidity) often make laboratory rearing with excised foliage more practical or useful. The stems or petioles of excised foliage used in laboratory trials are often inserted into a source o f water to maintain leaf turgor and leaf shape (Scriber, 1977, 1978; Hough and Pimentel, 1978; Lawson et al., 1982, 1984). Although this procedure maintains the visible aspects of leaf freshness, stability in the dietary components of excised foliage has received little consideration (Miller, 1987). Changes in quantities or composition o f leaf constituents, e.g., allelochemicals, in excised foliage could influence the validity of laboratory results. 1243 0098-0331/91/0600-1243506.50/0 9 1991 Plenum Publishing Corporation
1244
KLEINER
Phenolics comprise a class of putative allelochemicals that is virtually ubiquitous in plants. They have been shown to have both positive (Bernays, 1981; Bernays et al., 1983) and negative (Bernays, 1981; Berenbaum, 1983; Manuwoto et al., 1985; Karowe, 1989) effects on the feeding behavior and performance of insect herbivores. For example, the secondary chemistry of oaks and other tree species favored by the gypsy moth (Lymantria dispar L.) is comprised primarily of phenolics (Gibbs, 1974), and several classes of these phenolics have been shown to influence gypsy moth growth, fecundity, and mortality (Keating et al., 1988; Kleiner, 1989). In this study, stability in four phenolic measures and one protein measure in foliage from two oak species was examined over a range of handling and treatment stages identical to those used in laboratory rearing trials. METHODS AND MATERIALS
Sampling Experiment I (June 1986). One branch (0.5-1.0 m) was cut from each of five mature chestnut oaks (Quercus prinus L.) and five mature red oaks (Q. rubra L.) and recut under water in 19-liter buckets. Prior to transporting the branches to the lab, one foliage sample (four to five leaves) was taken from each branch (=tree) and flash frozen in liquid nitrogen. In the lab, one bouquet of foliage was made per tree by inserting the petioles of four to five leaves into a florists' Aqua-pic (Syndicate Sales, Kokomo, Indiana). These foliar bouquets are identical to those used in our lab for gypsy moth rearing trials (e.g., Kleiner, 1989). In addition, four pairs of single leaf bouquets were made per tree in the same manner and fed to gypsy moth larvae for 42 hr, then frozen for chemical analysis. All bouquets used in this experiment were kept at ambient conditions (26~ After a portion of the foliage from each branch had been removed to make the foliar bouquet, the remaining branch was kept for additional foliage sampling. These branches were kept in the sample buckets in an unlit cooler at 5~ Samples were frozen for analysis in the order shown in Table 1. Multileaf bouquets were chemically analyzed as separate samples. Combining the paired single leaf bouquets (sample 7) for chemical analysis produced 20 samples per species (four per tree). Experiment 2 (June 1987). One branch (0.5-1.0 m) was cut from each of five mature chestnut oak and five mature red oak trees and two samples of five to six leaves each were taken from each branch (day 1, zero hr). The branches were recut under water and transported to the lab in 19-liter buckets. Ten bouquets of foliage (five to six leaves in each bouquet) were prepared from each branch as described above. Foliar bouquets from each branch were placed in separate covered 31 x 17 x 9-cm plastic boxes in a growth chamber under
1245
OAK PHENOLICS AND PROTEIN TABLE 1. DESCRIPTION OF OAK LEAF SAMPLES FOR EXPERIMENT 1
Sample
Source
N/species
Day
Hours
1 2 3 4 5 6 7
Field Buckets Aqua-pics Buckets Buckets Buckets Aqua-pics (fed to larvae)
5 5 5 5 5 5 20
1 1 2 2 2 3 3
0 5 18 18 30 42 42
conditions used for gypsy moth rearing (24~ with a 14 : l0 light-dark photoperiod). Two foliar bouquets were sampled from each branch every day for five days (days 2-6) for a total of 50 bouquets per species sampled throughout a 120-hr period. All samples were flash frozen in liquid nitrogen and stored at -34~
Statistical Analyses To avoid pseudoreplication with sample 7 in experiment 1, the mean of the four samples per tree was used for statistical analysis, resulting in a balanced design of N = 5 per species. In experiment 2, red oak samples 2 and 3 were lost due to mishandling in the laboratory. For both experiments, differences among samples were analyzed by species, using one-way ANOVA (GLM, SAS Institute) with individual trees as the randomized blocks and sample time as the main effect. Samples different from sample 1 (the field sample) were detected with Tukey's studentized range test.
Chemical Analyses Frozen leaf samples were lyophilized and thoroughly ground in a UDY Cyclone Sample Mill and a subsample was extracted in 0.1 N NaOH to reduce interference from phenolics (Jones et al., 1989). Protein concentration was analyzed using a modification of the micro method of Bradford (1976) (0.04 ml of extract was diluted with 0.76 ml of deionized water, then mixed with 0.20 ml of Coomassie brilliant blue G-250; Biorad dye reagent). Samples were read within 10 min at 595 nm against a distilled water blank, which resulted in higher protein values than reported elsewhere (Faeth, 1985; Jones et al., 1989). A portion of the remaining ground sample was extracted in 70 % acetone, reduced to an aqueous extract via rotary evaporation, and analyzed for proanthocyanidins (vanillin assay; Swain and Hillis, 1959; Schultz and Baldwin 1982), gal-
1246
KLEINER
lotannins (potassium iodate; Bate-Smith, 1977, as modified by Schultz and Baldwin, 1982), total phenolics (Folin-Denis), and protein-binding capacity using hemoglobin as a substrate (Schultz et al., 1981). Protein concentration is reported as percent dry weight bovine serum albumin equivalents (%BSAE); protein binding, Folin-Denis active phenolics, and hydrolyzable tannins are reported as percent dry weight tannic acid equivalents (%TAE; Sigma, Lot 11F0559); and proanthocyanidins as percent dry weight wattle tannin equivalents (%WTE; Acacia sp., from Leon Monnier, Inc. Peabody, Massachusetts), derived from standard curves. RESULTS AND DISCUSSION
Experiment 1. For both red and chestnut oak foliage, there was only one significant change in the five measures of leaf quality made among the seven samples taken over a 42-hr period (Figure 1). After 42 hr, chestnut oak foliage PROTEIN CONTENT 24
9
-
Red Oak from
branches
LJ < 03 r~
22
o
-
Red Oak from
aqua-p]cs
9
-
Chestnut
oak from
branches
z~ -
Chestnut
oak from
aqua-pics
o'~
18
20
16 I
I
I
r
r
I
TOTAL PHENOLICS
I
GALLOTANNINS
10
3O
9
2~
8N
2o o~
t-rl
Vq
6
e 5 12
I
e
O T
~ I
t I
t I
10
~ ]
I
5
PROANTHOCYANIDINS
PROTEIN BINDING
11
N 20 9
o'~
15
8
10 i
I
[
HOURS AFTER FIELD SAMPLE
HOURS AFTER FIELD SAMPLE
FIc. 1. Levels of protein content and phenolics in excised foliage of red oak and chestnut oak subjected to laboratory treatment to simulate handling during rearing trials. Each point is mean ___SE of five trees. * indicates sample is significantly different from sample 1 at P < 0.05. % BSAE = percent dry weight bovine serum albumin equivalents; % TAE = percent dry weight tannic acid equivalents; WTE = dry weight wattle tannin equivalents. See text for explanation of samples.
OAKPrmNOLICSAND PROTEIN
1247
in Aqua-pics that had been fed to gypsy moth larvae had greater measures of total phenolics than the initial field sample (8.99% TAE vs. 7.68% TAE; P _< 0.05). Interestingly, the smallest difference among means that the statistical analysis could have detected (regardless of species) ranged from low (1.27% TAE, protein binding; 1.29% TAE, total phenolics; 1.9% TAE gallotannins) to medium (4.4 % BSAE protein) to high (8.17 % WTE proanthocyanidins). In this study, significant differences between sample times only indicates changes in the measures of foliage quality. Because of the differential tolerance to tannins by various herbivores (Bernays, 1978; Berenbaum, 1983; Karowe, 1989), the biological impact of even the smallest change in phenolics will be species specific. The results of experiment 1 show that after 42 hr of gypsy moth feeding, only small increases in phenolics were observed in the remaining foliage. This is surprising in light of many studies that have shown increases in secondary metabolites within hours of damage or insect feeding on intact foliage (Baldwin and Schultz, 1983; TaUamy, 1985; Niemeyer et al., 1989). Some inducible responses are limited to the site of damage, while others occur systemically and may require the response of the whole plant (Bowles, 1990). The limited response of excised foliage observed in this study suggests the latter, but without proper controls, this conclusion cannot be assumed. Experiment 2. Gallotannin was the only measure for both red and chestnut oak that did not change significantly over a five-day period (120 hr). The measures of protein content, proanthocyanidin, total phenolics, and protein-binding capacity all displayed increases (with the exception of red oak protein content and protein-binding capacity) within 48-72 hr, followed by subsequent declines (Figure 2). Protein measures in chestnut oak foliage were significantly greater 72 and 96 hr following the field sample (P < 0.0001 from ANOVA), but were below field sample measures by 120 hr. The measure of red oak protein content displayed a marked decrease at 72 hr, a considerable increase at 96 hr (P < 0.0001 from ANOVA), and a return to near field sample levels by 120 hr. The measures of total phenolics in red oak and chestnut oak foliage were significantly greater 72, 96, and for red oak, 120 hr after field sampling (P < 0.0001 from ANOVA for each species). The results of experiment 1 indicate that oak foliage can be collected, returned to the laboratory, and stored under refrigeration for up to 42 hr without significantly changing these measures of protein content and phenolics. Both experiments indicate that the most stable period for the measures of protein and phenolics in excised foliage is during the first 24 hr following field sampling. In experiment 2, some of these measures (protein content, total phenolics) did not differ significantly until 48 or 72 hr. The patterns of change observed in this study are not readily explained by the natural variation among samples. Although there was significant variation
1248
Ld .< (.'9 G3
o~
KLEINER 30 28 26 24 22 20 18 16
PROTEIN CONTENT *i
I
I
I
f
I
9
-
A
-- C h e s t n u t
Red
OGk Oak
I
TOTAL PHENOLICS
t GALLOTAN N INS
16
50 45 40 35
14
30 y.
20 18
*O 0
LLI
o~
PROTEIN B~INDI'NG '
F
'
14
18 16 14 12 10
10
8
16
I
F
I
i
!
25 20 15 10
~2 Vq
PROANTHOCYANIDINS
{ "5 {
6 4 0
8
72
96
120
HOURS AFTER RELD SAMPLE
I 0
t 24
;8
7P2
916
I 120
HOURS AFTER FIELD SAMPLE
FIG. 2. Levels of protein content and phenolics in excised foliage of red oak and chestnut oak from the field (0 hr) and at five 24-hr intervals while under insect rearing conditions. Each point is mean ___SE of 10 trees. * indicates sample is significantly different from field sample at P < 0.05. Axis labels defined in Figure 1.
among trees of both species within any sample period, this variation was exceeded by the magnitude of change in the measures over the 120-hr period (Figure 2). The increases in the measures of total phenolics after 120 hr were similar to those observed by Miller (1987) in excised birch and willow foliage after 144 hr. However, the increases in some of the phenolic measures cannot be interpreted simply as changes in the quantity of the compounds (proanthocyanidins and gallotannins) that were measured in the initial field samples. The results of assays that are specific for a class of tannins (potassium iodate and vanillin assays) indicate that no change occurred in the quantity of red oak gallotannins or chestnut oak proanthocyanidins. However, the measures of total phenolics and protein-binding capacity, which are sensitive to both proanthocyanidins and gallotannins, suggest that these tannins did increase. Because the Folin-Denis and protein binding assays are sensitive to a wide array of phenolics, they may reflect changes in other foliar constituents not measured in the more specific assays. These changes could include the synthesis and/or restructuring in excised foliage of nontannin phenolics or tannins not measured by the
OAK PHENOLICS AND PROTEIN
1249
analyses used here. Alternatively, the patterns observed in this study may reflect a process of phenolic degradation, which may involve oxidation and restructuring, followed by the complete breakdown of these products. This would result in the greater measures of phenolic activity observed at 72-96 hr, followed by the lower phenolic measures observed by 120 hr. As a consequence, it would appear that after 120 hr, hydrated, excised foliage is quantitatively the same as samples taken from the first 48 hr. However, it cannot be assumed that such foliage is qualitatively the same. The maintenance of leaf turgot for insect-rearing trials cannot assure the integrity of some dietary constituents for more than 48 hr. Such an assumption may lead to uninterpretable or incorrect biological indices, particularly when results are compared across different host species or foliage treatments (Scriber, 1977; Hough and Pimentel, 1978). Cautious interpretations should be made from larval performance and/or leaf quality studies in which foliage used for chemical analyses may have been handled differently from foliage for rearing trials (Fox and MacCauley, 1977; Wint, 1983; Barbosa et al., 1983, 1986; Puttick, 1986). Researchers who hydrate excised foliage and change it within 48 hr or use refrigerated foliage within 48 hr (Raupp et al., 1988) are likely to maintain the foliage traits that were measured in this study. Acknowledgments--This study was supported by United States Forest Service Cooperative Agreement FS-23-155 and National Science Foundation grants BSR 8813433 and BSR 8400284. M. Briggs helped analyze samples. Thanks to J. Schultz and M. Hunter and two anonymous reviewers for comments on earlier drafts of this manuscript.
REFERENCES BALDWIN,I.T., and SCHULTZ,J.C. 1983. Rapid changes in tree leaf chemistry induced by damage: Evidence for communication between plants, Science 221:277-279. BARBOSA,P., WALDVOGEL,M., MARTINAT,P., and DOUGLASS,W. 1983. Development and reproductive performance of the gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae), on selected hosts common to mid-atlantic and southern forests. Environ. Entomol. 12:18581862. BARBOSA,P., MARTINAT,P., and WALDVOGEL,M. 1986. Development, fecundity and survival of the herbivore Lymantria dispar and the number of plant species in its diet. Ecol. Entomol. 11:1-6. BATE-SMITH, E.C. 1977. Astringent tannins of Acer species. Phytochemistry 16:1421-1426. BERENBAUM,M.R. 1983. Effects of tannins on growth and digestion in two species of papilionids. Entomol. Exp. Appl. 34:245-250. BERNAYS, E.A. 1978. Tannins: An alternative viewpoint. Entomol. Exp. Appl. 24:44-53. BERNAYS, E.A. 1981. Plant tannins and insect herbivores: An appraisal. EcoL EntomoL 6:353360. BERNAYS, E.A., CHAMBERLAIN,D.J., and WOODHEAD, S. 1983. Phenols as nutrients for a phytophagous insect Anacridium melanorhodon. J. Insect Physiol. 29:535-539. BOWLES, D. 1990. Signals in the wounded plant. Nature 343:314-315.
1250
KLEINER
BRADFORD,M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principles of protein-dye binding. Anal. Biochem. 72:248-254. FAETH, S.H. 1985. Quantitative defense theory and patterns of feeding by oak insects. Oecologia 68:34-40. Fox, R.L., and MACAULEY, B.J. 1977. Insect grazing on Eucalyptus in response to variation in leaf tannins and nitrogen. Oecologia 29:145-162. GIBBS, R.O. 1974. Chemotaxonomy of Flowering Plants. McGill-Queens University Press, Montreal, Quebec, Canada. 1200 pp. HOUGH, J.A., and PIMENTEL, D. 1978. Influence of host foliage on development, survival and fecundity of the gypsy moth. Environ. Entomol. 7:97-102. JONES, C.G., HARE, T.D., and COMPTON, S.J. 1989. Measuring plant protein with the Bradford assay. J. Chem. Ecol. 15:979-992. KAROWE, D.N. 1989. Differential effect of tannic acid on two tree-feeding Lepidoptera: Implications for theories of plant anti-herbivore chemistry. Oecologia 80:507-512. KEATING, S.T., YENDOL, W.G., and SCHULTZ, J.C. 1988. Relationship between susceptibility of gypsy moth larvae (Lepidoptera: Lymantriidae) to a baculovirus and host plant foliage constituents. Environ. Entomol. 17:952-958. KLEINER, K.W. 1989. Sources of variation in oak leaf quality as food for the gypsy moth: implications for forest stand susceptibility. Unpublished PhD thesis. Pennsylvania State University, University Park, Pennsylvania. LAWSON, D.L., MERRITT, R.W., KLUG, M.J., and MARTIN, J.S. 1982. The utilization of late season foliage by the orange striped oakworm, Anisota senatoria. Entomol. Exp. Appl. 32:242248. LAWSON, D.L., MERRITT, R.W., MARTIN, M.M., and MARTIN, J.S., and KUKOR, J.J. 1984. The nutritional ecology of larvae of Alsophila pometaria and Anisota senatoria feeding on earlyand late-season oak foliage. Entomol. Exp. Appl. 35:105-114. MANUWOTO,S., SCRIBER,J.M., HSIA, M.T., and SUNARJO,P. 1985. Antibiosis/antixenosis in tulip tree and quaking aspen leaves against the polyphagous southern armyworm, Spodoptera eridania. Oecologia 67:1-7. MILLER, W.E. 1987. Changes in nutritional quality of detached aspen and willow foliage used as insect food in the laboratory. Great Lakes Entomoli 20:41-45. NIEMEYER, H.M., PESEL, E., COPAJA, S.V., BRAVO, H.R., FRANKE, S., and FRANCKE, W. 1989. Changes in hydroxamic acid levels of wheat plants induced by aphid feeding. Phytochemistry 28:447-449. PUTTICK, G.M. 1986. Utilization of evergreen and deciduous oaks by the Californian oak moth Phryganidia californica. Oecologia 68:589-594. RAUPP, M.J., WERREN,J.H., and SADOF,C.S. 1988. Effects of short-term phenological changes in leaf suitability on the survivorship, growth and development of gypsy moth (Lepidoptera: Lymantriidae) larvae. Environ. Entomol. 17:316-319. ROSSITER, M.C., SCHULTZ, J.C. and BALDWIN, I.T. 1988. Relationships among defoliation, red oak phenolics, and gypsy moth growth and reproduction. Ecology 69:267--277. SCHULTZ, J.C., and BALDWIN,I.T. 1982. Oak leaf quality declines in response to defoliation by gypsy moth larvae. Science 217:149-151. SCHULTZ, J.C., BALDWIN,I.T., and NOTHNAGLE, P.J. 1981. Hemoglobin as a binding substrate in the quantitative analysis of plant tannins. J. Agric. Food Chem. 29:823-826. SCRIBER, J.M. 1977. Limiting effects of low leaf-water content on the nitrogen utilization, energy budget, and larval growth of Hyalophora cecropia (Lepidoptera: Saturniidae). Oecologia 28:269-287. SCRmER, J.M. 1978. The effects of larval feeding specialization and plant growth form on the
OAK PHENOLICS AND PROTEIN
125 1
consumption and utilization of plant biomass and nitrogen: An ecological consideration. Entomol. Exp. Appl. 24:494-510. SWAIN, T., and HILLIS, W.E. 1959. The phenolic constituents of Prunus domestica. J. Sci. Food Agric. 10:63-68. TALLAMY, D.W. 1985. Squash beetle feeding behavior: An adaptation against induced cucurbit defenses. Ecology 66:1574-1579. WINT, G.R.W. 1983. The effect of foliar nutrients upon the growth and feeding of a Lepidopteran larva, pp. 301-320, in J.A. Lee, S. McNeill, and I.H. Rorison (eds.). Nitrogen as an Ecological Factor. 22nd Symposium of the British Ecological Society, Oxford 1981. Blackwell Scientific, Oxford.
STABILITY OF PHENOLIC A N D PROTEIN MEASURES IN EXCISED OAK FOLIAGE
K A R L W. K L E I N E R Department of Entomology, Pesticide Research Lab The Pennsylvania State University University Park, Pennsylvania 16802
(Received December 12, 1990; accepted February 19, 1991) Abstract--The stability of protein and phenolic measures in excised foliage from two oak species was measured under conditions that simulated the handling and treatment of foliage during insect rearing trials. Excised foliage kept hydrated under refrigeration or insect-rearing conditions maintained stable levels of protein content, proanthocyanidins, gallotannins, total phenolics, and protein-binding capacity for up to 48 hr following field sampling. Measures of protein content, total phenolics, protein-binding capacity, and proanthocyanidins were significantly greater 48-72 hr after field sampling, followed by declines to near field levels within 120 hr. Key Words--Quercus rubra, Quercus prinus, phenolics, tannins, foliage quality, insect-rearing trials. INTRODUCTION Investigations o f the suitability of food plants for herbivorous insects usually require rearing trials with foliage. Ideally, such trials should be performed in situ, but logistical constraints (access to foliage on mature trees, location o f food plants) and environmental variability (temperature, humidity) often make laboratory rearing with excised foliage more practical or useful. The stems or petioles of excised foliage used in laboratory trials are often inserted into a source o f water to maintain leaf turgor and leaf shape (Scriber, 1977, 1978; Hough and Pimentel, 1978; Lawson et al., 1982, 1984). Although this procedure maintains the visible aspects of leaf freshness, stability in the dietary components of excised foliage has received little consideration (Miller, 1987). Changes in quantities or composition o f leaf constituents, e.g., allelochemicals, in excised foliage could influence the validity of laboratory results. 1243 0098-0331/91/0600-1243506.50/0 9 1991 Plenum Publishing Corporation
1244
KLEINER
Phenolics comprise a class of putative allelochemicals that is virtually ubiquitous in plants. They have been shown to have both positive (Bernays, 1981; Bernays et al., 1983) and negative (Bernays, 1981; Berenbaum, 1983; Manuwoto et al., 1985; Karowe, 1989) effects on the feeding behavior and performance of insect herbivores. For example, the secondary chemistry of oaks and other tree species favored by the gypsy moth (Lymantria dispar L.) is comprised primarily of phenolics (Gibbs, 1974), and several classes of these phenolics have been shown to influence gypsy moth growth, fecundity, and mortality (Keating et al., 1988; Kleiner, 1989). In this study, stability in four phenolic measures and one protein measure in foliage from two oak species was examined over a range of handling and treatment stages identical to those used in laboratory rearing trials. METHODS AND MATERIALS
Sampling Experiment I (June 1986). One branch (0.5-1.0 m) was cut from each of five mature chestnut oaks (Quercus prinus L.) and five mature red oaks (Q. rubra L.) and recut under water in 19-liter buckets. Prior to transporting the branches to the lab, one foliage sample (four to five leaves) was taken from each branch (=tree) and flash frozen in liquid nitrogen. In the lab, one bouquet of foliage was made per tree by inserting the petioles of four to five leaves into a florists' Aqua-pic (Syndicate Sales, Kokomo, Indiana). These foliar bouquets are identical to those used in our lab for gypsy moth rearing trials (e.g., Kleiner, 1989). In addition, four pairs of single leaf bouquets were made per tree in the same manner and fed to gypsy moth larvae for 42 hr, then frozen for chemical analysis. All bouquets used in this experiment were kept at ambient conditions (26~ After a portion of the foliage from each branch had been removed to make the foliar bouquet, the remaining branch was kept for additional foliage sampling. These branches were kept in the sample buckets in an unlit cooler at 5~ Samples were frozen for analysis in the order shown in Table 1. Multileaf bouquets were chemically analyzed as separate samples. Combining the paired single leaf bouquets (sample 7) for chemical analysis produced 20 samples per species (four per tree). Experiment 2 (June 1987). One branch (0.5-1.0 m) was cut from each of five mature chestnut oak and five mature red oak trees and two samples of five to six leaves each were taken from each branch (day 1, zero hr). The branches were recut under water and transported to the lab in 19-liter buckets. Ten bouquets of foliage (five to six leaves in each bouquet) were prepared from each branch as described above. Foliar bouquets from each branch were placed in separate covered 31 x 17 x 9-cm plastic boxes in a growth chamber under
1245
OAK PHENOLICS AND PROTEIN TABLE 1. DESCRIPTION OF OAK LEAF SAMPLES FOR EXPERIMENT 1
Sample
Source
N/species
Day
Hours
1 2 3 4 5 6 7
Field Buckets Aqua-pics Buckets Buckets Buckets Aqua-pics (fed to larvae)
5 5 5 5 5 5 20
1 1 2 2 2 3 3
0 5 18 18 30 42 42
conditions used for gypsy moth rearing (24~ with a 14 : l0 light-dark photoperiod). Two foliar bouquets were sampled from each branch every day for five days (days 2-6) for a total of 50 bouquets per species sampled throughout a 120-hr period. All samples were flash frozen in liquid nitrogen and stored at -34~
Statistical Analyses To avoid pseudoreplication with sample 7 in experiment 1, the mean of the four samples per tree was used for statistical analysis, resulting in a balanced design of N = 5 per species. In experiment 2, red oak samples 2 and 3 were lost due to mishandling in the laboratory. For both experiments, differences among samples were analyzed by species, using one-way ANOVA (GLM, SAS Institute) with individual trees as the randomized blocks and sample time as the main effect. Samples different from sample 1 (the field sample) were detected with Tukey's studentized range test.
Chemical Analyses Frozen leaf samples were lyophilized and thoroughly ground in a UDY Cyclone Sample Mill and a subsample was extracted in 0.1 N NaOH to reduce interference from phenolics (Jones et al., 1989). Protein concentration was analyzed using a modification of the micro method of Bradford (1976) (0.04 ml of extract was diluted with 0.76 ml of deionized water, then mixed with 0.20 ml of Coomassie brilliant blue G-250; Biorad dye reagent). Samples were read within 10 min at 595 nm against a distilled water blank, which resulted in higher protein values than reported elsewhere (Faeth, 1985; Jones et al., 1989). A portion of the remaining ground sample was extracted in 70 % acetone, reduced to an aqueous extract via rotary evaporation, and analyzed for proanthocyanidins (vanillin assay; Swain and Hillis, 1959; Schultz and Baldwin 1982), gal-
1246
KLEINER
lotannins (potassium iodate; Bate-Smith, 1977, as modified by Schultz and Baldwin, 1982), total phenolics (Folin-Denis), and protein-binding capacity using hemoglobin as a substrate (Schultz et al., 1981). Protein concentration is reported as percent dry weight bovine serum albumin equivalents (%BSAE); protein binding, Folin-Denis active phenolics, and hydrolyzable tannins are reported as percent dry weight tannic acid equivalents (%TAE; Sigma, Lot 11F0559); and proanthocyanidins as percent dry weight wattle tannin equivalents (%WTE; Acacia sp., from Leon Monnier, Inc. Peabody, Massachusetts), derived from standard curves. RESULTS AND DISCUSSION
Experiment 1. For both red and chestnut oak foliage, there was only one significant change in the five measures of leaf quality made among the seven samples taken over a 42-hr period (Figure 1). After 42 hr, chestnut oak foliage PROTEIN CONTENT 24
9
-
Red Oak from
branches
LJ < 03 r~
22
o
-
Red Oak from
aqua-p]cs
9
-
Chestnut
oak from
branches
z~ -
Chestnut
oak from
aqua-pics
o'~
18
20
16 I
I
I
r
r
I
TOTAL PHENOLICS
I
GALLOTANNINS
10
3O
9
2~
8N
2o o~
t-rl
Vq
6
e 5 12
I
e
O T
~ I
t I
t I
10
~ ]
I
5
PROANTHOCYANIDINS
PROTEIN BINDING
11
N 20 9
o'~
15
8
10 i
I
[
HOURS AFTER FIELD SAMPLE
HOURS AFTER FIELD SAMPLE
FIc. 1. Levels of protein content and phenolics in excised foliage of red oak and chestnut oak subjected to laboratory treatment to simulate handling during rearing trials. Each point is mean ___SE of five trees. * indicates sample is significantly different from sample 1 at P < 0.05. % BSAE = percent dry weight bovine serum albumin equivalents; % TAE = percent dry weight tannic acid equivalents; WTE = dry weight wattle tannin equivalents. See text for explanation of samples.
OAKPrmNOLICSAND PROTEIN
1247
in Aqua-pics that had been fed to gypsy moth larvae had greater measures of total phenolics than the initial field sample (8.99% TAE vs. 7.68% TAE; P _< 0.05). Interestingly, the smallest difference among means that the statistical analysis could have detected (regardless of species) ranged from low (1.27% TAE, protein binding; 1.29% TAE, total phenolics; 1.9% TAE gallotannins) to medium (4.4 % BSAE protein) to high (8.17 % WTE proanthocyanidins). In this study, significant differences between sample times only indicates changes in the measures of foliage quality. Because of the differential tolerance to tannins by various herbivores (Bernays, 1978; Berenbaum, 1983; Karowe, 1989), the biological impact of even the smallest change in phenolics will be species specific. The results of experiment 1 show that after 42 hr of gypsy moth feeding, only small increases in phenolics were observed in the remaining foliage. This is surprising in light of many studies that have shown increases in secondary metabolites within hours of damage or insect feeding on intact foliage (Baldwin and Schultz, 1983; TaUamy, 1985; Niemeyer et al., 1989). Some inducible responses are limited to the site of damage, while others occur systemically and may require the response of the whole plant (Bowles, 1990). The limited response of excised foliage observed in this study suggests the latter, but without proper controls, this conclusion cannot be assumed. Experiment 2. Gallotannin was the only measure for both red and chestnut oak that did not change significantly over a five-day period (120 hr). The measures of protein content, proanthocyanidin, total phenolics, and protein-binding capacity all displayed increases (with the exception of red oak protein content and protein-binding capacity) within 48-72 hr, followed by subsequent declines (Figure 2). Protein measures in chestnut oak foliage were significantly greater 72 and 96 hr following the field sample (P < 0.0001 from ANOVA), but were below field sample measures by 120 hr. The measure of red oak protein content displayed a marked decrease at 72 hr, a considerable increase at 96 hr (P < 0.0001 from ANOVA), and a return to near field sample levels by 120 hr. The measures of total phenolics in red oak and chestnut oak foliage were significantly greater 72, 96, and for red oak, 120 hr after field sampling (P < 0.0001 from ANOVA for each species). The results of experiment 1 indicate that oak foliage can be collected, returned to the laboratory, and stored under refrigeration for up to 42 hr without significantly changing these measures of protein content and phenolics. Both experiments indicate that the most stable period for the measures of protein and phenolics in excised foliage is during the first 24 hr following field sampling. In experiment 2, some of these measures (protein content, total phenolics) did not differ significantly until 48 or 72 hr. The patterns of change observed in this study are not readily explained by the natural variation among samples. Although there was significant variation
1248
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KLEINER 30 28 26 24 22 20 18 16
PROTEIN CONTENT *i
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TOTAL PHENOLICS
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FIG. 2. Levels of protein content and phenolics in excised foliage of red oak and chestnut oak from the field (0 hr) and at five 24-hr intervals while under insect rearing conditions. Each point is mean ___SE of 10 trees. * indicates sample is significantly different from field sample at P < 0.05. Axis labels defined in Figure 1.
among trees of both species within any sample period, this variation was exceeded by the magnitude of change in the measures over the 120-hr period (Figure 2). The increases in the measures of total phenolics after 120 hr were similar to those observed by Miller (1987) in excised birch and willow foliage after 144 hr. However, the increases in some of the phenolic measures cannot be interpreted simply as changes in the quantity of the compounds (proanthocyanidins and gallotannins) that were measured in the initial field samples. The results of assays that are specific for a class of tannins (potassium iodate and vanillin assays) indicate that no change occurred in the quantity of red oak gallotannins or chestnut oak proanthocyanidins. However, the measures of total phenolics and protein-binding capacity, which are sensitive to both proanthocyanidins and gallotannins, suggest that these tannins did increase. Because the Folin-Denis and protein binding assays are sensitive to a wide array of phenolics, they may reflect changes in other foliar constituents not measured in the more specific assays. These changes could include the synthesis and/or restructuring in excised foliage of nontannin phenolics or tannins not measured by the
OAK PHENOLICS AND PROTEIN
1249
analyses used here. Alternatively, the patterns observed in this study may reflect a process of phenolic degradation, which may involve oxidation and restructuring, followed by the complete breakdown of these products. This would result in the greater measures of phenolic activity observed at 72-96 hr, followed by the lower phenolic measures observed by 120 hr. As a consequence, it would appear that after 120 hr, hydrated, excised foliage is quantitatively the same as samples taken from the first 48 hr. However, it cannot be assumed that such foliage is qualitatively the same. The maintenance of leaf turgot for insect-rearing trials cannot assure the integrity of some dietary constituents for more than 48 hr. Such an assumption may lead to uninterpretable or incorrect biological indices, particularly when results are compared across different host species or foliage treatments (Scriber, 1977; Hough and Pimentel, 1978). Cautious interpretations should be made from larval performance and/or leaf quality studies in which foliage used for chemical analyses may have been handled differently from foliage for rearing trials (Fox and MacCauley, 1977; Wint, 1983; Barbosa et al., 1983, 1986; Puttick, 1986). Researchers who hydrate excised foliage and change it within 48 hr or use refrigerated foliage within 48 hr (Raupp et al., 1988) are likely to maintain the foliage traits that were measured in this study. Acknowledgments--This study was supported by United States Forest Service Cooperative Agreement FS-23-155 and National Science Foundation grants BSR 8813433 and BSR 8400284. M. Briggs helped analyze samples. Thanks to J. Schultz and M. Hunter and two anonymous reviewers for comments on earlier drafts of this manuscript.
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consumption and utilization of plant biomass and nitrogen: An ecological consideration. Entomol. Exp. Appl. 24:494-510. SWAIN, T., and HILLIS, W.E. 1959. The phenolic constituents of Prunus domestica. J. Sci. Food Agric. 10:63-68. TALLAMY, D.W. 1985. Squash beetle feeding behavior: An adaptation against induced cucurbit defenses. Ecology 66:1574-1579. WINT, G.R.W. 1983. The effect of foliar nutrients upon the growth and feeding of a Lepidopteran larva, pp. 301-320, in J.A. Lee, S. McNeill, and I.H. Rorison (eds.). Nitrogen as an Ecological Factor. 22nd Symposium of the British Ecological Society, Oxford 1981. Blackwell Scientific, Oxford.
