Coniferyl benzoate in quaking aspen A ruffed grouse feeding deterrent

Quaking aspen (Populus tremuloides Michx.) staminate flower buds and catkins are important food resources for ruffed grouse (Bonasa umbellus); however, ruffed grouse select only certain quaking aspen to feed upon. Earlier studies indicate that the primary difference between quaking aspen that ruffed grouse feed upon and those not used is the level of coniferyl benzoate in the flower buds. Bioassays show that coniferyl benzoate is a feeding deterrent for ruffed grouse; its effect on ruffed grouse after ingestion has not been tested. Possible physiological effects, based on the chemical properties of coniferyl benzoate and its oxidation products, include inhibition of protein digestion, toxic effects, and antiestrogenic effects.Minnesota Agricultural Experiment Station Journal Series No. 17,079.     

Physiological and behavioral effects of coniferyl benzoate on avian reproduction

Various plant secondary metabolites related to cinnamic acid are of interest because of their repellency to birds and their occurrence in ecologically important food items. Coniferyl benzoate (CB), a phenylpropanoid ester that occurs in quaking aspen (Populus tremuloides) is of particular ecological interest because of its effect on ruffed grouse (Bonasa umbellus) feeding behavior and its possible influence on the population dynamics of this bird. During detoxification processes, CB and other analogous compounds are metabolized into by-products, such as ferulic acid (FA), that can cause anti-reproductive effects. We tested whether consumption of CB produces antire-productive effects similar to FA using male and female Japanese quail (Coturnix coturnix) as avian models for ruffed grouse. The parameters we investigated included: the production, morphology, and development of eggs; reproductive characteristics influenced by estrogen; serum prolactin levels; and male reproductive behavior. Dietary CB did not produce antireproductive effects similar to FA at intake levels that Japanese quail and ruffed grouse would freely consume. Consumption of CB by Japanese quail significantly reduced egg production and body mass but did not affect male reproductive performance. Coniferyl benzoate's effect on egg production may be explained by lower energy acquisition and retention rather than endocrine changes per se. Contrary to previous reports, it is unlikely that FA, or similar compounds act directly as estrogen mimics or antagonists. Although, CB did reduce egg production in quail, it is unlikely that it would affect egg production in wild ruffed grouse. Detoxification costs and the effects of CB on nutrient utilization may explain why ruffed grouse avoid high dietary levels of CB.     

Phytochemical Variation in Quaking Aspen: Effects on Gypsy Moth Susceptibility to Nuclear Polyhedrosis Virus

The performance of gypsy moths (Lymantria dispar) feeding on quaking aspen (Populus tremuloides) is strongly influenced by host foliar chemistry and susceptibility to a nuclear polyhedrosis virus (LdNPV), but the relationship of susceptibility to chemistry is poorly understood. We investigated the effects of genetic and resource-mediated variation in phytochemistry on viral pathogenicity. Trees were grown in pots in a common garden. Disks were punched from aspen leaves, inoculated with LdNPV and fed to third instars. Additional leaves were analyzed for levels of nitrogen, starch, phenolic glycosides, and condensed tannins. Despite marked variation among trees in levels of phenolic glycosides and tannins, we observed minimal variation in larval susceptibility to LdNPV. Viral pathogenicity was only weakly (inversely) correlated with tannin concentrations in one of two experiments. These results suggest that differential defoliation of aspen by gypsy moths in the field is due to the direct effects of host chemistry on larval performance rather than to the indirect effects of host chemistry on efficacy of this natural enemy.     

Consequences of Enriched Atmospheric CO2 and Defoliation for Foliar Chemistry and Gypsy Moth Performance

Elevated concentrations of atmospheric CO₂ are likely to interact with other factors affecting plant physiology to alter plant chemical profiles and plant–herbivore interactions. We evaluated the independent and interactive effects of enriched CO₂ and artificial defoliation on foliar chemistry of quaking aspen (Populus tremuloides) and sugar maple (Acer saccharum), and the consequences of such changes for short-term performance of the gypsy moth (Lymantria dispar). We grew aspen and maple seedlings in ambient (~360 ppm) and enriched (650 ppm) CO₂ environments at the University of Wisconsin Biotron. Seven weeks after budbreak, trees in half of the rooms were subjected to 50% defoliation. Afterwards, foliage was collected for chemical analyses, and feeding trials were conducted with fourth-stadium gypsy moths. Enriched CO₂ altered foliar levels of water, nitrogen, carbohydrates, and phenolics, and responses generally differed between the two tree species. Defoliation induced chemical changes only in aspen. We found no significant interactions between CO₂ and defoliation for levels of carbon-based defenses (phenolic glycosides and tannins). CO₂ treatment altered the performance of larvae fed aspen, but not maple, whereas defoliation had little effect on performance of insects. In general, results from this experimental system do not support the hypothesis that induction of carbon-based chemical defenses, and attendant effects on insects, will be stronger in a CO₂-enriched world.     

Role of avian trigeminal sensory system in detecting coniferyl benzoate,a plant allelochemical

Coniferyl benzoate, a secondary metabolite found in quaking aspen (Populus tremuloides) and other plants, is an avian feeding deterrent of ecological and potential commercial importance. This study was conducted to determine if coniferyl benzoate is a trigeminal stimulant for birds and to ascertain if trigeminal chemoreception of coniferyl benzoate can mediate avian feeding behavior. Five European starlings (Sturnus vulgaris) with bilateral nerve cuts (ophthalmic branch of the trigeminal nerve) and four starlings that had sham surgeries were fed a commercial diet treated with coniferyl benzoate. Birds receiving bilateral nerve cuts ate significantly more feed than intact birds, indicating trigeminal detection of coniferyl benzoate and trigeminal mediation of feeding behavior. In the past, trigeminal chemoreception has not been recognized as important in the detection of plant secondary metabolites despite the irritant or astringent properties of a number of them.     

Flower <Emphasis Type="Italic">vs.</Emphasis> Leaf Feeding by <Emphasis Type="Italic">Pieris brassicae</Emphasis>: Glucosinolate-Rich Flower Tissues are Preferred and Sustain Higher Growth Rate

Interactions between butterflies and caterpillars in the genus Pieris and plants in the family Brassicaceae are among the best explored in the field of insect–plant biology. However, we report here for the first time that Pieris brassicae, commonly assumed to be a typical folivore, actually prefers to feed on flowers of three Brassica nigra genotypes rather than on their leaves. First- and second-instar caterpillars were observed to feed primarily on leaves, whereas late second and early third instars migrated via the small leaves of the flower branches to the flower buds and flowers. Once flower feeding began, no further leaf feeding was observed. We investigated growth rates of caterpillars having access exclusively to either leaves of flowering plants or flowers. In addition, we analyzed glucosinolate concentrations in leaves and flowers. Late-second- and early-third-instar P. brassicae caterpillars moved upward into the inflorescences of B. nigra and fed on buds and flowers until the end of the final (fifth) instar, after which they entered into the wandering stage, leaving the plant in search of a pupation site. Flower feeding sustained a significantly higher growth rate than leaf feeding. Flowers contained levels of glucosinolates up to five times higher than those of leaves. Five glucosinolates were identified: the aliphatic sinigrin, the aromatic phenyethylglucosinolate, and three indole glucosinolates: glucobrassicin, 4-methoxyglucobrassicin, and 4-hydroxyglucobrassicin. Tissue type and genotype were the most important factors affecting levels of identified glucosinolates. Sinigrin was by far the most abundant compound in all three genotypes. Sinigrin, 4-hydroxyglucobrassicin, and phenylethylglucosinolate were present at significantly higher levels in flowers than in leaves. In response to caterpillar feeding, sinigrin levels in both leaves and flowers were significantly higher than in undamaged plants, whereas 4-hydroxyglucobrassicin leaf levels were lower. Our results show that feeding on flower tissues, containing higher concentrations of glucosinolates, provides P. brassicae with a nutritional benefit in terms of higher growth rate. This preference appears to be in contrast to published negative effects of volatile glucosinolate breakdown products on the closely related Pieris rapae.     

Methods and pitfalls of extracting condensed tannins and other phenolics from plants: Insights from investigations onEucalyptus leaves

Optimal conditions for extraction of tannins and other phenolics from tree foliage and their subsequent storage rarely have been investigated. We investigated methods of drying leaves, optimal solvents, and the effects of light and temperature on the extractability and stability of condensed tannins (proanthocyanidins) and total phenolics from leaves ofEucalyptus trees. Aqueous acetone was a better solvent than aqueous methanol for condensed tannins and total phenolics, but condensed tannins were less stable in aqueous acetone than aqueous methanol. Stability of condensed tannins also was decreased substantially by room temperature versus 4°C and by exposure to indirect sunlight, although the assay for total phenolics was unaffected. For quantitative estimation of condensed tannins, extraction with 50% acetone was better than methods of direct analysis of leaf tissue. The highest estimates of total condensed tannins were obtained by exhaustive extraction with 50% acetone followed by direct analysis of the residue. Lyophilization of fresh leaf increased yield of condensed tannin (although usually by less than 10%). Lyophilization and subsequent storage of extracts had little effect on assays for condensed tannins or total phenolics.     

Effects of large mammalian herbivores and ant symbionts on condensed tannins of Acacia drepanolobium in Kenya

Condensed tannins have been considered to be important inducible defenses against mammalian herbivory. We tested for differences in condensed tannin defenses in Acacia drepanolobium in Kenya over two years among different large mammalian herbivore treatments [total exclusion, antelope only, and megaherbivore (elephants and giraffes) + antelope] and with four different ant symbiont species on the trees. We predicted that (1) condensed tannin concentrations would be lowest in the mammal treatment with the lowest level of herbivory (total exclusion), (2) trees occupied by mutualist ants that protect the trees most aggressively would have lower levels of tannins, and (3) if chemical defense production is costly, there would be a trade-off between tannin concentrations, growth, and mechanical defenses. Mean tannin concentrations increased from total exclusion treatments to wildlife-only treatments to megaherbivore + antelope treatments. In 1997, condensed tannin concentrations were significantly lower in trees occupied by the ant Crematogaster nigriceps, the only ant species that actively removed axillary buds. Contrary to our prediction, trees occupied by ant species that protect the trees more aggressively against mammalian herbivores did not have lower overall levels of condensed tannins. There was no consistent evidence of a trade-off between tannin concentrations and growth rate, but there was a positive correlation between mean thorn length and mean tannin concentrations across species of ant inhabitants and across herbivore treatments in 1997. Contrary to our expectation, trees had higher tannin concentrations in the upper parts of the canopy where there is little herbivory by mammals.     

Feeding and oviposition deterrent activities of flower buds of globemallow,Sphaeralcea emoryi torrey,against boll weevil,Anthonomus grandis Boheman (Coleoptera: Curculionidae)

The globemallow,Sphaeralcea emoryi Torrey, a plant native to Arizona was evaluated as a source of feeding or oviposition deterrents to the boll weevil,Anthonomus grandis Boheman. Feeding and oviposition responses of reproductive weevils to the flower buds and artificial diets spiked with dry powder or extracts of the globemallow buds were determined. Boll weevils were deterred from feeding and ovipositing in the flower buds unless the calyxes were removed. Male and virgin female weevils were discouraged from feeding as much as gravid weevils. Secondary chemicals in the flower buds served primarily as feeding deterrents but also prevented oviposition. The concentration of these chemicals was highest in the calyxes of the buds, and potent deterrent activity could be extracted from the calyxes with methanol. Boll weevils were able to perceive the deterrents by contact chemosensory organs on the antennae, maxillary palps and labial palps.     

Phenolics in black oak bark and leaves

Catechin, quercitrin, robinin, quercetin 3-methyl ether, scopoletin, cholorogenic acid, several leucoanthocyanins, and condensed and hydrolyzable tannins were identified in bark and leaves ofQuercus velutina Lamarck. The concentrations of most phenolics in leaves increased as the growing season progressed, whereas those of most phenolics in bark remained essentially unchanged. Qualitative differences in bark and leaf phenolics among different trees were negligible.     

Red maple (Acer rubrum) inhibits feeding by beaver (Castor canadensis)

At many beaver (Castor canadensis) sites at Allegany State Park in New York State, red maple (Acer rubrum) is the only or one of the few tree species left standing at the ponds' edges. The relative palatability of red maple (RM) was studied in three ways. (1) At seven beaver sites, the available and utilized trees were recorded and an electivity index (E) computed. Of 15 tree species, RM ranked second or fourth lowest. (2) In experiment I, RM, sugar maple (A. saccharum, SM), and quaking aspen (Populus tremuloides) logs were presented cafeteria style at 10 colonies. RM was the least preferred. (3) Bark of RM was extracted with solvents. Aspen logs were painted (experiment II) or soaked (experiment III) with this RM extract and presented to beaver cafeteria-style, along with aspen and RM controls. This treatment rendered aspen logs less palatable, indicating that a chemical factor had been transferred.     

Effects of Light and Nutrient Availability on Aspen: Growth, Phytochemistry, and Insect Performance

This study explored the effect of resource availability on plant phytochemical composition within the framework of carbon–nutrient balance (CNB) theory. We grew quaking aspen (Populus tremuloides) under two levels of light and three levels of nutrient availability and measured photosynthesis, productivity, and foliar chemistry [water, total nonstructural carbohydrates (TNC), condensed tannins, and phenolic glycosides]. Gypsy moths (Lymantria dispar) and forest tent caterpillars (Malacosoma disstria) were reared on foliage from each of the treatments to determine effects on insect performance. Photosynthetic rates increased under high light, but were not influenced by nutrient availability. Tree growth increased in response to both the direct and interactive effects of light and nutrient availability. Increasing light reduced foliar nitrogen, while increasing nutrient availability increased foliar nitrogen. TNC levels were elevated under high light conditions, but were not influenced by nutrient availability. Starch and condensed tannins responded to changes in resource availability in a manner consistent with CNB theory; levels were highest under conditions where tree growth was limited more than photosynthesis (i.e., high light–low nutrient availability). Concentrations of phenolic glycosides, however, were only moderately influenced by resource availability. In general, insect performance varied relatively little among treatments. Both species performed most poorly on the high light–low nutrient availability treatment. Because phenolic glycosides are the primary factor determining aspen quality for these insects, and because levels of these compounds were minimally affected by the treatments, the limited response of the insects was not surprising. Thus, the ability of CNB theory to accurately predict allocation to defense compounds depends on the response of specific allelochemicals to changes in resource availability. Moreover, whether allelochemicals serve to defend the plant depends on the response of insects to specific allelochemicals. Finally, in contrast to predictions of CNB theory, we found substantial allocation to storage and defense compounds under conditions in which growth was carbon-limited (e.g., low light), suggesting a cost to defense in terms of reduced growth.     

Patterns and sources of leaf tannin variation in yellow birch (Betula allegheniensis) and sugar maple (Acer saccharum)

Leaves from forest-grown sugar maple (Acer saccharum Marsh) and yellow birch (Betula allegheniensis Britt.) trees were analyzed for four tannin measures (hydrolyzable and condensed tannins, total phenolics, and protein binding) at three times during the growing season. Fifteen-year-old half-sib sugar maples from four provenances, representing the geographical extremes of the sugar maple range and growing in a common garden, were examined for the same traits. We found no significant geographic or seed source component to variation in three of the four tannin measures. We found significant seasonal changes in both birch and maple leaf tannins. Withincanopy leaf tannin variation tended to obscure differences between trees in maple, but in birches between-tree differences in leaf tannin content were more readily found. We also found a significant negative correlation between leaf protein binding capacity and leaf wet weight.     

Immunological Memory of Mountain Birches: Effects of Phenolics on Performance of the Autumnal Moth Depend on Herbivory History of Trees

Plants have been suggested to have an immunological memory comparable to animals. The evidence for this, however, is scarce. In our study with the mountain birch—Epirrita autumnata system, we demonstrated that birches exposed as long as 5 yr to feeding of E. autumnata larvae (delayed induced resistance, DIR), responded more strongly to a new challenge than trees without an herbivory history. Pupal weights remained lower, and the duration of the larval period was prolonged in the DIR trees, although immunity, measured as an encapsulation rate, was not affected. We further demonstrated that the effects of birch phenolics on performance of E. autumnata were different in the exposed (DIR) trees from naive control trees, although we found only one significant change in chemistry. The quercetin:kaemferol ratio was increased in DIR trees, suggesting that herbivory caused oxidative stress in birches. In DIR trees, phenolics, especially hydrolyzable tannins (HTs), affected pupal weights negatively, whereas in control trees, the effects were either nonsignificant or positive. HTs also prolonged the duration of the larval period of females, whereas peroxidase (POD) activity prolonged that of males. We suggest that the causal explanation for the induced resistance was an enhanced oxidation of phenolic compounds from the DIR trees in the larval digestive tract. Phenolic oxidation produces semiquinones, quinones, free radicals, and ROS, which may have toxic, antinutritive, and/or repellent properties against herbivores.     

Factors affecting levels of some phenolic compounds,digestibility, and nitrogen content of the mature leaves ofBarteria fistulosa (Passifloraceae)

Levels of total phenolics, condensed tannins, acid detergent fiber, pepsin/cellulase digestibility, and nitrogen in mature leaves of 26 individuals of the ant-plant,Barteria fistulosa, have been determined. Analysis of the results in terms of the presence or absence of ants and the position of the branch from which the leaves were collected showed no relationship with concentrations of phenolics or fiber and only a weak relationship with digestibility and nitrogen. By contrast, light intensity strongly influenced levels of phenolics, notably condensed tannins, so that mature leaves of individuals growing in direct sunlight were less digestible and appeared to be of lower quality as food for herbivores than did mature leaves of individuals in shaded positions. Possible reasons for the variation in condensed tannin levels are discussed.     

Seasonal Changes in Foliar Terpenes Indicate Suitability Of Douglas-fir Buds for Western Spruce Budworm

The terpene composition of current-year buds of Douglas-fir, Pseudotsuga menziesii (Mirb.) Franco, was analyzed from before budburst to after buds were fully flushed. Terpene composition was measured at weekly intervals for several seasons at eight different locations in the southern interior of British Columbia, Canada. Discriminant functions relating terpene composition to suitability of buds for newly emerged western spruce budworm, Choristoneura occidentalis Freeman, were developed based on terpene profiles of the buds and bioassays measuring the suitability to budworms of a sister group of buds. Changes in percent composition of bud terpenes before and during budburst were closely associated with changes in the suitability of the buds to utilization by budworms at both the tree and site by date levels. Use of a degree-day scale for bud suitability removed much of the year-to-year variation, but remaining differences among sites suggest additional sources of variation influencing the insect-host plant relationship. The success of correctly classifying bud suitability using terpene profiles demonstrates the value of foliar terpenes as indicators of seasonal changes in suitability of Douglas-fir foliage during the critical spring emergence period of western spruce budworm. This indicator could be used to screen individual trees susceptible to budworm damage and identify sites at high risk of damaging defoliation.     

Tannin Composition Affects the Oxidative Activities of Tree Leaves

We examined whether tannin composition plays an important role in explaining the oxidative activities of tree leaves of Acer saccharum (sugar maple) and Quercus rubra (red oak). Sugar maple leaves contained substantial amounts of ellagitannins, condensed tannins, and galloyl glucoses, whereas red oak leaves contained almost exclusively condensed tannins. Oxidative activities of the crude phenolics from both species, and the phenolic fractions from sugar maple, were measured with electron paramagnetic resonance (EPR) spectrometry and UV-visible spectrophotometry. The two assays produced similar results: (1) sugar maple phenolics produced larger semiquinone radical concentrations,and higher semiquinone decay rates and browning rates than did red oak phenolics;(2) ellagitannin levels were positively associated with the three measures of oxidative activity; and (3) condensed tannin and galloyl glucose levels were negatively associated with these measures. The negative relationship between condensed tannin levels and oxidative activity resulted from the antioxidant effects of condensed tannins on hydrolyzable tannins; several purified condensed tannins significantly decreased the concentrations of semiquinone radicals and browning rates of pedunculagin (an ellagitannin) and pentagalloyl glucose. As expected, whole-leaf extracts from sugar maple produced elevated levels of semiquinone radicals, but none were observed in red oak extracts when the two species were compared with an EPR time-course assay. We conclude that the oxidative activities of tree leaves may be affected by tannin composition, and that the prooxidant activity of ellagitannins may be decreased by co-occurring condensed tannins.     

Effects of genotype, nutrient availability, and defoliation on aspen phytochemistry and insect performance

Genetic and environmental variability, and their interactions, influence phytochemical composition and, in turn, herbivore performance. We evaluated the independent and interactive effects of plant genotype, nutrient availability, and defoliation on the foliar chemistry of quaking aspen (Populus tremuloides) and consequences for performance of gypsy moths (Lymantria dispar). Saplings of four genotypes were grown under two conditions of nutrient availability and subjected to three levels of artificial defoliation. Concentrations of all secondary and primary metabolites evaluated responded to at least one or more of the experimental treatments. Of the secondary metabolites, phenolic glycosides were affected strongly by genotype, less so by nutrient availability, and not induced by defoliation. Condensed tannins were strongly dependent upon genotype, soil nutrient availability, and their interaction, and, in contrast to phenolic glycosides, were induced by artificial defoliation. Of the primary metabolites, foliar nitrogen was affected by genotype and soil nutrient availability. Starch concentrations were affected by genotype, nutrient availability, defoliation and interactions among these factors. Foliar water content responded to genotype, nutrient availability, and defoliation, and the effect of nutrient availability depended on genotype. Herbivore performance on these plants was strongly influenced by plant genotype and soil nutrient availability, but much less so by defoliation. Although several of the compound types (condensed tannins, starch, and water) responded to defoliation, quantitative variation in these compounds did not contribute to substantive changes in herbivore performance. Rather, the primary source of variation in insect performance was due to plant genotype (phenolic glycoside levels), while nutrient availability (foliar nitrogen levels) was of secondary importance. These results suggest that genetic variation in aspen plays a major role in determining patterns of insect performance, whereas environmental variation, such as was tested, here is of negligible importance.     

Preservation of salicaceae leaves for phytochemical analyses: Further assessment

The chemistry of the plant family Salicaceae has been of interest to researchers as diverse as chemical ecologists, chemosystematists, and paper chemists. Continuing the debate on proper methods for preservation of plant material prior to analysis, vacuum-drying was recently advocated, because freeze-drying may cause degradation of phenolic glycosides. This study was conducted to clarify the consequences of freeze-drying for foliar secondary chemicals and to evaluate the consequences of vacuum-drying for primary compounds (protein and carbohydrates). Leaves of quaking aspen (Populus tremuloides) were flash-frozen in liquid nitrogen and freeze-dried or vacuum-dried at room temperature. We then analyzed samples for levels of salicortin and tremulacin (phenolic glycosides), condensed tannins, nitrogen, soluble protein, sugars, and starch. Freeze-drying did not alter the concentrations of phenolic glycosides or tannins, relative to vacuum-drying. Freeze-drying did cause a small and inexplicable decline in nitrogen and soluble protein. Vacuum-drying, however, reduced starch concentrations by 38%. We suggest that the vacuum-drying method be used in studies in which carbohydrates are of no interest. For studies measuring carbohydrates, however, freeze-drying is a better alternative, and should effect no changes in levels of secondary compounds if samples are not allowed to thaw during the drying process.     

Feeding behavior of graminivorous grasshoppers in response to host-plant extracts,alkaloids, and tannins

Secondary metabolites exhibit the potential to direct food selection by grass-feeding (graminivorous) grasshoppers. We examined the effects of plant extracts and representative secondary metabolites on the feeding behavior of two such grasshoppers,Ageneotettix deorum (Scudder) andPhoetaliotes nebrascensis (Scudder). Three alkaloids and two tannins were bioassayed for their activity as feeding deterrent allelochemicals, as were extracts from the foliage of the graminoids commonly eaten by these grasshoppers:Agropyron smithii Rydb.,Andropogon hallii Hack.,Andropogon scoparius Michx.,Bouteloua gracilis (H. B. K) Lag. ex Griffiths,Carex heliophila Mack. andStipa comata Trin. & Rupr. Alkaloids strongly deterred feeding but tannins only exhibited a weak effect, even when present at four times the concentration of total phenolics typical for these graminoids. Host-plant extracts also exhibited weak effects, such that we found no evidence for either strong deterrence or phagostimulation. Our results for alkaloids and host-plant extracts are consistent with the view that grass-feeding grasshoppers may be restricted to graminoids because of: (1) the presence of deterrents in nonhosts and (2) the absence of deterrents in hosts. However, our data for tannins show that these are unlikely to be effective barriers to herbivory by these grasshoppers.