Antifungal leaf-surface metabolites correlate with fungal abundance in sagebrush populations

A central component in understanding plant–enemy interactions is to determine whether plant enemies, such as herbivores and pathogens, mediate the evolution of plant secondary metabolites. Using 26 populations of a broadly distributed plant species, sagebrush (Artemisia tridentata), we examined whether sagebrush populations in habitats with a greater prevalence of fungi contained antifungal secondary metabolites on leaf surfaces that were more active and diverse than sagebrush populations in habitats less favorable to fungi. Because moisture and temperature play a key role in the epidemiology of most plant–pathogen interactions, we also examined the relationship between the antifungal activity of secondary metabolites and the climate of a site. We evaluated the antifungal activity of sagebrush secondary metabolites against two fungi, a wild Penicillium sp. and a laboratory yeast, Saccharomyces cerevisiae, using a filter-paper disk assay and bioautography. Comparing the 26 sagebrush populations, we found that fungal abundance was a good predictor of both the activity (r ² = 0.36 for Saccharomyces, r ² = 0.37 for Penicillium) and number (r ² = 0.34 for Saccharomyces) of antifungal secondary metabolites. This suggests that selection imposed by fungal pathogens has led to more effective antifungal secondary metabolites. We found that the antifungal activity of sagebrush secondary metabolites was negatively related to average vapor pressure deficit of the habitat (r ² = 0.60 for Saccharomyces, r ² = 0.61 for Penicillium). Differences in antifungal activity among populations were not due to the amount of secondary metabolites, but rather to qualitative differences in the composition of antifungal compounds. Although all populations in habitats with high fungal prevalence had secondary metabolites with high antifungal activity, different suites of compounds were responsible for this activity, suggesting independent outcomes of selection on plants by fungal pathogens. The location of antifungal secondary metabolites on the leaf surface is consistent with their putative defense role, and we found no evidence supporting other functions, such as protection from ultraviolet light or oxidation. That the antifungal activity of sagebrush secondary metabolites was similar for two different fungi provides support for broad antifungal defenses. The incidence and severity of fungal disease in the field (caused by Puccinia tanaceti) were similar in moist and dry habitats, possibly reflecting an equilibrium between plant defense and fungal attack, as sites with greater fungal abundance compensated with more effective secondary metabolites. The geographic correlation between fungal abundance and antifungal secondary metabolites of sagebrush, coupled with our other data showing heritable variation in these metabolites, suggests that pathogenic fungi have selected for antifungal secondary metabolites in sagebrush.     

Degradation of Leaf Litter Phenolics by Aquatic and Terrestrial Isopods

To investigate species-specific decomposition rates of litter from native (Quercus faginea) and introduced (Eucalyptus globulus) tree species in Portugal, we monitored changes in the phenolic signature of leaf litter during decomposition as mediated by an aquatic, Proasellus coxalis (Isopoda: Asellota), and two terrestrial, Porcellio dispar and Eluma caelatum (Isopoda: Oniscidea), detritivores. Although the litter of Eucalyptus and Quercus did not differ in overall protein precipitation capacity, we detected differences in terms of contents of particular phenolic compounds and phenol oxidation products. Accordingly, we observed food-specific consumption rates in Proasellus, but not in the terrestrial isopods. Proasellus digested Eucalyptus at significantly higher rates than Quercus, whereas the opposite was the case for Eluma, and Porcellio digested both litter types equally well. Despite slight differences in detail, effects of Proasellus on changes in the signature of litter phenolics were similar for both litter types, whereas terrestrial isopods—Porcellio and Eluma, although they differed from each other—digestively degraded phenolic compounds in Eucalyptus and Quercus litter, respectively, in different ways. Overall, however, degradation of litter phenolics was similarly effective on both litter types. From these data, we conclude that decomposition of Eucalyptus litter does not proceed more slowly than of litter from native Portuguese trees.     

Systemic,genotype-specific induction of two herbivore-deterrent iridoid glycosides in Plantago lanceolata L. in response to fungal infection by Diaporthe adunca (Rob.) Niessel

Iridoid glycosides are a group of terpenoid secondary plant compounds known to deter generalist insect herbivores. In ribwort plantain (Plantago lanceolata), the iridoid glycosides aucubin and catalpol can be induced following damage by insect herbivores. In this study, we investigated whether the same compounds can be induced following infection by the fungal pathogen Diaporthe adunca, the causal agent of a stalk disease in P. lanceolata. Significant induction of aucubin and catalpol was observed in two of the three plant genotypes used in this study following inoculation with the pathogen. In one of the genotypes, induction occurred within 6 hr after inoculation, and no decay was observed within 8 days. The highest level of induction was observed in reproductive tissues (spikes and stalks) where infection took place. In these tissues, iridoid glycoside levels in infected plants were, on average, 97% and 37% higher than the constitutive levels in the corresponding control plants, respectively. Significant induction was also observed in leaves (24%) and roots (17%). In addition to significant genotypic variation in the level of induction, we found genetic variation for the tissue-specific pattern of induction, further broadening the scope for evolutionary fine-tuning of induced responses. Recent studies have revealed a negative association between iridoid glycoside levels in P. lanceolata genotypes and the amount of growth and reproduction of D. adunca that these genotypes support. However, for the three genotypes used in the present study, differences in resistance were not related to their constitutive or induced levels of iridoid glycosides, suggesting that additional resistance mechanisms are important in this host-pathogen system. We conclude that iridoid glycosides in P. lanceolata can be induced both by arthropods and pathogenic micro-organisms. Pathogen infection could, therefore, potentially enhance resistance to generalist insect herbivores in this species.     

Exposure of Lima Bean Leaves to Volatiles from Herbivore-Induced Conspecific Plants Results in Emission of Carnivore Attractants: Active or Passive Process?

There is increasing evidence that volatiles emitted by herbivore-damaged plants can cause responses in downwind undamaged neighboring plants, such as the attraction of carnivorous enemies of herbivores. One of the open questions is whether this involves an active (production of volatiles) or passive (adsorption of volatiles) response of the uninfested downwind plant. This issue is addressed in the present study. Uninfested lima bean leaves that were exposed to volatiles from conspecific leaves infested with the spider mite Tetranychus urticae, emitted very similar blends of volatiles to those emitted from infested leaves themselves. Treating leaves with a protein-synthesis inhibitor prior to infesting them with spider mites completely suppressed the production of herbivore-induced volatiles in the infested leaves. Conversely, inhibitor treatment to uninfested leaves prior to exposure to volatiles from infested leaves did not affect the emission of volatiles from the exposed, uninfested leaves. This evidence supports the hypothesis that response of the exposed downwind plant is passive. T. urticae-infested leaves that had been previously exposed to volatiles from infested leaves emitted more herbivore-induced volatiles than T. urticae-infested leaves previously exposed to volatiles from uninfested leaves. The former leaves were also more attractive to the predatory mite, Phytoseiulus persimilis, than the latter. This shows that previous exposure of plants to volatiles from herbivore-infested neighbors results in a stronger response of plants in terms of predator attraction when herbivores damage the plant. This supports the hypothesis that the downwind uninfested plant is actively involved. Both adsorption and production of volatiles can mediate the attraction of carnivorous mites to plants that have been exposed to volatiles from infested neighbors.     

Plant Cyanogenesis of <Emphasis Type="Italic">Phaseolus lunatus</Emphasis> and its Relevance for Herbivore–Plant Interaction: The Importance of Quantitative Data

Quantitative experimental results on the antiherbivorous effect of cyanogenesis are rare. In our analyses, we distinguished between the total amount of cyanide-containing compounds stored in a given tissue [cyanogenic potential (HCNp)] and the capacity for release of HCN per unit time (HCNc) from these cyanogenic precursors as a reaction to herbivory. We analyzed the impact of these cyanogenic features on herbivorous insects using different accessions of lima beans (Phaseolus lunatus L.) with different cyanogenic characteristics in their leaves and fourth instars of the generalist herbivore Schistocerca gregaria Forskål (Orthoptera, Acrididae). Young leaves exhibit a higher HCNp and HCNc than mature leaves. This ontogenetic variability of cyanogenesis was valid for all accessions studied. In no-choice bioassays, feeding of S. gregaria was reduced on high cyanogenic lima beans compared with low cyanogenic beans. A HCNp of about 15 μmol cyanide/g leaf (fresh weight) with a corresponding HCNc of about 1 μmol HCN released from leaf material within the first 10 min after complete tissue disintegration appears to be a threshold at which the first repellent effects on S. gregaria were observed. The repellent effect of cyanogenesis increased above these thresholds of HCNp and HCNc. No repellent action of cyanogenesis was observed on plants with lower HCNp and HCNc. These low cyanogenic accessions of P. lunatus were consumed extensively—with dramatic consequences for the herbivore. After consumption, locusts showed severe symptoms of intoxication. Choice assays confirmed the feeding preference of locusts for low over high cyanogenic leaf material of P. lunatus. The bioassays revealed total losses of HCN between 90 and 99% related to the estimated amount of ingested cyanide-containing compounds by the locusts. This general finding was independent of the cyanogenic status (high or low) of the leaf material.     

Problems Encountered in Feeding Microencapsulated Oils to a Folivorous Marsupial, the Common Ringtail Possum (Pseudocheirus peregrinus)

In our study of interactions between marsupial folivores and Eucalyptus, we have examined the role of terpenes, including the use of bioassay experiments where terpenes are added to an artificial diet. In pursuit of greater ecological realism in these experiments, we have sought means of packaging terpenes more realistically, by using microencapsulation to simulate oil glands of leaves. We report here on a preliminary experiment with microencapsulated olive oil (intended for use as an experimental control) in which the food intake of the animals was substantially reduced, to the point that starvation appeared imminent and we aborted the experiment. We discuss why this occurred and recommend caution to others intending to use microencapsulated terpenes (or other oils) in herbivore diets.     

Induction of Hypericins and Hyperforins in Hypericum perforatum in Response to Damage by Herbivores

Plants respond to herbivore and pathogen attack by a variety of direct and indirect mechanisms that include the induction of secondary metabolites. The phytomedicinal plant Hypericum perforatum L. produces two different classes of secondary metabolites: hyperforins, a family of antimicrobial acylphloroglucinols; and hypericins, a family of phototoxic anthraquinones exhibiting antimicrobial, antiviral, and antiherbivore properties in vitro. To determine whether these compounds are part of the herbivore-specific inducible plant defense system, we used an in vitro detached assay to assess the effects of specialist and generalist herbivore damage on the levels of hypericins and hyperforin. Greenhouse-grown H. perforatum plant sections were challenged with the specialist, Chrysolina quadrigemina, or with one of the following generalist feeders: Spilosoma virginica, Spilosoma congrua, or Spodoptera exigua. Feeding by the specialist beetle or mechanical wounding caused little change in phytochemical levels in plant tissue, whereas the small amount of feeding by the generalists caused 30-100% increases in hypericins and hyperforin as compared to control levels. Although the leaf damage index of the specialist feeding was 2.7 times greater, C. quadrigemina had little effect on H. perforatum chemical defenses in response to feeding damage in comparison to generalist feeding.     

GENDER DIMORPHISM AND ALTITUDINAL VARIATION OF SECONDARY COMPOUNDS IN LEAVES OF THE GYNODIOECIOUS SHRUB <Emphasis Type="Italic">Daphne laureola</Emphasis>

In this article, we analyzed the concentration of coumarins in leaves of female and hermaphrodite individuals of the gynodioecious shrub Daphne laureola, along an elevational gradient in southern Spain. Combining HPLC and NMR techniques, we identified three different glycosides of 7-methoxy-coumarin in leaves of this species. Total coumarin concentration averaged between 60 and 120 mg/g dry weight for mature summer leaves of D. laureola growing at six different populations. As predicted by optimal theory, females tended to have a higher concentration of coumarins than hermaphrodites, thus upholding the idea that male reproductive function is costly for hermaphrodites. Furthermore, concentrations in females but not hermaphrodites were positively correlated with increasing population altitude, and the magnitude of gender divergence in coumarin concentration varied among populations, suggesting that the cost of the male function may be context dependent. To our knowledge, this is the first evidence of gender differences in chemical defenses of a gynodioecious species in the field.     

Leaf Physical and Chemical Features Influence Selection of Plant Genotypes by Hessian Fly

To identify features of plants that mediate host selection behavior in the Hessian fly, we established the ranking of six grasses by ovipositing females and then ran choice tests with plant models that incorporated physical and/or chemical features of the six grasses. In tests with real plants, egg counts revealed the following ranking: 18ITSN triticale > Otane hexaploid bread wheat > Fleet barley > PND tetraploid durum wheat > 3424 hexaploid bread wheat > Awapuni oat. On all six grasses, the adaxial side of the leaf received more eggs than the abaxial side. In tests with green paper models treated with extracts of the six grasses, egg counts were similar to egg counts on real plants. In tests with models that incorporated a molded resin imprint of an abaxial or adaxial leaf surface, egg counts on adaxial models, but not abaxial models, again were similar to egg counts on real plants. In two final tests a factorial design was used to compare the effects of the chemical and physical features of two pairs of grasses: (1) two bread wheats, Otane and 3424; and (2) a bread wheat and an oat, Otane and Awapuni. In the two tests, the effects of the physical features of the models were at least as important as the effects of the chemical features.     

Do Enemies of Herbivores Influence Plant Growth and Chemistry? Evidence from a Seminatural Experiment

It is predicted that enemies of insect herbivores may influence the effects of herbivores on their host plants by affecting the choice of plant genotypes. To examine the effect of predators, we conducted two experiments, each with a different caterpillar species (Junonia coenia and Pyrrharctia isabella). Under seminatural conditions, we provided a choice between two genotypes of plantain (Plantago lanceolata) with different levels of iridoid glycosides and used Podisus maculiventris stinkbugs as predators. There were four treatments: no herbivores and no predators, low density of herbivores and no predators, high density of herbivores and no predators, and high density of herbivores plus predators. The caterpillars had little effect on plant growth but did influence the iridoid glycoside concentration. For the Junonia experiment, the concentration of iridoid glycosides was less for plots with a low density of caterpillars (with no predators) compared to the other treatments of caterpillar density. In the Pyrrharctia experiment, catalpol was induced by a high density of caterpillars (with no predators). There were no increases in total iridoid glycosides associated with either herbivore species. The presence of predators had no effect on plant growth or total iridoid glycoside pattern. The lack of effect by predators seems to reflect the relatively large variation in iridoid glycoside concentration among leaf ages, and the herbivores ability to respond to that variation, such that the difference in iridoid glycoside concentrations in the plant genotypes was less important.     

Organic Sulfur Compounds from Dictyopteris spp. Deter Feeding by an Herbivorous Amphipod (Ampithoe longimana) but Not by an Herbivorous Sea Urchin (Arbacia punctulata)

Brown seaweeds in the genus Dictyopteris produce several C₁₁ sulfur metabolites that appear biosynthetically related to the C₁₁ compounds known to attract sperm to female gametes of many brown algae. All four of the C₁₁ sulfur compounds that we tested strongly deterred feeding by the amphipod Ampithoe longimana but had no effect on feeding by the sea urchin Arbacia punctulata, even when tested at concentrations that were two to eight times greater than those that deterred amphipods. In numerous previous investigations, a variety of seaweed compounds have been shown to deter feeding by large mobile herbivores such as fishes and urchins but to be relatively ineffective against mesograzers, such as the amphipod of our study. Our results for the C₁₁ sulfur compounds from Dictyopteris thus contrast sharply with patterns from previous studies and suggest that these metabolites may be defenses specifically targeted against small mesograzers such as amphipods. The occurrence of C₁₁ metabolites in brown algal eggs could allow these defenses to be especially important in defending gametes, zygotes, or young sporelings from herbivorous mesograzers.     

Feeding and oviposition preferences of sweet potato weevil,Cylas formicarius elegantulus (Summers), on storage roots of sweet potato cultivars with differing surface chemistries

Cores from sweet potato [Ipomoea batatas (L.) Lam.] storage roots (Centennial, Jewel, Resisto, and Regal cultivars) were presented to sweet potato weevils [Cylas formicarius elegantulus (Summers) (Coleoptera; Curculionidae)] in multiple-choice, limited-choice, and no-choice bioassays. Centennial, a susceptible cultivar in field-plot experiments, was preferred for feeding and oviposition by female weevils in choice bioassays, and for ovi-position in no-choice bioassays, compared to three other cultivars. Analysis of root surface chemistry showed a tentatively identified triterpenol acetate in Centennial, which was not found in the more resistant cultivars; another root surface component was found in higher concentrations in the more resistant cultivars.     

Extracts of Flourensia cernua Reduce Consumption of Alfalfa Pellets by Sheep

Effects of three extracts (hexanes, ether, and ethanol) from tarbush (Flourensia cernua) on intake of alfalfa pellets by lambs were examined. Forty-five ewe lambs were fed one of five treatments for five days (randomized complete block, three lambs per block on each treatment). Treatments were alfalfa pellets (CON) or alfalfa pellets plus ethanol carrier (CAR), hexanes extract (HEX), ether extract (ETH), or ethanol extract (ETOH). Extracts were applied to alfalfa pellets at the same concentration as in an equivalent amount of tarbush (as fed basis) in experiment 1 and at 10-fold dilutions of that concentration in experiment 2. Treatments were isolated from tarbush leaves by using a sequential extraction with hexanes, diethyl ether, and 100% ethanol. Lambs received 640 g of alfalfa pellets (dry matter basis) each morning and intake was monitored during a 20-min interval. Lambs were maintained and fed alfalfa pellets (4.7% of body weight) as one group except during this interval. In experiment 1, mean intake by lambs during the 20-min interval was 361, 393, 204, 212, and 228 g for CON, CAR, HEX, ETH, and ETOH, respectively (SEM = 28.9). All three extracts decreased intake (P < 0.001) compared to CON or CAR. Intake did not differ among the three extracts (HEX, ETH, and ETOH) or between the two controls (CON and CAR). Mean intake did not differ among treatments in experiment 2 (468, 455, 389, 381, and 431 g for CON, CAR, HEX, ETH, and ETOH, respectively; SEM = 30.5; P = 0.187). Several compounds are probably responsible for the low palatability and differential use of tarbush typically exhibited by livestock.     

Chemical Defenses of Freshwater Macrophytes Against Crayfish Herbivory

We measured feeding preferences of the crayfish Procambarus clarkii for fresh tissue from four species of freshwater macrophytes (Habenaria repens, Saururus cernuus, Ceratophyllum demersum and Typha angustifolia). We then determined the role of plant chemical defenses in generating these preferences by incorporating crude aqueous and organic extracts from each species into palatable foods and comparing feeding on these foods to feeding on control foods lacking these extracts. Tissue toughness, dry mass and ash-free mass per volume, and percentages of carbon, nitrogen, and phenolics were also measured for each of the four macrophytes. Although it had a low nutritional value, Ceratophyllum was the preferred food when it was offered as fresh tissue; it did not produce a chemically deterrent extract. The lipophilic crude extract from Typha significantly deterred crayfish feeding, but this highly nutritious plant was preferred when offered in an agar-based diet lacking structural defenses. Habenaria and Saururus were low preference foods that did not appear to be structurally defended; each species contained both lipophilic and water-soluble extracts that significantly deterred feeding. Fractionation of the lipophilic crude extract from Saururus indicated the presence of at least three deterrent compounds. From the orchid Habenaria, we isolated and identified a novel bis-p-hydroxybenzyl-2-alkyl-2-hydroxysuccinoate metabolite, habenariol, that appeared to explain most of the feeding deterrent activity present in the lipophilic extract of this species. The concentration of the metabolite in frozen collections of this plant doubled if we allowed the material to thaw before placing it in extraction solvents.     

Combined Effect of Intercropping and Turnip Root Fly (<Emphasis Type="Italic">Delia floralis</Emphasis>) Larval Feeding on the Glucosinolate Concentrations in Cabbage Roots and Foliage

The effects of plant competition and herbivory on glucosinolate concentrations in cabbage root and foliage were investigated in a cabbage-red clover intercropping system. Cabbage plants were grown under different competitive pressures and with varying degrees of attack by root-feeding Delia floralis larvae. Glucosinolate concentrations in cabbage were affected both by intercropping and by D. floralis density. Glucosinolate concentrations in foliage generally decreased as a response to intercropping, while the responses to insect root damage of individual glucosinolates were weaker. Root glucosinolates responded more strongly to both intercropping and egg density. Total root glucosinolate concentration decreased with clover density, but only at high egg densities. Increased egg density led to opposite reactions by the indole and aliphatic glucosinolates in roots. The responses of individual root glucosinolates to competition and root damage were complex and, on occasion, nonlinear. Reduced concentrations of several glucosinolates and the tendency towards a decrease in total concentration in cabbage foliage caused by intercropping and larval damage suggest that competing plants or plants with root herbivory do not allocate the same resources as unchallenged plants towards sustaining levels of leaf defensive compounds. This could also be true for root glucosinolate concentrations at high egg densities. In addition, the results suggest that changes occurring within a structural group of glucosinolates may be influenced by changes in a single compound, e.g., glucobrassicin (indol-3-ylmethyl) in foliage or sinigrin (2-propenyl) in roots.     

Geographic Variation in Alkaloid Production in <Emphasis Type="Italic">Conium maculatum</Emphasis> Populations Experiencing Differential Herbivory by <Emphasis Type="Italic">Agonopterix alstroemeriana</Emphasis>

Conium maculatum, a Eurasian weed naturalized in North America, contains high concentrations of piperidine alkaloids that act as chemical defenses against herbivores. C. maculatum was largely free from herbivory in the United States, until approximately 30 yr ago, when it was reassociated via accidental introduction with a monophagous European herbivore, the oecophorid caterpillar Agonopterix alstroemeriana. At present, A. alstroemeriana is found in a continuum of reassociation time and intensities with C. maculatum across the continent; in the Pacific Northwest, A. alstroemeriana can cause severe damage, resulting in some cases in complete defoliation. Studies in biological control and invasion biology have yet to determine whether plants reassociated with a significant herbivore from the area of indigeneity increase their chemical defense investment in areas of introduction. In this study, we compared three locations in the United States (New York, Washington, and Illinois) where C. maculatum experiences different levels of herbivory by A. alstroemeriana to determine the association between the intensity of the interaction, as measured by damage, and chemical defense production. Total alkaloid production in C. maculatum was positively correlated with A. alstroemeriana herbivory levels: plants from New York and Washington, with higher herbivory levels, invested two and four times more N to alkaloid synthesis than did plants from Illinois. Individual plants with lower concentrations of alkaloids from a single location in Illinois experienced more damage by A. alstroemeriana, indicative of a preference on the part of the insect for plants with less chemical defense. These results suggest that A. alstroemeriana may act either as a selective agent or inducing agent for C. maculatum and increase its toxicity in its introduced range.     

Effect of nitrogen and water treatment on leaf chemistry in horsenettle (Solanum carolinense), and relationship to herbivory by flea beetles (Epitrix spp.) and tobacco hornworm (Manduca sexta)

We studied the interaction between plants (horsenettle; Solanum carolinense) and herbivorous insects (flea beetles; Epitrix spp., and tobacco hornworm; Manduca sexta) by focusing on three questions: (1) Does variation in nitrogen availability affect leaf chemistry as predicted by the carbon-nutrient balance (CNB) hypothesis? (2) Does variation in plant treatment and leaf chemistry affect insect feeding? (3) Is there an interaction between the insect herbivores that is mediated by variation in leaf chemistry? For three successive years (1998-2001), we grew a set of clones of 10 maternal plants under two nitrogen treatments and two water treatments. For each plant in the summer of 2000, we assayed herbivory by hornworms in both indoor (detached leaf) and outdoor (attached leaf) assays, as well as ambient flea beetle damage. Estimates of leaf material consumed were made via analysis of digitized leaf images. We also assayed leaves for total protein, phenolic, and glycoalkaloid content, and for trypsin inhibitor, polyphenol oxidase, and peroxidase activity. Despite strong effects of nitrogen treatment on growth and reproduction, only total protein responded as predicted by CNB. Leaf phenolic levels were increased by nitrogen treatment, polyphenol oxidase activity was decreased, and other leaf parameters were unaffected. Neither hornworm nor flea beetle herbivory could be related to plant treatment or genotype or to variation in any of the six leaf chemical parameters. A negative relationship between flea beetle and hornworm herbivory was found, but was not apparently mediated by any of the measured leaf chemicals. Because leaf resistance was maintained in low nitrogen plants at the apparent expense of growth and reproduction, our results support the concept of a fitness cost of defense, as predicted by the optimal defense hypothesis.     

Differences in Effects of Pyrrolizidine Alkaloids on Five Generalist Insect Herbivore Species

The evolution of the diversity in plant secondary compounds is often thought to be driven by insect herbivores, although there is little empirical evidence for this assumption. To investigate whether generalist insect herbivores could play a role in the evolution of the diversity of related compounds, we examined if (1) related compounds differ in their effects on generalists, (2) there is a synergistic effect among compounds, and (3) effects of related compounds differed among insect species. The effects of pyrrolizidine alkaloids (PAs) were tested on five generalist insect herbivore species of several genera using artificial diets or neutral substrates to which PAs were added. We found evidence that structurally related PAs differed in their effects to the thrips Frankliniella occidentalis, the aphid Myzus persicae, and the locust Locusta migratoria. The individual PAs had no effect on Spodoptera exigua and Mamestra brassicae caterpillars. For S. exigua, we found indications for synergistic deterrent effects of PAs in PA mixtures. The relative effects of PAs differed between insect species. The PA senkirkine had the strongest effect on the thrips, but had no effect at all on the aphids. Our results show that generalist herbivores could potentially play a role in the evolution and maintenance of the diversity of PAs.     

Chemical Stimulants of Leaf-Trenching by Cabbage Loopers: Natural Products, Neurotransmitters, Insecticides, and Drugs

Larvae of the cabbage looper, Trichoplusia ni (Lepidoptera: Noctuidae), often transect leaves with a narrow trench before eating the distal section. The trench reduces larval exposure to exudates, such as latex, during feeding. Plant species that do not emit exudate, such as Plantago lanceolata, are not trenched. However, if exudate is applied to a looper's mouth during feeding on P. lanceolata, the larva will often stop and cut a trench. Dissolved chemicals can be similarly applied and tested for effectiveness at triggering trenching. With this assay, I have documented that lactucin from lettuce latex (Lactuca sativa), myristicin from parsley oil (Petroselinum crispum), and lobeline from cardinal flower (Lobelia cardinalis) elicit trenching. These compounds are the first trenching stimulants reported. Several other constituents of lettuce and parsley, including some phenylpropanoids, monoterpenes, and furanocoumarins had little or no activity. Cucurbitacin E glycoside found in cucurbits, another plant family trenched by cabbage loopers, also was inactive. Lactucin, myristicin, and lobeline all affect the nervous system of mammals, with lobeline acting specifically as an antagonist of nicotinic acetylcholine receptors. To determine if cabbage loopers respond selectively to compounds active at acetylcholine synapses, I tested several neurotransmitters, insecticides, and drugs with known neurological activity, many of which triggered trenching. Active compounds included dopamine, serotonin, the insecticide imidacloprid, and various drugs such as ipratropium, apomorphine, buspirone, and metoclopramide. These results document that noxious plant chemicals trigger trenching, that loopers respond to different trenching stimulants in different plants, that diverse neuroactive chemicals elicit the behavior, and that feeding deterrents are not all trenching stimulants. The trenching assay offers a novel approach for identifying defensive plant compounds with potential uses in agriculture or medicine. Cabbage loopers in the lab and field routinely trench and feed on plants in the Asteraceae and Apiaceae. However, first and third instar larvae enclosed on Lobelia cardinalis (Campanulaceae) failed to develop, even though the third instar larvae attempted to trench. Trenching ability does not guarantee effective feeding on plants with canal-borne exudates. Cabbage loopers must not only recognize and respond to trenching stimulants, they must also tolerate exudates during the trenching procedure to disable canalicular defenses.     

Systemic Induction of Terpenoid Aldehydes in Cotton Pigment Glands by Feeding of Larval Spodoptera exigua

Pigment glands in cotton contain terpenoid aldehydes that are toxic and deterrent to feeding of several generalist lepidopteran insects. We hypothesized that previously observed systemically induced feeding deterrence may be associated with pigment glands. We conducted experiments to determine the dynamics and chemical nature of inducible feeding deterrents in leaves of cotton, Gossypium hirsutum L, to larvae of the beet armyworm, Spodoptera exigua. Production and/or filling of pigment glands was influenced by physiological age of Deltapine 90 cotton plants. In undamaged plants, successively formed leaves contained more pigment glands, up to the seventh or eighth true-leaf developmental stage. Feeding choice tests conducted one or seven days after initial feeding damage revealed that third instars of S. exigua consumed more of the two youngest leaves from control cotton plants than from plants whose two oldest leaves had been fed on previously for 24 hr by S. exigua. The preference for leaves from control plants was significant one day after initial damage and highly significant seven days after damage. Consumption of mature foliage (leaf immediately above initially damaged leaves) from control plants and damaged plants did not differ. More pigment glands were counted on the youngest leaf of damaged plants than on the youngest leaf of control plants one day after initial damage. HPLC analysis revealed greater amounts of hemigossypolone, heliocides 1 and 2 (H₁ and H₂), and total terpenoid aldehydes per gland in young foliage of damaged plants than control plants one day after initial injury. By seven days after initial injury, greater quantities of hemigossypolone and all heliocides except H₄ were detected in young foliage from damaged plants compared to control plants. Concentrations of H₁ per gland in young leaves from damaged plants increased the most of all terpenoid aldehydes measured (3.4× the amount found in leaves from control plants). Mature leaves from damaged plants did not contain more terpenoid aldehydes than mature leaves from control plants. We suggest that systemically induced feeding deterrence to S. exigua in young leaves of glanded cotton was due to increased amounts of terpenoid aldehydes in pigment glands.