Bacterial-barnacle interaction: Potential of using juncellins and antibiotics to alter structure of bacterial communities

In preparation for studies using natural products to probe interactions between bacterial consortia and settlement stage barnacles, we isolated 16 strains of bacteria associated with barnacles and examined: (1) effects of films of bacterial isolates on barnacle settlement, and (2) bacteriostatic effects of juncellins and standard antibiotics. Bacteria were isolated from the biofilm associated withBalanus amphitrite. On the basis of morphological and biochemical characteristics, bacteria were classified into five major groups:Aeromonas, Alcaligenes, Flavobacterium, Pseudomonas, andVibrio. Barnacle settlement was inhibited by allVibrio films and 64% of the other isolates. No film stimulated barnacle settlement. Juncellins were approximately as potent as standard antibiotics for all bacterial species tested.Vibrio spp. were most resistant to juncellins.     

Shield Defense of a Larval Tortoise Beetle

Larvae of the folivorous tortoise beetle, Plagiometriona clavata, carry shields formed from feces and exuviae above their bodies. We used an ecologically relevant predatory ant, Formica subsericea, in a bioassay to determine if shields functioned as simple barriers, as previous studies indicated, or whether they were chemical defenses. Shields were necessary for larval survival; shield removal rendered larvae vulnerable. Shields produced by larvae reared on a substitute diet failed to provide protection. Solvent-leached shields also failed to deter ants, indicating the shield had a host-derived chemical component likely located in the feces, not in the exuviae. Solanum dulcamara, the larval host plant, contained free phytol, steroidal glycoalkaloids, and saponins. Shields contained partially deglycosylated metabolites of host steroidal glycoalkaloids and saponins, a suite of fatty acids, and derivatives of phytol, which together formed a deterrent barrier against ant attack. We compared the mobile shield of P. clavata to the stationary shield of another S. dulcamara-feeding leaf beetle, Lema trilinea. Both larval shield defenses were formed from a very similar array of host-derived compounds with deterrent properties. We concluded that convergent patterns of limited chemical transformation and selective incorporation of particular deterrent metabolites in shield defenses of two unrelated taxa represented responses to selection from invertebrate predators.     

Antibacterial Activity of the Sponge Ircinia Ramosa: Importance of its Surface-Associated Bacteria

Variations in the antibacterial activity of the sponge Ircinia ramosa were evaluated during two collection periods (January and May) against vicinity fouling bacteria (VFB) and sponge surface-associated bacteria (SAB). The density of fouling bacteria in the water column, as well as epibacterial abundance on the sponge surface, was enumerated during both collections, and both increased in the warmer month of May. The extracts obtained from SAB were also tested for their role in antibacterial activity of the host. Sponge-associated bacteria are capable of producing antibacterial metabolites. The antibacterial activity that originated from polar fractions in the cooler month of January shifted towards the nonpolar fractions in May. Nonpolar fractions were more useful for the sponge when threatened with increased bacterial density. Thus, the chemical nature and production of antibacterial compounds produced by sponge or its associated bacteria appears to be governed by the environment. An inverse relationship was observed between the epibacterial abundance over the sponge surface in nature and the antibacterial activity displayed by the sponge extracts in laboratory bioassays. This investigation reveals the importance of collection period as well as the role of associated bacteria in the evaluation of antibacterial activity.     

Inhibition of larval settlement by natural products from the ascidian,Eudistoma olivaceum (Van Name)

Settlement bioassays with larvae of the cheilostome bryozoanBugula neritina were used to isolate and purify antifouling compounds from the ascidianEudistoma olivaceum (Van Name). Three inhibitors of larval settlement, two toxic and one nontoxic, were investigated. The toxic compounds accounted for approximately 0.4% of the organic fraction (on a dry weight basis) and were identified as eudistomins g and h, two of a number of alkaloids possessed byE. olivaceum. The nontoxic inhibitor of settlement was not characterized. Eudistomins g and h were effective inhibitors of larval settlement at concentrations as low as 0.5% of that present in the living animal.     

Amphibian Chemical Defense: Antifungal Metabolites of the Microsymbiont <Emphasis Type="Italic">Janthinobacterium lividum</Emphasis> on the Salamander <Emphasis Type="Italic">Plethodon cinereus</Emphasis>

Disease has spurred declines in global amphibian populations. In particular, the fungal pathogen Batrachochytrium dendrobatidis has decimated amphibian diversity in some areas unaffected by habitat loss. However, there is little evidence to explain how some amphibian species persist despite infection or even clear the pathogen beyond detection. One hypothesis is that certain bacterial symbionts on the skin of amphibians inhibit the growth of the pathogen. An antifungal strain of Janthinobacterium lividum, isolated from the skin of the red-backed salamander Plethodon cinereus, produces antifungal metabolites at concentrations lethal to B. dendrobatidis. Antifungal metabolites were identified by using reversed phase high performance liquid chromatography, high resolution mass spectrometry, nuclear magnetic resonance, and UV-Vis spectroscopy and tested for efficacy of inhibiting the pathogen. Two metabolites, indole-3-carboxaldehyde and violacein, inhibited the pathogen’s growth at relatively low concentrations (68.9 and 1.82 μM, respectively). Analysis of fresh salamander skin confirmed the presence of J. lividum and its metabolites on the skin of host salamanders in concentrations high enough to hinder or kill the pathogen (51 and 207 μM, respectively). These results support the hypothesis that cutaneous, mutualistic bacteria play a role in amphibian resistance to fungal disease. Exploitation of this biological process may provide long-term resistance to B. dendrobatidis for vulnerable amphibians and serve as a model for managing future emerging diseases in wildlife populations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Chemically induced metamorphosis of polychaete larvae in both the laboratory and ocean environment

Planktonic larvae of the marine polychaetePhragmatopoma californica preferentially attach to substrata and metamorphose to the adult form upon contact with cement in tubes built by conspecifics. This gregarious settlement and metamorphosis contributes to the formation of large aggregations or reefs. Larvae also metamorphose upon contact with 2,6-di-tert-butyl-4-methylphenol (DBMP), a possible aromatic analog of cross-linked dihydrox-yphenylalanine (DOPA) residues (present in the polyphenolic protein cement as 2.6% of the amino acid residues). Morphogenesis occurs in the laboratory when larvae are exposed to DBMP either adsorbed to solid surfaces or when dissolved in dimethyl sulfoxide (DMSO) to render it soluble in seawater. Larvae in the ocean were induced to settle and metamorphose on plates coated with DBMP prior to their deployment in the ocean. This is the first report in which a defined organic molecule, identified as an inducer (or precursor to an inducer) of larval settlement and metamorphosis in the laboratory, has been shown to induce these processes in the ocean. Both forskolin and isobutylmethylxanlhine (IBMX) induce metamorphosis ofP. californica larvae, presumably by causing increases in intracellular cyclic AMP (cAMP). A discussion of the pathway controlling chemically mediated metamorphosis and evidence suggesting the possible role of cAMP in the process are presented. Other compounds known to increase intracellular cAMP levels, including arachidonic, linoleic, and palmitoleic acids, found by other workers to induce settlement and metamorphosis ofP. californica, may exert this activity by direct modification of internal cAMP levels in the larvae.     

Phthalide-based host-plant resistance toSpodoptera exigua andTrichoplusia ni inApium graveolens

A chemical basis for the difference in suitability between two celeriac (Apium graveolens var.rapaceum) cultivars for the survival and growth ofSpodoptera exigua (Hübner) andTrichoplusia ni (Hübner) was identified as sedanenolide (3-n-butyl-4,5-dihydro-isobenzofuranone). Sedanenolide was isolated using a bioassay-driven extraction and purification procedure and was identified using several spectrometric methods. Foliar concentrations of sedanenolide were negatively correlated with larval performance and were significantly higher in the cultivar less suitable for larval survival and growth. Sedanenolide andBacillus thuringiensis Berliner acted additively in reducing larval growth when combined in artificial diets, a result that is consistent with previous studies in which the combined effect of host plant cultivar andB. thuringiensis on larval survival and growth were additive.     

The Antimicrobial Peptide Gad-1 Clears Pseudomonas aeruginosa Biofilms under Cystic Fibrosis Conditions

Bacterial infections in cystic fibrosis (CF) patients are an emerging health issue and lead to a premature death. CF is a hereditary disease that creates a thick mucus in the lungs that is prone to bacterial biofilm formation, specifically Pseudomonas aeruginosa biofilms. These biofilms are very difficult to treat because many of them have antibiotic resistance that is worsened by the presence of extracellular DNA (eDNA). eDNA helps to stabilize biofilms and can bind antimicrobial compounds to lessen their effects. The metallo-antimicrobial peptide Gaduscidin-1 (Gad-1) eradicates established P. aeruginosa biofilms through a combination of modes of action that includes nuclease activity that can cleave eDNA in biofilms. In addition, Gad-1 exhibits synergistic activity when used with the antibiotics kanamycin and ciprofloxacin, thus making Gad-1 a new lead compound for the potential treatment of bacterial biofilms in CF patients.     

Thermophiles as Potential Source of Novel Endotoxin Antagonists: the Full Structure and Bioactivity of theLipo‐oligosaccharide from Thermomonas hydrothermalis

Thermomonas hydrothermalis is a Gram‐negative thermophilic bacterium that is able to live at 50 °C. This ability is attributed to chemical modifications, involving those to bacterial cell‐wall components, such as proteins and (glyco)lipids. As the main component of the outer membrane of Gram‐negative bacteria, lipopolysaccharides (LPSs) are exposed to the environment, thus they can undergo structural chemical changes to allow thermophilic bacteria to live at their optimal growth temperature. Furthermore, as one of the major target of the eukaryotic innate immune system, LPS elicits host immune response in a structure‐dependent mode; thus the uncommon chemical features of thermophilic bacterial LPSs might exert a different biological action on the innate immune system—an antagonistic effect, as shown in studies of LPS structure–activity relationship in the ongoing research into antagonist LPS candidates. Here, we report the complete structural and biological activity analysis of the lipo‐oligosaccharide isolated from Thermomonas hydrothermalis, achieved by a multidisciplinary approach (chemical analysis, NMR, MALDI MS and cellular immunology). We demonstrate a tricky and interesting structure combined with a very interesting effect on human innate immunity.     

Use of chemical variation and predation as plant defenses byEncelia farinosa against a specialist herbivore

Larvae of the monophagous herbivore,Trirhabda geminata, selectively eat particular plants and plant parts of its natural host,Encelia farinosa. Measurements of leaf damage and larval positions on branches through time support this observation. Time-lapse movie photography revealed that larvae are sufficiently mobile to search most of a plant in a 48-hr period and that aggregations were the result of larval activity and not directly the result of oviposition. Experiments withT. geminata larvae on artificial diets containing a range of natural concentrations of chemical extracts fromE. farinosa leaves showed that the larvae grew significantly slower and had a lower overall survivorship at the high concentration. Combining the results of all choice tests, larvae appeared unable to distinguish between high- and low-concentration agar diets. Considered individually, larval preferences for natural production concentrations changed as the season progressed. Early-season larvae preferred low-concentration leaves, while late-season larvae preferred high-concentrations. Measurements of chemical and nitrogen content of leaves selected by larvae in the field confirmed this pattern. Percent parasitism in field-collected larvae increased with season as the larval population decreased. This combination of slowed growth and increasing parasitism and predation is a putative defense strategy ofEncelia farinosa to prevent adaptation by a specialist herbivore to the total range of compounds elaborated.     

Antagonistic Interactions Mediated by Marine Bacteria: The Role of Small Molecules

Marine bacteria are known to produce a wide variety of structurally diverse and biologically active secondary metabolites. Considerably less is known about the ecological functions of these compounds, in part due to methodological challenges associated with this field of research. Here, we review the antagonistic activities mediated by marine bacteria with a focus on activities linked to structurally defined secondary metabolites. Bacterial antagonism has been documented against other marine bacteria as well as eukaryotes, and includes antibiosis, the inhibition of quorum sensing, larval settlement deterrence, and defense against predation. These compounds likely play important ecological roles that ultimately affect ecosystem structure and function, however, much remains to be learned before these roles can be fully appreciated. Recent technological advances coupled with a better understanding of the diverse processes mediated by secondary metabolites provide new opportunities to expand our understanding of the chemical ecology of bacterial antagonism in the marine environment.     

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.     

Anti-Biofilm Performance of Three Natural Products against Initial Bacterial Attachment

Marine bacteria contribute significantly towards the fouling consortium, both directly (modern foul release coatings fail to prevent “slime” attachment) and indirectly (biofilms often excrete chemical cues that attract macrofouling settlement). This study assessed the natural product anti-biofilm performance of an extract of the seaweed, Chondrus crispus, and two isolated compounds from terrestrial sources, (+)-usnic acid and juglone, against two marine biofilm forming bacteria, Cobetia marina and Marinobacter hydrocarbonoclasticus. Bioassays were developed using quantitative imaging and fluorescent labelling to test the natural products over a range of concentrations against initial bacterial attachment. All natural products affected bacterial attachment; however, juglone demonstrated the best anti-biofilm performance against both bacterial species at a concentration range between 5–20 ppm. In addition, for the first time, a dose-dependent inhibition (hormetic) response was observed for natural products against marine biofilm forming bacteria.     

Antagonism between jasmonate- and salicylate-mediated induced plant resistance: effects of concentration and timing of elicitation on defense-related proteins,herbivore, and pathogen performance in tomato

The jasmonate (JA) and salicylate (SA) signaling pathways in plants provide resistance to herbivorous insects and pathogens. It is known that these pathways interact, sometimes resulting in antagonism between the pathways. We tested how the timing and concentration of elicitation of each pathway influenced the interaction between the jasmonate and salicylate pathways measured in terms of five biochemical responses and biological resistance to caterpillars and bacteria. The salicylate pathway had a stronger effect on the jasmonate pathway than did the reverse. The negative signal interaction was generated by two distinct paths in the plant. A negative interaction in the biochemical expression of the two pathways was most consistent in the simultaneous elicitation experiments compared to when the elicitors were temporally separated by two days. Herbivore bioassays with Spodoptera exigua also consistently reflected an interaction between the two pathways in the simultaneous elicitation experiments. The negative signal interaction reducing biological resistance to the herbivore was also demonstrated in some temporally separated treatment combinations where attenuation of the biochemical response was not evident. Concentration of the elicitors had an effect on the pathway interaction with consistent biochemical and biological antagonism in the high concentration experiments and inconsistent antagonism in the low concentration experiments. The bacterial pathogen, Pseudomonas syringae pv. tomato (Pst), consistently showed reduced lesion development on plants with SA responses activated and, in some experiments, on JA-elicited plants. Resistance to Pst was not reduced or enhanced in dual-elicited plants. Thus, signal interaction is most consistent when elicitors are applied at the same time or when applied at high doses. Signal interaction affected the herbivore S. exigua, but not the pathogen Pst.     

Chemistry and chemical ecology of the Bahamian spongeAplysilla glacialis

Chemical investigation of the secondary metabolites of the marine spongeAplysilla glacialis collected at French Wells, Crooked Island, Bahamas, resulted in the isolation and characterization of four diterpenes, two sterol endoperoxides, and two methylated primary metabolites. Some of these compounds, along with crude extracts of the sponge, were investigated for their ability to deter fish predation, affect the fouling of surfaces, and inhibit the growth of marine microorganisms. The diterpene manoöl (3), cholesterol endoperoxide (4), and the crude nonpolar extract ofA. glacialis were shown to deter feeding by a natural assemblage of fish predators in an in situ assay conducted at French Wells. Pure secondary metabolites and crude extracts ofA. glacialis also were tested in a laboratory fish feeding assay employing the wrasseThalassoma lunare. A mixture of sterol endoperoxides was isolated from the mucus that coats the surface ofA. glacialis and is exuded in large quantities when the sponge is disturbed. These compounds are thereby distributed in a manner in which they can best serve a defensive role for the sponge. An in situ assay was designed to determine the effect that pure secondary metabolites and crude extracts have on the fouling of surfaces. Manoöl (3) and cholesterol endoperoxide (4) were determined to increase the rate of fouling when compared to control surfaces. 1-Methyladenine (5) was identified as an antimicrobial constituent ofA. glacialis that inhibited the growth of four marine bacteria isolated from seawater samples collected at French Wells.This work was presented at the Gordon Research Conferences (Marine Natural Products Chemistry), February 1990, Oxnard, California.     

The Desert Locust, <Emphasis Type="Italic">Schistocerca gregaria</Emphasis>, Detoxifies the Glucosinolates of <Emphasis Type="Italic">Schouwia purpurea</Emphasis> by Desulfation

Desert locusts (Schistocerca gregaria) occasionally feed on Schouwia purpurea, a plant that contains tenfold higher levels of glucosinolates than most other Brassicaceae. Whereas this unusually high level of glucosinolates is expected to be toxic and/or deterrent to most insects, locusts feed on the plant with no apparent ill effects. In this paper, we demonstrate that the desert locust, like larvae of the diamondback moth (Plutella xylostella), possesses a glucosinolate sulfatase in the gut that hydrolyzes glucosinolates to their corresponding desulfonated forms. These are no longer susceptible to cleavage by myrosinase, thus eliminating the formation of toxic glucosinolate hydrolysis products. Sulfatase is found throughout the desert locust gut and can catalyze the hydrolysis of all of the glucosinolates present in S. purpurea. The enzyme was detected in all larval stages of locusts as well as in both male and female adults feeding on this plant species. Glucosinolate sulfatase activity is induced tenfold when locusts are fed S. purpurea after being maintained on a glucosinolate-free diet, and activity declines when glucosinolates are removed from the locust diet. A detoxification system that is sensitive to the dietary levels of a plant toxin may minimize the physiological costs of toxin processing, especially for a generalist insect herbivore that encounters large variations in plant defense metabolites while feeding on different species.     

Dental Chatter: Bacterial Cross-Talk in the Biofilm of the Oral Cavity

Dental biofilms are composed of hundreds of bacterial species. These biofilms are diverse biological structures due to the heterogeneity of the many different types of supports in the oral cavity. The bacteria immobilized in these biofilms are exposed to rapid environmental changes such as pH, temperature, nutrition and anti-plaque agents. One mode in which these bacteria adapt in the dental biofilm is by quorum sensing. This cell-cell communication regulates diverse sets of adhesion modes, physiological changes, virulence properties, allowing the bacteria to persist in the dental biofilm under rapid environmental changes. In this review, we will concentrate mostly on the cariogenic bacterium Streptococcus mutans as one of the pivotal microorganisms in the supra-gingival biofilm that plays a major role in dental caries.     

No Evidence for the Induction of Brown Algal Chemical Defense by the Phytohormones Jasmonic Acid and Methyl Jasmonate

Induced chemical defense reactions are widespread in marine brown algae. Despite the evidence that the biosynthesis of defense metabolites can be up-regulated upon herbivory, we do not know how this regulation of biosynthetic pathways to secondary metabolites is achieved in brown algae. In higher plants, the phytohormone jasmonic acid (JA) is crucial for the mediation of induced chemical defenses, and several findings of this metabolite from marine sources have been reported. We tested the hypothesis that JA or related metabolites play a role in induced brown algal defense. Quantification of oxylipins with a detection limit around 20 ng g⁻¹ algal tissue did not reveal the presence of JA in the seven examined brown algal species Dictyota dichotoma, Colpomenia peregrina, Ectocarpus fasciculatus, Fucus vesiculosus, Himanthalia elongata, Saccharina latissima (formerly Laminaria saccharina), and Sargassum muticum. Moreover, treatment with ecologically relevant concentrations of JA and methyl jasmonate did not lead to a significant change in the profile of medium- and non-polar metabolites of the tested algae. Only when high concentrations of ≥500 μg ml⁻¹ medium of the phytohormones were applied that a metabolic response which could be attributed to unspecific stress was observed. Bioassays with D. dichotoma that focused on medium- and non-polar compounds confirmed the lack of a biological role of JA and methyl jasmonate in the induction of algal induced chemical defenses. The phytohormone-treated samples did not exhibit any increased defense potential towards the amphipod Ampithoe longimana and the isopod Paracerceis caudata. JA and related phytohormones, known to be active in higher plants, thus appear to play no role in brown algae for induction of the defense chemicals studied here.     

Palatability of Macroalgae that Use Different Types of Chemical Defenses

This study compared algal palatability and chemical defenses from subtropical green algae that may use different types of defense systems that deter feeding by the rock-boring sea urchin Echinometra lucunter. The potential defense systems present include (1) the terpenoid caulerpenyne and its activated products from Caulerpa spp., and (2) dimethylsulfoniopropionate (DMSP)-related defenses in Ulva spp. Secondary metabolites from these chemical groups have been shown to deter feeding by various marine herbivores, including tropical and temperate sea urchins. Live algal multiple-choice feeding assays and assays incorporating algal extracts or isolated metabolites into an artificial diet were conducted. Several green algae, including Ulva lactuca, Caulerpa prolifera, and Cladophora sp., were unpalatable. Nonpolar extracts from U. lactuca deterred feeding, whereas nonpolar extracts from C. prolifera had no effect on feeding. Polar extracts from both species stimulated feeding. Caulerpenyne deterred feeding at approximately 4% dry mass; however, dimethyl sulfide and acrylic acid had no effect at natural and elevated concentrations. E. lucunter is more tolerant than other sea urchins to DMSP-related defenses and less tolerant to caulerpenyne than many reef fish. Understanding the chemical defenses of the algae tested in this study is important because they, and related species, frequently are invasive or form blooms, and can significantly modify marine ecosystems.     

Ecological adaptation of an Aristolochiaceae-feeding swallowtail butterfly,Atrophaneura alcinous,to aristolochic acids

Seven analogs of aristolochic acids (I, II, III, B, C, D, and E) were isolated from the leaves ofAristolochia debilis and characterized as the larval feeding stimulants of an Aristolochiaceae-feeding swallowtail butterfly,Atrophaneura alcinous. Aristolochic acids showed synergistic activity in combination with the water-soluble components in the leaf extract. Aristolochic acids were detected in the body tissues and specialized organs throughout all life stages ofA. alcinous. Larval osmeterial fluid contained aristolochic acids as high as 2% of the secretions, the compositions of which were similar to that found in the leaf extracts. In contrast, the eggs selectively contained aristolochic acids I and II. These two acids were present both in the egg yolk and egg coating material as well as in the collateral glands (glandulae sebaceae) of the adult females. The cannibalistic activity of the larvae against the eggs and pupae seemed to be triggered by aristolochic acids present on the egg surface and pupal cuticle, suggesting a possible adaptive mechanism in this species. Aristolocic acid I deterred feeding of tree sparrows, which suggested a defensive role against vertebrate predators.