Specific recognition, detoxification and metabolism of pyrrolizidine alkaloids by the polyphagous arctiid Estigmene acrea

Evidence is presented that the polyphagous arctiid Estigmene acrea is well adapted to sequester and specifically handle pyrrolizidine alkaloids of almost all known structural types representative of the major plant families with pyrrolizidine alkaloid-containing species, i.e. Asteraceae with the tribes Senecioneae and Eupatorieae, Boraginaceae, Fabaceae, Apocynaceae and Orchidaceae. The adaptation of E. acrea to pyrrolizidine alkaloids includes a number of specialized characters: (i) highly sensitive recognition of alkaloid sources by pyrrolizidine alkaloid-specific taste receptors; (ii) detoxification of pyrrolizidine alkaloids by N-oxidation catalyzed by a specific flavin-dependent monooxygenase; (iii) transfer and maintenance of all types of pyrrolizidine N-oxides through all developmental stages; (iv) conversion of the various structures into the male courtship pheromone hydroxydanaidal most probably through retronecine and insect specific retronecine esters (creatonotines) as common intermediates; (v) specific integration into mating behavior and defense strategies. Toxic otonecine derivatives, e.g. the senecionine analogue senkirkine, which often accompany the common retronecine derivatives and which cannot be detoxified by N-oxidation do not affect the development of E. acrea larvae. Senkirkine is not sequestered at all. Non-toxic 1,2-saturated platynecine derivatives that frequently occur together with toxic retronecine esters are sequestered and metabolized to hydroxydanaidal, indicating the ability of E. acrea to aromatize saturated pyrrolizidines. Although pyrrolizidine alkaloids, even if they are offered continuously at a high level (2%) in the larval diet, are non-toxic, E. acrea larvae are not able to develop exclusively on a pyrrolizidine alkaloid-containing plant like Crotalaria. Therefore, E. acrea appears to be specifically adapted to exploit pyrrolizidine alkaloid-containing plants as "drug source" but not as a food source.     

Uptake and metabolism of pyrrolizidine alkaloids in<Emphasis Type="Italic"> Longitarsus</Emphasis> flea beetles (Coleoptera: Chrysomelidae) adapted and non-adapted to alkaloid-containing host plants

Several Longitarsus flea beetle species sequester pyrrolizidine alkaloids acquired from their Asteraceae and Boraginaceae host plants. We carried out feeding and injection experiments using radioactively labeled pyrrolizidine alkaloids to investigate the physiological mechanisms of uptake, metabolism and storage of alkaloids in adult beetles. We examined six Longitarsus species belonging to different phylogenetic clades in a comparative approach. All species that accepted pyrrolizidine alkaloids in a preceding food choice study showed the ability both to store pyrrolizidine alkaloid N-oxides and to metabolize tertiary pyrrolizidine alkaloids into their N-oxides. Regardless of whether the beetles' natural host plants contain pyrrolizidine alkaloids or not, these species were found to possess an oxidizing enzyme. This oxygenase appears to be specific to pyrrolizidine alkaloids: [³H]Atropine and [¹⁴C]nicotine, two alkaloids not related to pyrrolizidine alkaloids, were neither stored nor N-oxidized by any of the tested species. One species, L. australis, that strictly avoids pyrrolizidine alkaloids behaviorally, exhibited a lack of adaptations to pyrrolizidine alkaloids on a physiological level as well. After injection of tertiary [¹⁴C]senecionine, beetles of this species neither N-oxidized nor stored the compounds, in contrast to L. jacobaeae, an adapted species that underwent the same treatment. L. jacobaeae demonstrated the same efficiency in N-oxidation and storage when fed or injected with tertiary [¹⁴C]senecionine.Communicated by G. Heldmaier     

Acquisition, transformation and maintenance of plant pyrrolizidine alkaloids by the polyphagous arctiid Grammia geneura

The polyphagous arctiid Grammia geneura appears well adapted to utilize for its protection plant pyrrolizidine alkaloids of almost all known structural types. Plant-acquired alkaloids that are maintained through all life-stages include various classes of macrocyclic diesters (typically occurring in the Asteraceae tribe Senecioneae and Fabaceae), macrocyclic triesters (Apocynaceae) and open-chain esters of the lycopsamine type (Asteraceae tribe Eupatorieae, Boraginaceae and Apocynaceae). As in other arctiids, all sequestered and processed pyrrolizidine alkaloids are maintained as non-toxic N-oxides. The only type of pyrrolizidine alkaloids that is neither sequestered nor metabolized are the pro-toxic otonecine-derivatives, e.g. the senecionine analog senkirkine that cannot be detoxified by N-oxidation. In its sequestration behavior, G. geneura resembles the previously studied highly polyphagous Estigmene acrea. Both arctiids are adapted to exploit pyrrolizidine alkaloid-containing plants as "drug sources". However, unlike E. acrea, G. geneura is not known to synthesize the pyrrolizidine-derived male courtship pheromone, hydroxydanaidal, and differs distinctly in its metabolic processing of the plant-acquired alkaloids. Necine bases obtained from plant acquired pyrrolizidine alkaloids are re-esterified yielding two distinct classes of insect-specific ester alkaloids, the creatonotines, also present in E. acrea, and the callimorphines, missing in E. acrea. The creatonotines are preferentially found in pupae; in adults they are largely replaced by the callimorphines. Before eclosion the creatonotines are apparently converted into the callimorphines by trans-esterification. Open-chain ester alkaloids such as the platynecine ester sarracine and the orchid alkaloid phalaenopsine, that do not possess the unique necic acid moiety of the lycopsamine type, are sequestered by larvae but they need to be converted into the respective creatonotines and callimorphines by trans-esterification in order to be transferred to the adult stage. In the case of the orchid alkaloids, evidence is presented that during this processing the necine base (trachelanthamidine) is converted into its 7-(R)-hydroxy derivative (turneforcidine), indicating the ability of G. geneura to introduce a hydroxyl group at C-7 of a necine base. The creatonotines and callimorphines display a striking similarity to plant necine monoesters of the lycopsamine type to which G. geneura is well adapted. The possible function of insect-specific trans-esterification in the acquisition of necine bases derived from plant acquired alkaloids, especially from those that cannot be maintained through all life-stages, is discussed.     

Morphogenetic effects of alkaloidal metabolites on the development of the coremata in the salt marsh moth, Estigmene acrea (Dru.) (Lepidoptera: Arctiidae)

Pyrrolizidine alkaloids (PAs) play a fundamental role in the sexual biology of the salt marsh moth Estigmene acrea. They are precursors for the male courtship pheromone hydroxydanaidal and they stimulate the growth and development of male pheromone-disseminating organs called coremata. Yet larval Estigmene are polyphagous and feed only sporadically on PA-containing plants and those they utilize contain different classes of PAs. The various PAs ingested are hydrolyzed to the common necine metabolite retronecine and re-esterified to insect-specific alkaloids from which the male pheromone hydroxydanaidal is synthesized. Given this complex metabolic pathway, we investigated the role of retronecine and the insect-specific alkaloids that stem from it as morphogens stimulating corematal growth. Retronecine fed to terminal instar larvae in a standard caterpillar diet stimulated corematal growth. It also stimulated corematal growth when it was injected into the hemolymph of larvae. These results indicate that this common PA metabolite, and/or the insect specific alkaloids produced from it, function as corematal morphogens. The parental forms (alkaloids ingested from the plant) are not strictly necessary for corematal growth. Stimulation of the PA receptors on the galea and ingestion process are also not critical to corematal development. Since the insect-specific alkaloids are the direct precursors for the male courtship pheromone, it is argued that their level is the best indicator of the ultimate pheromone titer and would provide the most accurate developmental signal. The effects of alkaloidal metabolites as morphogens in E. acrea are compared to those for the South Asian arctiines Creatonotus gangis and C. transiens in which the developmental role of PAs was first discovered.     

A taste receptor neurone dedicated to the perception of pyrrolizidine alkaloids in the medial galeal sensillum of two polyphagous arctiid caterpillars

It is shown that pyrrolizidine alkaloids are phagostimulants for the caterpillars of two polyphagous arctiid caterpillars, Estigmene acrea and Grammia geneura. The caterpillars will also eat dry glass‐fibre discs containing only pyrrolizidine alkaloid ? an example of pharmacophagy. The tip‐recording technique is used to demonstrate that each species has a neurone in the medial galeal styloconic taste sensillum responding to pyrrolizidine alkaloids, although the species differ in their sensitivities. This neurone responds to at least four different pyrrolizidine alkaloids and their N‐oxides, and experiments indicate that it is dedicated to perception of these compounds. The sensory response is phasic–tonic and during the tonic phase remains unchanged for at least 500 ms, resembling the type of response often seen in neurones that are sensitive to plant secondary compounds producing deterrent effects.     

Complex effects of parasitoids on pharmacophagy and diet choice of a polyphagous caterpillar

This study investigates complex effects of parasitoid infection on herbivore diet choice. Specifically, we examine how immunological resistance, parasitoid infection stage, and parasitoid taxonomic identity affect the pharmacophagous behavior of the polyphagous caterpillar, Grammia incorrupta (Arctiidae). Using a combination of lab and field experiments, we test the caterpillar’s pharmacophagous response to pyrrolizidine alkaloids (PAs) over the course of parasitoid infection, as well as the effect of dietary PAs on the caterpillar’s immunological response. Previous work from other systems gave the prediction that dietary PAs would be detrimental to the immune response and thus less acceptable to feeding early in the infection, when encapsulation of the parasitoid is most crucial. We found that the feeding acceptability of PAs was indeed low for caterpillars with early-stage parasitoid infections; however, this was not explained by PA interference with immune function. When allowed to choose among three host plant species, individuals harboring early-stage parasitoids increased their consumption of a nutritious plant containing antioxidants. This result was driven by wasp-parasitized caterpillars, whereas fly-parasitized caterpillars increased their consumption of plants containing iridoid glycosides. Individuals in the later time phase of infection exhibited an increase in PA intake that was consistent with previously reported self-medication behavior during late-stage parasitoid infection. This study reveals the depth of complexity and the dynamic nature of herbivore host plant choice, and underscores the importance of considering multitrophic interactions when studying insect diet choice.     

Effects of pyrrolizidine alkaloids and sesquiterpenes on snail feeding

Summary We determined in the laboratory the feeding response of two populations of the generalist herbivorous snail Arianta arbustorum (Helicidae) towards the composite Adenostyles alliariae and towards various allelochemicals. These were: a pyrrolizidine alkaloid (PA) extract of Adenostyles leaves; senecionine (a PA present in Adenostyles); retrorsine (a PA not present in Adenostyles) and two sesquiterpene (ST) fractions from Adenostyles: a mixture of the STs adenostylone and neoadenostylone, and deacyladenostylone. Tertiary PAs and PA N-oxides were tested separately. For each allelochemical, we tested whether it was deterrent or whether it induced changes of feeding behaviour (i.e. whether it had pre- or postingestive effects), and whether the effects were more pronounced with younger (smaller) snails. The tertiary PA extract from Adenostyles was deterrent, especially for young snails, but did not induce changes of feeding behaviour. Tertiary PA senecionine was deterrent for young snails only and induced changes of feeding behaviour. Also, consumption of untreated Petasites was higher after this treatment. Tertiary PA retrorsine was not deterrent, but induced changes of feeding behaviour. The PA N-oxides showed no activity against the snails. The mixture of adenostylone and neoadenostylone was deterrent and induced feeding aversions. Deacyladenostylone was highly deterrent, but did not induce changes of feeding behaviour. At the Jura site, PA content of Adenostyles was lower than at the Black Forest site. The snails from Jura consumed much less Adenostyles than the snails from Black Forest, and also ate a little less of the treated leaf discs. The PAs which are encountered by the snails in their natural food plants (PA extract and senecionine) were more deterrent than retrorsine (a novel compound). This suggests that the snails have mechanisms for the rejection of allelochemicals which they encounter in their natural food plants, but not for novel allelochemicals. The results suggest two hypotheses regarding the function of the allelochemicals in Adenostyles: (1) The allelochemicals act mainly on very young snails. (2) PAs render Adenostyles toxic, while STs act as feeding deterrents.     

Preference for high concentrations of plant pyrrolizidine alkaloids in the specialist arctiid moth Utetheisa ornatrix depends on previous experience

Secondary metabolites are one the most pervasive defensive mechanisms in plants. Many specialist herbivores have evolved adaptations to overcome these defensive compounds. Some herbivores can even take advantage of these compounds by sequestering them for protection and/or mate attraction. One of the most studied specialist insects that sequesters secondary metabolites is the arctiid moth Utetheisa ornatrix. This species sequesters pyrrolizidine alkaloids (PAs) from its host plant, the legume Crotalaria spp. The sequestered PAs are used as a predator repellent and as a mating pheromone. We used this species to test larval preference for different concentrations of PAs. We purified PAs from plant material and added them at different concentrations to an artificial diet. Larvae of U. ornatrix previously feeding on low and high PA concentration artificial diets were allowed to choose between two new artificial diets with different PA concentrations. The amount of PAs sequestered and larval preference were dependent on their previous exposure to low or high PA content in the diet. Larvae that were pretreated with a low PA diet significantly consumed more diet with the high PA concentration, while larvae that were pretreated with a high PA diet showed no discrimination between future feeding of different PA concentration diets. We discuss our results using mechanistic and evolutionary approaches. Finally, we discuss how these results have important implications on the evolution of plant herbivore interactions and how specialist herbivores may decrease the levels of chemical defenses on plant populations.     

Tritrophic interactions between aphids (Aphis jacobaeae Schrank), ant species,Tyria jacobaeae L., and Senecio jacobaea L. lead to maintenance of genetic variation in pyrrolizidine alkaloid concentration

Summary We hypothesize that the tritrophic interaction between ants, the aphid Aphis jacobaeae, the moth Tyria jacobaeae, and the plant Senecio jacobaea can explain the genetic variation observed in pyrrolizidine alkaloid concentration in natural populations of S. jacobaea. The ant Lasius niger effectively defends S. jacobaea plants infested with A. jacobaeae against larvae of T. jacobaeae. S. jacobaea plants with A. jacobaeae which are defended by ants escape regular defoliation by T. jacobaeae. Plants with aphids and ants have a lower pyrrolizidine alkaloid concentration than plants without aphids and ants. When these data are fitted to an existing theoretical model for temporal variation in fitness it is shown that varying herbivore pressure by T. jacobaeae in interaction with ants defending aphid-infested plants with a low pyrrolizidine alkaloid concentration can lead to a stable polymorphism in pyrrolizidine alkaloid concentration. Costs of the production and maintenance of pyrrolizidine alkaloids are not accounted for in the model.Publication of the Meijendel-comité, new series no. 114     

Pheromone-mediated sexual selection in the mothUtetheisa ornatrix: Olfactory receptor neurons responsive to a male-produced pheromone

Male Utetheisa ornatrixhave a pair of eversible glandular brushes (coremata) which are displayed during precopulatory interactions with the female. Earlier studies have shown that a pheromone associated with the coremata, hydroxydanaidal (HD), is derived by the males from pyrrolizidine alkaloids (PAs) that they sequester as larvae from their foodplants (Crotalariaspp.) The PAs impart a distastefulness upon Utetheisathat protects both larvae and adults against predation. The receptor neurons specialized for detection of HD are housed in sensilla whose morphological features, as revealed by scanning electron microscopy, classify them as sensilla basiconica. The sensitivity and dynamic range of these receptor neurons were largely unaffected by whether the females were raised on an alkaloid-free diet or on a diet supplemented with Crotalariaseeds. Acetylation of the hydroxyl group of HD substantially reduced the activity of the molecule. None of the antennal sensilla examined contained receptor neurons sensitive to a PA (monocrotaline) or its N-oxide.     

Pyrrolizidine alkaloids: different acquisition and use patterns in Apocynaceae and Solanaceae feeding ithomiine butterflies (Lepidoptera: Nymphalidae)

Pyrrolizidine alkaloids (PAs) often serve as chemical mediators of plant-herbivore-predator interactions. Butterflies (Danainae and Ithomiinae) and moths (Arctiidae) usually acquire PAs from plant sources (larval host plants, flowers or withered leaves visited by adults—pharmacophagy) and thereby become chemically protected against predators; they also use PAs as pheromone precursors. Study by GC-MS of PAs in three species of Ithomiinae butterflies, their larval host plants and adult alkaloid sources showed three different acquisition patterns: (1) larvae of the primitive Tithorea harmonia sequester PAs from their food plant Prestonia acutifolia (Apocynaceae: Echitoideae), and adults may also acquire these alkaloids from plant sources; (2) larvae of the more derived Aeria olena feed on Prestonia coalita , in whose leaves no PAs were detected, but freshly emerged adults sometimes contain PAs and males intensively seek and sequester these alkaloids in plant sources; and (3) larvae of the still more advanced Mechanitis polymnia feed on several PA-free Solanum species, and adult males sequester the alkaloids from various plant sources. Males and females of all three species contain mostly two PAs, the diastereoisomeric retronecine monoesters lycopsamine and intermedine, stored in the N-oxide form. Larval host plants and adult plant sources showed a large array of PA structures, the most abundant and frequent being lycopsamine and its diastereoisomers intermedine, echinatine, rinderine and indicine, and the deoxy-analogues supinine and amabiline. Bioassays with wild caught and freshly emerged adults suggest that protection against predation by the orb weaving spider Nephila clavipes may be dependent on PA concentration and maybe some spider idiosyncrasies, but freshly emerged Aeria olena without PAs are also liberated by Nephila , suggesting other protective compounds. The role of this spider as a selective pressure for PA acquisition by ithomiines is not clear.     

Experience influences gustatory responsiveness to pyrrolizidine alkaloids in the polyphagous caterpillar,Estigmene acrea

Electrophysiological recordings from taste sensilla of the caterpillar Estigmene acrea with the pyrrolizidine alkaloid, seneciphylline N-oxide, demonstrated that prior feeding on plants with pyrrolizidine alkaloids caused an increase in responsiveness of the PA-sensitive cells in two sensilla, relative to feeding on plants without such chemicals. Rearing on synthetic diet without pyrrolizidine alkaloids for up to seven generations caused a continuous decline in responsiveness, that could be reversed by experience with powdered Crotalaria pumila in the diet or by pure pyrrolizidine alkaloid, monocrotaline, in the diet. Response to the cardiac glycoside, ouabain, that stimulates one of the two pyrrolizidine alkaloid-sensitive cells, showed a similar decline. Pyrrolizidine alkaloids had no measurable effect on growth and development. Responses in all other taste cells were unaffected. The data are discussed in relation to the possible adaptive significance and the possible mechanisms involved.R.F. Chapman has died since this article was written     

Pyrrolizidine alkaloids quantitatively regulate both scent organ morphogenesis and pheromone biosynthesis in maleCreatonotos moths (Lepidoptera: Arctiidae)

Summary Males ofCreatonotos gangis andC. transiens possess coremata (scent organs) of drastically varying sizes (Figs. 2, 3), which release R(-)-hydroxydanaidal (Fig. 1A) in varying amounts. Both the size of the organs and their pheromone content depend on the ingestion of pyrrolizidine alkaloids (PAs; Fig. 1 B, C) by the larvae. There is a direct correlation between amounts of PAs ingested and the size of the organs (Fig. 4). It is the absolute amount of PAs ingested which determines the expression of corematal size, structurally different PAs have identical effects (Table 2); PAs are no essential dietary factors for the general development of the moths (Table 1), and the morphogenetic effect is restricted to the coremata. The findings are discussed in terms of developmental, ecological and functional aspects.Abbreviation PAs pyrrolizidine alkaloids     

Evolutionary trends in the male pheromone systems of arctiid moths: evidence from studies of courtship in Phragmatobia fuliginosa and Pyrrharctia isabella (Lepidoptera: Arctiidae)

Male Phragmatobia fuliginosa and Pyrrharctia Isabella evert tubular abdominal scent-organs (coremata: during courtship. The coremata secrete a pheromone that is derived from pyrrolizidine alkaloids (PAs) obtained during larval feeding. Field-collected males vary widely in the amount of pheromone they possess. The courtship behaviour of these species was studied to determine whether this variation among males serves as a basis for mate choice by females, in a wind-tunnel a male approaches a pheromone-releasing female from downwind with his coremata everted in flight. A female perceiving the pheromone responds by fluttering her wings and emitting a clicking sound. This response did not occur in all courtships, and successful copulation was not dependent on its occurrence. Males denied access as larvae to the PA-precursors of the corematal pheromone (PA-denicd males' mated as rapidly as males allowed access to pheromone precursor (PA-supplied males). Moreover, antennectomized females mated as rapidly as normal females. Comparative analyses of transitional probabilities indicated no differences among courtships involving ! I': PA-denicd males and normal females, (II) PA-supplied males and normal females, or antennectomized females and normal males of unspecified feeding history. Field observations of courtship interactions between laboratory-reared females and wild males indicated that males having little or no pheromone succeed in mating in a natural setting as do males having a relativelv large amount of pheromone. It is hypothesized that the failure of female Ph. fuliginosa and Py, Isabella to enforce the display of PA-derived pheromone is related to an cvolutionarv trend away from an ancestral larval host-range restricted to PA-containing plants toward the polyphagv shown by manv temperate and arctic arctiids in the lineage (subfamily Arctiinae: tribe Arctiini) to which Ph. fuliginosa and Py. Isabella belong. Parallels in other moth groups are cited to support the notion that a plesiomorphic courtship pheromone system could persist in a vestigial state, despite the loss of its adaptive raison dêtre.     

Pyrrolizidine alkaloids of probable host-plant origin in the pronotal and elytral secretion of the leaf beetle Oreina cacaliae

Oreina cacaliae (Coleoptera, Chrysomelidae) produces in its elytral and pronotal defensive secretion seneciphylline N-oxide together with small amounts of another pyrrolizidine alkaloid tentatively identified as senecionine N-oxide. This is a strong departure from the chemical composition of the defensive secretions in related species, characterized by complex mixtures of cardenolides, synthesized by the beetles from cholesterol. It is suggested that O. cacaliae sequesters the alkaloids from its host-plant, Adenostyles leucophylla. Other specimens of O. cacaliae from far distant populations feeding on Senecio nemorensis, Petasites paradoxus or P. album also produced pyrrolizidine alkaloids, but not O. speciosissima feeding on the same food plants and producing cardenolides. In addition to pyrrolizidine alkaloids, O. cacaliae secretes ethanolamine, which is also found in all the cardenolide-producing species.     

Detection and Toxicity Evaluation of Pyrrolizidine Alkaloids in Medicinal Plants Gynura bicolor and Gynura divaricata Collected from Different Chinese Locations

Two edible plants in Southeast Asia, Gynura bicolor and G . divaricata , are not only known to be nutritive but also useful as medicinal herbs. Previous phytochemical investigation of Gynura species showed the presence of hepatotoxic pyrrolizidine alkaloids (PA s), indicating the toxic risk of using these two plants. The present study was designed to analyze the distribution of PA components and tried to evaluate the preliminary toxicity of these two Gynura species. Eight samples of G . bicolor and G . divaricata from five different Chinese locations were collected and their specific PA s were qualitatively characterized by applying an UPLC /MS /MS spectrometry method. Using a pre‐column derivatization HPLC method, the total retronecine ester‐type PA s in their alkaloids extracts were quantitatively estimated as well. Finally, their genotoxicity was investigated with an effective high‐throughput screening method referred to as Vitotox ? test and their potential cytotoxicity was tested on HepG2 cells. It was found that different types of PA s were widely present in Gynura species collected from south of China. Among them, no significant genotoxic effects were detected with serial concentrations through the present in vitro assay. However, the cytotoxicity assay of Gynura plants collected from Jiangsu displayed weak activity at the concentration of 100 mg/ml. It is important to note that this research validates in part the indication that the use of Gynura species requires caution.     

A free lunch? No cost for acquiring defensive plant pyrrolizidine alkaloids in a specialist arctiid moth (Utetheisa ornatrix)

Many herbivorous insects sequester defensive chemicals from their host plants. We tested sequestration fitness costs in the specialist moth Utetheisa ornatrix (Lepidoptera: Arctiidae). We added pyrrolizidine alkaloids (PAs) to an artificial diet at different concentrations. Of all the larval and adult fitness components measured, only development time was negatively affected by PA concentration. These results were repeated under stressful laboratory conditions. On the other hand, the amount of PAs sequestered greatly increased with the diet PA concentration. Absence of a detectable negative effect does not necessarily imply a lack of costs if all individuals express the biochemical machinery of detoxification and sequestration constitutively. Therefore, we used qPCR to show that expression of the gene used to detoxify PAs, pyrrolizidine‐alkaloid‐N‐oxygenase (pno), increased 41‐fold in our highest PA treatment. Nevertheless, fitness components were affected only slightly or not at all, suggesting that sequestration in this species does not incur a strong cost. The apparent lack of costs has important implications for our understanding of the evolution of ecological interactions; for example, it implies that selection by specialist herbivores may decrease the levels of certain chemical defences in plant populations.     

Pyrrolizidine alkaloids in and on the leaf surface of Senecio jacobaea L

This is the first study showing that alkaloids are present on the leaf surface of plants. A concentration of 30-230 pmol/cm2 pyrrolizidine alkaloids (PA's) was detected in 8 different samples taken from Senecio jacobaea. PA concentration on the leaves was marginally correlated with PA concentration of the total leaf tissues. The PA spectrum on the leaf differed from the PA spectrum of the total leaf.     

Impact of the dual defence system of Plantago lanceolata (Plantaginaceae) on performance,nutrient utilisation and feeding choice behaviour of Amata mogadorensis larvae (Lepidoptera,Erebidae)

Iridoid glycosides are plant defence compounds with potentially detrimental effects on non-adapted herbivores. Some plant species possess β-glucosidases that hydrolyse iridoid glycosides and thereby release protein-denaturing aglycones. To test the hypothesis that iridoid glycosides and plant β-glucosidases form a dual defence system, we used Plantago lanceolata and a polyphagous caterpillar species. To analyse the impact of leaf-age dependent differences in iridoid glycoside concentrations and β-glucosidase activities on insect performance, old or young leaves were freeze-dried and incorporated into artificial diets or were provided freshly to the larvae. We determined larval consumption rates and the amounts of assimilated nitrogen. Furthermore, we quantified β-glucosidase activities in artificial diets and fresh leaves and the amount of iridoid glycosides that larvae feeding on fresh leaves ingested and excreted. Compared to fresh leaves, caterpillars grew faster on artificial diets, on which larval weight gain correlated positively to the absorbed amount of nitrogen. When feeding fresh young leaves, larvae even lost weight and excreted only minute proportions of the ingested iridoid glycosides intact with the faeces, indicating that the hydrolysis of these compounds might have interfered with nitrogen assimilation and impaired larval growth. To disentangle physiological effects from deterrent effects of iridoid glycosides, we performed dual choice feeding assays. Young leaves, their methanolic extracts and pure catalpol reduced larval feeding in comparison to the respective controls, while aucubin had no effect on larval consumption. We conclude that the dual defence system of P. lanceolata consisting of iridoid glycosides and β-glucosidases interferes with the nutrient utilisation via the hydrolysis of iridoid glycosides and also mediates larval feeding behaviour in a concentration- and substance-specific manner.     

The effect of varying alkaloid concentrations on the feeding behavior of gypsy moth larvae, Lymantria dispar (L.) (Lepidoptera: Lymantriidae)

Nine alkaloids (acridine, aristolochic acid, atropine, berberine, caffeine, nicotine, scopolamine, sparteine, and strychnine) were evaluated as feeding deterrents for gypsy moth larvae (Lymantria dispar (L.); Lepidoptera: Lymantriidae). Our aim was to determine and compare the taste threshold concentrations, as well as the ED₅₀ values, of the nine alkaloids to determine their potency as feeding deterrents. The alkaloids were applied to disks cut from red oak leaves (Quercus rubra) (L.), a plant species highly favored by larvae of this polyphagous insect species. We used two-choice feeding bioassays to test a broad range of biologically relevant alkaloid concentrations spanning five logarithmic steps. We observed increasing feeding deterrent responses for all the alkaloids tested and found that the alkaloids tested exhibited different deterrency threshold concentrations ranging from 0.1 to 10 mM. In conclusion, it appears that this generalist insect species bears a relatively high sensitivity to these alkaloids, which confirms behavioral observations that it avoids foliage containing alkaloids. Berberine and aristolochic acid were found to have the lowest ED₅₀ values and were the most potent antifeedants. Handling Editor: Joseph Dickens.