Triterpene saponin hemi-biosynthesis of a leaf beetle’s (<Emphasis Type="Italic">Platyphora kollari</Emphasis>) defensive secretion

The adults of the leaf beetle Platyphora kollari (Chrysomelidae) are able to metabolise the oleanane triterpene β-amyrin (1) into the glycoside 3-O-β-d-glucopyranosyl-(1→4)-β-d-glucuronopyranosyl-hederagenin (2) that is stored in their defensive glands. The aim of this study was to test the hypothesis that oleanolic acid (3) is an intermediate in the conversion of 1 into 2 and to check whether the sequestration of pentacyclic triterpenes is selective in favour of β-amyrin (1). To this end, adults of P. kollari were fed with Ipomoea batatas leaf disks painted with a solution of [2,2,3-²H₃]oleanolic acid or [2,2,3-²H₃]α-amyrin and the secretion of their defensive glands analysed by HPLC–ESIMS. The data presented in this work indicated that the first step of the transformation of β-amyrin (1) into the sequestered glycoside 2 is its oxidation into oleanolic acid (3) and that this conversion is selective but not specific in favour of β-amyrin (1).     

Short- and medium-term effects of experimental nitrogen fertilization on arthropods associated with Calluna vulgaris heathlands in north-west Spain

We studied the short- and medium-term effects of experimental nitrogen fertilization (3 and 15 months after the treatment) on the arthropods of Calluna vulgaris heathlands in NW Spain. Three heathland sites were selected with two permanent plots per site: control and fertilized. Ammonium nitrate fertilizer (56 kg N ha(-1) yr(-1)) was applied monthly and insects were caught using pitfall traps. We found mainly species-level responses to nitrogen addition. Seven species (e.g. Lochmaea suturalis) showed a consistent trend (benefited or harmed) in both periods and were proposed as possible reliable indicators of the effects of nitrogen deposition in these ecosystems. We also found variable arthropod trophic-group responses: (a) herbivores (leaf beetles, true bugs) increased in abundance on a short-term scale; (b) predators (carabid beetles, true bugs) showed opposite and less clear responses in both periods. Further long-term studies are needed to determine the mechanisms underlying the observed arthropod responses.     

Larval chemical defence and evolution of host shifts in Chrysomela leaf beetles

Summary. Larvae of Chrysomela leaf beetles release for defence volatile compounds belonging to various chemical families. This study focuses on the defensive strategy based on the esterification of isobutyric acid and 2-methylbutyric acid with a wide variety of alcohols taken up from the host plant. To date, only two species are known to produce these repellents C. interrupta, which is associated with Betulaceae and C. lapponica which occurs either on Betulaceae or Salicaceae.? In order to know if other species have developed this chemical defence and how the food plant influences the secretion of these toxins, we targeted by mass spectrometry the presence of iso- and 2-methylbutyric acids and esters of them in the defensive secretions of Chrysomela larvae exclusively associated with Betulaceae or Salicaceae. ?Screening analyses reveal that the synthesis of these compounds is a common character restricted to all the members belonging to the C. interrupta group sensu Brown (1956) regardless of the host-plant family. These results suggest that the biochemical mechanism leading to the synthesis of these compounds could be considered as a synapomorphy meaning that the group is probably monophyletic. ?Defensive secretions of the members of the interrupta group are quantitatively assayed for iso- and 2-methylbutyric acids and their (Z)-3-hexenyl esters. Results reveal a chemical plasticity developed by Chrysomela species associated with Salicaceae. The amounts of iso- and 2-methylbutyric acids derivatives and of salicylaldehyde in their larval secretions depend on the food plant and on its content in phenolglucosides. Received 5 October 1998; accepted 25 November 1998.     

Time course of pyrrolizidine alkaloid sequestration in <Emphasis Type="Italic">Longitarsus</Emphasis> flea beetles (Coleoptera,Chrysomelidae)

Summary. Several species of Longitarsus take up, metabolize and store pyrrolizidine alkaloids (PAs) from their host plants. In feeding experiments using radioactively labeled PAs of different types we examined the time course of the sequestration process in L. jacobaeae and L. aeruginosus. We found that adapted species efficiently store PAs for at least two weeks without major losses. During that time, there is virtually no change in the ratio of tertiary alkaloids to stored non-toxic N-oxides, regardless of chemical form fed to the beetles. This implies a transient N-oxidation process where the alkaloids are only temporarily accessible to the enzyme. A dissection experiment with L. aeruginosus six days after uptake of labeled PAs showed that the tertiary alkaloids are not found in the hemolymph but are stored in the elytra and other body compartments. This conforms with earlier experiments that localized the enzymes site of action in the hemolymph. Furthermore we show that different total alkaloid doses in the diet of L. jacobaeae and the potentially less adapted L. succineus do not affect the ratio of recovered N-oxides to tertiary molecules. Thus, the efficiency of the N-oxidizing enzyme is not dependent on the concentration of alkaloids offered.     

Preferences of six leaf beetle species among qualitatively different leaf age classes of three Salicaceous host species

Summary. As Salicaceous plants produce new leaves for a prolonged period of time, they expose a wide range of differentially aged leaves to herbivores during the growing season. In this work, I show that young leaves of three Salicaceous species, Populus tremula L., Salix phylicifolia L. and S. pentandra L., contain more nitrogen than conspecific old leaves. In P. tremula and S. pentandra young leaves also contained more low-molecular weight secondary compounds, phenolic glucosides. Leaves of S. phylicifolia did not contain phenolic glucosides in detectable amounts. Furthermore, in P. tremula and S. pentandra young leaves contained less polymeric digestability-reducing phenolics, condensed tannins, than old leaves. In S. phylicifolia, higher concentrations of condensed tannins were found in young leaves. In laboratory feeding trials with six leaf beetle species, young leaves of the studied plants were invariably preferred in all tested herbivore × host species combinations. In particular, it is remarkable that three leaf beetle species with known different overall relationships to phenolic glucosides equally preferred more glucoside-containing young S. pentandra leaves over conspecific old ones. Four beetle species were found to prefer young leaves of S. phylicifolia despite the higher content of condensed tannins in young leaves. These results indicate that the general preference of leaf beetles for young leaves of Salicaceous plants probably does not primarily result from variable distribution of secondary compounds. Apparently, the preference for young leaves is fundamentally due to variation in leaf nutritive traits, such as nitrogen content. Received 9 February 2001.     

Pyrrolizidine alkaloids on three trophic levels – evidence for toxic and deterrent effects on phytophages and predators

Summary. Pyrrolizidine alkaloids (PAs) present a model system in the investigation of tritrophic interactions mediated by plant secondary compounds. However, their toxicity for insect herbivores has never been experimentally proven. Here, we demonstrate the toxic effects of a PA on growth and survival of the eri silk moth Philosamia ricini. In a feeding experiment, larvae of this generalist herbivore fed with an artificial PA diet gained weight significantly slower than control animals, and died as pupae. We suggest that derivatives of the ingested PA N-oxide damage developmental functions during metamorphosis. A tracer test with [¹⁴C]senecionine N-oxide revealed that the caterpillars lack adaptations that would prevent conversion of the chemical into the pro-toxic free base. In contrast, the PA adapted leaf beetle Longitarsus anchusae accumulates PAs as N-oxides. We tested the purpose of sequestration in this species as defence against predators. Through a series of prey choice experiments with three carabid predator species, chemically non-protected bark beetle pupae were chosen almost uniformly over L. anchusae pupae. In a following choice test with one of these predators, artificially PA-treated mealworm segments deterred the predator from feeding. Overall the study corroborates the immediate toxic effect of PAs on non-adapted herbivores and the protective effect that adapted insects may gain by sequestering them. It thereby underlines the potential for PAs to play a central role in multitrophic interactions between plants, phytophages and their predators.     

Quantitative analysis of pyrrolizidine alkaloids sequestered from diverse host plants in Longitarsus flea beetles (Coleoptera, Chrysomelidae)

Summary. Several species of the flea beetles genus Longitarsus are able to sequester pyrrolizidine alkaloids (PAs) from their host plants. In five Longitarsus species we compare the concentration of PAs present in their host plants belonging to the Asteraceae or Boraginaceae with those found in the beetles. To get an estimate of the intrapopulation variability, three samples of five beetles each and five individual plants were analyzed for each comparison. A strong intrapopulation variability could be detected both among plant and beetle samples. The total concentration found in the beetles varied strongly between species. The local host plant and its phenology influence the concentrations present in the beetles as evidenced in comparisons of a single beetle species from two different hosts and of one beetle species collected at the same site at different times of the year. In addition, different beetle species apparently vary in their capacity to sequester the alkaloids, at the lowest extreme the mean PA concentration in the beetles (0.034 μg PA/mg dry weight) was 1/30 of the mean concentration found in the plant leaves (L. aeruginosus from Eupatorium cannabinum), at the highest extreme (2.098 μg PA/mg dw) the concentration in the beetles was a 1000 fold higher than in the plant leaves (L. nasturtii from Symphytum officinale). The highest mean concentration found in the beetles was 3.446 μg/mg dw (L. exoletus from Cynoglossum officinale). The absolute concentrations found in the beetles are comparable to other insects which have been shown to be effectively defended against their potential predators. Received 22 June 1999; accepted 25 August 1999     

Sequestration of plant pyrrolizidine alkaloids by chrysomelid beetles and selective transfer into the defensive secretions

Summary Oreina cacaliae andO. speciosissima (Coleoptera, Chrysomelidae) sequester in their elytral and pronotal defensive secretions pyrrolizidine alkaloids (PAs) as Noxides (PA N-oxides). The PA N-oxide patterns found in the beetles and their host plants were evaluated qualitatively and quantitatively by capillary gas chromatography/mass spectrometry (GC-MS). Of the three host plantsAdenostyles alliariae (Asteraceae) is the exclusive source for PA N-oxide sequestration in the defensive secretions of the beetles. With the exception of O-acetylseneciphylline the N-oxides of all PAs ofA. alliariae, i.e. senecionine, seneciphylline, spartioidine, integerrimine, platyphylline and neoplatyphylline were identified in the secretion. PA N-oxides typical ofSenecio fuchsii (Asteraceae) were detected in the bodies of the beetles but not in their secretion. No PAs were found in the leaves of the third host plant,Petasites paradoxus (Asteraceae). The results suggest the existence of two distinctive storage compartments for PA N-oxides in the beetle: (1) the defensive secretion, containing specifically PA N-oxides acquired fromA. alliariae; (2) the body of the beetle, sequestering additionally but less selectively PA N-oxides from other sources,e.g. S. fuchsii or monocrotaline N-oxide fed in the laboratory. The concentration of PA N-oxides in the defensive secretion is in the range of 0.1 to 0.3 mol/1, which is more than 2.5 orders of magnitude higher than that found in the body of the beetle. No significant differences exist in the ability of the two species of beetles to sequester PA N-oxides fromA. alliariae, althoughO. speciosissima, but notO. cacaliae, produces autogenous cardenolides. A negative correlation seems to exist between the concentrations of plant-derived PA N-oxides andde novo synthesized cardenolides in the defensive secretion ofO. speciosissima.     

Leaf surface compounds of the potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis>) and their influence on Colorado potato beetle (<Emphasis Type="Italic">Leptinotarsa decemlineata</Emphasis>) feeding

Summary.  The sampling behavior of the Colorado potato beetle Leptinotarsa decemlineata Say (CPB) involves examination of the surface of potato leaves. It has been suggested that leaf surface compounds (volatiles and cuticular waxes) may be involved in host-plant recognition, acceptance or discrimination. Here we report on the effect of leaf surface extracts of six Polish commercial potato varieties on CPB feeding. We tested the influence of potato leaf surface extracts on CPB adult and larval feeding, then separated the extracts with HPLC, and finally tested the effect of the HPLC-separated fractions on CPB feeding. The bioassays were performed using potato leaf discs deprived of their original surface compounds. Applied to test discs at concentrations ten times higher than natural (10 leaf area equivalent), the extracts deterred CPB adults and larvae from feeding. HPLC-separated fractions composed of alkanes, sesquiterpene hydrocarbons, wax esters, benzoic acid esters, fatty acid methyl, ethyl, isopropyl and phenylethyl esters, aldehydes, ketones, methyl ketones, fatty acids, primary alcohols, β-amyrin and sterols did not affect adult CPB feeding. Similarly, alkanes, sesquiterpene hydrocarbons, wax esters, methyl ketones, sesquiterpene alcohols and secondary alcohols had no effect on larval CPB feeding. The sterol fraction (cholesterol, β-sitosterol and stigmasterol) acted as a phagostimulant to CPB larvae. We isolated a fraction demonstrating a phagodeterrent effect on CPB adults and larvae. The qualitative composition of the deterrent fraction was quite similar in all potato extracts, but there were quantitative differences between the varieties. Much further work is needed to identify the compounds that can produce the deterrent effect.     

Sequestration of cucurbitacin analogs by New and Old World chrysomelid leaf beetles in the tribe Luperini

Summary Two South American polyphagous leaf beetles,Diabrotica speciosa andCerotoma arcuata, selectively accumulated the bitter tasting compound 23,24-dihydrocucurbitacin D in their body after ingesting root tissues of cucurbit plants. Similarly, three Asian Cucurbitaceae-feeding specialists in the genusAulacophora were found to sequester the same compound. Cucurbitacin analogs were shown to deter feeding by a bird predator, indicating an allomonal role for these compounds in cucurbitacin-associated chrysomelid leaf beetles both of New and Old Worlds. The strong affinity to cucurbitacins, selective sequestration of the analogs and consequent protection from predators suggested an ecological adaptation mechanism developed in common among these two geographically isolated subtribes in the Luperini.