Host plant shifts affect a major defense enzyme in Chrysomela lapponica.

Chrysomelid leaf beetles use chemical defenses to overcome predatory attack and microbial infestation. Larvae of Chrysomela lapponica that feed on willow sequester plant-derived salicin and other leaf alcohol glucosides, which are modified in their defensive glands to bioactive compounds. Salicin is converted into salicylaldehyde by a consecutive action of a ß-glucosidase and salicyl alcohol oxidase (SAO). The other leaf alcohol glucosides are not oxidized, but are deglucosylated and esterified with isobutyric- and 2-methylbutyric acid. Like some other closely related Chrysomela species, certain populations of C. lapponica shift host plants from willow to salicin-free birch. The only striking difference between willow feeders and birch feeders in terms of chemical defense is the lack of salicylaldehyde formation. To clarify the impact of host plant shifts on SAO activity, we identified and compared this enzyme by cloning, expression, and functional testing in a willow-feeding and birch-feeding population of C. lapponica. Although the birch feeders still demonstrated defensive gland-specific expression, their SAO mRNA levels were 1,000-fold lower, and the SAO enzyme was nonfunctional. Obviously, the loss of catalytic function of the SAO of birch-adapted larvae is fixed at the transcriptional, translational, and enzyme levels, thus avoiding costly expression of a highly abundant protein that is not required in the birch feeders.

Beetles do it differently: two stereodivergent cyclisation modes in iridoid-producing leaf-beetle larvae.

Larvae of the Chrysomelina species Phaedon cochleariae, Hydrothassa marginella, Phratora vulgatissima, Gastrophysa viridula, Gastrophysa atrocyanea, Gastrophysa cyanea and Gastrophysa polygoni produce the iridoid chrysomelidial (1) to defend themselves against predators. Feeding experiments with a deuterated precursor ([(2)H(5)]8-hydroxygeraniol 9) and in vitro isotope exchange experiments with defensive secretion in (2)H(2)O revealed differences in the cyclisation of the ultimate precursor 8-oxogeranial (8) to 1, between members of the genus Gastrophysa and all other species. In P. cochleariae, H. marginella and P. vulgatissima 1 is most likely produced by a Rauhut-Currier-type cyclisation via a "transoid dienamine", with loss of a single deuterium atom from C(4) of the precursor. In contrast, members of the genus Gastrophysa cyclise 8 via a "cisoid dienamine" intermediate, with exchange of all three deuterium atoms from the methyl group at C(3). To study whether the different cyclisation modes influence the stereochemistry of 1, the absolute configuration of 1 of the larvae was determined by GC-MS on a chiral column. In accordance with literature (J. Meinwald, T. H. Jones, J. Am. Chem. Soc. 1978, 100, 1883 and N. Shimizu, R. Yakumaru, T. Sakata, S. Shimano, Y. Kuwahara, J. Chem. Ecol. 2012, 38, 29), we found (5S,8S)-chrysomelidial (1) in H. marginella and P. vulgatissima, but P. cochleariae and all investigated members of the genus Gastrophysa synthesise (5R,8R)-chrysomelidial (1).

To be or not to be convergent in salicin-based defence in chrysomeline leaf beetle larvae: evidence from Phratora vitellinae salicyl alcohol oxidase.

Glandular chemical defence relying on the action of salicylaldehyde is characteristic for Chrysomela leaf beetle larvae. The salicylaldehyde precursor salicin, sequestered from salicaceous host plants, is deglucosylated and the aglycon further oxidized by a salicyl alcohol oxidase (SAO) to the respective aldehyde. SAOs, key enzymes in salicin-based glandular chemical defence, were previously identified and shown to be of a single evolutionary origin in Chrysomela species. We here identified and characterized SAO of Phratora vitellinae, the only species outside the genus Chrysomela that produce salicylaldehyde as a defensive compound. Although Chrysomela and Phratora are not closest relatives, their SAOs share glucose-methanol-choline oxidoreductase (GMC) affiliation, a specific GMCi subfamily ancestor, glandular tissue-specific expression and almost identical gene architectures. Together, this strongly supports a single origin of SAOs of both Chrysomela and Phratora. Closely related species of Chrysomela and P. vitellinae use iridoids as defensive compounds, which are like salicylaldehyde synthesized by the consecutive action of glucosidase and oxidase. However, we elucidated SAO-like sequences but no SAO proteins in the glandular secretion of iridoid producers. These findings support a different evolutionary history of SAO, related genes and other oxidases involved in chemical defence in the glandular system of salicylaldehyde and iridoid-producing leaf beetle larvae.

Conflicting mitochondrial and nuclear phylogeographic signals and evolution of host-plant shifts in the boreo-montane leaf beetle Chrysomela lapponica.

We conducted a phylogeographic study on the cold-adapted leaf beetle Chrysomela lapponica, that feeds on willow or birch, by sampling several populations throughout most of the geographic distribution of the species, and by sequencing for each individual one mitochondrial and two nuclear DNA fragments. Patterns of DNA sequence variation from the mitochondrial and nuclear loci, as displayed in the median-joining networks, appear to display contradicting historical signal: a deep genealogical divergence is observed with the mitochondrial genome between the Alpine population and all other populations found in the Euro-Siberian distribution of the species, that is completely absent with both nuclear loci. We use coalescence simulations of DNA sequence evolution to test the hypothesis that this apparent conflict is compatible with a neutral model of sequence evolution (i.e., to check whether the stochastic nature of the coalescence process can explain these patterns). Because the simulations show that this is highly unlikely, we consider two alternative hypotheses: (1) introgression of the mitochondrial genome of another species and (2) the effect of natural selection. Although introgression is the most plausible explanation, we fail to identify the source species of the introgressed mitochondrial genome among all known species closely related to C. lapponica. We therefore suggest that the putative introgression event is ancient and the source species is either extinct or currently outside the geographic range of C. lapponica explored in this study. The observed DNA sequence variation also suggests that a host-plant shift from willow to birch has occurred recently and independently in each of the three birch-feeding populations. This emphasizes further the relative ease with which these beetles can escape their ancestral host-plant specialization on willow, but shows at the same time that host-plant shifts are highly constrained, as they only occur between willow and birch.

Glucose and glucose esters in the larval secretion of Chrysomela lapponica; selectivity of the glucoside import system from host plant leaves.

Larvae of Chrysomela lapponica (Coleoptera: Chrysomelidae) sequester characteristic O-glucosides from the leaves of their food plants, namely Betula and/or Salix The present study focuses on birch-feeding larvae of C. lapponica from the Altai region in East Kazakhstan. As in other sequestering leaf beetle larvae, the compounds are transported intact via different membrane barriers into the defensive system, followed by glucoside cleavage and subsequent transformations of the plant-derived aglycones. Unlike previous studies with model compounds, we studied the sequestration of phytogenic precursors by analyzing the complex pattern of glucosides present in food plant Betula rotundifolia (39 compounds) and compared this composition with the aglycones present as butyrate esters in the defensive secretion. In addition to the analytic approach, the insect's ability, to transport individual glucosides was tested by using hydrolysis-resistant thioglucoside analogs, applied onto the leaf surface. The test compounds reach the defensive system intact and without intermediate transformation. No significant difference of the transport capacity and selectivity was observed between larvae of birch-feeding population from Kazakhstan, and previous results for larvae of birch-feeding population from the Czech Republic or willow-feeding populations. Overall, the transport of the phytogenic glucosides is highly selective and highly efficient, since only minor compounds of the spectrum of phytogenic glucoside precursors contribute to the limited number of aglycones utilized in the defensive secretion. Interestingly, salicortin 44 and tremulacin 60 were found in the leaves, but no aldehyde or esters of salicylalcohol. Surprisingly, we observed large amounts of free glucose, together with small amounts of 6-O-butyrate esters of glucose (27a/b and 28a/b).

A versatile transport network for sequestering and excreting plant glycosides in leaf beetles provides an evolutionary flexible defense strategy.

The larval defenses of chrysomeline leaf beetles comprise components that are either synthesized de novo or sequestered from their food plants. Both biosynthetic modes are based on glucosides that serve as substrates and forms of transport. The defensive glands import the compounds through highly selective glucoside transporters from a circulating pool in the hemolymph. Here we address the selectivity of the different transport systems with larvae of Chrysomela populi, an obligate sequestering species, and with larvae of Phaedon cochleariae, producing monoterpene [corrected] iridoids. Both species possess an interconnected network of transport systems for uptake and excretion. The glucosides are imported by the gut membrane with low selectivity. Their excretion by the Malpighian tubules is similarly unselective, but the uptake of the glucosides from the hemolymph into the defensive system is specific. Only the genuine glucoside precursors made de novo or sequestered from the plant are imported. The successful combination of the precursor-adapted pathways of excretion and defense has probably allowed many leaf beetle species to adaptively radiate onto, and coevolve with plants that offer appropriate glucoside precursors.

A subset of chemosensory genes differs between two populations of a specialized leaf beetle after host plant shift.

Due to its fundamental role in shaping host selection behavior, we have analyzed the chemosensory repertoire of Chrysomela lapponica. This specialized leaf beetle evolved distinct populations which shifted from the ancestral host plant, willow (Salix sp., Salicaceae), to birch (Betula rotundifolia, Betulaceae). We identified 114 chemosensory candidate genes in adult C. lapponica: 41 olfactory receptors (ORs), eight gustatory receptors, 17 ionotropic receptors, four sensory neuron membrane proteins, 32 odorant binding proteins (OBPs), and 12 chemosensory proteins (CSP) by RNA-seq. Differential expression analyses in the antennae revealed significant upregulation of one minus-C OBP (Clap OBP27) and one CSP (Clap CSP12) in the willow feeders. In contrast, one OR (Clap OR17), four minus-C OBPs (Clap OBP02, 07, 13, 20), and one plus-C OBP (Clap OBP32) were significantly upregulated in birch feeders. The differential expression pattern in the legs was more complex. To narrow down putative ligands acting as cues for host discrimination, the relative abundance and diversity of volatiles of the two host plant species were analyzed. In addition to salicylaldehyde (willow-specific), both plant species differed mainly in their emission rate of terpenoids such as (E,E)-α-farnesene (high in willow) or 4,8-dimethylnona-1,3,7-triene (high in birch). Qualitatively, the volatiles were similar between willow and birch leaves constituting an "olfactory bridge" for the beetles. Subsequent structural modeling of the three most differentially expressed OBPs and docking studies using 22 host volatiles indicated that ligands bind with varying affinity. We suggest that the evolution of particularly minus-C OBPs and ORs in C. lapponica facilitated its host plant shift via chemosensation of the phytochemicals from birch as novel host plant.

Switchable reflector in the Panamanian tortoise beetle Charidotella egregia (Chrysomelidae: Cassidinae).

The tortoise beetle Charidotella egregia is able to modify the structural color of its cuticle reversibly, when disturbed by stressful external events. After field observations, measurements of the optical properties in the two main stable color states and scanning electron microscope and transmission electron microscope investigations, a physical mechanism is proposed to explain the color switching of this insect. It is shown that the gold coloration displayed by animals at rest arises from a chirped multilayer reflector maintained in a perfect coherent state by the presence of humidity in the porous patches within each layer, while the red color displayed by disturbed animals results from the destruction of this reflector by the expulsion of the liquid from the porous patches, turning the multilayer into a translucent slab that leaves an unobstructed view of the deeper-lying, pigmented red substrate. This mechanism not only explains the change of hue but also the change of scattering mode from specular to diffuse. Quantitative modeling is developed in support of this analysis.

Structural and physical evidence for an endocuticular gold reflector in the tortoise beetle, Charidotella ambita.

Charidotella ambita offers a unique opportunity for unambiguously locating its gold reflector by comparing the structure of reflecting and non-reflecting cuticle of the elytron and pronotum. Using light microscopy and TEM, the reflector was located underneath the macrofiber endocuticle just above the epidermis. The reflector is a multilayer comprising up to 50 bilayers alternating high and low density layers parallel to the surface of the cuticle. It is chirped, i.e., showing a progressive decrease in layer thickness from approximately 150 nm-100 nm across its depth. The high density layers in contact with the endocuticle fuse to the last macrofiber when the reflector is interrupted by a trabecula, demonstrating their cuticular nature. Simulated reflectance spectra from models of the multilayer matched the reflection spectra measured on the major gold patch of the elytron of living specimens. Previous reports in adult insects exhibiting metallic colors located their reflector in the upper strata and structures of the cuticle, i.e., epicuticle, exocuticle, scales and hairs. Thus, the endocuticular location of the reflector in C. ambita (and other tortoise beetles) appears unique for adult insects. Gold reflection appears in C. ambita only when the synthesis of the macrolayer endocuticle is complete, which may take up to 2 weeks. The development of the gold reflector coincides with the start of mating behavior, possibly suggesting a signaling function in conspecific recognition once sexual maturity has been reached.

Dual chemical sequestration: a key mechanism in transitions among ecological specialization.

Platyphora leaf beetles form a vast group of tropical species each feeding on a restricted set of host plants and exhibiting bright coloration warning predators against their chemical protection. These beetles offer an exceptional opportunity for understanding the evolution of phytochemical sequestration. Indeed, qualitative studies of defensive secretions indicate that Platyphora species acquire toxicity via sequestration of plant secondary metabolites. All produce pentacyclic triterpene saponins from sequestered plant amyrins, but our analyses also indicate that many Platyphora species produce a dual chemical defence, that is, they are additionally protected by lycopsamine-type pyrolyzidine alkaloids that they also sequester from their host. This paper reports on the evolution of chemical defence and host affiliation in Platyphora leaf beetles as reconstructed on a molecular phylogeny of mitochondrial and nuclear DNA sequences. The analyses indicate that dual sequestration could be the key mechanistic means by which transitions among ecological specializations (i.e. restricted host-plant affiliations) are made possible.

In vitro production of adaline and coccinelline, two defensive alkaloids from ladybird beetles (Coleoptera: Coccinellidae).

In vitro experiments using [1-(14)C] and [2-(14)C]acetate were devised to study the biosynthesis of the defensive coccinellid alkaloids adaline and coccinelline in Adalia 2-punctata and Coccinella 7-punctata, respectively. The labelled alkaloids obtained in these experiments had a specific activity about ten times higher than that of the samples obtained in feeding experiments. This in vitro assay has enabled us to demonstrate that these two alkaloids are most likely biosynthesised through a fatty acid rather than a polyketide pathway, that glutamine is the preferred source of the nitrogen atom and that alkaloid biosynthesis takes place in the insect fat body.

Subsocial Neotropical Doryphorini (Chrysomelidae, Chrysomelinae): new observations on behavior, host plants and systematics.

A summary of literature, documented observations and field studies finds evidence that mothers actively defend offspring in at least eight species and three genera of Neotropical Chrysomelinae associated with two host plant families. Reports on three Doryphora species reveal that all are oviparous and feed on vines in the Apocyanaceae. Mothers in the two subsocial species defend eggs and larvae by straddling, blocking access at the petiole and greeting potential predators with leaf-shaking and jerky advances. A less aggressive form of maternal care is found in two Platyphora and four Proseicela species associated with Solanaceae, shrubs and small trees. For these and other morphologically similar taxa associated with Solanaceae, genetic distances support morphology-based taxonomy at the species level, reveal one new species, but raise questions regarding boundaries separating genera. We urge continued study of these magnificent insects, their enemies and their defenses, both behavioral and chemical, especially in forests along the eastern versant of the Central and South American cordillera.

Testing phylogeographic hypotheses in a Euro-Siberian cold-adapted leaf beetle with coalescent simulations.

Few studies to date have investigated the impact of Pleistocene climatic oscillations on the genetic diversity of cold-adapted species. We focus on the geographic distribution of genetic diversity in a Euro-Siberian boreo-montane leaf beetle, Gonioctena pallida. We present the molecular variation from three independent gene fragments over the entire geographic range of this insect. The observed sequence variation identifies a genetic diversity hot spot in the Carpathian Mountains, in central Europe, which reveals the presence of (1) an ancestral refuge population or (2) a secondary contact zone in this area. Modeling of population evolution in a coalescent framework allowed us to favor the ancestral refuge hypothesis. These analyses suggest that the Carpathian Mountains served as a refuge for G. pallida, whereas the rest of the species distribution, that spans a large portion of Europe and Asia, experienced a dramatic reduction in genetic variation probably associated to bottlenecks and/or founder events. We estimated the time of isolation of the ancestral refuge population, using an approximate Bayesian method, to be larger than 90,000 years. If true, the current pattern of genetic variation in this cold-adapted organism was shaped by a climatic event predating by far the end of the last ice age.

Triterpene saponin hemi-biosynthesis of a leaf beetle's (Platyphora kollari) defensive secretion.

The adults of the leaf beetle Platyphora kollari (Chrysomelidae) are able to metabolise the oleanane triterpene beta-amyrin (1) into the glycoside 3-O-beta-D: -glucopyranosyl-(1-->4)-beta-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 beta-amyrin (1). To this end, adults of P. kollari were fed with Ipomoea batatas leaf disks painted with a solution of [2,2,3-(2)H(3)]oleanolic acid or [2,2,3-(2)H(3)]alpha-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 beta-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 beta-amyrin (1).

Differentiation of worker-derived intercastes and precocious imagoes after queen removal in the Neo-Guinean termite Nasutitermes princeps (Desneux).

Numerous female workers underwent a beginning of sexualization in an experimentally orphaned colony of the Neo-Guinean termite N. princeps (Desneux). This led to the development of intercastes, displaying variable mosaics of worker, nymph, and soldier characteristics. The developmental pathways of these intercastes were studied with the help of multivariate techniques that revealed two main lines, one ending in a new "giant soldier" morph, the other proceeding toward the differentiation of nymphal characters, i.e., wing pads and compound eyes. It is not known whether these individuals might ultimately have become functional. Precocious adults resulting from a shortened nymphal course were also present, and one young replacement queen had already begun laying eggs.

Sequestration and metabolism of protoxic pyrrolizidine alkaloids by larvae of the leaf beetle Platyphora boucardi and their transfer via pupae into defensive secretions of adults.

Several neotropical leaf-beetles of the genus Platyphora ingest and specifically metabolize plant acquired pyrrolizidine alkaloids (PAs) of the lycopsamine type (e.g., rinderine or intermedine) and enrich the processed alkaloids in their exocrine defensive secretions. In contrast to the related palaearctic leaf beetles of the genus Oreina, which absorb and store only the non-toxic alkaloid N-oxides, Platyphora sequesters PAs exclusively as protoxic tertiary amines. In this study, the ability of P. boucardi larvae to accumulate PAs was investigated. Tracer studies with [14C]rinderine and its N-oxide revealed that P. boucardi larvae, like adult beetles, utilize the two alkaloidal forms with the same efficiency, but accumulate the alkaloid as a tertiary amine exclusively. Ingested rinderine is rapidly epimerized to intermedine, which is localized in the hemolymph and all other tissues; it is also detected on the larval surface. Like adults, larvae are able to synthesize their own alkaloid esters (beetle PAs) from orally administered [14C]retronecine and endogenous aliphatic 2-hydroxy acids. These retronecine esters show the same tissue distribution as intermedine. A long-term feeding experiment lasting for almost four months revealed that retronecine esters synthesized from [14C]retronecine in the larvae are transferred from larvae via pupae into the exocrine glands of adult beetles. Pupae contain ca. 45% of the labeled retronecine originally ingested, metabolized, and stored by larvae; ca. 12% of larval radioactivity could be recovered from the defensive secretions of adults sampled successively over two and a half months. Almost all of this radioactivity is found in the insect-made retronecine esters that are highly enriched in the defensive secretions, i.e., more than 200-fold higher concentration compared to pupae.

HOST-PLANT SWITCHES AND THE EVOLUTION OF CHEMICAL DEFENSE AND LIFE HISTORY IN THE LEAF BEETLE GENUS OREINA.

Insect-plant interactions have played a prominent role in investigating phylogenetic constraints in the evolution of ecological traits. The patterns of host association among specialized insects have often been described as highly conservative, yet not all specialized herbivorous insect lineages display the same degree of fidelity to their host plants. In this paper, we present an estimate of the evolutionary history of the leaf beetle genus Oreina. This genus displays an amazing flexibility in several aspects of its ecology and life history: (1) host plant switches in Oreina occurred between plant families or distantly related tribes within families and thereby to more distantly related plants than in several model systems that have contributed to the idea of parallel cladogenesis; (2) all species of the genus are chemically defended, but within the genus a transition between autogenous production of defensive toxins and sequestration of secondary plant compounds has occurred; and (3) reproductive strategies in the genus range from oviparity to viviparity including all intermediates that could allow the gradual evolution of viviparity. Cladistic analysis of 18 allozyme loci found two most parsimonious trees that differ only in the branching of one species. According to this phylogeny estimate, Oreina species were originally associated with Asteraceae, with an inclusion of Apiaceae in the diet of one oligophagous species and an independent switch to Apiaceae in a derived clade. The original mode of defense appears to be the autogenous production of cardenolides as previously postulated; the additional sequestration of pyrrolizidine alkaloids could have either originated at the base of the genus or have arisen three times independently in all species that switched to plants containing these compounds. Viviparity apparently evolved twice in the genus, once without matrotrophy, through a retention of the eggs inside the female's oviducts, and once in combination with matrotrophy. We hypothesize that the combination of autogenous defense and a life history that involves mobile externally feeding larvae allowed these beetles to switch host plants more readily than has been reported for highly conservative systems.

Selective transport systems mediate sequestration of plant glucosides in leaf beetles: a molecular basis for adaptation and evolution.

Chrysomeline larvae respond to disturbance and attack by everting dorsal glandular reservoirs, which release defensive secretions. The ancestral defense is based on the de novo synthesis of monoterpene iridoids. The catabolization of the host-plant O-glucoside salicin into salicylaldehyde is a character state that evolved later in two distinct lineages, which specialized on Salicaceae. By using two species producing monoterpenes (Hydrothassa marginella and Phratora laticollis) and two sequestering species (Chrysomela populi and Phratora vitellinae), we studied the molecular basis of sequestration by feeding the larvae structurally different thioglucosides resembling natural O-glucosides. Their accumulation in the defensive systems demonstrated that the larvae possess transport systems, which are evolutionarily adapted to the glycosides of their host plants. Minor structural modifications in the aglycon result in drastically reduced transport rates of the test compounds. Moreover, the ancestral iridoid-producing leaf beetles already possess a fully functional import system for an early precursor of the iridoid defenses. Our data confirm an evolutionary scenario in which, after a host-plant change, the transport system of the leaf beetles may play a pivotal role in the adaptation on new hosts by selecting plant-derived glucosides that can be channeled to the defensive system.

THE EVOLUTION OF HOST-PLANT USE AND SEQUESTRATION IN THE LEAF BEETLE GENUS PHRATORA (COLEOPTERA: CHRYSOMELIDAE).

Leaf beetles in the genus Phratora differ in host plant use and in the chemical composition of their larval defensive secretion. Most species specialize on either poplars or willows (family Salicaceae), but two species feed on birch (family Betulaceae). Phratora vitellinae utilizes salicylates from the host plant to produce its larval secretion, which contains salicylaldehyde, while other Phratora species produce an autogenous secretion. To reconstruct the evolutionary history of host plant use and the larval secretion chemistry in this genus, we sequenced 1383 base pairs of the mt cytochrome oxidase I gene for six European and one North American Phratora species and three outgroup taxa. Bootstrap values of the complete nucleotide sequence were 99-100% for six of eight nodes in the maximum parsimony tree. They were 71% and 77% for the two other nodes. The maximum parsimony tree and the maximum likelihood tree based on nucleotide sequence showed the same relationships as a maximum parsimony tree based on the amino acid sequence. Beetle phylogeny overlapped broadly with host plant taxonomy and chemistry, and it revealed historical constraints influencing host plant use. However, there was one host shift from the willow family (Salicaceae) to the birch family (Betulaceae). The use of host plant phenol glycosides for the larval defensive secretion evolved along the lineage that led to P. vitellinae. Phratora vitellinae feeds on the taxonomically widest range of host plants, which are characterized by moderate to high levels of salicylates. The results support the hypothesis that the use of salicylates for the larval secretion evolved twice independently in chrysomeline leaf beetles.

Recycling plant wax constituents for chemical defense: hemi-biosynthesis of triterpene saponins from beta-amyrin in a leaf beetle.

Several species of Doryphorina leaf beetles from Central- and South America produce oleanane triterpene glycosides in their defensive glands. The presence of pentacyclic triterpenes in insects is intriguing since they lack the key enzymes necessary to synthesize these compounds. Since beta-amyrin is a common constituent of leaf waxes, we hypothesized that these leaf beetles use this compound as a precursor to their oleanane glycosides. To test this hypothesis we first confirmed the presence of beta-amyrin in Ipomoea batatas, the food plant of Platyphora kollari. Next, adults of P. kollari were fed for 10 days with I. batatas leaf disks painted with a solution of [2,2,3-(2)H(3)]beta-amyrin ([2,2,3-(2)H(3)]-1). The secretion from their defensive glands was collected and analyzed by HPLC-ESIMS. The results demonstrated that the secretion of beetles fed with an amount of [2,2,3-(2)H(3)]beta-amyrin corresponding to the quantity of unlabeled (natural) beta-amyrin present in the leaf disks contained on average 5.1% of [2,2,3-(2)H(3)]-3- O-beta- d-glucopyranosyl-(1-->4)-beta- d-glucuronopyranosyl-hederagenin ([2,2,3-(2)H(3)]-2), whereas the secretions of beetles fed with 10 times this amount of [2,2,3-(2)H(3)]beta-amyrin contained on average 23.9% of [2,2,3-(2)H(3)]-2. In both series of experiments, the percentage of labeled versus unlabeled triterpene glycoside in the secretion was positively correlated with the amount of deuterated beta-amyrin ingested. These results demonstrate for the first time that some leaf beetles are able to metabolize a widespread triterpenic constituent of leaf wax into more complex glycosides that are stored in their defensive glands.