Two Defensive Lines in Juvenile Leaf Beetles; Esters of 3-nitropropionic Acid in the Hemolymph and Aposematic Warning.

Juveniles of the leaf beetles in subtribe Chrysomelina have efficient defense strategies against predators. When disturbed, they transiently expose volatile deterrents in large droplets from nine pairs of defensive glands on their back. Here, we report on an additional line of defense consisting of the non-volatile isoxazolin-5-one glucoside and its 3-nitropropanoyl ester in the larval hemolymph. Because isoxazolin-5-one derivatives were not detectable in related leaf beetle taxa, they serve as a diagnostic marker for the Chrysomelina subtribe. Conjugation of isotopically labelled 3-nitropropionic acid to isoxazolin-5-one glucoside in vivo demonstrates its function as a carrier for the 3-nitropropanoyl esters. The previous identification of characteristic glucosides as precursors of the volatile deterrents underlines the general importance of glucosides for sequestration from food plants, and the subsequent transport in the hemolymph to the defense system. The combination of repellent volatiles with non-volatile toxic compounds in the hemolymph has the potential to create synergistic effects since the odorant stimulus may help predators learn to avoid some foods. The combination of the two defense lines has the advantage, that the hemolymph toxins provide reliable and durable protection, while the repellents may vary after a host plant change.

Metal ions control product specificity of isoprenyl diphosphate synthases in the insect terpenoid pathway.

Isoprenyl diphosphate synthases (IDSs) produce the ubiquitous branched-chain diphosphates of different lengths that are precursors of all major classes of terpenes. Typically, individual short-chain IDSs (scIDSs) make the C10, C15, and C20 isoprenyl diphosphates separately. Here, we report that the product length synthesized by a single scIDS shifts depending on the divalent metal cofactor present. This previously undescribed mechanism of carbon chain-length determination was discovered for a scIDS from juvenile horseradish leaf beetles, Phaedon cochleariae. The recombinant enzyme P. cochleariae isoprenyl diphosphate synthase 1 (PcIDS1) yields 96% C10-geranyl diphosphate (GDP) and only 4% C15-farnesyl diphosphate (FDP) in the presence of Co(2+) or Mn(2+) as a cofactor, whereas it yields only 18% C10 GDP but 82% C15 FDP in the presence of Mg(2+). In reaction with Co(2+), PcIDS1 has a Km of 11.6 µM for dimethylallyl diphosphate as a cosubstrate and 24.3 µM for GDP. However, with Mg(2+), PcIDS1 has a Km of 1.18 µM for GDP, suggesting that this substrate is favored by the enzyme under such conditions. RNAi targeting PcIDS1 revealed the participation of this enzyme in the de novo synthesis of defensive monoterpenoids in the beetle larvae. As an FDP synthase, PcIDS1 could be associated with the formation of sesquiterpenes, such as juvenile hormones. Detection of Co(2+), Mn(2+), or Mg(2+) in the beetle larvae suggests flux control into C10 vs. C15 isoprenoids could be accomplished by these ions in vivo. The dependence of product chain length of scIDSs on metal cofactor identity introduces an additional regulation for these branch point enzymes of terpene metabolism.

Novel Set-Up for Low-Disturbance Sampling of Volatile and Non-volatile Compounds from Plant Roots.

Most studies on rhizosphere chemicals are carried out in substrate-free set-ups or in artificial substrates using sampling methods that require an air flow and may thus cause disturbance to the rhizosphere. Our study aimed to develop a simplified and inexpensive system that allows analysis of rhizosphere chemicals at experimentally less disturbed conditions. We designed a mesocosm in which volatile rhizosphere chemicals were sampled passively (by diffusion) without air- and water flow on polydimethylsiloxane-(PDMS) tubes. Dandelion (Taraxacum sect. ruderalia) was used as model plant; roots were left undamaged. Fifteen volatiles were retrieved from the sorptive material by thermal desorption for analysis by gas chromatography/mass spectrometry (GC/MS). Furthermore, three sugars were collected from the rhizosphere substrate by aqueous extraction and derivatized prior to GC/MS analysis. In order to study how the quantity of detected rhizosphere compounds depends on the type of soil or substrate, we determined the matrix-dependent recovery of synthetic rhizosphere chemicals. Furthermore, we compared sorption of volatiles on PDMS tubes with and without direct contact to the substrate. The results show that the newly designed mesocosm is suitable for low-invasive extraction of volatile and non-volatile compounds from rhizospheres. We further highlight how strongly the type of substrate and contact of PDMS tubes to the substrate affect the detectability of compounds from rhizospheres.

Synthesis of isoxazolin-5-one glucosides by a cascade reaction.

A novel synthetic route was developed for the construction of isoxazolin-5-one glucosides using a cascade reaction. An X-ray crystal structure analysis of a isoxazolin-5-one glucoside confirmed the structure and stereochemistry of the heterocycle. The properties of the α- and ß-anomers of the isoxazolin-5-one glucosides were compared. The first synthesis of 2-[6'-(3″-nitropropanoyl)-ß-D-glucopyranosyl]-3-isoxazolin-5-one was realized by direct enzymatic esterification without need of further protective groups.

Immune modulation enables a specialist insect to benefit from antibacterial withanolides in its host plant.

The development of novel plant chemical defenses and counter adaptations by herbivorous insect could continually drive speciation, producing more insect specialists than generalists. One approach to test this hypothesis is to compare closely related generalist and specialist species to reveal the associated costs and benefits of these different adaptive strategies. We use the specialized moth Heliothis subflexa, which feeds exclusively on plants in the genus Physalis, and its close generalist relative H. virescens. Specialization on Physalis plants necessitates the ability to tolerate withanolides, the secondary metabolites of Physalis species that are known to have feeding deterrent and immune inhibiting properties for other insects. Here we find that only H. subflexa benefits from the antibacterial properties of withanolides, and thereby gains a higher tolerance of the pathogen Bacillus thuringiensis. We argue that the specialization in H. subflexa has been guided to a large extent by a unique role of plant chemistry on ecological immunology.

Spodoptera littoralis detoxifies neurotoxic 3-nitropropanoic acid by conjugation with amino acids.

Spodoptera littoralis is a phytophagous generalist. Its host range includes more than 40 plant species, some of which produce 3-nitropropanoic acid (3-NPA), an irreversible inhibitor of mitochondrial succinate dehydrogenase. Growth in larvae fed an artificial diet with a sublethal admixture of 3-NPA (4.2 µmol per g) was slowed significantly, but larvae experienced no increase in mortality. In contrast, larvae injected with 25.2 µmol/g (bodyweight) 3-NPA experienced acute toxicity and death. To study the detoxification mechanism of 3-NPA in S. littoralis, the insect frass was analyzed by HPLC-MS. Comparative analysis of 3-NPA-treated and -untreated control samples using HR-MS(2) revealed a group of differential signals that were identified as amino acid amides of 3-NPA with glycine, alanine, serine, and threonine. When sublethal amounts of stable isotope-labeled 3-NPA were injected into a larva's hemolymph, 3-NPA amino acid conjugates were identified as putative detoxification products. Bioassays with synthetic standards confirmed that the toxicity of the amides was negligible in comparison to the toxicity of free 3-NPA, demonstrating that amino acid conjugation in S. littoralis represents an efficient way to detoxify 3-NPA. Furthermore, biosynthetic studies using crude fractions of the gut tissue indicated that conjugation of 3-NPA with amino acids occurs in epithelial cells of the insect's gut. Taken together, these results suggest that the detoxification of 3-NPA in S. littoralis proceeds via conjugation to specific amino acids within the epithelial cells followed by export of the nontoxic amino acid conjugates to the hemolymph via as yet uncharacterized mechanisms, most likely involving the Malpighian tubules.

Glandular ß-glucosidases in juvenile Chrysomelina leaf beetles support the evolution of a host-plant-dependent chemical defense.

Plant-feeding insects are spread across the entire plant kingdom. Because they chew externally on leaves, leaf beetle of the subtribe Chrysomelina sensu stricto are constantly exposed to life-threatening predators and parasitoids. To counter these pressures, the juveniles repel their enemies by displaying glandular secretions that contain defensive compounds. These repellents can be produced either de novo (iridoids) or by using plant-derived precursors. The autonomous production of iridoids pre-dates the evolution of phytochemical-based defense strategies. Both strategies include hydrolysis of the secreted non-toxic glycosides in the defensive exudates. By combining in vitro as well as in vivo experiments, we show that iridoid de novo producing as well as sequestering species rely on secreted ß-glucosidases to cleave the pre-toxins. Our phylogenetic analyses support a common origin of chrysomeline ß-glucosidases. The kinetic parameters of these ß-glucosidases demonstrated substrate selectivity which reflects the adaptation of Chrysomelina sensu stricto to the chemistry of their hosts during the course of evolution. However, the functional studies also showed that the broad substrate selectivity allows building a chemical defense, which is dependent on the host plant, but does not lead to an "evolutionary dead end".