The multiple strategies of an insect herbivore to overcome plant cyanogenic glucoside defence

Cyanogenic glucosides (CNglcs) are widespread plant defence compounds that release toxic hydrogen cyanide by plant β-glucosidase activity after tissue damage. Specialised insect herbivores have evolved counter strategies and some sequester CNglcs, but the underlying mechanisms to keep CNglcs intact during feeding and digestion are unknown. We show that CNglc-sequestering Zygaena filipendulae larvae combine behavioural, morphological, physiological and biochemical strategies at different time points during feeding and digestion to avoid toxic hydrolysis of the CNglcs present in their Lotus food plant, i.e. cyanogenesis. We found that a high feeding rate limits the time for plant β-glucosidases to hydrolyse CNglcs. Larvae performed leaf-snipping, a minimal disruptive feeding mode that prevents mixing of plant β-glucosidases and CNglcs. Saliva extracts did not inhibit plant cyanogenesis. However, a highly alkaline midgut lumen inhibited the activity of ingested plant β-glucosidases significantly. Moreover, insect β-glucosidases from the saliva and gut tissue did not hydrolyse the CNglcs present in Lotus. The strategies disclosed may also be used by other insect species to overcome CNglc-based plant defence and to sequester these compounds intact

Cyanogenesis in Arthropods: From Chemical Warfare to Nuptial Gifts.

Chemical defences are key components in insect⁻plant interactions, as insects continuously learn to overcome plant defence systems by, e.g., detoxification, excretion or sequestration. Cyanogenic glucosides are natural products widespread in the plant kingdom, and also known to be present in arthropods. They are stabilised by a glucoside linkage, which is hydrolysed by the action of β-glucosidase enzymes, resulting in the release of toxic hydrogen cyanide and deterrent aldehydes or ketones. Such a binary system of components that are chemically inert when spatially separated provides an immediate defence against predators that cause tissue damage. Further roles in nitrogen metabolism and inter- and intraspecific communication has also been suggested for cyanogenic glucosides. In arthropods, cyanogenic glucosides are found in millipedes, centipedes, mites, beetles and bugs, and particularly within butterflies and moths. Cyanogenic glucosides may be even more widespread since many arthropod taxa have not yet been analysed for the presence of this class of natural products. In many instances, arthropods sequester cyanogenic glucosides or their precursors from food plants, thereby avoiding the demand for de novo biosynthesis and minimising the energy spent for defence. Nevertheless, several species of butterflies, moths and millipedes have been shown to biosynthesise cyanogenic glucosides de novo, and even more species have been hypothesised to do so. As for higher plant species, the specific steps in the pathway is catalysed by three enzymes, two cytochromes P450, a glycosyl transferase, and a general P450 oxidoreductase providing electrons to the P450s. The pathway for biosynthesis of cyanogenic glucosides in arthropods has most likely been assembled by recruitment of enzymes, which could most easily be adapted to acquire the required catalytic properties for manufacturing these compounds. The scattered phylogenetic distribution of cyanogenic glucosides in arthropods indicates that the ability to biosynthesise this class of natural products has evolved independently several times. This is corroborated by the characterised enzymes from the pathway in moths and millipedes. Since the biosynthetic pathway is hypothesised to have evolved convergently in plants as well, this would suggest that there is only one universal series of unique intermediates by which amino acids are efficiently converted into CNglcs in different Kingdoms of Life. For arthropods to handle ingestion of cyanogenic glucosides, an effective detoxification system is required. In butterflies and moths, hydrogen cyanide released from hydrolysis of cyanogenic glucosides is mainly detoxified by β-cyanoalanine synthase, while other arthropods use the enzyme rhodanese. The storage of cyanogenic glucosides and spatially separated hydrolytic enzymes (β-glucosidases and α-hydroxynitrile lyases) are important for an effective hydrogen cyanide release for defensive purposes. Accordingly, such hydrolytic enzymes are also present in many cyanogenic arthropods, and spatial separation has been shown in a few species. Although much knowledge regarding presence, biosynthesis, hydrolysis and detoxification of cyanogenic glucosides in arthropods has emerged in recent years, many exciting unanswered questions remain regarding the distribution, roles apart from defence, and convergent evolution of the metabolic pathways involved

Lepidopteran defence droplets - a composite physical and chemical weapon against potential predators

Insects often release noxious substances for their defence. Larvae of Zygaena filipendulae (Lepidoptera) secrete viscous and cyanogenic glucoside-containing droplets, whose effectiveness was associated with their physical and chemical properties. The droplets glued mandibles and legs of potential predators together and immobilised them. Droplets were characterised by a matrix of an aqueous solution of glycine-rich peptides (H-WG(11)-NH(2)) with significant amounts of proteins and glucose. Among the proteins, defensive proteins such as protease inhibitors, proteases and oxidases were abundant. The neurotoxin β-cyanoalanine was also found in the droplets. Despite the presence of cyanogenic glucosides, which release toxic hydrogen cyanide after hydrolysis by a specific β-glucosidase, the only β-glucosidase identified in the droplets (ZfBGD1) was inactive against cyanogenic glucosides. Accordingly, droplets did not release hydrogen cyanide, unless they were mixed with specific β-glucosidases present in the Zygaena haemolymph. Droplets secreted onto the cuticle hardened and formed sharp crystalline-like precipitates that may act as mandible abrasives to chewing predators. Hardening followed water evaporation and formation of antiparallel β-sheets of the peptide oligomers. Consequently, after mild irritation, Zygaena larvae deter predators by viscous and hardening droplets that contain defence proteins and β-cyanoalanine. After severe injury, droplets may mix with exuding haemolymph to release hydrogen cyanide

454 pyrosequencing based transcriptome analysis of Zygaena filipendulae with focus on genes involved in biosynthesis of cyanogenic glucosides

<p>Abstract</p> <p>Background</p> <p>An essential driving component in the co-evolution of plants and insects is the ability to produce and handle bioactive compounds. Plants produce bioactive natural products for defense, but some insects detoxify and/or sequester the compounds, opening up for new niches with fewer competitors. To study the molecular mechanism behind the co-adaption in plant-insect interactions, we have investigated the interactions between <it>Lotus corniculatus </it>and <it>Zygaena filipendulae</it>. They both contain cyanogenic glucosides which liberate toxic hydrogen cyanide upon breakdown. Moths belonging to the <it>Zygaena </it>family are the only insects known, able to carry out both <it>de novo </it>biosynthesis and sequestration of the same cyanogenic glucosides as those from their feed plants. The biosynthetic pathway for cyanogenic glucoside biosynthesis in <it>Z. filipendulae </it>proceeds using the same intermediates as in the well known pathway from plants, but none of the enzymes responsible have been identified. A genomics strategy founded on 454 pyrosequencing of the <it>Z. filipendulae </it>transcriptome was undertaken to identify some of these enzymes in <it>Z. filipendulae</it>.</p> <p>Results</p> <p>Comparisons of the <it>Z. filipendulae </it>transcriptome with the sequenced genomes of <it>Bombyx mori</it>, <it>Drosophila melanogaster</it>, <it>Tribolium castaneum</it>, <it>Apis mellifera </it>and <it>Anopheles gambiae </it>indicate a high coverage of the <it>Z. filipendulae </it>transcriptome. 11% of the <it>Z. filipendulae </it>transcriptome sequences were assigned to Gene Ontology categories. Candidate genes for enzymes functioning in the biosynthesis of cyanogenic glucosides (cytochrome P450 and family 1 glycosyltransferases) were identified based on sequence length, number of copies and presence/absence of close homologs in <it>D. melanogaster</it>, <it>B. mori </it>and the cyanogenic butterfly <it>Heliconius</it>. Examination of biased codon usage, GC content and selection on gene candidates support the notion of cyanogenesis as an "old" trait within Ditrysia, as well as its origins being convergent between plants and insects.</p> <p>Conclusion</p> <p>Pyrosequencing is an attractive approach to gain access to genes in the biosynthesis of bio-active natural products from insects and other organisms, for which the genome sequence is not known. Based on analysis of the <it>Z. filipendulae </it>transcriptome, promising gene candidates for biosynthesis of cyanogenic glucosides was identified, and the suitability of <it>Z. filipendulae </it>as a model system for cyanogenesis in insects is evident.</p