Influence of two selective factors on cyanogenesis polymorphism ofTrifolium repens L. in Darjeeling Himalaya

Cyanogenesis-the production of toxic hydrogen cyanide (HCN) by damaged tissue-inTrifolium repens L. (white clover), a type of most important pasture legume, has been studied at different elevations of Darjeeling Himalaya (latitude-27° 2′ 57″ N, longitude-88° 15′ 45″ E). Release of HCN takes place due to reaction between cyanogenic glucosides stored in vacuoles of the leaf cell and the corresponding enzyme β-glucosidase present in another compartment, often cell wall. Cyanogenesis, a defense system in plant, protects the clover from herbivore and inhibits grazing. Biochemical analysis showed the presence and absence of the cyanogenesis trait within the population in different proportions at different elevations. Acyanogenic individuals also showed variations with respect to presence or absence of either cyanogenic glucosides or β-glucosidase enzyme or both. The distribution of cyanogenic and acyanogenic plants was found in all places, but at lower altitudes (2084–2094 m) the dominating plants were cyanogenic whereas in higher altitude (2560 m) the dominating plants were acyanogenic. It was observed that blister beetle (Mylabris pustalata Thunb.) and the mollusc (Macrochlamys tusgurium Benson.) were the most common consumer of leaflets ofT. repens. Six categories of damage on white clover leaf by these animals were recorded. Our results suggest that the two selective factors or forces i.e. very cold temperature (harmful to cyanogenic plants) at higher altitude as well as indiscriminate but preferential predation (harmful to acyanogenic plants) interact to affect the system of cyanogenesis and also to cause the stable and protective polymorphism inT. repens rather than genotypic differences present among the plants.     

Cyanogenic glycosides in Lotus corniculatus

Summary Two genotypes (one cyanogenic and the other acyanogenic) of birds-foot trefoil, Lotus corniculatus L., were used to study the effects of cyanogenic glycosides in leaf tissues upon a polyphagous herbivore, the southern armyworm, Spodoptera eridania Cram. (Lepidoptera). No differences were observed in consumption rate, assimilation efficiency, utilization of plant biomass, or metabolic costs in terms of expended calories between larvae fed acyanogenic or cyanogenic leaves. Similarly no differences were seen in the nitrogen or caloric utilization efficiencies, or in the nitrogen accumulation rate or growth rate of larvae on cyanogenic versus acyanogenic plants. Larval performance and growth on 20-week old plants was generally poorer than on 4 week old plants, however. This was reflected in slower growth, smaller pupal weights, lower nitrogen utilization efficiencies (N.U.E.) and biomass assimilation efficiencies (A.D.) on both the cyanogenic and acyanogenic plants.Although useful as a deterrent to some herbivores, cyanogenesis does not seem to provide an effective defense against this adapted herbivore. This study supports current hypotheses of insect/plant coevolution, and suggests that the metabolic costs of processing cyanogenic plant biomass are small in comparison to those imposed by the nutritional status of the plant leaves.     

Effects of cyanogenesis in bracken fern (Pteridium aquilinum) on associated insects

Abstract. 1. The relationship between cyanogenesis in bracken fern and the insect fauna feeding on the plant was investigated over a 3 year period. The most common insects between May and July, while cyanide levels were high, were the sawflies Strongylogaster impressata Provancher, S.multicincta Norton, Aneug-menus fzavipes (Norton), the aphid Macrosiphum euphorbiae (Thomas) and a microlepidopteran species of Monochroa .
2. Collections of insects from cyanogenic and acyanogenic fronds showed significantly fewer sawflies on the cyanogenic fronds. The aphid and the microlepidopteran were randomly distributed with respect to cyanogenicity.
3. Feeding tests for two of the sawfly species ( Simpressata and Smulticincta ) showed that larvae grew more slowly and had a higher mortality when raised on cyanogenic fronds than on acyanogenic ones.
4. Field collected cyanogenic bracken fronds were found to have sustained less damage from chewing herbivores compared with acyanogenic fronds.     

An investigation of the responses of herbivores to cyanogenesis in Lotus corniculatus L.

In laboratory feeding choice experiments several species of insects and molluscs eat acyanogenic Lotus corniculatus leaves or petals in preference to cyanogenic alternatives. These species can be characterized as generalist feeders for which L. corniculatus forms only an occasional dietary item for any particular individual. Some of the insects that were found to display selective eating were maintained on exclusive diets of either cyanogenic or acyanogenic L. corniculatus . Survival times varied considerably between species, but there was no evidence that mortalities were higher amongst the groups exposed to cyanide. These results suggest that the major role of cyanogenesis may be as a feeding inhibitor, not as a toxin.     

Positive effects of cyanogenic glycosides in food plants on larval development of the common blue butterfly

Cyanogenesis is a widespread chemical defence mechanism in plants against herbivory. However, some specialised herbivores overcome this protection by different behavioural or metabolic mechanisms. In the present study, we investigated the effect of presence or absence of cyanogenic glycosides in birdsfoot trefoil (Lotus corniculatus, Fabaceae) on oviposition behaviour, larval preference, larval development, adult weight and nectar preference of the common blue butterfly (Polyommatus icarus, Lycaenidae). For oviposition behaviour there was a female-specific reaction to cyanogenic glycoside content; i.e. some females preferred to oviposit on cyanogenic over acyanogenic plants, while other females behaved in the opposite way. Freshly hatched larvae did not discriminate between the two plant morphs. Since the two plant morphs differed not only in their content of cyanogenic glycoside, but also in N and water content, we expected these differences to affect larval growth. Contrary to our expectations, larvae feeding on cyanogenic plants showed a faster development and stronger weight gain than larvae feeding on acyanogenic plants. Furthermore, female genotype affected development time, larval and pupal weight of the common blue butterfly. However, most effects detected in the larval phase disappeared for adult weight, indicating compensatory feeding of larvae. Adult butterflies reared on the two cyanogenic glycoside plant morphs did not differ in their nectar preference. But a gender-specific effect was found, where females preferred amino acid-rich nectar while males did not discriminate between the two nectar mimics. The presented results indicate that larvae of the common blue butterfly can metabolise the surplus of N in cyanogenic plants for growth. Additionally, the female-specific behaviour to oviposit preferably on cyanogenic or acyanogenic plant morphs and the female-genotype-specific responses in life history traits indicate the genetic flexibility of this butterfly species and its potential for local adaptation.     

Cyanogenic glycosides and the systematics of the Flacourtiaceae

A survey of 682 accessions including 550 species and most genera of the Flacourtiaceae for cyclopentenoid cyanogenic glycosides showed the presence of only three compounds. These were found only in the tribes Berberidopsideae, Oncobeae, Pangieae and Banaraeae. The family is thus divided into cyanogenic and acyanogenic members. The former group possess compounds of similar structure to those of the Passifloraceae and have been considered primitive morphologically. It seems improbable that the Passifloraceae are derived from the acyanogenic, and putatively evolutionarily more advanced, group of Flacourtiaceae.     

Mollusc grazing may constrain the ecological niche of the old forest lichen Pseudocyphellaria crocata

This study reports on mollusc grazing of two epiphytic cyanobacterial lichens (Pseudocyphellaria crocata and Lobaria pulmonaria) transplanted within three Picea abies-dominated boreal rain forest stands (clear-cut, young and old forests) in west central Norway. Grazing was particularly high in transplants located in the old forest and was almost absent in clear-cut transplants. Grazing marks were absent on natural thalli on nearby spruce twigs (required creeping distance for mollusc from the ground >4 m). Transplantation of lichens from twigs to artificial transplantation frames reduced the creeping distance to 1.2 m, and caused a significant increase in grazing damage in P. crocata. Given a paired choice under transplantation, molluscs consistently preferred P. crocata and avoided L. pulmonaria, implying species-specific differences in herbivore defence. Pseudocyphellaria crocata has a much lower content of the medullary depsidones stictic and constictic acid than L. pulmonaria. Heavy grazing occurred in the P. crocata thalli lowest in these two depsidones. The upper part of the medulla hosting the photobiont was the preferred fodder for grazing molluscs. Molluscs avoided the yellow soralia in P. crocata (localised pulvinic acid), suggesting a role for pulvinic acid in preventing grazing of detached soredia and early establishment stages. The preference of P. crocata for thin spruce twigs is probably a result of a lower grazing pressure on twigs compared to e.g. deciduous stems that frequently support the better defended L. pulmonaria. Ongoing climate changes with increased annual rainfall and milder winters have presumably increased mollusc grazing, particularly in SW parts of Norway which have more species of lichen-feeding molluscs than the boreal sites studied. These temperate areas lacking natural spruce populations have recently experienced reported extinctions of the poorly defended P. crocata from rocks and deciduous stems prone to mollusc grazing. Lichen-feeding molluscs have likely played a role in these extinctions, causing spruce twigs in Atlantic boreal forests to be a last strong foothold for P. crocata in Scandinavia.     

Herbivores and plant defences affect selection on plant reproductive traits more strongly than pollinators

Pollinators and herbivores can both affect the evolutionary diversification of plant reproductive traits. However, plant defences frequently alter antagonistic and mutualistic interactions, and therefore, variation in plant defences may alter patterns of herbivore‐ and pollinator‐mediated selection on plant traits. We tested this hypothesis by conducting a common garden field experiment using 50 clonal genotypes of white clover (Trifolium repens) that varied in a Mendelian‐inherited chemical antiherbivore defence—the production of hydrogen cyanide (HCN). To evaluate whether plant defences alter herbivore‐ and/or pollinator‐mediated selection, we factorially crossed chemical defence (25 cyanogenic and 25 acyanogenic genotypes), herbivore damage (herbivore suppression) and pollination (hand pollination). We found that herbivores weakened selection for increased inflorescence production, suggesting that large displays are costly in the presence of herbivores. In addition, herbivores weakened selection on flower size but only among acyanogenic plants, suggesting that plant defences reduce the strength of herbivore‐mediated selection. Pollinators did not independently affect selection on any trait, although pollinators weakened selection for later flowering among cyanogenic plants. Overall, cyanogenic plant defences consistently increased the strength of positive directional selection on reproductive traits. Herbivores and pollinators both strengthened and weakened the strength of selection on reproductive traits, although herbivores imposed ~2.7× stronger selection than pollinators across all traits. Contrary to the view that pollinators are the most important agents of selection on reproductive traits, our data show that selection on reproductive traits is driven primarily by variation in herbivory and plant defences in this system.     

Cyanogenic glycosides: a case study for evolution and application of cytochromes P450

Cyanogenic glycosides are ancient biomolecules found in more than 2,650 higher plant species as well as in a few arthropod species. Cyanogenic glycosides are amino acid-derived β-glycosides of α-hydroxynitriles. In analogy to cyanogenic plants, cyanogenic arthropods may use cyanogenic glycosides as defence compounds. Many of these arthropod species have been shown to de novo synthesize cyanogenic glycosides by biochemical pathways that involve identical intermediates to those known from plants, while the ability to sequester cyanogenic glycosides appears to be restricted to Lepidopteran species. In plants, two atypical multifunctional cytochromes P450 and a soluble family 1 glycosyltransferase form a metabolon to facilitate channelling of the otherwise toxic and reactive intermediates to the end product in the pathway, the cyanogenic glycoside. The glucosinolate pathway present in Brassicales and the pathway for cyanoalk(en)yl glucoside synthesis such as rhodiocyanosides A and D in Lotus japonicus exemplify how cytochromes P450 in the course of evolution may be recruited for novel pathways. The use of metabolic engineering using cytochromes P450 involved in biosynthesis of cyanogenic glycosides allows for the generation of acyanogenic cassava plants or cyanogenic Arabidopsis thaliana plants as well as L. japonicus and A. thaliana plants with altered cyanogenic, cyanoalkenyl or glucosinolate profiles.     

Spectrophotometer-aided evaluation of cyanogenic potential in white clover (Trifolium repens L.)

In comparison with ordinary methods of colorimetric evaluation of cyanogenic potential based on visual evaluation of the alkaline picrate reaction, a spectrophotometer-aided method could be more accurate (since it determines the exact amount of hydrogen cyanide released by the plant material), and less time-consuming as it can be performed on bulk material rather than on a number of individual plants. Ten white clover populations were evaluated by a spectrophotometer-aided method and by two visual evaluation criteria. All methods indicated the presence of large variation between populations. Visual methods gave almost identical results and allowed only for the distinction between cyanogenic and substantially acyanogenic populations. The results were only moderately consistent with those obtained by the spectrophotometer-aided method, which could detect the presence of variation also between cyanogenic populations. The effects of various incubation times (from 4 to 48 h) and of the addition of β-glucosidase on hydrogen cyanide release were also investigated. Comparable results for ranking of populations could be obtained over a range of incubation times, but at least 24 h were needed for a reliable estimation of the hydrogen cyanide produced by plants. The addition of enzyme did not increase the released cyanide. The effect of season and/or conditions of evaluation was marked on mean cyanogenic potential but limited on ranking of populations. Copyright © 2000 John Wiley & Sons, Ltd.     

Cassava plants with a depleted cyanogenic glucoside content in leaves and tubers. Distribution of cyanogenic glucosides, their site of synthesis and transport, and blockage of the biosynthesis by RNA interference technology

Transgenic cassava (Manihot esculenta Crantz, cv MCol22) plants with a 92% reduction in cyanogenic glucoside content in tubers and acyanogenic (<1% of wild type) leaves were obtained by RNA interference to block expression of CYP79D1 and CYP79D2, the two paralogous genes encoding the first committed enzymes in linamarin and lotaustralin synthesis. About 180 independent lines with acyanogenic (<1% of wild type) leaves were obtained. Only a few of these were depleted with respect to cyanogenic glucoside content in tubers. In agreement with this observation, girdling experiments demonstrated that cyanogenic glucosides are synthesized in the shoot apex and transported to the root, resulting in a negative concentration gradient basipetal in the plant with the concentration of cyanogenic glucosides being highest in the shoot apex and the petiole of the first unfolded leaf. Supply of nitrogen increased the cyanogenic glucoside concentration in the shoot apex. In situ polymerase chain reaction studies demonstrated that CYP79D1 and CYP79D2 were preferentially expressed in leaf mesophyll cells positioned adjacent to the epidermis. In young petioles, preferential expression was observed in the epidermis, in the two first cortex cell layers, and in the endodermis together with pericycle cells and specific parenchymatic cells around the laticifers. These data demonstrate that it is possible to drastically reduce the linamarin and lotaustralin content in cassava tubers by blockage of cyanogenic glucoside synthesis in leaves and petioles. The reduced flux to the roots of reduced nitrogen in the form of cyanogenic glucosides did not prevent tuber formation.     

Phylogenetic analysis of Zygaenoidea small-subunit rRNA structural variation implies initial oligophagy on cyanogenic host plants in larvae of the moth genus Zygaena (Insecta: Lepidoptera)

Cyanogenesis, the release of toxic cyanide from living cells, plays an important role in the defence system of certain plant (e.g. Fabaceae) and animal (e.g. Zygaenidae) taxa. The larvae of a significant number of Zygaena moth species (Zygaenidae) preferentially feed on cyanogenic Fabaceae and some of them are able to sequester cyanogenic compounds of their host plants. Using secondary structure variation of the small-subunit rRNA, we tested the currently accepted evolutionary hypothesis explaining species diversification in the genus Zygaena . We derived secondary structures considering evidence from covariation patterns and thermodynamic folding and applied structural information in a phylogenetic analysis. Contrary to previous assumptions, our results suggest that the use of cyanogenic larval host plants is an ancient trait and that the ability to feed on cyanogenic plants was probably already present in the most recent common ancestor of Zygaena . The utilization of acyanogenic plants in Zygaena species appears to be the result of a single secondary, reverse, larval host-plant shift. © 2006 The Linnean Society of London, Zoological Journal of the Linnean Society , 2006, 147 , 367–381.     

Evidence on the molecular basis of the Ac/ac adaptive cyanogenesis polymorphism in white clover (Trifolium repens L)

White clover is polymorphic for cyanogenesis, with both cyanogenic and acyanogenic plants occurring in nature. This chemical defense polymorphism is one of the longest-studied and best-documented examples of an adaptive polymorphism in plants. It is controlled by two independently segregating genes: Ac/ac controls the presence/absence of cyanogenic glucosides; and Li/li controls the presence/absence of their hydrolyzing enzyme, linamarase. Whereas Li is well characterized at the molecular level, Ac has remained unidentified. Here we report evidence that Ac corresponds to a gene encoding a cytochrome P450 of the CYP79D protein subfamily (CYP79D15), and we describe the apparent molecular basis of the Ac/ac polymorphism. CYP79D orthologs catalyze the first step in cyanogenic glucoside biosynthesis in other cyanogenic plant species. In white clover, Southern hybridizations indicate that CYP79D15 occurs as a single-copy gene in cyanogenic plants but is absent from the genomes of ac plants. Gene-expression analyses by RT-PCR corroborate this finding. This apparent molecular basis of the Ac/ac polymorphism parallels our previous findings for the Li/li polymorphism, which also arises through the presence/absence of a single-copy gene. The nature of these polymorphisms may reflect white clover's evolutionary origin as an allotetraploid derived from cyanogenic and acyanogenic diploid progenitors.     

Mollusc grazing limits growth and early development of the old forest lichen Lobaria pulmonaria in broadleaved deciduous forests

This study aims: (1) to quantify mollusc grazing on juvenile and mature thalli of the foliose epiphytic lichen Lobaria pulmonaria, and (2) to test the hypothesis inferring a herbivore defensive role of lichen depsidones in forests with indigenous populations of lichen-feeding molluscs. Lichens were transplanted in shaded and less shaded positions in each of two calcareous broadleaved deciduous forests, one poor in lichens, one with a rich Lobarion community. Preventing the access of molluscs significantly reduced the loss of juvenile L. pulmonaria, particularly in the naturally lichen-poor forest. Molluscs also severely grazed mature thalli in the lichen-poor forest, especially thalli placed under the more shading canopies. Furthermore, reducing the natural concentration of depsidones by pre-rinsing with acetone increased subsequent grazing significantly, showing that lichen depsidones function as herbivore defence in natural habitats. Our results suggest that mollusc grazing may play important roles in shaping the epiphytic vegetation in calcareous deciduous forests, and that recently established juvenile L. pulmonaria thalli seem to be particularly vulnerable.     

Molecular characterization of a new leucine-rich repeat-containing G protein-coupled receptor from a bivalve mollusc: evolutionary implications

The family of leucine-rich repeat-containing G protein-coupled receptors (LGRs) shows members in both vertebrates and invertebrates including the most ancestral ones. Although this suggests an early evolutionary origin of this family of receptors, little is known about their diversity in molluscs, a major phylum of bilaterian invertebrates. Based on sequences of mammalian and insect LGRs, we have cloned and characterized a new typical LGR in the bivalve mollusc Crassostrea gigas. This receptor named Cg-LGRB exhibits high degree of amino acid sequence identity with both mammalian and Drosophila LGRs. Phylogenetic analysis indicates that Cg-LGRB belongs to the cluster of type B orphan LGRs and suggests that molluscs likely express the three LGR subgroups identified previously in other animals. Quantitative RT-PCR shows that Cg-LGRB is expressed mainly in the digestive gland and only at moderate levels in other organs and developmental stages. A possible involvement in the control of cytological changes occurring in bivalve mollusc digestive gland is discussed.     

The effect of mollusc grazing on seedling recruitment in artificially created grassland gaps

Two experiments conducted in spring and autumn 1992 examined the effect of mollusc grazing on seedling regeneration from natural grassland seedbanks by creating artificial gaps in plots in a grassland sward. Molluscs were excluded from half the gaps by application of molluscicide. Mollusc grazing in both the spring and autumn experiment significantly reduced seedling recruitment, though the intensity of grazing was greatest in autumn. Recruitment of five species was markedly influenced by molluscicide application. In spring, plots from which molluscs were excluded contained significantly more seedlings of Chenopodium polyspermum and Ranunculus acris. In the autumn, exclusion of molluscs resulted in increased populations of R. acris, Stellaria graminea and Rumex acetosa. Cerastium holosteoides populations were greatest in autumn grazed plots. Other species, notably the grasses Holcus lanatus and Agrostis capillaris and the legume Trifolium repens were unaffected by molluscicide application. Species diversity was significantly decreased by molluscicide application in the autumn. Gap size significantly affected the recruitment of two species. Ranunculus acris populations were significantly higher in small gaps in both spring and summer, while Chenopodium recruitment in the spring was greater in small gaps. Gap size also significantly influenced the risk of mollusc attack on Ranunculus as molluscs appeared to show an aggregative feeding response in the high seedling density small gaps. Selective grazing of vulnerable seedlings by molluscs may influence the eventual relative proportions of the species present and so provide a potent mechanism in shaping community composition in grasslands.     

Contrasting effects of insect and molluscan herbivores on plant diversity in a long-term field experiment

The importance of invertebrate herbivores in regulating plant communities remains unclear, due to the absence of long-term exclusion experiments. An experiment in an English grassland involving long-term exclusions of insect and mollusc herbivores, along with rabbit fencing, showed strong, but opposing, effects of the invertebrate herbivores. Plant species richness declined and biomass increased following insect exclusion, due to increased dominance by a grass species, whereas mollusc exclusion led to increased herbs abundance. The two herbivores had a compensatory interaction: molluscs had no effects in the absence of insects and large insect effects depended on the absence of molluscs. The effects of invertebrate exclusion became apparent only after 8 years, and would have been seriously underestimated in shorter studies. Our results suggest that theorists and conservation managers need to shift from their historic focus on vertebrate herbivory, to a recognition that invertebrates can be equally important drivers of plant community structure.     

Rapid evolution of an adaptive cyanogenesis cline in introduced North American white clover (Trifolium repens L.)

White clover is polymorphic for cyanogenesis (HCN production after tissue damage), and this herbivore defence polymorphism has served as a classic model for studying adaptive variation. The cyanogenic phenotype requires two interacting biochemical components; the presence/absence of each component is controlled by a simple Mendelian gene (Ac/ac and Li/li). Climate-associated cyanogenesis clines occur in both native (Eurasian) and introduced populations worldwide, with cyanogenic plants predominating in warmer locations. Moreover, previous studies have suggested that epistatic selection may act within populations to maintain cyanogenic (AcLi) plants and acyanogenic plants that lack both components (acli plants) at the expense of plants possessing a single component (Acli and acLi plants). Here, we examine the roles of selection, gene flow and demography in the evolution of a latitudinal cyanogenesis cline in introduced North American populations. Using 1145 plants sampled across a 1650 km transect, we determine the distribution of cyanogenesis variation across the central United States and investigate whether clinal variation is adaptive or an artefact of population introduction history. We also test for the evidence of epistatic selection. We detect a clear latitudinal cline, with cyanogenesis frequencies increasing from 11% to 86% across the transect. Population structure analysis using nine microsatellite loci indicates that the cline is adaptive and not a by-product of demographic history. However, we find no evidence for epistatic selection within populations. Our results provide strong evidence for rapid adaptive evolution in these introduced populations, and they further suggest that the mechanisms maintaining adaptive variation may vary among populations of a species.     

Endogenous evolution of HCN during pre-germination periods in many seed species

Both cyanogenic ( Malus pumila Mill) and acyanogenic ( Oryza sativa L., Hordeum vulgare L., Zea mays L., Glycine max Merr., Lactuca sativa L., and Xanthium pennsylvanicum Wallr. etc.) seeds evolve HCN gas during the early periods of water imbibition. All tested seeds contained reserve cyanogens which liberated HCN upon hydrolysis with H₂SO₄ and with β-glucosidase and/or lipase. The amounts of liberated HCN were roughly comparable to those of unidentified cyanogens. It is thus conceivable that the cyanogens within seeds are available as precursors for free HCN evolved in the pre-germination period. The amounts of HCN evolved in the acyanogenic seeds were only 0.002 to 1% of that in apple, but the contents of the cyanogenic compounds in rice and cocklebur increased temporarily during the pre-germination period, then decreased and, finally, disappeared completely with the start of germination.     

The cyanogenic glucoside composition of Zygaena filipendulae (Lepidoptera: Zygaenidae) as effected by feeding on wild-type and transgenic lotus populations with variable cyanogenic glucoside profiles

Zygaena larvae sequester the cyanogenic glucosides linamarin and lotaustralin from their food plants (Fabaceae) as well as carry out de novo biosynthesis of these compounds. In this study, Zygaena filipendulae were reared on wild-type Lotus corniculatus and wild-type and transgenic L. japonicus plants with differing content and ratios of the cyanogenic glucosides linamarin and lotaustralin and of the cyanoalkenyl glucosides rhodiocyanoside A and D. LC-MS analyses, free choice feeding experiments and developmental studies were used to examine the effect of varying content and ratios of these secondary metabolites on the feeding preferences, growth and development of Z. filipendulae. Larvae reared on cyanogenic L. corniculatus developed faster compared to larvae reared on L. japonicus although free choice feeding trials demonstrated that the latter plant source was the preferred food plant. Larvae reared on acyanogenic L. corniculatus showed decelerated development. Analysis of different life stages and tissues demonstrate that Z. filipendulae strive to maintain certain threshold content and ratios of cyanogenic glucosides regardless of the composition of the food plants. Despite this, the ratios of cyanogenic glucosides in Z. filipendulae remain partly affected by the ratio of the food plant due to the high proportion of sequestering that takes place.