Plant Cyanogenesis of <Emphasis Type="Italic">Phaseolus lunatus</Emphasis> and its Relevance for Herbivore–Plant Interaction: The Importance of Quantitative Data

Quantitative experimental results on the antiherbivorous effect of cyanogenesis are rare. In our analyses, we distinguished between the total amount of cyanide-containing compounds stored in a given tissue [cyanogenic potential (HCNp)] and the capacity for release of HCN per unit time (HCNc) from these cyanogenic precursors as a reaction to herbivory. We analyzed the impact of these cyanogenic features on herbivorous insects using different accessions of lima beans (Phaseolus lunatus L.) with different cyanogenic characteristics in their leaves and fourth instars of the generalist herbivore Schistocerca gregaria Forskål (Orthoptera, Acrididae). Young leaves exhibit a higher HCNp and HCNc than mature leaves. This ontogenetic variability of cyanogenesis was valid for all accessions studied. In no-choice bioassays, feeding of S. gregaria was reduced on high cyanogenic lima beans compared with low cyanogenic beans. A HCNp of about 15 μmol cyanide/g leaf (fresh weight) with a corresponding HCNc of about 1 μmol HCN released from leaf material within the first 10 min after complete tissue disintegration appears to be a threshold at which the first repellent effects on S. gregaria were observed. The repellent effect of cyanogenesis increased above these thresholds of HCNp and HCNc. No repellent action of cyanogenesis was observed on plants with lower HCNp and HCNc. These low cyanogenic accessions of P. lunatus were consumed extensively—with dramatic consequences for the herbivore. After consumption, locusts showed severe symptoms of intoxication. Choice assays confirmed the feeding preference of locusts for low over high cyanogenic leaf material of P. lunatus. The bioassays revealed total losses of HCN between 90 and 99% related to the estimated amount of ingested cyanide-containing compounds by the locusts. This general finding was independent of the cyanogenic status (high or low) of the leaf material.     

Preference for Acyanogenic White Clover (Trifolium repens) in the Vole Arvicola terrestris: I. Experiments with Two Varieties

We report experimental results showing that, under both laboratory conditions as well as in outdoor enclosures, the fossorial vole Arvicola terrestris preferentially feeds on acyanogenic white clover (Trifolium repens) when offered the choice between two varieties (Ladino and Aran) differing highly in their content in cyanogenic glycosides. We also observed that the voles adapted their diet and reduced their relative consumption of the cyanogenic variety during experiments conducted for two to three weeks in outdoor enclosures as compared to shorter tests conducted for 48 hr in laboratory cages. In addition, we report a similar preference for the acyanogenic Ladino variety for the slugs Arion ater and A. subfuscus.     

Quantitative Variability of Direct Chemical Defense in Primary and Secondary Leaves of Lima Bean (<Emphasis Type="Italic">Phaseolus lunatus</Emphasis>) and Consequences for a Natural Herbivore

Ontogenetic variability in chemical plant defenses against herbivores is a common phenomenon, but the effects of this variability on herbivore–plant interactions are little understood. In a previous study on lima bean (Phaseolus lunatus), we found a trade-off between cyanogenesis, a direct defense, and the release of herbivore-induced volatile organic compounds (VOCs; mainly functioning as an indirect defense). Moreover, the expression of these two defenses could change during plant ontogeny. The present study aimed at elucidating whether such ontogenetic changes in plant defense can affect herbivore–plant interactions. We quantified feeding rates of a natural insect herbivore, the Mexican bean beetle (Epilachna varivestis), on primary and secondary leaves of individual lima bean plants. These insects strongly preferred low cyanogenic primary leaves over high cyanogenic secondary leaves. Although weakly defended by cyanogenesis, lima beans’ primary leaves showed protein concentrations and photosynthetic activities that did not differ significantly from secondary leaves at the time of analysis. Based on our findings, we suggest that lima beans’ long-lived primary leaves function as efficient source organs, even beyond the stage of seedlings. This hypothesis may explain why primary leaves express a strong indirect defense by the release of herbivore induced VOCs.     

Phenotypic Plasticity of Cyanogenesis in Lima Bean Phaseolus lunatus—Activity and Activation of β-Glucosidase

Cyanogenesis, the release of toxic HCN from damaged plant tissues, is generally considered as a constitutive plant defense. We found phenotypic plasticity of cyanogenesis in young leaves of lima bean Phaseolus lunatus based on increased activity of the β-glucosidase in response to herbivore attack. Two aspects of plant cyanogenesis have to be considered in ecological analyses: (1) the cyanogenic potential (HCNp), which indicates the total amount of cyanide-containing compounds present in a given tissue, and (2) the cyanogenic capacity (HCNc), representing the release of HCN per unit time. This release is catalyzed by specific β-glucosidases, whose activity is a crucial parameter determining overall toxicity. Enzymatic activity of β-glucosidase—and, in consequence, the rate of HCN release—was increased significantly after 72 hr of incubation with spider mites as compared to noninfested leaves. Feeding by L1 larvae of Mexican bean beetles also led to enhanced enzymatic activity, whereas mechanical damage of leaf tissue had no effect on β-glucosidase activity and the release of HCN. The results place plant cyanogenesis in the group of induced resistance traits, whose degree of activity depends on the feeding by a particular herbivore.     

Kinetics of the Natural Evolution of Hydrogen Cyanide in Plants in Neotropical <Emphasis Type="Italic">Pteridium arachnoideum</Emphasis> and its Ecological Significance

The time-dependent natural release of hydrogen cyanide (HCN) was studied quantitatively using young croziers of the neotropical bracken fern Pteridium arachnoideum. HCN production was quantified in crushed tissue using a flow reactor at 30.0 ± 0.1C. Released HCN was carried into appropriate traps with a moist air flow. Aliquots were drawn from the traps at fixed time intervals, and the HCN concentration was evaluated spectroscopically. All available prunasin (Pru), the only cyanogenic glycoside present, underwent decomposition into HCN in less than 1200 min. Fiddleheads (N = 76) contained 1.84–107.70 mg Pru g^–1 dw in a continuous fashion suggesting genetic polymorphism. Acyanogenic morphs were rare (1/77). From the kinetics of the samples with Pru content near the median histographic distribution (N = 46), accumulated HCN formation as a function of time, initial velocities, average HCN production rate, and corresponding rate equations were obtained. Initial and average velocities correlated well with total Pru content. The yield of cyanide liberation varied widely between 0.51 and 47.86 g HCN min^–1 g^–1 dw and was a linear function of [Pru]_t. However, the -glucosidase enzyme involved in this reaction was not rate limiting and occurs in excess in the natural system. Enzyme activity was found to be independent of [Pru]_t. The contribution of HCN as an allomone-upon-request against herbivores was assessed quantitatively. Bracken fiddleheads produced a pulse of HCN soon after tissue injury that waned rapidly, leaving a large portion of intact prunasin to decompose more slowly in the herbivores lumen. The balance between the external and internal courses was found to depend on the concentration of prunasin in the plant, the amount of crozier eaten, and the time used to consume it.     

Aposematism in Archips cerasivoranus Not Linked to the Sequestration of Host-derived Cyanide

This study addressed the question of how caterpillars of Archips cerasivoranus feeding upon Prunus virginiana cope with the cyanogenic compounds of their food. Analysis by ion chromatography showed that young and aged leaves of P. virginiana consumed by the caterpillars during spring have hydrogen cyanide potentials (HCN-ps) of 2,473 +/- 130 ppm and 1,058 +/- 98 ppm, respectively. Although less than 3% of the cyanide released as the caterpillars feed escapes into the atmosphere, the larva's bright-yellow aposematic coloration and conspicuous activity can not be attributed to the sequestration of cyanide. Only six of 25 samples of the caterpillars' defensive regurgitants collected from 12 field colonies contained cyanide (17.6 +/- 6.54 ppm), less than 5% of the quantity previously reported to occur in the regurgitant of the tent caterpillar M. americanum. Only seven of 13 caterpillars assayed had detectable quantities of cyanide in their bodies (3.9 +/- 0.9 ppm). The fecal pellets that encase the cocoon contained no cyanide, nor did the frass that litters the leaf shelters. The small quantities of cyanide that occur in the caterpillar compared to the HCN-p of ingested plant material appear attributable to paced bouts of feeding and the maintenance of a highly alkaline foregut that inhibits cyanogenesis.     

Oviposition Choice of Mexican Bean Beetle (Epilachna varivestis) Depends on Host Plants Cyanogenic Capacity

The choice of insect females as to where to lay their eggs strongly affects progeny survival and, thus, fitness. We conducted choice experiments with female Mexican bean beetles, which were offered lima bean plants differing in their cyanogenic capacity (HCNc), i.e., in the intensity of hydrogen cyanide release per time unit from damaged plant tissue. Females preferred to lay their eggs on plants with low HCNc. In contrast, the mere concentration of cyanide-containing precursors (the cyanogenic potential, HCNp) did not affect oviposition choices. Plant cyanogenesis occurs after tissue damage, which brings specific β-glucosidases in contact with their substrates, cyanogenic glycosides, from which they are separated by compartmentation in the intact plant tissue. Plants commonly store toxic compounds in an inactive form. Our results demonstrate that for cyanogenesis, which is widespread in plants, it is the toxic product itself rather than the precursor that affects oviposition choices of a specialized herbivore.