Chemical Defenses of Freshwater Macrophytes Against Crayfish Herbivory

We measured feeding preferences of the crayfish Procambarus clarkii for fresh tissue from four species of freshwater macrophytes (Habenaria repens, Saururus cernuus, Ceratophyllum demersum and Typha angustifolia). We then determined the role of plant chemical defenses in generating these preferences by incorporating crude aqueous and organic extracts from each species into palatable foods and comparing feeding on these foods to feeding on control foods lacking these extracts. Tissue toughness, dry mass and ash-free mass per volume, and percentages of carbon, nitrogen, and phenolics were also measured for each of the four macrophytes. Although it had a low nutritional value, Ceratophyllum was the preferred food when it was offered as fresh tissue; it did not produce a chemically deterrent extract. The lipophilic crude extract from Typha significantly deterred crayfish feeding, but this highly nutritious plant was preferred when offered in an agar-based diet lacking structural defenses. Habenaria and Saururus were low preference foods that did not appear to be structurally defended; each species contained both lipophilic and water-soluble extracts that significantly deterred feeding. Fractionation of the lipophilic crude extract from Saururus indicated the presence of at least three deterrent compounds. From the orchid Habenaria, we isolated and identified a novel bis-p-hydroxybenzyl-2-alkyl-2-hydroxysuccinoate metabolite, habenariol, that appeared to explain most of the feeding deterrent activity present in the lipophilic extract of this species. The concentration of the metabolite in frozen collections of this plant doubled if we allowed the material to thaw before placing it in extraction solvents.     

Prevalence of Chemical Defenses among Freshwater Plants

Although macrophyte–herbivore interactions in freshwater systems were generally disregarded for many years, recent data suggest that herbivory can be intense and important in structuring freshwater communities. This has led to the hypothesis that chemical defenses should be common among freshwater plants, but few studies have reported such chemical defenses, and no previous studies have assessed the frequency of chemical defenses among a substantial number of freshwater plant species. In a study of 21 macrophyte species co-occurring with the omnivorous crayfish Procambarus acutus in a southeastern USA wetland environment, we found that extracts of 11 species (52%) deterred feeding by P. acutus when tested in artificial foods at natural concentrations. Of these 11 chemically defended species, one species, Eupatorium capillifolium, consistently had a more unpalatable extract following mechanical damage to plant tissue, indicative of an activated chemical defense. Because herbivores are commonly nitrogen-limited and select food based on several plant traits, including plant nutritional value, it might be expected that chemical defenses would be especially important for protein-rich plants. However, we found no relationship between soluble protein concentration and deterrence of plant extracts.     

Cotton Plant, Gossypium hirsutum L., Defense in Response to Nitrogen Fertilization

Plants respond to insect herbivory by producing dynamic changes in an array of defense-related volatile and nonvolatile secondary metabolites. A scaled response relative to herbivory levels and nutrient availability would be adaptive, particularly under nutrient-limited conditions, in minimizing the costs of expressed defensive pathways and synthesis. In this study, we investigated effects of varying nitrogen (N) fertilization (42, 112, 196, and 280 ppm N) on levels of cotton plant (Gossypium hirsutum) phytohormones [jasmonic acid (JA) and salicylic acid (SA)], terpenoid aldehydes (hemigossypolone, heliocides H₁, H₂, H₃, and H₄), and volatile production in response to beet armyworm (Spodoptera exigua) herbivory. Additional bioassays assessed parasitoid (Cotesia marginiventris) host-searching success in response to cotton plants grown under various N fertilizer regimes. At low N input (42 ppm N), herbivore damage resulted in significant increases in local leaf tissue concentrations of JA and volatiles and in systemic accumulation of terpenoid aldehydes. However, increased N fertilization of cotton plants suppressed S. exigua-induced plant hormones and led to reduced production of various terpenoid aldehydes in damaged mature leaves and undamaged young leaves. While increased N fertilization significantly diminished herbivore-induced leaf volatile concentrations, the parasitism of S. exigua larvae by the parasitoid C. marginiventris in field cages did not differ among N treatments. This suggests that, despite significant N fertilization effects on herbivore-induced plant defenses, at short range, the parasitoids were unable to differentiate between S. exigua larvae feeding on physiologically different cotton plants that share large constitutive volatile pools releasable when damaged by herbivores.     

Palatability of Macroalgae that Use Different Types of Chemical Defenses

This study compared algal palatability and chemical defenses from subtropical green algae that may use different types of defense systems that deter feeding by the rock-boring sea urchin Echinometra lucunter. The potential defense systems present include (1) the terpenoid caulerpenyne and its activated products from Caulerpa spp., and (2) dimethylsulfoniopropionate (DMSP)-related defenses in Ulva spp. Secondary metabolites from these chemical groups have been shown to deter feeding by various marine herbivores, including tropical and temperate sea urchins. Live algal multiple-choice feeding assays and assays incorporating algal extracts or isolated metabolites into an artificial diet were conducted. Several green algae, including Ulva lactuca, Caulerpa prolifera, and Cladophora sp., were unpalatable. Nonpolar extracts from U. lactuca deterred feeding, whereas nonpolar extracts from C. prolifera had no effect on feeding. Polar extracts from both species stimulated feeding. Caulerpenyne deterred feeding at approximately 4% dry mass; however, dimethyl sulfide and acrylic acid had no effect at natural and elevated concentrations. E. lucunter is more tolerant than other sea urchins to DMSP-related defenses and less tolerant to caulerpenyne than many reef fish. Understanding the chemical defenses of the algae tested in this study is important because they, and related species, frequently are invasive or form blooms, and can significantly modify marine ecosystems.     

Induction of Plant Volatiles by Herbivores with Different Feeding Habits and the Effects of Induced Defenses on Host-Plant Selection by Thrips

Induced plant responses to attack by chewing insects have been intensively studied, but little is known about plant responses to nonchewing insects or to attack by multiple herbivores with different feeding habits. We examined volatile emissions by tobacco, Nicotiana tabacum, in response to feeding by the piercing–sucking insect western flower thrips (WFT), Frankliniella occidentalis, the chewing herbivore Heliothis virescens, and both herbivores simultaneously. In addition, we examined the effects of herbivore-induced plant defenses on host-plant selection by WFT. Plants responded to thrips feeding by consistently releasing five compounds. Simultaneous feeding by WFT and H. virescens elicited the same 11 compounds emitted in response to caterpillar feeding alone; however, two compounds, α-humulene and caryophyllene oxide, were produced in greater amounts in response to simultaneous herbivory. In choice tests, thrips consistently preferred uninduced plants over all other treatments and preferred plants damaged by caterpillars and those treated with caterpillar saliva over those treated with caterpillar regurgitant. The results are consistent with a previous finding that caterpillar regurgitant induces the release of significantly more volatile nicotine than plants damaged by caterpillars or plants treated with caterpillar saliva. A repellent effect of nicotine on WFT was confirmed by encircling unwounded plants with septa releasing volatile nicotine. Our results provide the first direct evidence that thrips feeding induces volatile responses and indicates that simultaneous herbivory by insects with different feeding habits can alter volatile emissions. In addition, the findings demonstrate that induced plant responses influence host-plant selection by WFT and suggest that the induction of volatile nicotine may play a role in this process.     

Belowground Chemical Signaling in Maize: When Simplicity Rhymes with Efficiency

Maize roots respond to feeding by larvae of the beetle Diabrotica virgifera virgifera by releasing (E)-β-caryophyllene. This sesquiterpene, which is not found in healthy maize roots, attracts the entomopathogenic nematode Heterorhabditis megidis. In sharp contrast to the emission of virtually only this single compound by damaged roots, maize leaves emit a blend of numerous volatile organic compounds in response to herbivory. To try to explain this difference between roots and leaves, we studied the diffusion properties of various maize volatiles in sand and soil. The best diffusing compounds were found to be terpenes. Only one other sesquiterpene known for maize, α-copaene, diffused better than (E)-β-caryophyllene, but biosynthesis of the former is far more costly for the plant than the latter. The diffusion of (E)-β-caryophyllene occurs through the gaseous rather than the aqueous phase, as it was found to diffuse faster and further at low moisture level. However, a water layer is needed to prevent complete loss through vertical diffusion, as was found for totally dry sand. Hence, it appears that maize has adapted to emit a readily diffusing and cost-effective belowground signal from its insect-damaged roots.     

Defoliation Affects Chemical Defenses in All Plant Parts of Rye Seedlings

The effect of defoliation and consecutive defoliation (condefoliation) of rye seedlings on the allocation patterns of biomass and hydroxamic acids (Hx) was evaluated five days after treatment. Growth of condefoliated seedlings was lower than that of defoliated and nondefoliated ones. Concentration of Hx decreased in shoots of condefoliated seedlings compared to nondefoliated ones, while concentration of Hx in root exudates increased. Allocation of Hx to roots and root exudates increased at the expense of allocation to the shoots in condefoliated seedlings. The ratio of Hx-aglucone to Hx-glucoside was higher in shoots of defoliated and condefoliated seedlings. The decrease in quantity of defense in shoots was accompanied by an increase in its quality, given that aglucones are more toxic than glucosides. The increase in concentration of Hx—an allelopathic compound also involved in mineral uptake by roots—in root exudates of condefoliated seedlings was suggested to lead to an advantage in the acquisition of resources for the regeneration of lost biomass.     

Chemical Defenses of Cryptic and Aposematic Gastropterid Molluscs Feeding on their Host Sponge Dysidea granulosa

Numerous opisthobranchs are known to sequester chemical defenses from their prey and use them for their own defense. Information on feeding biology is critical for understanding the ecology and evolution of molluscs, yet information on feeding biology is still scarce for many groups. Gastropterid molluscs are often found on sponges, but there is controversy as to whether they are true sponge feeders. On Guam, we found the gastropterids Sagaminopteron nigropunctatum and S. psychedelicum on the sponge Dysidea granulosa. They seem to rely on contrasting defense strategies as S. psychedelicum has vivid colors, consistent with the warning coloration found in many chemically defended opisthobranchs, whereas S. nigropunctatum is highly cryptic on the sponge. S. nigropunctatum is avoided by the pufferfish Canthigaster solandri in aquarium assays. We analyzed the secondary metabolites of the two species and found that both share polybrominated diphenyl ethers (BDEs) with their host sponge D. granulosa. S. psychedelicum and S. nigropunctatum sequester the major BDE in the sponge and accumulate it in the mantle at approximately the same concentration as in the sponge (4.03 and 2.37%, respectively), and concentrate it in their parapodia at over twice the sponge concentration (7.97 and 10.10%, respectively). We also detected trace amounts in the mucus secretion of S. psychedelicum, and quantified significant amounts in the mucus (1.84%) and egg masses (2.22%) of S. nigropunctatum. Despite contrasting color patterns displayed by the two gastropterid species, they seem to share a similar chemical defense strategy, i.e., they feed on D. granulosa and accumulate the major BDE of the sponge in their tissues.     

Combined Effect of Intercropping and Turnip Root Fly (<Emphasis Type="Italic">Delia floralis</Emphasis>) Larval Feeding on the Glucosinolate Concentrations in Cabbage Roots and Foliage

The effects of plant competition and herbivory on glucosinolate concentrations in cabbage root and foliage were investigated in a cabbage-red clover intercropping system. Cabbage plants were grown under different competitive pressures and with varying degrees of attack by root-feeding Delia floralis larvae. Glucosinolate concentrations in cabbage were affected both by intercropping and by D. floralis density. Glucosinolate concentrations in foliage generally decreased as a response to intercropping, while the responses to insect root damage of individual glucosinolates were weaker. Root glucosinolates responded more strongly to both intercropping and egg density. Total root glucosinolate concentration decreased with clover density, but only at high egg densities. Increased egg density led to opposite reactions by the indole and aliphatic glucosinolates in roots. The responses of individual root glucosinolates to competition and root damage were complex and, on occasion, nonlinear. Reduced concentrations of several glucosinolates and the tendency towards a decrease in total concentration in cabbage foliage caused by intercropping and larval damage suggest that competing plants or plants with root herbivory do not allocate the same resources as unchallenged plants towards sustaining levels of leaf defensive compounds. This could also be true for root glucosinolate concentrations at high egg densities. In addition, the results suggest that changes occurring within a structural group of glucosinolates may be influenced by changes in a single compound, e.g., glucobrassicin (indol-3-ylmethyl) in foliage or sinigrin (2-propenyl) in roots.     

Chemically Mediated Host-Plant Selection by the Milfoil Weevil: A Freshwater Insect–Plant Interaction

The milfoil weevil Euhrychiopsis lecontei is a specialist aquatic herbivore that feeds, oviposits, and mates on the invasive freshwater macrophyte Myriophyllum spicatum. We characterized the weevil's preference for M. spicatum, and through bioassay-driven fractionation, isolated and identified two chemicals released by M. spicatum that attract E. lecontei. Mass spectrometry and nuclear magnetic resonance spectroscopy were used to identify the attractive compounds as glycerol and uracil. Dose-response curves for glycerol and uracil indicated that weevil preference increased as sample concentration increased. Weevils were attracted to a crude sample of M. spicatum-released chemicals from 0.17 to 17 mg/l, to glycerol from 18 to 1800 μM (0.0017–0.17 mg/l), and to uracil from 0.015 to 15 μM (0.00014–1.4 mg/l). Although glycerol and uracil are ubiquitous, weevils are likely responding to high concentrations that are released as a result of the rapid growth of M. spicatum. Uracil concentration was greater in the exudates of M.spicatum than other Myriophyllum spp. E. lecontei was attracted to glycerol at a concentration similar to that at which terrestrial insects are attracted to sugar alcohols. This is the first example of a freshwater specialist insect being attracted to chemicals released by its host plant. Analysis of the water milfoil–weevil interaction provides further understanding as to how insects locate their host plants in aquatic systems.     

Geographic and Seasonal Variation in Alkaloid-Based Chemical Defenses of Dendrobates pumilio from Bocas del Toro, Panama

Poison frogs contain an alkaloid-based chemical defense that is derived from a diet of certain alkaloid-containing arthropods, which include mites, ants, beetles, and millipedes. Variation in population-level alkaloid profiles among species has been documented, and more than 800 different alkaloids have been identified. In the present study, we examine individual alkaloid variation in the dendrobatid poison frog Dendrobates pumilio among seven populations and between two seasons on Isla Bastimentos, located in the Bocas del Toro archipelago of Panama. Alkaloid profiles vary among populations and between seasons, illustrating that chemical defense in this species can vary on a small spatial and temporal scale. Alkaloid variation among populations is marginally correlated with geographic distance, and close populations have profiles more similar to each other than to distant populations. Individuals within populations also vary in alkaloid profiles. Differences are attributed to both spatial and temporal variations in the availability of alkaloid-containing arthropods. Many of the alkaloids present in the skin of D. pumilio appear likely to be of ant origin, supporting the importance of myrmecophagy in chemical defense among poison frogs. However, a variety of frog skin alkaloids was recently detected in mites, suggesting that mites may also play an important role in chemical defense.     

Quantitative Effects of Cyanogenesis on an Adapted Herbivore

Plant cyanogenesis means the release of gaseous hydrogen cyanide (HCN) in response to cell damage and is considered as an effective defense against generalist herbivores. In contrast, specialists are generally believed not to be affected negatively by this trait. However, quantitative data on long-term effects of cyanogenesis on specialists are rare. In this study, we used lima bean accessions (Fabaceae: Phaseolus lunatus L.) with high quantitative variability of cyanogenic features comprising cyanogenic potential (HCNp; concentration of cyanogenic precursors) and cyanogenic capacities (HCNc; release of gaseous HCN per unit time). In feeding trials, we analyzed performance of herbivorous Mexican bean beetle (Coleoptera: Coccinellidae: Epilachna varivestis Mulsant) on selected lines characterized by high (HC-plants) and low HCNp (LC-plants). Larval and adult stages of this herbivore feed on a narrow range of legumes and prefer cyanogenic lima bean as host plant. Nevertheless, we found that performance of beetles (larval weight gain per time and body mass of adult beetles) was significantly affected by lima bean HCNp: Body weight decreased and developmental period of larvae and pupae increased on HC-plants during the first generation of beetles and then remained constant for four consecutive generations. In addition, we found continuously decreasing numbers of eggs and larval hatching as inter-generational effects on HC-plants. In contrast to HC-plants, constantly high performance was observed among four generations on LC-plants. Our results demonstrate that Mexican bean beetle, although preferentially feeding on lima bean, is quantitatively affected by the HCNp of its host plant. Effects can only be detected when considering more than one generation. Thus, cyanide-containing precursors can have negative effects even on herbivores adapted to feed on cyanogenic plants.     

Chemical Constituents of the Aquatic Plant Schoenoplectus lacustris: Evaluation of Phytotoxic Effects on the Green Alga Selenastrum capricornutum

Forty-nine secondary metabolites were isolated from aqueous and alcoholic extracts of the aquatic plant Shoenoplectus lacustris. All compounds were characterized based on spectroscopic data. Eleven free and glycosylated low-molecular polyphenols, 17 cinnamic acid and dihydrocinnamic acid derivatives, 11 flavonoids, and 10 C₁₃ nor-isoprenoids were identified. The structure of the new compound, 1-benzoyl-glycerol-2-α-l-arabinopyranoside, was elucidated by 2D NMR experiments (COSY, HSQC, HMBC, NOESY). To evaluate potential phytotoxic effects, all compounds were tested on the green alga Selenastrum capricornutum, a unicellular organism commonly used in tests of toxicity as a bioindicator of eutrophic sites. The most active compound was (−)-catechin, showing an inhibition similar to that of the algaecide CuSO₄.     

Inter- and Intraspecific Comparisons of Antiherbivore Defenses in Three Species of Rainforest Understory Shrubs

Plants defend themselves against herbivores and pathogens with a suite of morphological, phenological, biochemical, and biotic defenses, each of which is presumably costly. The best studied are allocation costs that involve trade-offs in investment of resources to defense versus other plant functions. Decreases in growth or reproductive effort are the costs most often associated with antiherbivore defenses, but trade-offs among different defenses may also occur within a single plant species. We examined trade-offs among defenses in closely related tropical rain forest shrubs (Piper cenocladum, P. imperiale, and P. melanocladum) that possess different combinations of three types of defense: ant mutualists, secondary compounds, and leaf toughness. We also examined the effectiveness of different defenses and suites of defenses against the most abundant generalist and specialist Piper herbivores. For all species examined, leaf toughness was the most effective defense, with the toughest species, P. melanocladum, receiving the lowest incidence of total herbivory, and the least tough species, P. imperiale, receiving the highest incidence. Although variation in toughness within each species was substantial, there were no intraspecific relationships between toughness and herbivory. In other Piper studies, chemical and biotic defenses had strong intraspecific negative correlations with herbivory. A wide variety of defensive mechanisms was quantified in the three Piper species studied, ranging from low concentrations of chemical defenses in P. imperiale to a complex suite of defenses in P. cenocladum that includes ant mutualists, secondary metabolites, and moderate toughness. Ecological costs were evident for the array of defensive mechanisms within these Piper species, and the differences in defensive strategies among species may represent evolutionary trade-offs between costly defenses.     

Influence of Induced Plant Defenses in Cotton and Tomato on the Efficacy of Baculoviruses on Noctuid Larvae

Constitutive phenolase activity of plants has a profound ability to modulate disease in insects caused by baculoviruses. We investigated the influence of damage-induced plant phenolic oxidases in cotton and tomato on mortality caused by two different baculoviruses in their respective hosts, Heliothis virescens (L.) and Helicoverpa zea (Boddie). For both plant species, peroxidase (POD) and phenolic levels were predictive of larval mortality caused by baculoviruses. The higher the POD activity, the lower the mortality in both hosts. Different classes of phenolics (e.g., monohydroxyphenolics vs. catecholic phenolics) in combination with POD activity had different effects on the severity of viral disease depending upon mixture, which implies that viral efficacy is predictable only if total chemical content of the plants is specified. Inhibition of baculoviral disease by plant phenolase activity has potential implications for the compatibility of baculoviruses with induced resistance in IPM programs.     

Genetic and Environmental Sources of Variation in the Autogenous Chemical Defense of a Leaf Beetle

Chemical defense plays a central role for many herbivorous insects in their interactions with predators and host plants. The leaf beetle genus Oreina (Coleoptera, Chrysomelidae) includes species able to both sequester pyrrolizidine alkaloids and autogenously produce cardenolides. Sequestered compounds are clearly related to patterns of host-plant use, but variation in de novo synthesized cardenolides is less obviously linked to the environment. In this study, intraspecific variation in cardenolide composition was examined by HPLC–MS analysis in 18 populations of Oreina speciosa spanning Europe from the Massif Central to the Balkans. This revealed the defense secretion to be a complex blend of up to 42 compounds per population. There was considerable geographical variation in the total sample of 50 compounds detected, with only 14 found in all sites. The environmental and genetic influences on defense chemistry were investigated by correlation with distance matrices based on habitat factors, host-plant use, and genetics (sequence data from COI, COII, and 16s rRNA). This demonstrated an influence of both genetics and host-plant use on the overall blend of cardenolides and on the presence of some of the individual compounds. The implications of this result are discussed for the evolution of defense chemistry and for the use of cardenolide compounds as markers of the evolutionary history of the species. Triponez and Naisbit contributed equally to this work and are considered joint first authors. An erratum to this article can be found at     

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.     

Intraspecific Variation in Plant Defense Alters Effects of Root Herbivores on Leaf Chemistry and Aboveground Herbivore Damage

Root herbivores can indirectly affect aboveground herbivores by altering the food quality of the plant. However, it is largely unknown whether plant genotypes differ in their response to root herbivores, leading to variable defensive phenotypes. In this study, we investigated whether root-feeding insect larvae (Agriotes sp. larvae, wireworms) induce different responses in Plantago lanceolata plants from lines selected for low and high levels of iridoid glycosides (IG). In the absence of wireworms, plants of the “high-IG line” contained approximately twofold higher levels of total IG and threefold higher levels of catalpol (one of the IG) in leaves than plants from the “low-IG line,” whereas both lines had similar levels of IG in roots. In response to wireworms, roots of plants from both lines showed increased concentrations of catalpol. Leaves of “low-IG line” plants increased catalpol concentrations in response to wireworms, whereas catalpol concentrations of leaves of “high-IG line” plants decreased. In contrast, glucose concentrations in roots of “low-IG” plants decreased, while they increased in “high-IG” plants after feeding by wireworms. The leaf volatile profile differed between the lines, but was not affected by root herbivores. In the field, leaf damage by herbivores was higher in wireworm-induced compared to noninduced “low-IG” plants and lower in wireworm-induced compared to noninduced “high-IG” plants, despite induction of catalpol in leaves of the “low-IG” plants and reduction in “high-IG” plants. This pattern might arise if damage is caused mainly by specialist herbivores for which catalpol may act as feeding stimulant rather than as deterrent. The present study documents for the first time that intraspecific variation in plant defense affects the outcome of plant-mediated interactions between root and shoot herbivores.     

<Emphasis Type="Italic">De novo</Emphasis> Synthesis vs. Sequestration: Negatively Correlated Metabolic Traits and the Evolution of Host Plant Specialization in Cyanogenic Butterflies

Larvae of Heliconius butterflies (Nymphalidae: Heliconiinae) feed exclusively on cyanogenic leaves of Passiflora (passion vine). Most Heliconius manufacture cyanogenic glycosides (cyanogens) and some species sequester cyanogens from host plants. We compare ability to sequester simple monoglycoside cyclopentenyl (SMC) cyanogens and manufacture aliphatic cyanogens in 12 Heliconius species, including larvae that are specialized (single host species) and generalized (many host species). All butterflies tested higher for cyanide concentrations when reared on plants that larvae can sequester from (SMC plants) than when reared on plants that larvae do not sequester from (non-SMC plants). Specialists in the sara–sapho clade sequestered SMC cyanogens from specific host plants at seven times that of Passiflora generalists fed the same hosts. In contrast, sara–sapho clade species reared on non-SMC plants had significantly lower cyanide concentrations from de novo synthesis than generalists fed the same plants. Furthermore, cyanogen analyses indicated that Heliconius sara butterflies reared on an SMC host had a greater proportion of sequestered SMC cyanogens (95.0%) than de novo-synthesized aliphatic cyanogens (5.0%). Thus, sequestration and de novo synthesis are negatively correlated traits. Results suggest that losing the ability to synthesize cyanogens has restricted sara–sapho clade species to specific hosts containing SMC cyanogens and explains dietary restriction in this clade. We dedicate our paper to the memory of the Honorable Miriam Rothschild, F.R.S., a friend, colleague and supporter of many young scientists. Her enthusiasm and delight in chemical ecology and natural history continued to the last weeks of her life.