Cardenolide Concentrations Research Articles

Potent Nitrogen-containing Milkweed Toxins are Differentially Regulated by Soil Nitrogen and Herbivore-induced Defense.

Theories have been widely proposed and tested for impacts of soil nitrogen (N) on phytochemical defenses. Among the hundreds of distinct cardenolide toxins produced by milkweeds (Asclepias spp.), few contain N, yet these appear to be the most toxic against specialist herbivores. Because N- and non-N-cardenolides coexist in milkweed leaves and likely have distinct biosynthesis, they present an opportunity to address hypotheses about drivers of toxin expression. We tested effects of soil N and herbivore-damage on cardenolide profiles of two milkweed species differing in life-history strategies (Asclepias syriaca and A. curassavica), and the toxicity of their leaves. In particular leaf extracts were tested against the target enzymes (Na+/K+-ATPase extracted from neural tissue) from both monarch butterflies (Danaus plexippus) as well as less cardenolide-resistant queen butterflies, D. gilippus. Increasing soil N enhanced biomass of Asclepias syriaca but had weak effects on cardenolides, including causing a significant reduction in the N-cardenolide labriformin; feeding by monarch caterpillars strongly induced N-cardenolides (labriformin), its precursors, and total cardenolides. Conversely, soil N had little impact on A. curassavica biomass, but was the primary driver of increasing N-cardenolides (voruscharin, uscharin and their precursors); caterpillar induction was weak. Butterfly enzyme assays revealed damage-induced cardenolides substantially increased toxicity of both milkweeds to both butterflies, swamping out effects of soil N on cardenolide concentration and composition. Although these two milkweed species differentially responded to soil N with allocation to growth and specific cardenolides, leaf toxicity to butterfly Na+/K+-ATPases was primarily driven byherbivore-induced defense. Thus, both biotic and abiotic factors shape the composition of phytochemical defense expression, and their relative importance may be dictated by plant life-history differences.

Plant secondary metabolite has dose‐dependent effects on bumblebees

The presence of secondary metabolites in flower nectar can mediate interactions between plants, pollinators, herbivores, and microbes. Milkweeds range in concentrations of cardenolides in flower nectar from ~ 1 to 100 ng μl–1. Using three different behavioral assays with bumblebees Bombus impatiens, we examined the impacts of the commercially available cardenolide ouabain at the range of concentrations at which cardenolides naturally occur in milkweeds. We show that after four days of exposure bees in consumption assays drank more of a nectar solution with a low ouabain concentration of 10 ng μl–1 than the control sucrose nectar, and over the course of the experiment bees consumed less of the 100 ng μl–1 ouabain solution than the control. Bee activity levels in Petri dish arena assays were not impacted by ouabain consumption, even at the highest concentrations; however, in free‐flying choice assays, bees preferentially visited artificial flowers containing 10 ng μl–1 ouabain more than flowers with sucrose control, or flowers with 100 ng μl–1 of ouabain. We therefore conclude that cardenolides may provide plants advantages to pollination at the low end of the naturally occurring range of concentrations for cardenolides, but may be costly to plants at the high end of the range. This research highlights that secondary metabolites in nectar may be under selection to be maintained at low concentrations, and species with high concentrations of secondary metabolites may be under conflicting selection pressures to maintain high circulating levels to deter herbivores at the cost of reduced pollination.

Tissue and toxin-specific divergent evolution in plant defense.

A major predicted constraint on the evolution of anti-herbivore defense in plants is the non-independent expression of traits mediating resistance. Since herbivore attack can be highly variable across plant tissues, we hypothesized that correlations in toxin expression within and between plant tissues may limit population differentiation and, thus, plant adaptation. Using full-sib families from two nearby (<1 km) common milkweed (Asclepias syriaca) populations, we investigated genetic correlations among 28 distinct cardenolide toxins within and between roots, leaves and seeds, and examined signatures of tissue-specific divergent selection between populations by QST-FST comparisons. The prevalence, direction and strength of genetic correlations among cardenolides were tissue-specific, and concentrations of individual cardenolides were moderately correlated between tissues; nonetheless, the direction and strength of correlations were population-specific. Population divergence in the cardenolide chemistry was stronger in roots than in leaves and seeds. Divergent selection on individual cardenolides was tissue- and toxin-specific, except for a single highly toxic cardenolide (labriformin), that showed divergent selection across all plant tissues. Heterogeneous evolution of cardenolides within and between tissues across populations appears possible due to their highly independent expression. This independence may be common in nature, especially in specialized interactions in which distinct herbivores feed on different plant tissues.

No physiological costs of dual sequestration of chemically different plant toxins in the milkweed bug Spilostethus saxatilis (Heteroptera: Lygaeidae)

Many herbivorous insects not only cope with plant toxins but also sequester them as a defense against predators and parasitoids. Sequestration is a product of the evolutionary arms race between plants and herbivorous insects and has been hypothesized to incur physiological costs due to specific adaptations required. Contradictory evidence about these costs exists for insects sequestering only one class of toxin, but very little is known about the physiological implications for species sequestering structurally different classes of compounds. Spilostethus saxatilis is a milkweed bug belonging to the cardenolide-sequestering heteropteran subfamily Lygaeinae (Heteroptera: Lygaeidae) that has shifted to the colchicine-containing plant Colchicum autumnale, a resource of chemically unrelated alkaloids. Using feeding-assays on artificial diet and chemical analysis, we assessed whether S. saxatilis is still able to sequester cardenolides apart from colchicine and related metabolites (colchicoids), and tested the effect of (1) either a natural cardenolide concentration (using ouabain as a model compound) or a natural colchicine concentration, (2) an increased concentration of both toxins, and (3) seeds of either Asclepias syriaca (cardenolides) or C. autumnale (colchicoids) on a set of life-history traits. For comparison, we assessed the same life-history traits in the milkweed bug Oncopeltus fasciatus exposed to cardenolides only. Although cardenolides and colchicoids have different physiological targets (Na+/K+-ATPase vs tubulin) and thus require different resistance traits, chronic exposure and sequestration of both isolated toxins caused no physiological costs such as reduced growth, increased mortality, lower fertility, or shorter adult life span in S. saxatilis. Indeed, an increased performance was observed in O. fasciatus and an according trend was found in S. saxatilis when feeding on isolated ouabain and isolated colchicine, respectively. Positive effects were even more pronounced when insects were provided with natural toxic seeds (i.e. C. autumnale for S. saxatilis and A. syriaca for O. fasciatus), especially in O. fasciatus. Our findings suggest, that S. saxatilis can sequester two chemically unrelated classes of plant compounds at a cost-free level, and that colchicoids may even play a beneficial role in terms of fertility.

Host plant specificity of the monarch butterfly Danaus plexippus: A systematic review and meta-analysis.

The preference-performance hypothesis explains host specificity in phytophagous insects, positing that host plants chosen by adults confer the greatest larval fitness. However, adults sometimes oviposit on plants supporting low larval success because the components of host specificity (adult preference, plant palatability, and larval survival) are non-binary and not necessarily correlated. Palatability (willingness to eat) is governed by chemical cues and physical barriers such as trichomes, while survival (ability to complete development) depends upon nutrition and toxicity. Absence of a correlation between the components of host specificity results in low-performance hosts supporting limited larval development. Most studies of specificity focus on oviposition behavior leaving the importance and basis of palatability and survival under-explored. We conducted a comprehensive review of 127 plant species that have been claimed or tested to be hosts for the monarch butterfly Danaus plexippus to classify them as non-hosts, low performance, or high performance. We performed a meta-analysis to test if performance status could be explained by properties of neurotoxic cardenolides or trichome density. We also conducted a no-choice larval feeding experiment to identify causes of low performance. We identified 34 high performance, 42 low performance, 33 non-hosts, and 18 species with unsubstantiated claims. Mean cardenolide concentration was greater in high- than low-performance hosts and a significant predictor of host status, suggesting possible evolutionary trade-offs in monarch specialization. Other cardenolide properties and trichome density were not significant predictors of host status. In the experiment, we found, of the 62% of larvae that attempted to eat low-performance hosts, only 3.5% survived to adult compared to 85% of those on the high-performance host, demonstrating that multiple factors affect larval host plant specificity. Our study is the first to classify all known host plants for monarchs and has conservation implications for this threatened species.

Functional evidence supports adaptive plant chemical defense along a geographical cline

Environmental clines in organismal defensive traits are usually attributed to stronger selection by enemies at lower latitudes or near the host's range center. Nonetheless, little functional evidence has supported this hypothesis, especially for coevolving plants and herbivores. We quantified cardenolide toxins in seeds of 24 populations of common milkweed (Asclepias syriaca) across 13 degrees of latitude, revealing a pattern of increasing cardenolide concentrations toward the host's range center. The unusual nitrogen-containing cardenolide labriformin was an exception and peaked at higher latitudes. Milkweed seeds are eaten by specialist lygaeid bugs that are even more tolerant of cardenolides than the monarch butterfly, concentrating most cardenolides (but not labriformin) from seeds into their bodies. Accordingly, whether cardenolides defend seeds against these specialist bugs is unclear. We demonstrate that Oncopeltus fasciatus (Lygaeidae) metabolized two major compounds (glycosylated aspecioside and labriformin) into distinct products that were sequestered without impairing growth. We next tested several isolated cardenolides invitro on the physiological target of cardenolides (Na+/K+-ATPase); there was little variation among compounds in inhibition of an unadapted Na+/K+-ATPase, but tremendous variation in impacts on that of monarchs and Oncopeltus. Labriformin was the most inhibitive compound tested for both insects, but Oncopeltus had the greater advantage over monarchs in tolerating labriformin compared to other compounds. Three metabolized (and stored) cardenolides were less toxic than their parent compounds found in seeds. Our results suggest that a potent plant defense is evolving by natural selection along a geographical cline and targets specialist herbivores, but is met by insect tolerance, detoxification, and sequestration.

Evidence for tissue-specific defence-offence interactions between milkweed and its community of specialized herbivores.

Coevolution between plants and herbivores often involves escalation of defence-offence strategies, but attack by multiple herbivores may obscure the match of plant defence to any one attacker. As herbivores often specialize on distinct plant parts, we hypothesized that defence-offence interactions in coevolved systems may become physiologically and evolutionarily compartmentalized between plant tissues. We report that roots, leaves, flower buds and seeds of the tropical milkweed (Asclepias curassavica) show increasing concentrations of cardenolide toxins acropetally, with latex showing the highest concentration. In vitro assays of the physiological target of cardenolides, the Na+ /K+ -ATPase (hereafter "sodium pump"), of three specialized milkweed herbivores (root-feeding Tetraopes tetrophthalmus, leaf-feeding Danaus plexippus, and seed-feeding Oncopeltus fasciatus) show that they are proportionally tolerant to the cardenolide concentrations of the tissues they eat. Indeed, molecular substitutions in the insects' sodium pumps predicted their tolerance to toxins from their target tissues. Nonetheless, the relative inhibition of the sodium pumps of these specialists by the concentration versus composition (inhibition controlled for concentration, what we term "potency") of cardenolides from their target versus nontarget plant tissues revealed different degrees of insect adaptation to tissue-specific toxins. In addition, a trade-off between toxin concentration and potency emerged across plant tissues, potentially reflecting coevolutionary history or plant physiological constraints. Our findings suggest that tissue-specific coevolutionary dynamics may be proceeding between the plant and its specialized community of herbivores. This novel finding may be common in nature, contributing to ways in which coevolution proceeds in multispecies communities.

Unraveling the roles of genotype and environment in the expression of plant defense phenotypes.

Phenotypic variability results from interactions between genotype and environment and is a major driver of ecological and evolutionary interactions. Measuring the relative contributions of genetic variation, the environment, and their interaction to phenotypic variation remains a fundamental goal of evolutionary ecology.In this study, we assess the question: How do genetic variation and local environmental conditions interact to influence phenotype within a single population? We explored this question using seed from a single population of common milkweed, Asclepias syriaca, in northern Michigan. We first measured resistance and resistance traits of 14 maternal lines in two common garden experiments (field and greenhouse) to detect genetic variation within the population. We carried out a reciprocal transplant experiment with three of these maternal lines to assess effects of local environment on phenotype. Finally, we compared the phenotypic traits measured in our experiments with the phenotypic traits of the naturally growing maternal genets to be able to compare relative effect of genetic and environmental variation on naturally occurring phenotypic variation. We measured defoliation levels, arthropod abundances, foliar cardenolide concentrations, foliar latex exudation, foliar carbon and nitrogen concentrations, and plant growth.We found a striking lack of correlation in trait expression of the maternal lines between the common gardens, or between the common gardens and the naturally growing maternal genets, suggesting that environment plays a larger role in phenotypic trait variation of this population. We found evidence of significant genotype‐by‐environment interactions for all traits except foliar concentrations of nitrogen and cardenolide. Milkweed resistance to chewing herbivores was associated more strongly with the growing environment. We observed no variation in foliar cardenolide concentrations among maternal lines but did observe variation among maternal lines in foliar latex exudation.Overall, our data reveal powerful genotype‐by‐environment interactions on the expression of most resistance traits in milkweed.

Effects of cardenolides of milkweed plants on immunity of the monarch butterfly

Plants and herbivores have co-existed for millions of years, leading to complex relationships. Recent studies of plant–insect interactions have focused on important implications of plant defenses on insect immunity. Plants express defenses against herbivores through the production of toxic secondary chemicals, which may alter immune responses. The monarch butterfly, Danaus plexippus, has been shown to use toxic secondary chemicals (cardenolides) of milkweed plants (Asclepias spp.) to help reduce parasitism. However, little is known about the interaction of these secondary chemicals on the insect immune response. Therefore, we reared monarch caterpillars on five different milkweed species with varying cardenolide levels and measured their immune response to an immune stimulus. In particular, we measured a humoral immune response, in the form of anti-microbial growth, following exposure to lipopolysaccharide (LPS), a bacterial cell wall component. We show that the immune challenge caused a strong humoral immune response in monarch caterpillars. However, the response did not vary with milkweed species and cardenolide concentrations. Our results suggest that the toxins of milkweeds do not directly impact the humoral immune response.

Elevated atmospheric concentrations of CO2 increase endogenous immune function in a specialist herbivore.

Animals rely on a balance of endogenous and exogenous sources of immunity to mitigate parasite attack. Understanding how environmental context affects that balance is increasingly urgent under rapid environmental change. In herbivores, immunity is determined, in part, by phytochemistry which is plastic in response to environmental conditions. Monarch butterflies Danaus plexippus, consistently experience infection by a virulent parasite Ophryocystis elektroscirrha, and some medicinal milkweed (Asclepias) species, with high concentrations of toxic steroids (cardenolides), provide a potent source of exogenous immunity. We investigated plant-mediated influences of elevated CO2 (eCO2 ) on endogenous immune responses of monarch larvae to infection by O. elektroscirrha. Recently, transcriptomics have revealed that infection by O. elektroscirrha does not alter monarch immune gene regulation in larvae, corroborating that monarchs rely more on exogenous than endogenous immunity. However, monarchs feeding on medicinal milkweed grown under eCO2 lose tolerance to the parasite, associated with changes in phytochemistry. Whether changes in milkweed phytochemistry induced by eCO2 alter the balance between exogenous and endogenous sources of immunity remains unknown. We fed monarchs two species of milkweed; A. curassavica (medicinal) and A. incarnata (non-medicinal) grown under ambient CO2 (aCO2 ) or eCO2 . We then measured endogenous immune responses (phenoloxidase activity, haemocyte concentration and melanization strength), along with foliar chemistry, to assess mechanisms of monarch immunity under future atmospheric conditions. The melanization response of late-instar larvae was reduced on medicinal milkweed in comparison to non-medicinal milkweed. Moreover, the endogenous immune responses of early-instar larvae to infection by O. elektroscirrha were generally lower in larvae reared on foliage from aCO2 plants and higher in larvae reared on foliage from eCO2 plants. When grown under eCO2 , milkweed plants exhibited lower cardenolide concentrations, lower phytochemical diversity and lower nutritional quality (higher C:N ratios). Together, these results suggest that the loss of exogenous immunity from foliage under eCO2 results in increased endogenous immune function. Animal populations face multiple threats induced by anthropogenic environmental change. Our results suggest that shifts in the balance between exogenous and endogenous sources of immunity to parasite attack may represent an underappreciated consequence of environmental change.

Effects of diet and temperature on monarch butterfly wing morphology and flight ability

While global change research has greatly expanded in recent years, it remains unclear how environmental change will impact the mobility of many organisms. Flight is an important mode of transportation that affects ecological functions, including mate location, foraging, and migration. However, the effects of increasing temperature and diet quality on flight remain largely unknown. Here, we explore the effects of rearing temperature and larval diet quality on the flight ability of an iconic and ecologically threatened migratory insect, the monarch butterfly, Danaus plexippus. Monarch larvae were reared at two temperatures (25 °C and 28 °C) and on three milkweed species with varying phytochemistry (Asclepias incarnata, Asclepias syriaca, and Asclepias curassavica) in a fully factorial experiment. We tested flight ability using an automated flight mill, which measured cumulative flight distance, duration, and instantaneous velocity. Higher rearing temperatures reduced monarch flight ability, and larval diet quality influenced forewing morphology. Dietary milkweed with higher cardenolide concentrations (A. curassavica) induced shorter, wider forewings whereas milkweed with low to intermediate cardenolides (A. incarnata and A. syriaca) induced longer, narrower forewings, which are considered better for gliding flight used during migration. Our results provide evidence that projected increases in temperature and the subsequent expansion of tropical milkweed (A. curassavica) into the central breeding range of eastern North American migratory monarchs could reduce migration success. Further research is needed to identify mechanisms explaining the effects of diet and temperature on monarch flight ability and fitness, to ensure that appropriate conservation strategies are employed to preserve migratory populations.

Interspecific variation and elevated CO2 influence the relationship between plant chemical resistance and regrowth tolerance.

To understand how comprehensive plant defense phenotypes will respond to global change, we investigated the legacy effects of elevated CO2 on the relationships between chemical resistance (constitutive and induced via mechanical damage) and regrowth tolerance in four milkweed species (Asclepias). We quantified potential resistance and tolerance trade‐offs at the physiological level following simulated mowing, which are relevant to milkweed ecology and conservation. We examined the legacy effects of elevated CO2 on four hypothesized trade‐offs between the following: (a) plant growth rate and constitutive chemical resistance (foliar cardenolide concentrations), (b) plant growth rate and mechanically induced chemical resistance, (c) constitutive resistance and regrowth tolerance, and (d) regrowth tolerance and mechanically induced resistance. We observed support for one trade‐off between plant regrowth tolerance and mechanically induced resistance traits that was, surprisingly, independent of CO2 exposure. Across milkweed species, mechanically induced resistance increased by 28% in those plants previously exposed to elevated CO2. In contrast, constitutive resistance and the diversity of mechanically induced chemical resistance traits declined in response to elevated CO2 in two out of four milkweed species. Finally, previous exposure to elevated CO2 uncoupled the positive relationship between plant growth rate and regrowth tolerance following damage. Our data highlight the complex and dynamic nature of plant defense phenotypes under environmental change and question the generality of physiologically based defense trade‐offs.

Transcriptomics of monarch butterflies (Danaus plexippus) reveals that toxic host plants alter expression of detoxification genes and down-regulate a small number of immune genes.

Herbivorous insects have evolved many mechanisms to overcome plant chemical defences, including detoxification and sequestration. Herbivores may also use toxic plants to reduce parasite infection. Plant toxins could directly interfere with parasites or could enhance endogenous immunity. Alternatively, plant toxins could favour down-regulation of endogenous immunity by providing an alternative (exogenous) defence against parasitism. However, studies on genomewide transcriptomic responses to plant defences and the interplay between plant toxicity and parasite infection remain rare. Monarch butterflies (Danaus plexippus) are specialist herbivores of milkweeds (Asclepias spp.), which contain toxic cardenolides. Monarchs have adapted to cardenolides through multiple resistance mechanisms and can sequester cardenolides to defend against bird predators. In addition, high-cardenolide milkweeds confer monarch resistance to a specialist protozoan parasite (Ophryocystis elektroscirrha). We used this system to study the interplay between the effects of plant toxicity and parasite infection on global gene expression. We compared transcriptional profiles between parasite-infected and uninfected monarch larvae reared on two milkweed species. Our results demonstrate that monarch differentially express several hundred genes when feeding on A.curassavica and A.incarnata, two species that differ substantially in cardenolide concentrations. These differentially expressed genes include genes within multiple families of canonical insect detoxification genes, suggesting that they play a role in monarch toxin resistance and sequestration. Interestingly, we found little transcriptional response to infection. However, parasite growth was reduced in monarchs reared on A.curassavica, and in these monarchs, several immune genes were down-regulated, consistent with the hypothesis that medicinal plants can reduce reliance on endogenous immunity.

Does chemistry make a difference? Milkweed butterfly sequestered cardenolides as a defense against parasitoid wasps

Plant allelochemicals have important roles in plant defense as well as ecological and co-evolutionary dynamics within tri-trophic systems of plants, herbivores, and natural enemies. Milkweed butterflies represent a model system for chemical ecology because they sequester cardenolides semi-proportionally to the concentration in their host plants, yet little is known about the role of sequestered cardenolides in interactions with invertebrate natural enemies. We experimentally tested the preference and performance of two species of parasitic wasps (Pteromalus cassotis Walker and Pteromalus puparum Linnaeus) on milkweed butterfly pupae (monarchs, Danaus plexippus Linnaeus, and Euploea core Cramer) reared on plants to contain variable concentrations of sequestered cardenolides. We measured host survival and parasitoid reproductive success to determine whether greater concentrations of herbivore-sequestered plant toxins provide a defensive benefit or influence parasitoid success. We found that P. puparum was unable to develop from monarchs, regardless of toxicity. Monarchs containing more cardenolides (those fed Asclepias curassavica) were more likely to survive encounters with P. cassotis than those containing fewer cardenolides (fed Asclepias incarnata), but only because this parasitoid was less likely to attack more toxic monarchs. Once attacked, host toxicity had no effect on the likelihood of monarch survival nor the emergence of parasitoids. Host toxicity affected parasitoid performance in more subtle ways, however, decreasing P. cassotis brood size and survival to adulthood. When attacking cardenolide-free E. core pupae, P. cassotis reproduced successfully, but P. puparum did not, suggesting that milkweed butterflies may employ other defenses against parasitoids, perhaps in addition to cardenolides.

Trade-offs constrain the evolution of an inducible defense within but not between plant species.

Inducible defense is a common form of phenotypic plasticity, and inducibility (change in defense after herbivore attack) has long been predicted to trade off with constitutive (or baseline) defense to manage resource allocation. Although such trade-offs likely constrain evolution within species, the extent to which they influence divergence among species is unresolved. We studied cardenolide toxins among genetic families in eight North American Asclepias species, spanning the full range of defense in the genus. Using common environment experiments and chemical assays, we report a consistent trade-off (negative genetic correlation) between concentrations of constitutive cardenolides and their inducibility within each species. However, no trade-off was found in a phylogenetic analysis across species. To investigate factors driving differences in defense allocation among species we used latitude as a proxy for growing season and herbivore pressure and found that divergence into lower latitudes resulted in evolution of higher cardenolides overall. Next we used an enzymatic assay of the cellular target of cardenolides (sodium-potassium ATPase) and confirm that higher cardenolides resulted in stronger toxicity to a sensitive species, but not to specialized monarch butterflies. Thus, plant speciation into biogeographic regions with alternative resources or pest pressure resulted in the macroevolution of cardenolide defense, especially against unspecialized herbivores. Nonetheless, trade-offs persist in the extent to which this defense is allocated constitutively or is inducible among genotypes within each species.

Mycorrhizae Alter Constitutive and Herbivore-Induced Volatile Emissions by Milkweeds.

Plants use volatile organic compounds (VOCs) to cue natural enemies to their herbivore prey on plants. Simultaneously, herbivores utilize volatile cues to identify appropriate hosts. Despite extensive efforts to understand sources of variation in plant communication by VOCs, we lack an understanding of how ubiquitous belowground mutualists, such as arbuscular mycorrhizal fungi (AMF), influence plant VOC emissions. In a full factorial experiment, we subjected plants of two milkweed (Asclepias) species under three levels of AMF availability to damage by aphids (Aphis nerii). We then measured plant headspace volatiles and chemical defenses (cardenolides) and compared these to VOCs emitted and cardenolides produced by plants without herbivores. We found that AMF have plant species-specific effects on constitutive and aphid-induced VOC emissions. High AMF availability increased emissions of total VOCs, two green leaf volatiles (3-hexenyl acetate and hexyl acetate), and methyl salicylate in A. curassavica, but did not affect emissions in A. incarnata. In contrast, aphids consistently increased emissions of 6-methyl-5-hepten-2-one and benzeneacetaldehyde in both species, independent of AMF availability. Both high AMF availability and aphids alone suppressed emissions of individual terpenes. However, aphid damage on plants under high AMF availability increased, or did not affect, emissions of those terpenes. Lastly, aphid feeding suppressed cardenolide concentrations only in A. curassavica, and AMF did not affect cardenolides in either plant species. Our findings suggest that by altering milkweed VOC profiles, AMF may affect both herbivore performance and natural enemy attraction.

Cardenolide Intake, Sequestration, and Excretion by the Monarch Butterfly along Gradients of Plant Toxicity and Larval Ontogeny.

Monarch butterflies, Danaus plexippus, migrate long distances over which they encounter host plants that vary broadly in toxic cardenolides. Remarkably little is understood about the mechanisms of sequestration in Lepidoptera that lay eggs on host plants ranging in such toxins. Using closely-related milkweed host plants that differ more than ten-fold in cardenolide concentrations, we mechanistically address the intake, sequestration, and excretion of cardenolides by monarchs. We show that on high cardenolide plant species, adult butterflies saturate in cardenolides, resulting in lower concentrations than in leaves, while on low cardenolide plants, butterflies concentrate toxins. Butterflies appear to focus their sequestration on particular compounds, as the diversity of cardenolides is highest in plant leaves, lower in frass, and least in adult butterflies. Among the variety of cardenolides produced by the plant, sequestered compounds may be less toxic to the butterflies themselves, as they are more polar on average than those in leaves. In accordance with this, results from an in vitro assay based on inhibition of Na+/K+ ATPase (the physiological target of cardenolides) showed that on two milkweed species, including the high cardenolide A. perennis, extracts from butterflies have lower inhibitory effects than leaves when standardized by cardenolide concentration, indicating selective sequestration of less toxic compounds from these host plants. To understand how ontogeny shapes sequestration, we examined cardenolide concentrations in caterpillar body tissues and hemolymph over the course of development. Caterpillars sequestered higher concentrations of cardenolides as early instars than as late instars, but within the fifth instar, concentration increased with body mass. Although it appears that large amounts of sequestration occurs in early instars, a host switching experiment revealed that caterpillars can compensate for feeding on low cardenolide host plants with substantial sequestration in the fifth instar. We highlight commonalities and striking differences in the mechanisms of sequestration depending on host plant chemistry and developmental stage, which have important implications for monarch defense.

Toxicity of Milkweed Leaves and Latex: Chromatographic Quantification Versus Biological Activity of Cardenolides in 16 Asclepias Species.

Cardenolides are classically studied steroidal defenses in chemical ecology and plant-herbivore coevolution. Although milkweed plants (Asclepias spp.) produce up to 200 structurally different cardenolides, all compounds seemingly share the same well-characterized mode of action, inhibition of the ubiquitous Na+/K+ ATPase in animal cells. Over their evolutionary radiation, milkweeds show a quantitative decline of cardenolide production and diversity. This reduction is contrary to coevolutionary predictions and could represent a cost-saving strategy, i.e. production of fewer but more toxic cardenolides. Here we test this hypothesis by tandem cardenolide quantification using HPLC (UV absorption of the unsaturated lactone) and a pharmacological assay (in vitro inhibition of a sensitive Na+/K+ ATPase) in a comparative study of 16 species of Asclepias. We contrast cardenolide concentrations in leaf tissue to the subset of cardenolides present in exuding latex. Results from the two quantification methods were strongly correlated, but the enzymatic assay revealed that milkweed cardenolide mixtures often cause stronger inhibition than equal amounts of a non-milkweed reference cardenolide, ouabain. Cardenolide concentrations in latex and leaves were positively correlated across species, yet latex caused 27% stronger enzyme inhibition than equimolar amounts of leaf cardenolides. Using a novel multiple regression approach, we found three highly potent cardenolides (identified as calactin, calotropin, and voruscharin) to be primarily responsible for the increased pharmacological activity of milkweed cardenolide mixtures. However, contrary to an expected trade-off between concentration and toxicity, later-diverging milkweeds had the lowest amounts of these potent cardenolides, perhaps indicating an evolutionary response to milkweed's diverse community of specialist cardenolide-sequestering insect herbivores.

The Effects of Milkweed Induced Defense on Parasite Resistance in Monarch Butterflies, Danaus plexippus.

Many plants express induced defenses against herbivores through increasing the production of toxic secondary chemicals following damage. Phytochemical induction can directly or indirectly affect other organisms within the community. In tri-trophic systems, increased concentrations of plant toxins could be detrimental to plants if herbivores can sequester these toxins as protective chemicals for themselves. Thus, through trophic interactions, induction can lead to either positive or negative effects on plant fitness. We examined the effects of milkweed (Asclepias spp.) induced defenses on the resistance of monarch caterpillars (Danaus plexippus) to a protozoan parasite (Ophryocystis elektroscirrha). Milkweeds contain toxic secondary chemicals called cardenolides, higher concentrations of which are associated with reduced parasite growth. Previous work showed that declines in foliar cardenolides caused by aphid attack render monarch caterpillars more susceptible to infection. Here, we ask whether cardenolide induction by monarchs increases monarch resistance to disease. We subjected the high-cardenolide milkweed A. curassavica and the low-cardenolide A. syriaca to caterpillar grazing, and reared infected and uninfected caterpillars on these plants. As expected, monarchs suffered less parasite growth and disease when reared on A. curassavica than on A. syriaca. We also found that herbivory increased cardenolide concentrations in A. curassavica, but not A. syriaca. However, cardenolide induction in A. curassavica was insufficient to influence monarch resistance to the parasite. Our results suggest that interspecific variation in cardenolide concentration is a more important driver of parasite defense than plasticity via induced defenses in this tri-trophic system.

Arbuscular mycorrhizal fungi mediate herbivore‐induction of plant defenses differently above and belowground

Plants are exposed to herbivores and symbionts above and below ground. Herbivores above ground alter plant defenses in both leaves and roots, affecting plant–herbivore interactions above and below ground. Root symbionts, such as arbuscular mycorrhizal fungi (AMF), also influence the defenses of leaves and roots, and alter plant responses to herbivory. However, we lack an understanding of how AMF mediate plant responses to herbivores simultaneously in above and belowground plant tissues, despite the ubiquity of such interactions and their consequences for ecological communities. In a full factorial experiment, we subjected plants of four milkweed (Asclepias) species under three levels of AMF inoculum availability to damage by aphids Aphis nerii, caterpillars Danaus plexippus, or no herbivores. We then measured foliar and root cardenolides (chemical defenses), leaf toughness, latex exudation (physical defenses), foliar carbon, nitrogen, and phosphorous concentrations, plant biomass, and levels of AMF colonization of roots.Plants inoculated with AMF generally produced tougher leaves with higher cardenolide concentrations than did plants without AMF. In contrast, root cardenolides were altered by AMF inoculum availability in a plant species‐specific manner. The relative induction or suppression of foliar cardenolides and leaf toughness by herbivores was altered strongly by the level of AMF inoculum available to plants. However, AMF did not influence caterpillar‐induction or aphid‐suppression of root cardenolides. In addition, herbivore feeding induced substantial changes in levels of AMF colonization of roots in a plant species‐specific manner. We demonstrate that the availability of AMF in soil alters herbivore induction and suppression of plant defenses strongly, and does so differently in above and belowground plant tissues. Furthermore, we show that herbivore feeding alters levels of AMF colonization substantially, completing a feedback loop between above‐ and belowground organisms. Our study suggests that indirect interactions between AMF and herbivores may have community‐wide consequences by altering plant phenotype above and below ground.