Foliar Cardenolide Research Articles

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.

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.

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.

What doesn’t kill you makes you stronger: The burdens and benefits of toxin sequestration in a milkweed aphid

Abstract Specialized insect herbivores commonly co‐opt plant defences for protection against predators, but the costs, benefits and mechanisms of sequestration are poorly understood. We quantified sequestration of toxic cardenolides by the specialist aphid Aphis nerii when reared on four closely related milkweed (Asclepias) species with >20‐fold variation in cardenolide content, and in the presence or absence of generalist ladybug predators. Increasing concentrations of apolar plant cardenolides increased sequestered amounts in aphids. High concentrations in plants impaired aphid population growth, but also reduced the top‐down effects of predators. An in vitro enzymatic assay of total cardenolides in aphid bodies using the cardenolides’ target (animal Na+/K+‐ATPase) revealed that the subset of sequestered cardenolides is disproportionally more toxic than cardenolides in leaves. All aphids accumulated two cardenolides not present in their host plant, even on plants with very low foliar cardenolide concentrations. Sequestration of potent cardenolides by A. nerii thus involves passive, concentration‐dependent uptake from the host plant, as well as a presumably more active mechanism of modification and up‐concentration of plant cardenolides. The concentration of toxins in the host plant thus not only determines the negative impacts on growth and performance of an aphid, but also the ease and efficiency by which toxins are sequestered for the aphid’s defence, making the costs and benefits of plant toxins highly context‐dependent for both the plant and the herbivore. Therefore, variation in plant toxins is of central importance for co‐evolutionary plant–insect interactions, particularly in environments with variable predator pressure. A plain language summary is available for this article.

Mycorrhizae Alter Toxin Sequestration and Performance of Two Specialist Herbivores

Multitrophic species interactions are shaped by both top-down and bottom-up factors. Belowground symbionts of plants, such as arbuscular mycorrhizal fungi (AMF), can alter the strength of these forces by altering plant phenotype. For example, AMF-mediated changes in foliar toxin and nutrient concentrations may influence herbivore growth and fecundity. In addition, many specialist herbivores sequester toxins from their host plants to resist natural enemies, and the extent of sequestration varies with host plant secondary chemistry. Therefore, by altering plant phenotype, AMF may affect both herbivore performance and their resistance to natural enemies. We examined how inoculation of plants with AMF influences toxin sequestration and performance of two specialist herbivores feeding upon four milkweed species (Asclepias incarnata, A. curassavica, A. latifolia, A. syriaca). We raised aphids (Aphis nerii) and caterpillars (Danaus plexippus) on plants for six days in a fully factorial manipulation of milkweed species and level of AMF inoculation (zero, medium, and high). We then assessed aphid and caterpillar sequestration of toxins (cardenolides) and performance, and measured defensive and nutritive traits of control plants. Aphids and caterpillars sequestered higher concentrations of cardenolides from plants inoculated with AMF across all milkweed species. Aphid per capita growth rates and aphid body mass varied nonlinearly with increasing AMF inoculum availability; across all milkweed species, aphids had the lowest performance under medium levels of AMF availability and highest performance under high AMF availability. In contrast, caterpillar survival varied strongly with AMF availability in a plant species-specific manner, and caterpillar growth was unaffected by AMF. Inoculation with AMF increased foliar cardenolide concentrations consistently among milkweed species, but altered aboveground biomasses and foliar phosphorous concentrations in a plant species-specific fashion. Increased herbivore sequestration of cardenolides followed AMF-mediated increases in foliar cardenolide concentrations. Aphid performance declined with increasing foliar cardenolide concentrations, while caterpillar survival increased with aboveground biomass. Our findings suggest that by altering plant phenotype, the availability of AMF in soil has the potential to influence both top-down (via sequestration) and bottom up (via plant defense and nutrition) forces that operate on herbivores.

Fitness costs of animal medication: antiparasitic plant chemicals reduce fitness of monarch butterfly hosts

The emerging field of ecological immunology demonstrates that allocation by hosts to immune defence against parasites is constrained by the costs of those defences. However, the costs of non-immunological defences, which are important alternatives to canonical immune systems, are less well characterized. Estimating such costs is essential for our understanding of the ecology and evolution of alternative host defence strategies. Many animals have evolved medication behaviours, whereby they use antiparasitic compounds from their environment to protect themselves or their kin from parasitism. Documenting the costs of medication behaviours is complicated by natural variation in the medicinal components of diets and their covariance with other dietary components, such as macronutrients. In the current study, we explore the costs of the usage of antiparasitic compounds in monarch butterflies (Danaus plexippus), using natural variation in concentrations of antiparasitic compounds among plants. Upon infection by their specialist protozoan parasite Ophryocystis elektroscirrha, monarch butterflies can selectively oviposit on milkweed with high foliar concentrations of cardenolides, secondary chemicals that reduce parasite growth. Here, we show that these antiparasitic cardenolides can also impose significant costs on both uninfected and infected butterflies. Among eight milkweed species that vary substantially in their foliar cardenolide concentration and composition, we observed the opposing effects of cardenolides on monarch fitness traits. While high foliar cardenolide concentrations increased the tolerance of monarch butterflies to infection, they reduced the survival rate of caterpillars to adulthood. Additionally, although non-polar cardenolide compounds decreased the spore load of infected butterflies, they also reduced the life span of uninfected butterflies, resulting in a hump-shaped curve between cardenolide non-polarity and the life span of infected butterflies. Overall, our results suggest that the use of antiparasitic compounds carries substantial costs, which could constrain host investment in medication behaviours.

Arbuscular mycorrhizal fungi affect plant tolerance and chemical defences to herbivory through different mechanisms

Summary Arbuscular mycorrhizal fungi (AMF) are ubiquitous plant symbionts that affect plant nutrient status, patterns of resource allocation and rates of plant growth. In addition, AMF influence the expression of plant defence, which may affect subsequent interactions between plants and higher trophic levels. Tolerance to herbivory and chemical defence represent two distinct defence strategies of plants, and there is accumulating evidence that AMF can influence each strategy separately. However, investigations on the simultaneous effects of AMF on tolerance and chemical defence mechanisms are lacking. Using six milkweed (Asclepias) species and three levels of AMF inoculum, we show that plant tolerance to herbivory increases with foliar P concentrations, while both foliar cardenolide concentration and latex exudation (major chemical defences in milkweeds) increase with foliar N concentrations, but decrease with plant growth rate. Additionally, foliar cardenolide concentration increases with foliar P concentration, and latex exudation increases with root biomass. Because AMF have significant effects on all of these traits (foliar N and P concentrations, root biomass and growth rate), AMF can change plant tolerance and chemical defence simultaneously, albeit through different mechanisms. Synthesis. Overall, plant tolerance and chemical defence to herbivores are affected by AMF through changes in basic plant physiology such as nutrient status, allocation patterns and growth rate, and our study suggests that it may be possible to predict effects of AMF on plant defences through these simple traits.

Tests of the coupled expression of latex and cardenolide plant defense in common milkweed (Asclepias syriaca)

The coexpression of plant resistance traits suggests the hypothesis that they may have complementary functions in defense against herbivory. To address the extent to which defensive traits are necessarily coupled in plants grown under various conditions, we focused on latex and cardenolides, two potent defenses of milkweeds. We measured defenses across ontogenetic stages, different biotic and abiotic environments, and across genetic families of the common milkweed Asclepias syriaca. We first addressed the extent to which foliar cardenolides are derived from latex because latex actively flows through canals in leaves. We rinsed latex out of shredded leaves, which had no impact on foliar cardenolides, suggesting cardenolides are allocated to leaves independently of latex. Accordingly, there is potential for independent expression of the two traits. We next followed a cohort of plants from germination over three years; expression of both latex exudation and cardenolides increased annually, with the exception of a second year dip in cardenolides. Damage by monarch caterpillars induced ≈50% increases of both latex and cardenolides, with the former occurring rapidly within a day and the latter taking five days of herbivory; these responses were preceded by an earlier peak of the signaling hormones jasmonic acid and abscisic acid. Endogenous jasmonic acid showed an instantaneous positive correlation with latex exudation and foliar cardenolides. Under drought stress, latex and cardenolide expression were reversed, with water stress suppressing latex exudation, but nearly doubling cardenolide concentrations. These drought effects were not driven by phytohormones in the expected manner, as jasmonic acid was unaffected, salicylic acid was strongly suppressed, and abscisic acid tripled in response to drought. Finally, a meta‐analysis of four previously published field studies representing 85 genetic families of A. syriaca revealed no evidence for a genetic correlation between latex exudation and foliar cardenolide concentrations. The same lack of a correlation was observed across 22 populations of A. syriaca when grown in a common environment. Thus, the two most important defensive traits of milkweeds, although often coexpressed, can become uncoupled during some ontogenetic stages, under some biotic and abiotic conditions, and there is no evidence that they evolve together.

Mycorrhizal abundance affects the expression of plant resistance traits and herbivore performance

Summary The effects of mutualistic interactions on partner phenotype and fitness can vary with many factors, including the abundance of interacting partners. Partner abundance may determine the relative costs and benefits associated with the interaction. Although arbuscular mycorrhizal fungi (AMF) can strongly influence plant phenotype and community interactions, the effects of AMF abundance on plant resistance traits and multitrophic interactions are not well understood. We tested the hypothesis that increasing AMF abundance in soil will increase mycorrhizal colonization and affect plant biomass, foliar phosphorus concentration, the expression of plant resistance and herbivore performance. We inoculated Asclepias syriaca seedlings with Glomus etunicatum, Scutellospora fulgida and a mix of the two species in 11 AMF abundance treatments. We quantified plant phosphorus (P), growth and resistance phenotype and the performance of a specialist herbivore, Danaus plexippus on plants associated with varying amounts of fungi. Increasing abundance of S. fulgida or G. etunicatum in soil increased the proportion of plant root colonized by AMF, but root colonization by a mix of the fungi was not related to inoculum density. The abundance of S. fulgida, but not G. etunicatum, increased per cent foliar P and trichome density, but decreased latex exudation. Abundance of all AMF treatments tended to decrease specific leaf mass (SLM), and the two single‐species treatments unimodally affected the expression of total foliar cardenolides. Increasing abundance of the mix of AMF species also increased above‐ground biomass, foliar P and trichome density, but had little effect on other traits. The presence of AMF, species identity and the AMF abundance all explained significant variation in the expression of plant traits, although their relative contribution varied depending on the trait examined. Mycorrhizal abundance strongly increased caterpillar growth rate, which was associated with a decline in SLM. Synthesis. Variation in mycorrhizal abundance can profoundly influence the expression of plant resistance and subsequent herbivore performance. AMF abundance may be a key, but overlooked factor in determining the outcome of mycorrhizal mutualisms.

Spectroscopic sensitivity of real‐time, rapidly induced phytochemical change in response to damage

An ecological consequence of plant-herbivore interactions is the phytochemical induction of defenses in response to insect damage. Here, we used reflectance spectroscopy to characterize the foliar induction profile of cardenolides in Asclepias syriaca in response to damage, tracked in vivo changes and examined the influence of multiple plant traits on cardenolide concentrations. Foliar cardenolide concentrations were measured at specific time points following damage to capture their induction profile. Partial least-squares regression (PLSR) modeling was employed to calibrate cardenolide concentrations to reflectance spectroscopy. In addition, subsets of plants were either repeatedly sampled to track in vivo changes or modified to reduce latex flow to damaged areas. Cardenolide concentrations and the induction profile of A. syriaca were well predicted using models derived from reflectance spectroscopy, and this held true for repeatedly sampled plants. Correlations between cardenolides and other foliar-related variables were weak or not significant. Plant modification for latex reduction inhibited an induced cardenolide response. Our findings show that reflectance spectroscopy can characterize rapid phytochemical changes in vivo. We used reflectance spectroscopy to identify the mechanisms behind the production of plant secondary metabolites, simultaneously characterizing multiple foliar constituents. In this case, cardenolide induction appears to be largely driven by enhanced latex delivery to leaves following damage.

Arbuscular mycorrhizal fungi alter above- and below-ground chemical defense expression differentially among Asclepias species

Below-ground (BG) symbionts of plants can have substantial influence on plant growth and nutrition. Recent work demonstrates that mycorrhizal fungi can affect plant resistance to herbivory and the performance of above- (AG) and BG herbivores. Although these examples emerge from diverse systems, it is unclear if plant species that express similar defensive traits respond similarly to fungal colonization, but comparative work may inform this question. To examine the effects of arbuscular mycorrhizal fungi (AMF) on the expression of chemical resistance, we inoculated 8 species of Asclepias (milkweed)—which all produce toxic cardenolides—with a community of AMF. We quantified plant biomass, foliar and root cardenolide concentration and composition, and assessed evidence for a growth-defense tradeoff in the presence and absence of AMF. As expected, total foliar and root cardenolide concentration varied among milkweed species. Importantly, the effect of mycorrhizal fungi on total foliar cardenolide concentration also varied among milkweed species, with foliar cardenolides increasing or decreasing, depending on the plant species. We detected a phylogenetic signal to this variation; AMF fungi reduced foliar cardenolide concentrations to a greater extent in the clade including A. curassavica than in the clade including A. syriaca. Moreover, AMF inoculation shifted the composition of cardenolides in AG and BG plant tissues in a species-specific fashion. Mycorrhizal inoculation changed the relative distribution of cardenolides between root and shoot tissue in a species-specific fashion, but did not affect cardenolide diversity or polarity. Finally, a tradeoff between plant growth and defense in non-mycorrhizal plants was mitigated completely by AMF inoculation. Overall, we conclude that the effects of AMF inoculation on the expression of chemical resistance can vary among congeneric plant species, and ameliorate tradeoffs between growth and defense.

Specificity and trade‐offs in the induced plant defence of common milkweed Asclepias syriaca to two lepidopteran herbivores

Summary 1. We studied induced defences in common milkweed (Asclepias syriaca) to two specialist caterpillars (Danaus plexippus and Euchaetes egle) to test several hypotheses about co‐evolution and defence allocation. 2. Latex, a potent defence of milkweed, showed nearly fivefold variation in the amount exuded among genetic families and more than doubled production in response to herbivory by D. plexippus; this induced latex response was fourfold stronger than the latex response to E. egle herbivory. In contrast, D. plexippus and E. egle equivalently induced foliar cardenolide concentration (an average increase of 26%). 3. We found broad‐sense heritable variation for the induced plant responses, and there was a genetic correlation between the responses induced by the two herbivores for cardenolides, but not for latex. We found no genetic correlation between investment in latex and cardenolides in any of the treatments. 4. Using a bias‐corrected Monte Carlo procedure, we found strong evidence for a trade‐off between constitutive and induced cardenolides, but not for latex. 5. Synthesis. As natural selection by specific herbivores varies in space and time, we expect that these defence traits in A. syriaca will evolve independently, with the expression of cardenolides showing less specificity, as well as being constrained by a trade‐off between constitutive and induced defence.

SOIL CHARACTERISTICS, MINERAL NUTRIENTS, BIOMASS, AND CARDENOLIDE PRODUCTION IN DIGITALIS OBSCURA WILD POPULATIONS

ABSTRACT Mature leaves of wild-growing Digitalis obscura plants and soil samples were collected in four different regions. Soil and leaf mineral nutrient contents as well as biomass and cardenolide productions were determined. The soils in the four collection sites, basic and highly calcareous, showed significant interpopulation differences in phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), manganese (Mn), copper (Cu), and zinc (Zn). Foliar mineral nutrients and cardenolides also varied among populations. Highest cardenolide yield was found in those Digitalis populations presenting a better development. The cardenolide content exhibited significant negative correlations with the levels of P in plants and those of Cu in soils, suggesting that these elements may affect cardenolide biosynthesis.