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Mark D Hunter - One of the best experts on this subject based on the ideXlab platform.
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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
Molecular Ecology, 2019Co-Authors: Wenhao Tan, James R. Walters, Mark D Hunter, Tarik Acevedo, Erica V Harris, Tiffanie Y Alcaide, Nicole M Gerardo, Jacobus C De RoodeAbstract: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.
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transcriptomics of monarch butterflies danaus plexippus reveals strong differential gene expression in response to host plant toxicity but weak response to parasite infection
bioRxiv, 2019Co-Authors: Wenhao Tan, James R. Walters, Mark D Hunter, Tarik Acevedo, Erica V Harris, Tiffanie Y Alcaide, Nicole M Gerardo, Jacobus C De RoodeAbstract:ABSTRACT Herbivorous insects have evolved many mechanisms to overcome plant chemical defenses, 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 favor down-regulation of endogenous immunity by providing an alternative (exogenous) defense against parasitism. However, studies on genome-wide transcriptomic responses to plant defenses and the interplay between host plant toxicity and parasite infection remain rare. Monarch butterflies (Danaus plexippus) are specialist herbivores that feed on 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 medicinal effects to monarchs against 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. Our results demonstrate that monarch larvae differentially express several hundred genes when feeding on A. curassavica and A. incarnata, two species that are similar in nutritional content but 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, a small number of immune genes were down-regulated, consistent with the hypothesis that medicinal plants can reduce reliance on endogenous immunity.
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elevated atmospheric concentrations of carbon dioxide reduce monarch tolerance and increase parasite virulence by altering the medicinal properties of milkweeds
Ecology Letters, 2018Co-Authors: Leslie E Decker, Jacobus C De Roode, Mark D HunterAbstract:Hosts combat their parasites using mechanisms of resistance and tolerance, which together determine parasite virulence. Environmental factors, including diet, mediate the impact of parasites on hosts, with diet providing nutritional and medicinal properties. Here, we present the first evidence that ongoing environmental change decreases host tolerance and increases parasite virulence through a loss of dietary medicinal quality. Monarch butterflies use dietary toxins (Cardenolides) to reduce the deleterious impacts of a protozoan parasite. We fed monarch larvae foliage from four milkweed species grown under either elevated or ambient CO2 , and measured changes in resistance, tolerance, and virulence. The most high-cardenolide milkweed species lost its medicinal properties under elevated CO2 ; monarch tolerance to infection decreased, and parasite virulence increased. Declines in medicinal quality were associated with declines in foliar concentrations of lipophilic Cardenolides. Our results emphasize that global environmental change may influence parasite-host interactions through changes in the medicinal properties of plants.
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the effects of milkweed induced defense on parasite resistance in monarch butterflies danaus plexippus
Journal of Chemical Ecology, 2018Co-Authors: Wenhao Tan, Mark D Hunter, Leiling Tao, Kevin M Hoang, Jacobus C De RoodeAbstract: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.
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DataSheet1_Mycorrhizae Alter Toxin Sequestration and Performance of Two Specialist Herbivores.docx
2018Co-Authors: Amanda R. Meier, Mark D HunterAbstract: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 6 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 non-linearly 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.
Jacobus C De Roode - One of the best experts on this subject based on the ideXlab platform.
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Effects of Cardenolides of milkweed plants on immunity of the monarch butterfly
Arthropod-Plant Interactions, 2021Co-Authors: Kandis L. Adams, Ahmed Aljohani, Joselyne Chavez, Jacobus C De RoodeAbstract: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.
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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
Molecular Ecology, 2019Co-Authors: Wenhao Tan, James R. Walters, Mark D Hunter, Tarik Acevedo, Erica V Harris, Tiffanie Y Alcaide, Nicole M Gerardo, Jacobus C De RoodeAbstract: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.
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transcriptomics of monarch butterflies danaus plexippus reveals strong differential gene expression in response to host plant toxicity but weak response to parasite infection
bioRxiv, 2019Co-Authors: Wenhao Tan, James R. Walters, Mark D Hunter, Tarik Acevedo, Erica V Harris, Tiffanie Y Alcaide, Nicole M Gerardo, Jacobus C De RoodeAbstract:ABSTRACT Herbivorous insects have evolved many mechanisms to overcome plant chemical defenses, 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 favor down-regulation of endogenous immunity by providing an alternative (exogenous) defense against parasitism. However, studies on genome-wide transcriptomic responses to plant defenses and the interplay between host plant toxicity and parasite infection remain rare. Monarch butterflies (Danaus plexippus) are specialist herbivores that feed on 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 medicinal effects to monarchs against 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. Our results demonstrate that monarch larvae differentially express several hundred genes when feeding on A. curassavica and A. incarnata, two species that are similar in nutritional content but 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, a small number of immune genes were down-regulated, consistent with the hypothesis that medicinal plants can reduce reliance on endogenous immunity.
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elevated atmospheric concentrations of carbon dioxide reduce monarch tolerance and increase parasite virulence by altering the medicinal properties of milkweeds
Ecology Letters, 2018Co-Authors: Leslie E Decker, Jacobus C De Roode, Mark D HunterAbstract:Hosts combat their parasites using mechanisms of resistance and tolerance, which together determine parasite virulence. Environmental factors, including diet, mediate the impact of parasites on hosts, with diet providing nutritional and medicinal properties. Here, we present the first evidence that ongoing environmental change decreases host tolerance and increases parasite virulence through a loss of dietary medicinal quality. Monarch butterflies use dietary toxins (Cardenolides) to reduce the deleterious impacts of a protozoan parasite. We fed monarch larvae foliage from four milkweed species grown under either elevated or ambient CO2 , and measured changes in resistance, tolerance, and virulence. The most high-cardenolide milkweed species lost its medicinal properties under elevated CO2 ; monarch tolerance to infection decreased, and parasite virulence increased. Declines in medicinal quality were associated with declines in foliar concentrations of lipophilic Cardenolides. Our results emphasize that global environmental change may influence parasite-host interactions through changes in the medicinal properties of plants.
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the effects of milkweed induced defense on parasite resistance in monarch butterflies danaus plexippus
Journal of Chemical Ecology, 2018Co-Authors: Wenhao Tan, Mark D Hunter, Leiling Tao, Kevin M Hoang, Jacobus C De RoodeAbstract: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.
Ernst Reinhard - One of the best experts on this subject based on the ideXlab platform.
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cardenolide biosynthesis in light and dark grown digitalis lanata shoot cultures
Plant Physiology and Biochemistry, 1999Co-Authors: Marina Eisenbeis, Wolfgang Kreis, Ernst ReinhardAbstract:Abstract Shoot cultures of the cardenolide-producing species Digitalis lanata Ehrh. accumulated up to 0.6 μmol Cardenolides per g dry mass when cultivated under continuous white light. After transfer to permanent dark, the cardenolide content of cultured shoots gradually decreased and reached non-detectable levels after 12 weeks. After transfer back to light conditions, Cardenolides started to accumulate and reached the levels of light-grown controls after 4 weeks. Radiolabelled pregnenolone and progesterone were incorporated into Cardenolides in both green light-grown and white dark-grown shoots. It was thus established that Cardenolides are synthesised de novo in chloroplast-free tissues without apparent cardenolide accumulation, indicating that these compounds are efficiently turned over in the dark and that tissue differentiation, but not intact chloroplasts, is essential for cardenolide formation. The time course of two late anabolic enzymes of cardenolide metabolism, acetyl-CoA:digitoxin 15′-O-acetyltransferase (DAT, EC 2.3.1.-) and UDP-glucose:digitoxin 16′-glucosyltransferase (DGT, EC 2.4.1.-) was established during transfer of shoots from light to dark and vice versa. Only DAT was affected and was not measurable any more under dark conditions. The DGT may not be down-regulated because of its important, maybe even vital, role as an enzyme providing the vacuolar storage forms of Cardenolides. Two catabolic cardenolide-specific enzymes, lanatoside 15′-O-acetylesterase (LAE, EC 3.1.1.6.) and cardenolide 16′-O-glucohydrolase I (CGH I, EC 3.2.1.21), were also investigated and it was demonstrated that CGH I is inactive in dark-grown shoots. These observations indicate that CGH I is not involved in cardenolide degradation in situ, but may instead play a role in cardenolide remetabolisation and activation after wounding or in developmental programs.
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effects of digitoxigenin digoxigenin and various cardiac glycosides on cardenolide accumulation in shoot cultures of digitalis lanata
Planta Medica, 1998Co-Authors: Christoph Theurer, Wolfgang Kreis, Ernst ReinhardAbstract:Various cardenolide genins and cardenolide glycosides were administered to light-grown and dark-grown Digitalis lanata shoot cultures to investigate conversion reactions related to the formation and rearrangement of the sugar side chain of Digitalis glycosides. Digitoxigenin was converted to digitoxigen-3-one, 3-epidigitoxigenin, and digoxigenin. In addition, various cardiac glycosides were formed, including monoglycosides with glucose, glucomethylose, fucose, and digitalose, as well as the corresponding diglycosides, all containing a terminal glucose. Digitoxosylated Cardenolides were not formed, although the light-grown shoot cultures were capable of producing these compounds. Exogenous cardenolide fucosides were not converted into cardenolide digitoxosides. Administration of evatromonoside (digitoxigenin monodigitoxoside) did not force the formation of cardenolide di- or tridigitoxosides. Our results support the hypothesis that cardenolide fucosides and digitoxosides are formed via different biosynthetic routes and that cardenolide genins can be fucosylated but not digitoxosylated, indicating that digitoxosylation may only occur at an earlier stage in the cardenolide pathway.
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effects of digitoxigenin digoxigenin and various cardiac glycosides on cardenolide accumulation in shoot cultures of digitalis lanata
Planta Medica, 1998Co-Authors: Christoph Theurer, Wolfgang Kreis, Ernst ReinhardAbstract:Various cardenolide genins and cardenolide glycosides were administered to light-grown and dark-grown Digitalis lanata shoot cultures to investigate conversion reactions related to the formation and rearrangement of the sugar side chain of Digitalis glycosides. Digitoxigenin was converted to digitoxigen-3-one, 3-epidigitoxigenin, and digoxigenin. In addition, various cardiac glycosides were formed, including monoglycosides with glucose, glucomethylose, fucose, and digitalose, as well as the corresponding diglycosides, all containing a terminal glucose. Digitoxosylated Cardenolides were not formed, although the light-grown shoot cultures were capable of producing these compounds. Exogenous cardenolide fucosides were not converted into cardenolide digitoxosides. Administration of evatromonoside (digitoxigenin monodigitoxoside) did not force the formation of cardenolide di- or tridigitoxosides. Our results support the hypothesis that cardenolide fucosides and digitoxosides are formed via different biosynthetic routes and that cardenolide genins can be fucosylated but not digitoxosylated, indicating that digitoxosylation may only occur at an earlier stage in the cardenolide pathway.
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effects of various pregnanes and two 23 nor 5 cholenic acids on cardenolide accumulation in cell and organ cultures of digitalis lanata
Planta Medica, 1997Co-Authors: Werner Haussmann, Wolfgang Kreis, Ursel Stuhlemmer, Ernst ReinhardAbstract:5-Pregnen-3beta-ol-20-one (pregnenolone), 4-pregnene-3,20-dione (progesterone), 5-pregnene-3beta,21-diol-20-one (21-hydroxypregnenolone), 4-pregnen-21 -ol-3,20-dione (cortexone), 5beta-pregnane-3,20-dione, 5alpha-pregnane-3,20-dione, 5beta-pregnan-3alpha-ol-20-one, 5beta-pregnan-3beta-ol-20-one, 5beta-pregnane-3beta,14beta, 21-triol-20-one 3-acetate, 23-nor-5-cholenic acid-3beta,20xi-diol, and 23-nor-3,20(22) E-choladienic acid-3beta-ol were administered to photomixotrophic shoot cultures of Digitalis lanata Ehrh. capable of synthesizing Cardenolides, as well as to cardenolide-free tissue cultures, such as auxotrophic, dark-grown shoot cultures and cell suspension cultures of the same plant species. None of the pregnane precursors was qualified to restore cardenolide biosynthesis in the cardenolide-free tissues. The cardenolide content of light-grown shoot cultures, on the other hand, increased by 161%, 240%, 30%, 430% and 80% when 100 mg l(-1) of 21-hydroxypregnenolone, 5beta-pregnane-3,20-dione, 5beta-pregnan-3beta-ol-20-one, 5beta-pregnane-3beta,14beta,21-triol-20-one, 23-nor-5,20 (22) E-choladienic acid-3beta-ol, respectively, were administered. Pregnenolone, progesterone, cortexone, 5alpha-pregnanes, 5beta -pregnan-21-ols, and 23-nor-5-cholenic acid-3beta,20xi-diol, on the other hand, had no visible effect. Two different types of Cardenolides (termed fucose-type Cardenolides and digitoxose-type Cardenolides) were identified which may be formed via different biosynthetic routes. The "norcholanic acid pathway" seems to be operative in D. lanata shoot cultures only in the formation of fucose-type Cardenolides.
Anurag A. Agrawal - One of the best experts on this subject based on the ideXlab platform.
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Cardenolides toxicity and the costs of sequestration in the coevolutionary interaction between monarchs and milkweeds
Proceedings of the National Academy of Sciences of the United States of America, 2021Co-Authors: Anurag A. Agrawal, Amy P Hastings, Katalin Boroczky, Meena Haribal, Ronald A White, Renwang Jiang, Christophe DuplaisAbstract:For highly specialized insect herbivores, plant chemical defenses are often co-opted as cues for oviposition and sequestration. In such interactions, can plants evolve novel defenses, pushing herbivores to trade off benefits of specialization with costs of coping with toxins? We tested how variation in milkweed toxins (Cardenolides) impacted monarch butterfly ( Danaus plexippus ) growth, sequestration, and oviposition when consuming tropical milkweed ( Asclepias curassavica ), one of two critical host plants worldwide. The most abundant leaf toxin, highly apolar and thiazolidine ring–containing voruscharin, accounted for 40% of leaf Cardenolides, negatively predicted caterpillar growth, and was not sequestered. Using whole plants and purified voruscharin, we show that monarch caterpillars convert voruscharin to calotropin and calactin in vivo, imposing a burden on growth. As shown by in vitro experiments, this conversion is facilitated by temperature and alkaline pH. We next employed toxin-target site experiments with isolated Cardenolides and the monarch’s neural Na + /K + -ATPase, revealing that voruscharin is highly inhibitory compared with several standards and sequestered Cardenolides. The monarch’s typical >50-fold enhanced resistance to Cardenolides compared with sensitive animals was absent for voruscharin, suggesting highly specific plant defense. Finally, oviposition was greatest on intermediate cardenolide plants, supporting the notion of a trade-off between benefits and costs of sequestration for this highly specialized herbivore. There is apparently ample opportunity for continued coevolution between monarchs and milkweeds, although the diffuse nature of the interaction, due to migration and interaction with multiple milkweeds, may limit the ability of monarchs to counteradapt.
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less is more a mutation in the chemical defense pathway of erysimum cheiranthoides brassicaceae reduces total cardenolide abundance but increases resistance to insect herbivores
bioRxiv, 2020Co-Authors: Mahdieh Mirzaei, Anurag A. Agrawal, Tobias Zuest, Amy P Hastings, Georg JanderAbstract:Many plants produce structurally related defensive metabolites with the same target sites in insect herbivores. Two possible drivers of this chemical diversity are: (i) interacting effects of structurally related compounds increase resistance against individual herbivores, and (ii) variants of the same chemical structures differentially affect diverse herbivore species or feeding guilds. Erysimum cheiranthoides L (Brassicaceae; wormseed wallflower) produces abundant and diverse cardenolide toxins, which are derived from digitoxigenin, cannogenol, and strophanthidin, all of which inhibit Na+/K+-ATPases in animal cells. Here we describe an E. cheiranthoides mutant with 66% lower cardenolide content, resulting from greatly decreased cannogenol- and strophanthidin-derived Cardenolides, partially compensated for by increases in digitoxigenin-derived Cardenolides. This compositional change created a more even cardenolide distribution, decreased the average cardenolide polarity, but did not impact glucosinolates, a different class of chemical defenses. Growth of generalist herbivores from two feeding guilds, Myzus persicae Sulzer (Hemiptera: Aphididae; green peach aphid) and Trichoplusia ni Hubner (Lepidoptera: Noctuidae; cabbage looper), was decreased on the mutant line compared to wildtype. Both herbivores accumulated Cardenolides in proportion to plant content, with T. ni accumulating higher total concentrations than M. persicae. Helveticoside, an abundant cardenolide in E. cheiranthoides, was absent in M. persicae, suggesting that this compound is not present in the phloem. Our results support the hypothesis that cardenolide diversity protects plants against different herbivores, with digitoxigenin-derived compounds providing better protection against insects like M. persicae and T. ni, whereas cannogenol and strophanthidin provide better protection against other herbivores of E. cheiranthoides. FundingThis research was funded by US National Science Foundation awards 1907491 to AAA and 1645256 to GJ and AAA, Swiss National Science Foundation grant PZ00P3-161472 to TZ, and a Triad Foundation grant to GJ.
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multidrug transporters and organic anion transporting polypeptides protect insects against the toxic effects of Cardenolides
Insect Biochemistry and Molecular Biology, 2017Co-Authors: Simon C Groen, Susanne Dobler, Anurag A. Agrawal, Erika R Laplante, Nicolas M Alexandre, Noah K WhitemanAbstract:In the struggle against dietary toxins, insects are known to employ target site insensitivity, metabolic detoxification, and transporters that shunt away toxins. Specialized insects across six taxonomic orders feeding on cardenolide-containing plants have convergently evolved target site insensitivity via specific amino acid substitutions in the Na/K-ATPase. Nonetheless, in vitro pharmacological experiments have suggested a role for multidrug transporters (Mdrs) and organic anion transporting polypeptides (Oatps), which may provide a basal level of protection in both specialized and non-adapted insects. Because the genes coding for these proteins are evolutionarily conserved and in vivo genetic evidence in support of this hypothesis is lacking, here we used wildtype and mutant Drosophila melanogaster (Drosophila) in capillary feeder (CAFE) assays to quantify toxicity of three chemically diverse, medically relevant Cardenolides. We examined multiple components of fitness, including mortality, longevity, and LD50, and found that, while the three Cardenolides each stimulated feeding (i.e., no deterrence to the toxin), all decreased lifespan, with the most apolar cardenolide having the lowest LD50 value. Flies showed a clear non-monotonic dose response and experienced high levels of toxicity at the cardenolide concentration found in plants. At this concentration, both Mdr and Oatp knockout mutant flies died more rapidly than wildtype flies, and the mutants also experienced more adverse neurological effects on high-cardenolide-level diets. Our study further establishes Drosophila as a model for the study of cardenolide pharmacology and solidifies support for the hypothesis that multidrug and organic anion transporters are key players in insect protection against dietary Cardenolides.
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Milkweed butterfly resistance to plant toxins is linked to sequestration, not coping with a toxic diet.
Proceedings. Biological sciences, 2015Co-Authors: Georg Petschenka, Anurag A. AgrawalAbstract:Insect resistance to plant toxins is widely assumed to have evolved in response to using defended plants as a dietary resource. We tested this hypothesis in the milkweed butterflies (Danaini) which have progressively evolved higher levels of resistance to cardenolide toxins based on amino acid substitutions of their cellular sodium-potassium pump (Na(+)/K(+)-ATPase). Using chemical, physiological and caterpillar growth assays on diverse milkweeds (Asclepias spp.) and isolated Cardenolides, we show that resistant Na(+)/K(+)-ATPases are not necessary to cope with dietary Cardenolides. By contrast, sequestration of Cardenolides in the body (as a defence against predators) is associated with the three levels of Na(+)/K(+)-ATPase resistance. To estimate the potential physiological burden of cardenolide sequestration without Na(+)/K(+)-ATPase adaptations, we applied haemolymph of sequestering species on isolated Na(+)/K(+)-ATPase of sequestering and non-sequestering species. Haemolymph Cardenolides dramatically impair non-adapted Na(+)/K(+)-ATPase, but had systematically reduced effects on Na(+)/K(+)-ATPase of sequestering species. Our data indicate that major adaptations to plant toxins may be evolutionarily linked to sequestration, and may not necessarily be a means to eat toxic plants. Na(+)/K(+)-ATPase adaptations thus were a potential mechanism through which predators spurred the coevolutionary arms race between plants and insects.
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growth defense tradeoffs for two major anti herbivore traits of the common milkweed asclepias syriaca
Oikos, 2015Co-Authors: Tobias Züst, Sergio Rasmann, Anurag A. AgrawalAbstract:Costs of plant defense are a key assumption in evolutionary ecology, yet their detection has remained challenging. Here we introduce a novel method for quantifying plant growth using the common milkweed Asclepias syriaca and repeated non-destructive size measurements to experimentally test for costs of defensive traits. We estimated mechanistic components of plant growth (relative growth rate, net assimilation rate, specific leaf area and leaf-mass ratio) at two levels of fertilization (high and low), and related them to production of toxic Cardenolides and exudation of sticky latex. We found negative genetic correlations between Cardenolides and growth (most strongly with net assimilation rate) at both nutrient levels. Additionally, plants varied in their cardenolide response to low nutrients, and genetic families maintaining higher cardenolide production at low nutrient availability suffered proportionally larger reductions in growth. In contrast, the amount of latex was positively correlated with plant growth. Because latex is instantly deployed from a plant-wide system of pressurized laticifers, larger plants may simply exude proportionally more latex when damaged and thus plant size is likely to mask potential costs of latex synthesis. Unbiased quantification of mechanistic growth processes, coupled with the manipulation of nutrient or stress levels, is thus an effective approach to demonstrate allocation to defense and tradeoffs with growth, especially in long-lived plant species.
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the new semisynthetic cardenolide analog 3β 2 1 amantadine 1 on ethylamine digitoxigenin amantadig efficiently suppresses cell growth in human leukemia and urological tumor cell lines
Anticancer Research, 2015Co-Authors: Elke Nolte, Anna Sobel, Sven Wach, Heidi Hertlein, Nadja Ebert, Frieder Mulleruri, Robert K Slany, Helge Taubert, Bernd Wullich, Wolfgang KreisAbstract:The use of Cardenolides in the treatment of cardiac insufficiency is well-established. However, the potential of Cardenolides in tumor therapy has not been comprehensively studied. The aim of the present study was to characterize the cytotoxic effects of the new semisynthetic cardenolide analog AMANTADIG (3β-[2-(1-amantadine)-1-on-ethylamine]-digitoxigenin), and the cardenolide digitoxin on leukemia and urological tumor cell lines.The anti-proliferative effects of AMANTADIG and digitoxin on leukemia and urological cancer cell lines were analyzed using (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction viability assay.AMANTADIG and digitoxin exhibited anti-proliferative activities against the leukemia cell lines in the low nanomolar range. The prostate cancer and renal cell carcinoma cell lines were equally sensitive to AMANTADIG and digitoxin, however, the leukemia cell lines were more sensitive to both Cardenolides.The new cardenolide analog AMANTADIG appears effective in cell growth inhibition of leukemia and urological tumor cell lines.
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antiherpes activity of glucoevatromonoside a cardenolide isolated from a brazilian cultivar of digitalis lanata
Antiviral Research, 2011Co-Authors: Jessica Wildgrube Bertol, Wolfgang Kreis, Rodrigo Maia De Padua, Caroline Rigotto, C R M Barardi, Fernao Castro Braga, Claudia Maria Oliveira SimoesAbstract:Abstract Cardiac glycosides, known ligands of the sodium pump, are widely used in the treatment of heart failure, such as digoxin and digitoxin. Besides this important activity, other biological activities, such as the antiviral activity, have been described for this group. HSV are responsible for many infections of oral, ocular and genital regions. Treatment with nucleoside analogs such as acyclovir is effective in most cases; however drug-resistance may arise due to prolonged treatment mainly in immunocompromised individuals. In this study, an antiherpes screening was performed with 65 cardenolide derivatives obtained from different sources, and one natural cardenolide, glucoevatromonoside, inhibited HSV-1 and HSV-2 replication at very low concentrations. This cardenolide showed viral inhibitory effects if added up to 12 h p.i. and these effects appear to take place by the inhibition of viral proteins synthesis (ICP27, U L 42, gB, gD), the blockage of virus release and the reduction of viral cell-to-cell spread. This compound also showed synergistic antiviral effects with acyclovir and anti-Na + K + ATPase activity, suggesting that cellular electrochemical gradient alterations might be involved in the mechanism of viral inhibition. These results suggest that Cardenolides might be promising for future antiviral drug design.
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expression of 3β hsd and p5βr genes respectively coding for δ5 3β hydroxysteroid dehydrogenase and progesterone 5β reductase in leaves and cell cultures of digitalis lanata ehrh
Planta Medica, 2010Co-Authors: Mona Ernst, Frieder Mulleruri, Rodrigo Maia De Padua, Vanessa Herl, Wolfgang KreisAbstract:Plants of the genus Digitalis produce 5 beta-Cardenolides that are used in the therapy of cardiac insufficiency in humans. 3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) and progesterone 5 beta-reductase (P5 betaR) are both supposed to be important enzymes in the biosynthesis of these natural products. Activity and gene expression were demonstrated for both enzymes in cardenolide-accumulating leaves of Digitalis lanata but also in cardenolide-free permanent cell suspension cultures initiated from D. lanata leaf tissue. Enzyme activities were determined and quantified by HPLC and GC-MS methods. Expression of the respective genes, namely AY585867.1 (P5betaR gene) and DQ466890.1 (3beta-HSD gene), was made evident by real-time polymerase chain reaction (qPCR) analysis. We demonstrate for the first time that the P5betaR gene, encoding an enzyme described as a key enzyme in cardenolide biosynthesis, is also expressed in cardenolide-free tissues of cardenolide-containing plants.
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cardenolide biosynthesis in light and dark grown digitalis lanata shoot cultures
Plant Physiology and Biochemistry, 1999Co-Authors: Marina Eisenbeis, Wolfgang Kreis, Ernst ReinhardAbstract:Abstract Shoot cultures of the cardenolide-producing species Digitalis lanata Ehrh. accumulated up to 0.6 μmol Cardenolides per g dry mass when cultivated under continuous white light. After transfer to permanent dark, the cardenolide content of cultured shoots gradually decreased and reached non-detectable levels after 12 weeks. After transfer back to light conditions, Cardenolides started to accumulate and reached the levels of light-grown controls after 4 weeks. Radiolabelled pregnenolone and progesterone were incorporated into Cardenolides in both green light-grown and white dark-grown shoots. It was thus established that Cardenolides are synthesised de novo in chloroplast-free tissues without apparent cardenolide accumulation, indicating that these compounds are efficiently turned over in the dark and that tissue differentiation, but not intact chloroplasts, is essential for cardenolide formation. The time course of two late anabolic enzymes of cardenolide metabolism, acetyl-CoA:digitoxin 15′-O-acetyltransferase (DAT, EC 2.3.1.-) and UDP-glucose:digitoxin 16′-glucosyltransferase (DGT, EC 2.4.1.-) was established during transfer of shoots from light to dark and vice versa. Only DAT was affected and was not measurable any more under dark conditions. The DGT may not be down-regulated because of its important, maybe even vital, role as an enzyme providing the vacuolar storage forms of Cardenolides. Two catabolic cardenolide-specific enzymes, lanatoside 15′-O-acetylesterase (LAE, EC 3.1.1.6.) and cardenolide 16′-O-glucohydrolase I (CGH I, EC 3.2.1.21), were also investigated and it was demonstrated that CGH I is inactive in dark-grown shoots. These observations indicate that CGH I is not involved in cardenolide degradation in situ, but may instead play a role in cardenolide remetabolisation and activation after wounding or in developmental programs.
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effects of digitoxigenin digoxigenin and various cardiac glycosides on cardenolide accumulation in shoot cultures of digitalis lanata
Planta Medica, 1998Co-Authors: Christoph Theurer, Wolfgang Kreis, Ernst ReinhardAbstract:Various cardenolide genins and cardenolide glycosides were administered to light-grown and dark-grown Digitalis lanata shoot cultures to investigate conversion reactions related to the formation and rearrangement of the sugar side chain of Digitalis glycosides. Digitoxigenin was converted to digitoxigen-3-one, 3-epidigitoxigenin, and digoxigenin. In addition, various cardiac glycosides were formed, including monoglycosides with glucose, glucomethylose, fucose, and digitalose, as well as the corresponding diglycosides, all containing a terminal glucose. Digitoxosylated Cardenolides were not formed, although the light-grown shoot cultures were capable of producing these compounds. Exogenous cardenolide fucosides were not converted into cardenolide digitoxosides. Administration of evatromonoside (digitoxigenin monodigitoxoside) did not force the formation of cardenolide di- or tridigitoxosides. Our results support the hypothesis that cardenolide fucosides and digitoxosides are formed via different biosynthetic routes and that cardenolide genins can be fucosylated but not digitoxosylated, indicating that digitoxosylation may only occur at an earlier stage in the cardenolide pathway.