Progesterone Metabolism In Digitalis And Other Plants - 60 Years Research And Recent Results.

5ß-Cardenolides are pharmaceutically important metabolites of the specialized metabolism of Digitalis lanata. They were used over decades to treat cardiac insufficiency and supraventricular tachycardia. Since the 1960s, plant scientists have known that progesterone is an essential precursor of cardenolide formation. Therefore, plant progesterone biosynthesis was mainly analyzed in species of the cardenolide-containing genus Digitalis during the following decades. Today, Digitalis enzymes catalyzing the main steps of progesterone biosynthesis are known. Most of them were found in a broad range of organisms. This review will summarize the findings of 60 years of research on plant progesterone metabolism with a particular focus on the recent results in Digitalis lanata and other plants.

Late-instar monarch caterpillars sabotage milkweed to acquire toxins, not to disarm plant defence.

Sabotaging milkweed by monarch caterpillars (Danaus plexippus) is a famous textbook example of disarming plant defence. By severing leaf veins, monarchs are thought to prevent the flow of toxic latex to their feeding site. Here, we show that sabotaging by monarch caterpillars is not only an avoidance strategy. While young caterpillars appear to avoid latex, late-instar caterpillars actively ingest exuding latex, presumably to increase sequestration of cardenolides used for defence against predators. Comparisons with caterpillars of the related but non-sequestering common crow butterfly (Euploea core) revealed three lines of evidence supporting our hypothesis. First, monarch caterpillars sabotage inconsistently and therefore the behaviour is not obligatory to feed on milkweed, whereas sabotaging precedes each feeding event in Euploea caterpillars. Second, monarch caterpillars shift their behaviour from latex avoidance in younger to eager drinking in later stages, whereas Euploea caterpillars consistently avoid latex and spit it out during sabotaging. Third, monarchs reared on detached leaves without latex sequestered more cardenolides when caterpillars imbibed latex offered with a pipette. Thus, we conclude that monarch caterpillars have transformed the ancestral 'sabotage to avoid' strategy into a 'sabotage to consume' strategy, implying a novel behavioural adaptation to increase sequestration of cardenolides for defence.

Mixtures of Milkweed Cardenolides Protect Monarch Butterflies against Parasites.

Plants have evolved a diverse arsenal of defensive secondary metabolites in their evolutionary arms race with insect herbivores. In addition to the bottom-up forces created by plant chemicals, herbivores face top-down pressure from natural enemies, such as predators, parasitoids and parasites. This has led to the evolution of specialist herbivores that do not only tolerate plant secondary metabolites but even use them to fight natural enemies. Monarch butterflies (Danaus plexippus) are known for their use of milkweed chemicals (cardenolides) as protection against vertebrate predators. Recent studies have shown that milkweeds with high cardenolide concentrations can also provide protection against a virulent protozoan parasite. However, whether cardenolides are directly responsible for these effects, and whether individual cardenolides or mixtures of these chemicals are needed to reduce infection, remains unknown. We fed monarch larvae the four most abundant cardenolides found in the anti-parasitic-milkweed Asclepias curassavica at varying concentrations and compositions to determine which provided the highest resistance to parasite infection. Measuring infection rates and infection intensities, we found that resistance is dependent on both concentration and composition of cardenolides, with mixtures of cardenolides performing significantly better than individual compounds, even when mixtures included lower concentrations of individual compounds. These results suggest that cardenolides function synergistically to provide resistance against parasite infection and help explain why only milkweed species that produce diverse cardenolide compounds provide measurable parasite resistance. More broadly, our results suggest that herbivores can benefit from consuming plants with diverse defensive chemical compounds through release from parasitism.

The price of defence: toxins, visual signals and oxidative state in an aposematic butterfly.

In a variety of aposematic species, the conspicuousness of an individual's warning signal and the quantity of its chemical defence are positively correlated. This apparent honest signalling is predicted by resource competition models which assume that the production and maintenance of aposematic defences compete for access to antioxidant molecules that have dual functions as pigments and in protecting against oxidative damage. To test for such trade-offs, we raised monarch butterflies (Danaus plexippus) on different species of their milkweed host plants (Apocynaceae) that vary in quantities of cardenolides to test whether (i) the sequestration of cardenolides as a secondary defence is associated with costs in the form of oxidative lipid damage and reduced antioxidant defences; and (ii) lower oxidative state is associated with a reduced capacity to produce aposematic displays. In male monarchs conspicuousness was explained by an interaction between oxidative damage and sequestration: males with high levels of oxidative damage became less conspicuous with increased sequestration of cardenolides, whereas those with low oxidative damage became more conspicuous with increased levels of cardenolides. There was no significant effect of oxidative damage or concentration of sequestered cardenolides on female conspicuousness. Our results demonstrate a physiological linkage between the production of coloration and oxidative state, and differential costs of sequestration and signalling in monarch butterflies.

Molecular Modes of Action of an Aqueous Nerium oleander Extract in Cancer Cells In Vitro and In Vivo.

Cancer drug resistance remains a major obstacle in clinical oncology. As most anticancer drugs are of natural origin, we investigated the anticancer potential of a standardized cold-water leaf extract from Nerium oleander L., termed Breastin. The phytochemical characterization by nuclear magnetic resonance spectroscopy (NMR) and low- and high-resolution mass spectrometry revealed several monoglycosidic cardenolides as major constituents (adynerin, neritaloside, odoroside A, odoroside H, oleandrin, and vanderoside). Breastin inhibited the growth of 14 cell lines from hematopoietic tumors and 5 of 6 carcinomas. Remarkably, the cellular responsiveness of odoroside H and neritaloside was not correlated with all other classical drug resistance mechanisms, i.e., ATP-binding cassette transporters (ABCB1, ABCB5, ABCC1, ABCG2), oncogenes (EGFR, RAS), tumor suppressors (TP53, WT1), and others (GSTP1, HSP90, proliferation rate), in 59 tumor cell lines of the National Cancer Institute (NCI, USA), indicating that Breastin may indeed bypass drug resistance. COMPARE analyses with 153 anticancer agents in 74 tumor cell lines of the Oncotest panel revealed frequent correlations of Breastin with mitosis-inhibiting drugs. Using tubulin-GFP-transfected U2OS cells and confocal microscopy, it was found that the microtubule-disturbing effect of Breastin was comparable to that of the tubulin-depolymerizing drug paclitaxel. This result was verified by a tubulin polymerization assay in vitro and molecular docking in silico. Proteome profiling of 3171 proteins in the NCI panel revealed protein subsets whose expression significantly correlated with cellular responsiveness to odoroside H and neritaloside, indicating that protein expression profiles can be identified to predict the sensitivity or resistance of tumor cells to Breastin constituents. Breastin moderately inhibited breast cancer xenograft tumors in vivo. Remarkably, in contrast to what was observed with paclitaxel monotherapy, the combination of paclitaxel and Breastin prevented tumor relapse, indicating Breastin's potential for drug combination regimens.

Effects of Lipophilicity and Structural Features on the Antiherpes Activity of Digitalis Cardenolides and Derivatives.

There is growing interest in exploring Digitalis cardenolides as potential antiviral agents. Hence, we herein investigated the influence of structural features and lipophilicity on the antiherpes activity of 65 natural and semisynthetic cardenolides assayed in vitro against HSV-1. The presence of an α,ß-unsaturated lactone ring at C-17, a ß-hydroxy group at C-14 and C-3ß-OR substituents were considered essential requirements for this biological activity. Glycosides were more active than their genins, especially monoglycosides containing a rhamnose residue. The activity enhanced in derivatives bearing an aldehyde group at C-19 instead of a methyl group, whereas inserting a C-5ß-OH improved the antiherpes effect significantly. The cardenolides lipophilicity was accessed by measuring experimentally their log P values (n-octanol-water partition coefficient) and disclosed a range of lipophilicity (log P 0.75±0.25) associated with the optimal antiherpes activity. In silico studies were carried out and resulted in the establishment of two predictive models potentially useful to identify and/or optimize novel antiherpes cardenolides. The effectiveness of the models was confirmed by retrospective analysis of the studied compounds. This is the first SAR study addressing the antiherpes activity of cardenolides. The developed computational models were able to predict the active cardenolides and their log P values.

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.

Antiproliferative activity of cardenolides on cell line A549: structure-activity relationship analysis.

Since the beginning, natural products have represented an important source of bioactive molecules for cancer treatment. Among them, cardenolides attract the attention of different research groups due to their cardiotonic and antitumor activity. The observed biological activity is closely related to their Na+/K+-ATPase inhibition potency. Currently, the discovery of new compounds against cancer is an urgent need in modern pharmaceutical research. Thus, the aim of this work is to determine the physicochemical properties and substituent effects that module the antiproliferative activity of cardenolides on the human lung cancer cell line A549. We build and curate a library with results obtained from literature; molecular descriptors were calculated in PaDEL software, and SAR/QSAR analysis was performed. The SAR results showed that cardenolides were sensitive to modifications in C and D steroidal ring and required substituent groups with the function of hydrogen bond acceptor at the C3 position. QSAR models to doubly linked-type cardenolides indicated that properties as lipoaffinity and atoms with the capacity to be hydrogen bond acceptors are involved in the increment of antiproliferative activity on A549 cell line. In contrast, the presence and position of very electro-negative atoms on the molecule decreased the antiproliferative effect on A549 cells. These results suggest that the antiproliferative capacity of cardenolides on the cell line A549 is strongly related to substituent groups on the C3 position, which must not be carbohydrate. Additionally, the steroidal rings C and D must remain without modifications.

New Cardenolides from Biotransformation of Gitoxigenin by the Endophytic Fungus Alternaria eureka 1E1BL1: Characterization and Cytotoxic Activities.

Microbial biotransformation is an important tool in drug discovery and for metabolism studies. To expand our bioactive natural product library via modification and to identify possible mammalian metabolites, a cytotoxic cardenolide (gitoxigenin) was biotransformed using the endophytic fungus Alternaria eureka 1E1BL1. Initially, oleandrin was isolated from the dried leaves of Nerium oleander L. and subjected to an acid-catalysed hydrolysis to obtain the substrate gitoxigenin (yield; ~25%). After 21 days of incubation, five new cardenolides 1, 3, 4, 6, and 8 and three previously- identified compounds 2, 5 and 7 were isolated using chromatographic methods. Structural elucidations were accomplished through 1D/2D NMR, HR-ESI-MS and FT-IR analysis. A. eureka catalyzed oxygenation, oxidation, epimerization and dimethyl acetal formation reactions on the substrate. Cytotoxicity of the metabolites were evaluated using MTT cell viability method, whereas doxorubicin and oleandrin were used as positive controls. Biotransformation products displayed less cytotoxicity than the substrate. The new metabolite 8 exhibited the highest activity with IC50 values of 8.25, 1.95 and 3.4 µM against A549, PANC-1 and MIA PaCa-2 cells, respectively, without causing toxicity on healthy cell lines (MRC-5 and HEK-293) up to concentration of 10 µM. Our results suggest that A. eureka is an effective biocatalyst for modifying cardenolide-type secondary metabolites.

ABCB transporters in a leaf beetle respond to sequestered plant toxins.

Phytophagous insects can tolerate and detoxify toxic compounds present in their host plants and have evolved intricate adaptations to this end. Some insects even sequester the toxins for their defence. This necessitates specific mechanisms, especially carrier proteins that regulate uptake and transport to specific storage sites or protect sensitive tissues from noxious compounds. We identified three ATP-binding cassette subfamily B (ABCB) transporters from the transcriptome of the cardenolide-sequestering leaf beetle Chrysochus auratus and analysed their functional role in the sequestration process. These were heterologously expressed and tested for their ability to interact with various potential substrates: verapamil (standard ABCB substrate), the cardenolides digoxin (commonly used), cymarin (present in the species's host plant) and calotropin (present in the ancestral host plants). Verapamil stimulated all three ABCBs and each was activated by at least one cardenolide, however, they differed as to which they were activated by. While the expression of the most versatile transporter fits with a protective role in the blood-brain barrier, the one specific for cymarin shows an extreme abundance in the elytra, coinciding with the location of the defensive glands. Our data thus suggest a key role of ABCBs in the transport network needed for cardenolide sequestration.

Semisynthetic Cardenolides Acting as Antiviral Inhibitors of Influenza A Virus Replication by Preventing Polymerase Complex Formation.

Influenza virus infections represent a major public health issue by causing annual epidemics and occasional pandemics that affect thousands of people worldwide. Vaccination is the main prophylaxis to prevent these epidemics/pandemics, although the effectiveness of licensed vaccines is rather limited due to the constant mutations of influenza virus antigenic characteristics. The available anti-influenza drugs are still restricted and there is an increasing viral resistance to these compounds, thus highlighting the need for research and development of new antiviral drugs. In this work, two semisynthetic derivatives of digitoxigenin, namely C10 (3ß-((N-(2-hydroxyethyl)aminoacetyl)amino-3-deoxydigitoxigenin) and C11 (3ß-(hydroxyacetyl)amino-3-deoxydigitoxigenin), showed anti-influenza A virus activity by affecting the expression of viral proteins at the early and late stages of replication cycle, and altering the transcription and synthesis of new viral proteins, thereby inhibiting the formation of new virions. Such antiviral action occurred due to the interference in the assembly of viral polymerase, resulting in an impaired polymerase activity and, therefore, reducing viral replication. Confirming the in vitro results, a clinically relevant ex vivo model of influenza virus infection of human tumor-free lung tissues corroborated the potential of these compounds, especially C10, to completely abrogate influenza A virus replication at the highest concentration tested (2.0 µM). Taken together, these promising results demonstrated that C10 and C11 can be considered as potential new anti-influenza drug candidates.

The demographic effects of functional traits: an integral projection model approach reveals population-level consequences of reproduction-defence trade-offs.

Quantitatively linking individual variation in functional traits to demography is a necessary step to advance our understanding of trait-based ecological processes. We constructed a population model for Asclepias syriaca to identify how functional traits affect vital rates and population growth and whether trade-offs in chemical defence and demography alter population growth. Plants with higher foliar cardenolides had lower fibre, cellulose and lignin levels, as well as decreased sexual and clonal reproduction. Average cardenolide concentrations had the strongest effect on population growth. In both the sexual and clonal pathway, the trade-off between reproduction and defence affected population growth. We found that both increasing the mean of the distribution of individual plant values for cardenolides and herbivory decreased population growth. However, increasing the variance in both defence and herbivory increased population growth. Functional traits can impact population growth and quantifying individual-level variation in traits should be included in assessments of population-level processes.

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.

Elevated atmospheric concentrations of carbon dioxide reduce monarch tolerance and increase parasite virulence by altering the medicinal properties of milkweeds.

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.

The function and evolutionary significance of a triplicated Na,K-ATPase gene in a toxin-specialized insect.

BACKGROUND: The Na,K-ATPase is a vital animal cell-membrane protein that maintains the cell's resting potential, among other functions. Cardenolides, a group of potent plant toxins, bind to and inhibit this pump. The gene encoding the α-subunit of the pump has undergone duplication events in some insect species known to feed on plants containing cardenolides. Here we test the function of these duplicated gene copies in the cardenolide-adapted milkweed bug, Oncopeltus fasciatus, which has three known copies of the gene: α1A, α1B and α1C. RESULTS: Using RT-qPCR analyses we demonstrate that the α1C is highly expressed in neural tissue, where the pump is generally thought to be most important for neuron excitability. With the use of in vivo RNAi in adult bugs we found that α1C knockdowns suffered high mortality, where as α1A and α1B did not, supporting that α1C is most important for effective ion pumping. Next we show a role for α1A and α1B in the handling of cardenolides: expression results find that both copies are primarily expressed in the Malpighian tubules, the primary insect organ responsible for excretion, and when we injected either α1A or α1B knockdowns with cardenolides this proved fatal (whereas not in controls). CONCLUSIONS: These results show that the Na,K-ATPα gene-copies have taken on diverse functions. Having multiple copies of this gene appears to have allowed the newly arisen duplicates to specialize on resistance to cardenolides, whereas the ancestral copy of the pump remains comparatively sensitive, but acts as a more efficient ion carrier. Interestingly both the α1A and α1B were required for cardenolide handling, suggesting that these two copies have separate and vital functions. Gene duplications of the Na,K-ATPase thus represent an excellent example of subfunctionalization in response to a new environmental challenge.

Plant chemical defense indirectly mediates aphid performance via interactions with tending ants.

The benefits of mutualistic interactions are often highly context dependent. We studied the interaction between the milkweed aphid Aphis asclepiadis and a tending ant, Formica podzolica. Although this interaction is generally considered beneficial, variation in plant genotype may alter it from mutualistic to antagonistic. Here we link the shift in strength and relative benefit of the ant-aphid interaction to plant genotypic variation in the production of cardenolides, a class of toxic defensive chemicals. In a field experiment with highly variable genotypes of the common milkweed (Asclepias syriaca), we show that plant cardenolides, especially polar forms, are ingested by aphids and excreted in honeydew proportionally to plant concentrations without directly affecting aphid performance. Ants consume honeydew, and aphids that excreted high amounts of cardenolides received fewer ant visits, which in turn reduced aphid survival. On at least some plant genotypes, aphid numbers per plant were reduced in the presence of ants to levels lower than in corresponding ant-exclusion treatments, suggesting antagonistic ant behavior. Although cardenolides appear ineffective as direct plant defenses against aphids, the multi-trophic context reveals an ant-mediated negative indirect effect on aphid performance and population dynamics.

Cytotoxic effects of the cardenolide convallatoxin and its Na,K-ATPase regulation.

Cardenolides are cardiac glycosides, mostly obtained from natural sources. They are well known for their inhibitory action on the Na,K-ATPase, an effect that regulates cardiovascular alterations such as congestive heart failure and atrial arrhythmias. In recent years, they have also sparked new interest in their anticancer potential. In the present study, the cytotoxic effects of the natural cardenolide convallatoxin (CON) were evaluated on non-small cell lung cancer (A549 cells). It was found that CON induced cytostatic and cytotoxic effects in A549 cells, showing essentially apoptotic cell death, as detected by annexin V-propidium iodide double-staining, as well as changes in cell form. In addition, it prompted cell cycle arrest in G2/M and reduced cyclin B1 expression. This compound also increased the number of cells in subG1 in a concentration- and time-dependent manner. At a long term, the reduction of cumulative population doubling was shown along with an increase of ß-galactosidase positive cells and larger nucleus, indicative of senescence. Subsequently, CON inhibited the Na,K-ATPase in A549 cells at nM concentrations. Interestingly, at the same concentrations, CON was unable to directly inhibit the Na,K-ATPase, either in pig kidney or in red blood cells. Additionally, results of docking calculations showed that CON binds with high efficiency to the Na,K-ATPase. Taken together, our data highlight the potent anticancer effects of CON in A549 cells, and their possible link with non-classical inhibition of Na,K-ATPase.

On the Many Actions of Ouabain: Pro-Cystogenic Effects in Autosomal Dominant Polycystic Kidney Disease.

Ouabain and other cardenolides are steroidal compounds originally discovered in plants. Cardenolides were first used as poisons, but after finding their beneficial cardiotonic effects, they were rapidly included in the medical pharmacopeia. The use of cardenolides to treat congestive heart failure remained empirical for centuries and only relatively recently, their mechanisms of action became better understood. A breakthrough came with the discovery that ouabain and other cardenolides exist as endogenous compounds that circulate in the bloodstream of mammals. This elevated these compounds to the category of hormones and opened new lines of investigation directed to further study their biological role. Another important discovery was the finding that the effect of ouabain was mediated not only by inhibition of the activity of the Na,K-ATPase (NKA), but by the unexpected role of NKA as a receptor and a signal transducer, which activates a complex cascade of intracellular second messengers in the cell. This broadened the interest for ouabain and showed that it exerts actions that go beyond its cardiotonic effect. It is now clear that ouabain regulates multiple cell functions, including cell proliferation and hypertrophy, apoptosis, cell adhesion, cell migration, and cell metabolism in a cell and tissue type specific manner. This review article focuses on the cardenolide ouabain and discusses its various in vitro and in vivo effects, its role as an endogenous compound, its mechanisms of action, and its potential use as a therapeutic agent; placing especial emphasis on our findings of ouabain as a pro-cystogenic agent in autosomal dominant polycystic kidney disease (ADPKD).

Different rates of defense evolution and niche preferences in clonal and nonclonal milkweeds (Asclepias spp.).

Given the dual role of many plant traits to tolerate both herbivore attack and abiotic stress, the climatic niche of a species should be integrated into the study of plant defense strategies. Here we investigate the impact of plant reproductive strategy and components of species' climatic niche on the rate of chemical defense evolution in the milkweeds using a common garden experiment of 49 species. We found that across Asclepias species, clonal reproduction repeatedly evolved in lower temperature conditions, in species generally producing low concentrations of a toxic defense (cardenolides). Additionally, we found that rates of cardenolide evolution were lower for clonal than for nonclonal species. We thus conclude that because the clonal strategy is based on survival, long generation times, and is associated with tolerance of herbivory, it may be an alternative to toxicity in colder ecosystems. Taken together, these results indicate that the rate of chemical defense evolution is influenced by the intersection of life-history strategy and climatic niches into which plants radiate.

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.