Escalation by duplication: Milkweed bug trumps Monarch butterfly.

The iconic Monarch butterfly is probably the best-known example of chemical defence against predation, as pictures of vomiting naive blue jays in countless textbooks vividly illustrate. Larvae of the butterfly take up toxic cardiac glycosides from their milkweed hostplants and carry them over to the adult stage. These compounds (cardiotonic steroids, including cardenolides and bufadienolides) inhibit the animal transmembrane sodium-potassium ATPase (Na,K-ATPase), but the Monarch enzyme resists this inhibition thanks to amino acid substitutions in its catalytic alpha-subunit. Some birds also have substitutions and can feast on cardiac glycoside-sequestering insects with impunity. A flurry of recent work has shown how the alpha-subunit gene has been duplicated multiple times in separate insect lineages specializing in cardiac glycoside-producing plants. In this issue of Molecular Ecology, Herbertz et al. toss the beta-subunit into the mix, by expressing all nine combinations of three alpha- and three beta-subunits of the milkweed bug Na,K-ATPase and testing their response to a cardenolide from the hostplant. The findings suggest that the diversification and subfunctionalization of genes allow milkweed bugs to balance trade-offs between resistance towards sequestered host plant toxins that protect the bugs from predators, and physiological costs in terms of Na,K-ATPase activity.

Preliminary investigation of the safe dose of oleandrin when administered orally to Beagles.

OBJECTIVE: The purpose of the study reported herein was to determine the dose of oleander extract and oleandrin (the key pharmacologically active constituent) that could be safely administered PO to dogs. ANIMALS: 42 purebred Beagle dogs were used to study an extract of Nerium oleander. METHODS: 3 studies were performed in 42 purebred young adult (ages 12 months or older) Beagle dogs using a supercritical fluid extract of N oleander leaves. The first study was an 8-day initial dose-ranging study in 2 dogs, a second 7-day repeat-dosing study was performed in 4 dogs, and the final study was performed in 32 dogs where test subjects were given extract or placebo once daily for 28 consecutive days via oral (gavage) administration followed by a 14-day recovery period. RESULTS: At 2.3 µg/kg of oleandrin, there were no observable adverse effects during the duration of the study. Adverse effects were not seen until doses exceeded 6.9 µg/kg of oleandrin, at which time mild, reversible clinical signs were noted. However, a dose > 460 µg of oleandrin/kg was fatal in 1 of 2 dogs in this study. CLINICAL RELEVANCE: The studies reported here, taken in totality, suggest that doses exceeding 6.9 µg/kg of oleandrin may be associated with cardiac abnormalities. An estimated no treatment effective adverse event oral dose of oleandrin appears to be 4.6 µg of oleandrin/kg. Higher doses may be tolerable but should be used with appropriate monitoring.

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.

Nectar cardenolides and floral volatiles mediate a specialized wasp pollination system.

Specialization in plant pollination systems can arise from traits that function as filters of flower visitors. This may involve chemical traits such as floral volatiles that selectively attract favoured visitors and non-volatile nectar constituents that selectively deter disfavoured visitors through taste or longer-term toxic effects or both. We explored the functions of floral chemical traits in the African milkweed Gomphocarpus physocarpus, which is pollinated almost exclusively by vespid wasps, despite having nectar that is highly accessible to other insects such as honeybees. We demonstrated that the nectar of wasp-pollinated G. physocarpus contains cardenolides that had greater toxic effects on Apis mellifera honeybees than on Vespula germanica wasps, and also reduced feeding rates by honeybees. Behavioural experiments using natural compositions of nectar compounds showed that these interactions are mediated by non-volatile nectar chemistry. We also identified volatile compounds with acetic acid as a main component in the floral scent of G. physocarpus that elicited electrophysiological responses in wasp antennae. Mixtures of these compounds were behaviourally effective for attraction of V. germanica wasps. The results show the importance of both volatile and non-volatile chemical traits as filters that lead to specialization in plant pollination systems.

Testing the selective sequestration hypothesis: Monarch butterflies preferentially sequester plant defences that are less toxic to themselves while maintaining potency to others.

Herbivores that sequester toxins are thought to have cracked the code of plant defences. Nonetheless, coevolutionary theory predicts that plants should evolve toxic variants that also negatively impact specialists. We propose and test the selective sequestration hypothesis, that specialists preferentially sequester compounds that are less toxic to themselves while maintaining toxicity to enemies. Using chemically distinct plants, we show that monarch butterflies sequester only a subset of cardenolides from milkweed leaves that are less potent against their target enzyme (Na+ /K+ -ATPase) compared to several dominant cardenolides from leaves. However, sequestered compounds remain highly potent against sensitive Na+ /K+ -ATPases found in most predators. We confirmed this differential toxicity with mixtures of purified cardenolides from leaves and butterflies. The genetic basis of monarch adaptation to sequestered cardenolides was also confirmed with transgenic Drosophila that were CRISPR-edited with the monarch's Na+ /K+ -ATPase. Thus, the monarch's selective sequestration appears to reduce self-harm while maintaining protection from enemies.

Fecal Deployment: An Alternative Way of Defensive Host Plant Cardenolide Use by Lilioceris merdigera Larvae.

The brilliant red Lilioceris merdigera (Coleoptera, Chrysomelidae) can spend its entire life cycle on the cardenolide-containing plant Convallaria majalis (lily of the valley) and forms stable populations on this host. Yet, in contrast to many other insects on cardenolide-containing plants L. merdigera does not sequester these plant toxins in the body but rather both adult beetles and larvae eliminate ingested cardenolides with the feces. Tracer feeding experiments showed that this holds true for radioactively labeled ouabain and digoxin, a highly polar and a rather apolar cardenolide. Both compounds or their derivatives are incorporated in the fecal shields of the larvae. The apolar digoxin, but not the polar ouabain, showed a deterrent effect on the generalist predatory ant Myrmica rubra, which occurs in the habitat of L. merdigera. The deterrent effect was detected for digoxin both in choice and feeding time assays. In a predator choice assay, a fecal shield derived from a diet of cardenolide-containing C. majalis offered L. merdigera larvae better protection from M. rubra than one derived from non-cardenolide Allium schoenoprasum (chives) or no fecal shield at all. Thus, we here present data suggesting a new way how insects may gain protection by feeding on cardenolide-containing plants.

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.

Tissue and toxin-specific divergent evolution in plant defense Evolución divergente específica de tejido y toxina en defensa de plantas.

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

Bioassay-Guided Fractionation and Biological Activity of Cardenolides from Streptocaulon juventas.

The discovery that Na/K-ATPase acts as a signal transducer led us to investigate the structural diversity of cardiotonic steroids and study their ligand effects. By applying Na/K-ATPase activity assay-guided fractionation, we isolated a total of 20 cardiotonic steroids from Streptocaulon juventas, including an undescribed juventasoside B (10: ) and 19 known cardiotonic steroids. Their structures have been elucidated. Using our platform of purified Na/K-ATPase and an LLC-PK1 cell model, we found that 10: , at a concentration that induces less than 10% Na/K-ATPase inhibition, can stimulate the Na/K-ATPase/Src receptor complex and selectively activate downstream pathways, ultimately altering prostate cancer cell growth. By assessing the ligand effect of the isolated cardiotonic steroids, we found that the regulation of cell viability by the isolated cardiotonic steroids was not associated with their inhibitory potencies against Na/K-ATPase activity but reflected their ligand-binding affinity to the Na/K-ATPase receptor. Based on this discovery, we identified a unique active cardiotonic steroid, digitoxigenin (1: ), and verified that it can protect LLC-PK1 cells from hypoxic injury, implicating its potential use in ischemia/reperfusion injury and inducing collagen synthesis in primary human dermal fibroblast cells, and implicating that compound 2: is the molecular basis of the wound healing activity of S. juventas.

Promiscuous CYP87A enzyme activity initiates cardenolide biosynthesis in plants.

Cardenolides are specialized, steroidal metabolites produced in a wide array of plant families1,2. Cardenolides play protective roles in plants, but these molecules, including digoxin from foxglove (Digitalis spp.), are better known for treatment of congenital heart failure, atrial arrhythmia, various cancers and other chronic diseases3-9. However, it is still unknown how plants synthesize 'high-value', complex cardenolide structures from, presumably, a sterol precursor. Here we identify two cytochrome P450, family 87, subfamily A (CYP87A) enzymes that act on both cholesterol and phytosterols (campesterol and ß-sitosterol) to form pregnenolone, the first committed step in cardenolide biosynthesis in the two phylogenetically distant plants Digitalis purpurea and Calotropis procera. Arabidopsis plants overexpressing these CYP87A enzymes ectopically accumulated pregnenolone, whereas silencing of CYP87A in D. purpurea leaves by RNA interference resulted in substantial reduction of pregnenolone and cardenolides. Our work uncovers the key entry point to the cardenolide pathway, and expands the toolbox for sustainable production of high-value plant steroids via synthetic biology.

Endogenous Digitalis-like Factors as a Key Molecule in the Pathophysiology of Pregnancy-Induced Hypertension and a Potential Therapeutic Target in Preeclampsia.

Preeclampsia (PE), the most severe presentation of hypertensive disorders of pregnancy, is the major cause of morbidity and mortality linked to pregnancy, affecting both mother and fetus. Despite advances in prophylaxis and managing PE, delivery of the fetus remains the only causative treatment available. Focus on complex pathophysiology brought the potential for new treatment options, and more conservative options allowing reduction of feto-maternal complications and sequelae are being investigated. Endogenous digitalis-like factors, which have been linked to the pathogenesis of preeclampsia since the mid-1980s, have been shown to play a role in the pathogenesis of various cardiovascular diseases, including congestive heart failure and chronic renal disease. Elevated levels of EDLF have been described in pregnancy complicated by hypertensive disorders and are currently being investigated as a therapeutic target in the context of a possible breakthrough in managing preeclampsia. This review summarizes mechanisms implicating EDLFs in the pathogenesis of preeclampsia and evidence for their potential role in treating this doubly life-threatening disease.

A nutrition-defence trade-off drives diet choice in a toxic plant generalist.

Plant toxicity shapes the dietary choices of herbivores. Especially when herbivores sequester plant toxins, they may experience a trade-off between gaining protection from natural enemies and avoiding toxicity. The availability of toxins for sequestration may additionally trade off with the nutritional quality of a potential food source for sequestering herbivores. We hypothesized that diet mixing might allow a sequestering herbivore to balance nutrition and defence (via sequestration of plant toxins). Accordingly, here we address diet mixing and sequestration of large milkweed bugs (Oncopeltus fasciatus) when they have differential access to toxins (cardenolides) in their diet. In the absence of toxins from a preferred food (milkweed seeds), large milkweed bugs fed on nutritionally adequate non-toxic seeds, but supplemented their diet by feeding on nutritionally poor, but cardenolide-rich milkweed leaf and stem tissues. This dietary shift corresponded to reduced insect growth but facilitated sequestration of defensive toxins. Plant production of cardenolides was also substantially induced by bug feeding on leaf and stem tissues, perhaps benefitting this cardenolide-resistant herbivore. Thus, sequestration appears to drive diet mixing in this toxic plant generalist, even at the cost of feeding on nutritionally poor plant tissue.

The cardiac glycoside ZINC253504760 induces parthanatos-type cell death and G2/M arrest via downregulation of MEK1/2 phosphorylation in leukemia cells.

Overcoming multidrug resistance (MDR) represents a major obstacle in cancer chemotherapy. Cardiac glycosides (CGs) are efficient in the treatment of heart failure and recently emerged in a new role in the treatment of cancer. ZINC253504760, a synthetic cardenolide that is structurally similar to well-known GCs, digitoxin and digoxin, has not been investigated yet. This study aims to investigate the cytotoxicity of ZINC253504760 on MDR cell lines and its molecular mode of action for cancer treatment. Four drug-resistant cell lines (P-glycoprotein-, ABCB5-, and EGFR-overexpressing cells, and TP53-knockout cells) did not show cross-resistance to ZINC253504760 except BCRP-overexpressing cells. Transcriptomic profiling indicated that cell death and survival as well as cell cycle (G2/M damage) were the top cellular functions affected by ZINC253504760 in CCRF-CEM cells, while CDK1 was linked with the downregulation of MEK and ERK. With flow cytometry, ZINC253504760 induced G2/M phase arrest. Interestingly, ZINC253504760 induced a novel state-of-the-art mode of cell death (parthanatos) through PARP and PAR overexpression as shown by western blotting, apoptosis-inducing factor (AIF) translocation by immunofluorescence, DNA damage by comet assay, and mitochondrial membrane potential collapse by flow cytometry. These results were ROS-independent. Furthermore, ZINC253504760 is an ATP-competitive MEK inhibitor evidenced by its interaction with the MEK phosphorylation site as shown by molecular docking in silico and binding to recombinant MEK by microscale thermophoresis in vitro. To the best of our knowledge, this is the first time to describe a cardenolide that induces parthanatos in leukemia cells, which may help to improve efforts to overcome drug resistance in cancer. A cardiac glycoside compound ZINC253504760 displayed cytotoxicity against different multidrug-resistant cell lines. ZINC253504760 exhibited cytotoxicity in CCRF-CEM leukemia cells by predominantly inducing a new mode of cell death (parthanatos). ZINC253504760 downregulated MEK1/2 phosphorylation and further affected ERK activation, which induced G2/M phase arrest.

Overexpression and RNAi-mediated Knockdown of Two 3ß-hydroxy-Δ5-steroid dehydrogenase Genes in Digitalis lanata Shoot Cultures Reveal Their Role in Cardenolide Biosynthesis.

3ß-hydroxy-Δ5-steroid dehydrogenases (3ßHSDs) are supposed to be involved in 5ß-cardenolide biosynthesis. Here, a novel 3ßHSD (Dl3ßHSD2) was isolated from Digitalis lanata shoot cultures and expressed in E. coli. Recombinant Dl3ßHSD1 and Dl3ßHSD2 shared 70% amino acid identity, reduced various 3-oxopregnanes and oxidised 3-hydroxypregnanes, but only rDl3ßHSD2 converted small ketones and secondary alcohols efficiently. To explain these differences in substrate specificity, we established homology models using borneol dehydrogenase of Salvia rosmarinus (6zyz) as the template. Hydrophobicity and amino acid residues in the binding pocket may explain the difference in enzyme activities and substrate preferences. Compared to Dl3ßHSD1, Dl3ßHSD2 is weakly expressed in D. lanata shoots. High constitutive expression of Dl3ßHSDs was realised by Agrobacterium-mediated transfer of Dl3ßHSD genes fused to the CaMV-35S promotor into the genome of D. lanata wild type shoot cultures. Transformed shoots (35S:Dl3ßHSD1 and 35S:Dl3ßHSD2) accumulated less cardenolides than controls. The levels of reduced glutathione (GSH), which is known to inhibit cardenolide formation, were higher in the 35S:Dl3ßHSD1 lines than in the controls. In the 35S:Dl3ßHSD1 lines cardenolide levels were restored after adding of the substrate pregnane-3,20-dione in combination with buthionine-sulfoximine (BSO), an inhibitor of GSH formation. RNAi-mediated knockdown of the Dl3ßHSD1 yielded several shoot culture lines with strongly reduced cardenolide levels. In these lines, cardenolide biosynthesis was fully restored after addition of the downstream precursor pregnan-3ß-ol-20-one, whereas upstream precursors such as progesterone had no effect, indicating that no shunt pathway could overcome the Dl3ßHSD1 knockdown. These results can be taken as the first direct proof that Dl3ßHSD1 is indeed involved in 5ß-cardenolide biosynthesis.

Tissue-specific plant toxins and adaptation in a specialist root herbivore.

In coevolution between plants and insects, reciprocal selection often leads to phenotype matching between chemical defense and herbivore offense. Nonetheless, it is not well understood whether distinct plant parts are differentially defended and how herbivores adapted to those parts cope with tissue-specific defense. Milkweed plants produce a diversity of cardenolide toxins and specialist herbivores have substitutions in their target enzyme (Na+/K+-ATPase), each playing a central role in milkweed-insect coevolution. The four-eyed milkweed beetle (Tetraopes tetrophthalmus) is an abundant toxin-sequestering herbivore that feeds exclusively on milkweed roots as larvae and less so on milkweed leaves as adults. Accordingly, we tested the tolerance of this beetle's Na+/K+-ATPase to cardenolide extracts from roots versus leaves of its main host (Asclepias syriaca), along with sequestered cardenolides from beetle tissues. We additionally purified and tested the inhibitory activity of dominant cardenolides from roots (syrioside) and leaves (glycosylated aspecioside). Tetraopes' enzyme was threefold more tolerant of root extracts and syrioside than leaf cardenolides. Nonetheless, beetle-sequestered cardenolides were more potent than those in roots, suggesting selective uptake or dependence on compartmentalization of toxins away from the beetle's enzymatic target. Because Tetraopes has two functionally validated amino acid substitutions in its Na+/K+-ATPase compared to the ancestral form in other insects, we compared its cardenolide tolerance to that of wild-type Drosophila and CRISPR-edited Drosophila with Tetraopes' Na+/K+-ATPase genotype. Those two amino acid substitutions accounted for >50% of Tetraopes' enhanced enzymatic tolerance of cardenolides. Thus, milkweed's tissue-specific expression of root toxins is matched by physiological adaptations in its specialist root herbivore.

Spatial metabolomics reveal divergent cardenolide processing in the monarch (Danaus plexippus) and the common crow butterfly (Euploea core).

Although being famous for sequestering milkweed cardenolides, the mechanism of sequestration and where cardenolides are localized in caterpillars of the monarch butterfly (Danaus plexippus, Lepidoptera: Danaini) is still unknown. While monarchs tolerate cardenolides by a resistant Na+ /K+ -ATPase, it is unclear how closely related species such as the nonsequestering common crow butterfly (Euploea core, Lepidoptera: Danaini) cope with these toxins. Using novel atmospheric-pressure scanning microprobe matrix-assisted laser/desorption ionization mass spectrometry imaging, we compared the distribution of cardenolides in caterpillars of D. plexippus and E. core. Specifically, we tested at which physiological scale quantitative differences between both species are mediated and how cardenolides distribute across body tissues. Whereas D. plexippus sequestered most cardenolides from milkweed (Asclepias curassavica), no cardenolides were found in the tissues of E. core. Remarkably, quantitative differences already manifest in the gut lumen: while monarchs retain and accumulate cardenolides above plant concentrations, the toxins are degraded in the gut lumen of crows. We visualized cardenolide transport over the monarch midgut epithelium and identified integument cells as the final site of storage where defences might be perceived by predators. Our study provides molecular insight into cardenolide sequestration and highlights the great potential of mass spectrometry imaging for understanding the kinetics of multiple compounds including endogenous metabolites, plant toxins, or insecticides in insects.

Compound-Specific Behavioral and Enzymatic Resistance to Toxic Milkweed Cardenolides in a Generalist Bumblebee Pollinator.

Plant secondary metabolites that defend leaves from herbivores also occur in floral nectar. While specialist herbivores often have adaptations providing resistance to these compounds in leaves, many social insect pollinators are generalists, and therefore are not expected to be as resistant to such compounds. The milkweeds, Asclepias spp., contain toxic cardenolides in all tissues including floral nectar. We compared the concentrations and identities of cardenolides between tissues of the North American common milkweed Asclepias syriaca, and then studied the effect of the predominant cardenolide in nectar, glycosylated aspecioside, on an abundant pollinator. We show that a generalist bumblebee, Bombus impatiens, a common pollinator in eastern North America, consumes less nectar with experimental addition of ouabain (a standard cardenolide derived from Apocynacid plants native to east Africa) but not with addition of glycosylated aspecioside from milkweeds. At a concentration matching that of the maximum in the natural range, both cardenolides reduced activity levels of bees after four days of consumption, demonstrating toxicity despite variation in behavioral deterrence (i.e., consumption). In vitro enzymatic assays of Na+/K+-ATPase, the target site of cardenolides, showed lower toxicity of the milkweed cardenolide than ouabain for B. impatiens, indicating that the lower deterrence may be due to greater tolerance to glycosylated aspecioside. In contrast, there was no difference between the two cardenolides in toxicity to the Na+/K+-ATPase from a control insect, the fruit fly Drosophila melanogaster. Accordingly, this work reveals that even generalist pollinators such as B. impatiens may have adaptations to reduce the toxicity of specific plant secondary metabolites that occur in nectar, despite visiting flowers from a wide variety of plants over the colony's lifespan.

Quantification of plant cardenolides by HPLC, measurement of Na+/K+-ATPase inhibition activity, and characterization of target enzymes.

The biosynthesis of cardiac glycosides, broadly classified as cardenolides and bufadienolides, has evolved repeatedly among flowering plants. Individual species can produce dozens or even hundreds of structurally distinct cardiac glycosides. Although all cardiac glycosides exhibit biological activity by inhibiting the function of the essential Na+/K+-ATPase in animal cells, they differ in their level of inhibitory activity. For within- and between-species comparisons of cardiac glycosides to address ecological and evolutionary questions, it is necessary to not only quantify their relative abundance, but also their effectiveness in inhibiting the activity of different animal Na+/K+-ATPases. Here we describe protocols for characterizing the amount and toxicity of cardenolides from plant samples and the degree of insect Na+/K+-ATPase tolerance to inhibition: (1) an HPLC-based assay to quantify the abundance of individual cardenolides in plant extracts, (2) an assay to quantify inhibition of Na+/K+-ATPase activity by plant extracts, and (3) extraction of insect Na+/K+-ATPases for inhibition assays.

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.

Cardenolide Increase in Foxglove after 2,1,3-Benzothiadiazole Treatment Reveals a Potential Link between Cardenolide and Phytosterol Biosynthesis.

Cardenolides are steroidal metabolites in Digitalis lanata with potent cardioactive effects on animals. In plants, cardenolides are likely involved in various stress responses. However, the molecular mechanism of cardenolide increase during stresses is mostly unknown. Additionally, cardenolides are proposed to arise from cholesterol, but indirect results show that phytosterols may also be substrates for cardenolide biosynthesis. Here, we show that cardenolides increased after methyl jasmonate (MJ), sorbitol, potassium chloride (KCl) and salicylic acid analog [2,1,3-benzothiadiazole (BTH)] treatments. However, the expression of three known genes for cardenolide biosynthesis did not correlate well with these increases. Specifically, the expression of progesterone-5ß-reductases (P5ßR and P5ßR2) did not correlate with the cardenolide increase. The expression of 3ß-hydroxysteroid dehydrogenase (3ßHSD) correlated with changes in cardenolide levels only during the BTH treatment. Mining the D. lanata transcriptome identified genes involved in cholesterol and phytosterol biosynthesis: C24 sterol sidechain reductase 1 (SSR1), C4 sterol methyl oxidase 1, and 3 (SMO1 and SMO3). Surprisingly, the expression of all three genes correlated well with the cardenolide increase after the BTH treatment. Phylogenetic analysis showed that SSR1 is likely involved in both cholesterol and phytosterol biosynthesis. In addition, SMO1 is likely specific to phytosterol biosynthesis, and SMO3 is specific to cholesterol biosynthesis. These results suggest that stress-induced increase of cardenolides in foxglove may correlate with cholesterol and phytosterol biosynthesis. In summary, this work shows that cardenolides are important for stress responses in D. lanata and reveals a potential link between phytosterol and cardenolide biosynthesis.