Abstract
Cardenolides are classically studied steroidal defenses in chemical ecology and plant-herbivore coevolution. Although milkweed plants (Asclepias spp.) produce up to 200 structurally different cardenolides, all compounds seemingly share the same well-characterized mode of action, inhibition of the ubiquitous Na+/K+ ATPase in animal cells. Over their evolutionary radiation, milkweeds show a quantitative decline of cardenolide production and diversity. This reduction is contrary to coevolutionary predictions and could represent a cost-saving strategy, i.e. production of fewer but more toxic cardenolides. Here we test this hypothesis by tandem cardenolide quantification using HPLC (UV absorption of the unsaturated lactone) and a pharmacological assay (in vitro inhibition of a sensitive Na+/K+ ATPase) in a comparative study of 16 species of Asclepias. We contrast cardenolide concentrations in leaf tissue to the subset of cardenolides present in exuding latex. Results from the two quantification methods were strongly correlated, but the enzymatic assay revealed that milkweed cardenolide mixtures often cause stronger inhibition than equal amounts of a non-milkweed reference cardenolide, ouabain. Cardenolide concentrations in latex and leaves were positively correlated across species, yet latex caused 27% stronger enzyme inhibition than equimolar amounts of leaf cardenolides. Using a novel multiple regression approach, we found three highly potent cardenolides (identified as calactin, calotropin, and voruscharin) to be primarily responsible for the increased pharmacological activity of milkweed cardenolide mixtures. However, contrary to an expected trade-off between concentration and toxicity, later-diverging milkweeds had the lowest amounts of these potent cardenolides, perhaps indicating an evolutionary response to milkweed’s diverse community of specialist cardenolide-sequestering insect herbivores.
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References
Agrawal AA (2011) Current trends in the evolutionary ecology of plant defence. Funct Ecol 25:420–432
Agrawal AA (2017) Monarchs and milkweed: a migrating butterfly, a poisonous plant, and their remarkable story of coevolution. Princeton University Press, Princeton
Agrawal AA, Fishbein M (2008) Phylogenetic escalation and decline of plant defense strategies. Proc Natl Acad Sci U S A 105:10057–10060
Agrawal AA, Konno K (2009) Latex: a model for understanding mechanisms, ecology, and evolution of plant defense against herbivory. Annu Rev Ecol Evol Syst 40:311–331
Agrawal AA, Lajeunesse MJ, Fishbein M (2008) Evolution of latex and its constituent defensive chemistry in milkweeds (Asclepias): a phylogenetic test of plant defense escalation. Entomol Exp Appl 128:126–138
Agrawal AA, Fishbein M, Halitschke R, Hastings AP, Rabosky DL, Rasmann S (2009a) Evidence for adaptive radiation from a phylogenetic study of plant defenses. Proc Natl Acad Sci U S A 106:18067–18072
Agrawal AA, Salminen JP, Fishbein M (2009b) Phylogenetic trends in phenolic metabolism of milkweeds (Asclepias): evidence for escalation. Evolution 63:663–673
Agrawal AA, Petschenka G, Bingham RA, Weber MG, Rasmann S (2012) Toxic cardenolides: chemical ecology and coevolution of specialized plant-herbivore interactions. New Phytol 194:28–45
Agrawal AA, Patrick ET, Hastings AP (2014) Tests of the coupled expression of latex and cardenolide plant defense in common milkweed (Asclepias syriaca). Ecosphere 5:1–11
Agrawal AA, Ali JG, Rasmann S, Fishbein M (2015) Macroevolutionary trends in the defense of milkweeds against monarchs: latex, cardenolides, and tolerance of herbivory. In: Oberhauser K, Altizer S, Nail K (eds) Monarchs in a changing world: biology and conservation of an iconic insect. Cornell University Press, Ithaca, pp 47–59
Benson JM, Seiber JN, Bagley CV, Keeler RF, Johnson AE, Young S (1979) Effects on sheep of the milkweeds Asclepias eriocarpa and Asclepias labriformis and of cardiac glycoside-containing derivative material. Toxicon 17:155–165
Berenbaum M (1978) Toxicity of furanocoumarin to armyworms: a case of biosynthetic escape from insect herbivores. Science 201:532–534
Berenbaum MR (1995) The chemistry of defense: theory and practice. Proc Natl Acad Sci U S A 92:2–8
Berenbaum MR (1999) Animal-plant warfare: molecular basis for cytochrome P450-mediated natural adaptation. In: Puga A, Wallace K (eds) Molecular biology of the toxic response. Taylor and Francis, Philadelphia, pp 553–571
Berenbaum MR, Zangerl AR (1993) Furanocoumarin metabolism in Papilio polyxenes: biochemistry, genetic variability, and ecological significance. Oecologia 95:370–375
Brower LP, Seiber JN, Nelson CJ, Lynch SP, Tuskes PM (1982) Plant-determined variation in the cardenolide content, thin-layer chromatography profiles, and emetic potency of monarch butterflies, Danaus plexippus reared on the milkweed Asclepias eriocarpa in California. J Chem Ecol 8:579–633
Cacho NI, Kliebenstein DJ, Strauss SY (2015) Macroevolutionary patterns of glucosinolate defense and tests of defense-escalation and resource availability hypotheses. New Phytol 208:915–927
Conner WE, Boada R, Schroeder FC, Gonzalez A, Meinwald J, Eisner T (2000) Chemical defense: bestowal of a nuptial alkaloidal garment by a male moth on its mate. Proc Natl Acad Sci U S A 97:14406–14411
Despres L, David JP, Gallet C (2007) The evolutionary ecology of insect resistance to plant chemicals. Trends Ecol Evol 22:298–307
Dobler S, Dalla S, Wagschal V, Agrawal AA (2012) Community-wide convergent evolution in insect adaptation to toxic cardenolides by substitutions in the Na,K-ATPase. Proc Natl Acad Sci U S A 109:13040–13045
Dobler S, Petschenka G, Wagschal V, Flacht L (2015) Convergent adaptive evolution - how insects master the challenge of cardiac glycoside-containing host plants. Entomol Exp Appl 157:30–39
Dzimiri N, Fricke U, Klaus W (1987) Influence of derivation on the lipophilicity and inhibitory actions of cardiac glycosides on myocardial Na+-K+-ATPase. Br J Pharmacol 91:31–38
Ehrlich PR, Raven PH (1964) Butterflies and plants: a study in coevolution. Evolution 18:586–608
Eisner T, Meinwald J (1995) The chemistry of sexual selection. Proc Natl Acad Sci U S A 92:50–55
Farrell BD, Mitter C (1998) The timing of insect-plant diversification: might Tetraopes (Coleoptera : Cerambycidae) and Asclepias (Asclepiadaceae) have co-evolved? Biol J Linn Soc 63:553–577
Firn RD, Jones CG (2003) Natural products - a simple model to explain chemical diversity. Nat Prod Rep 20:382–391
Fishbein M, Chuba D, Ellison C, Mason-Gamer RJ, Lynch SP (2011) Phylogenetic relationships of Asclepias (Apocynaceae) inferred from non-coding chloroplast DNA sequences. Syst Bot 36:1008–1023
Forbey JS, Dearing MD, Gross EM, Orians CM, Sotka EE, Foley WJ (2013) A pharm-ecological perspective of terrestrial and aquatic plant-herbivore interactions. J Chem Ecol 39:465–480
Fraenkel GS (1959) The raison d'être of secondary plant substances. Science 129:1466–1470
Futuyma DJ, Agrawal AA (2009) Macroevolution and the biological diversity of plants and herbivores. Proc Natl Acad Sci U S A 106:18054–18061
Jeschke V, Gershenzon J, Vassão DG (2015) Metabolism of glucosinolates and their hydrolysis products in insect herbivores. In: Jetter R (ed) The formation, structure, and activity of phytochemicals, vol 45. Recent advances in phytochemistry. Springer, pp 163–194
Jeschke V, Gershenzon J, Vassão DG (2016) A mode of action of glucosinolate-derived isothiocyanates: detoxification depletes glutathione and cysteine levels with ramifications on protein metabolism in Spodoptera littoralis. Insect Biochem Mol Biol 71:37–48
Klauck D, Luckner M (1995) In vitro measurement of digitalis-like compounds by inhibition of Na+/K+-ATPase: determination of the inhibitory effect. Pharmazie 50:395–399
Livshultz T et al (2018) Evolution of pyrrolizidine alkaloid biosynthesis in Apocynaceae: revisiting the defence de-escalation hypothesis. New Phytol 218:762–773
Malcom SB (1991) Cardenolide-mediated interactions between plants and herbivores. In: Rosenthal GA, Berenbaum MR (eds) Herbivores: their interactions with secondary plant metabolites, second edition, Vol. I: the chemical participants. Academic, San Diego, pp 251–296
Manson JS, Rasmann S, Halitschke R, Thomson JD, Agrawal AA (2012) Cardenolides in nectar may be more than a consequence of allocation to other plant parts: a phylogenetic study of Asclepias. Funct Ecol 26:1100–1110
Marty MA, Krieger RI (1984) Metabolism of uscharidin, a milkweed cardenolide, by tissue homogenates of monarch butterfly larvae, Danaus plexippus L. J Chem Ecol 10:945–956
Nelson CJ, Seiber JN, Brower LP (1981) Seasonal and intraplant variation of cardenolide content in the California milkweed, Asclepias eriocarpa, and implications for plant defense. J Chem Ecol 7:981–1010
Nishida R (2002) Sequestration of defensive substances from plants by Lepidoptera. Annu Rev Entomol 47:57–92
Petschenka G, Agrawal AA (2016) How herbivores coopt plant defenses: natural selection, specialization, and sequestration. Curr Opin Insect Sci 14:17–24
Petschenka G, Fandrich S, Sander N, Wagschal V, Boppré M, Dobler S (2013) Stepwise evolution of resistance to toxic cardenolides via genetic substitutions in the Na+/K+-ATPase of milkweed butterflies (Lepidoptera: Danaini). Evolution 67:2753–2761
Petschenka G, Wagschal V, von Tschirnhaus M, Donath A, Dobler S (2017) Convergently evolved toxic secondary metabolites in plants drive the parallel molecular evolution of insect resistance. Am Nat 190:S29–S43
Petschenka G, Fei CS, Araya JJ, Schröder S, Timmermann BN, Agrawal AA (2018) Relative selectivity of plant cardenolides for Na+/K+-ATPases from the monarch butterfly and non-resistant insects. Front Plant Sci 9:1424
Rasmann S, Agrawal AA (2009) Plant defense against herbivory: progress in identifying synergism, redundancy, and antagonism between resistance traits. Curr Opin Plant Biol 12:473–478
Rasmann S, Agrawal AA (2011) Latitudinal patterns in plant defense: evolution of cardenolides, their toxicity and induction following herbivory. Ecol Lett 14:476–483
Rasmann S, Agrawal AA, Cook SC, Erwin AC (2009a) Cardenolides, induced responses, and interactions between above- and belowground herbivores of milkweed (Asclepias spp.). Ecology 90:2393–2404
Rasmann S, Johnson MD, Agrawal AA (2009b) Induced responses to herbivory and jasmonate in three milkweed species. J Chem Ecol 35:1326–1334
Reichstein T, Von Euw J, Parsons JA, Rothschild M (1968) Heart poisons in the monarch butterfly - some aposematic butterflies obtain protection from cardenolides present in their food plant. Science 161:861–866
Richards LA, Glassmire AE, Ochsenrider KM, Smilanich AM, Dodson CD, Jeffrey CS, Dyer LA (2016) Phytochemical diversity and synergistic effects on herbivores. Phytochem Rev 15:1153–1166
Roeske CN, Seiber JN, Brower LP, Moffitt CM (1976) Milkweed cardenolides and their comparative processing by monarch butterflies (Danaus plexippus L.). In: Wallace JW, Mansell RL (eds) Biochemical interaction between plants and insects. Springer, Boston, pp 93–167
Romeo JT, Saunders JA, Barbosa P (eds) (1996) Phytochemical diversity and redundancy in ecological interactions. Plenum Press, New York
Rosenthal GA, Hughes CG, Janzen DH (1982) L-Canavanine, a dietary nitrogen source for the seed predator Caryedes brasiliensis (Bruchidae). Science 217:353–355
Seiber JN, Tuskes PM, Brower LP, Nelson CJ (1980) Pharmacodynamics of some individual milkweed cardenolides fed to larvae of the monarch butterfly (Danaus plexippus L.). J Chem Ecol 6:321–339
Seiber JN, Nelson CJ, Lee SM (1982) Cardenolides in the latex and leaves of seven Asclepias species and Calotropis procera. Phytochemistry 21:2343–2348
Seiber JN, Lee SM, Benson JM (1983) Cardiac glycosides (cardenolides) in species of Asclepias (Asclepiadaceae). In: Keeler RF, Tu AT (eds) Handbook of natural toxins, vol 1: plant and fungal toxins. Marcel Dekker, Amsterdam, pp 43–83
Taussky HH, Shorr E (1953) A microcolorimetric method for the determination of inorganic phosphorus. J Biol Chem 202:675–685
Von Euw J, Fishelson L, Parsons JA, Reichstein T, Rothschild M (1967) Cardenolides (heart poisons) in a grasshopper feeding on milkweeds. Nature 214:35–39
Zalucki MP, Brower LP, Alonso A (2001) Detrimental effects of latex and cardiac glycosides on survival and growth of first-instar monarch butterfly larvae Danaus plexippus feeding on the sandhill milkweed Asclepias humistrata. Ecol Entomol 26:212–224
Acknowledgements
We thank Eamonn Patrick for technical support and performing parts of the experiment, Katalin Böröczky, Steve Broyles, Ron White, Hongxing Xu, Navid Movahed, and Georg Jander for help with the purification and HRMS analysis of labriformin, uscharin, and voruscharin, Ivan Keresztes for performing the NMR analysis of labriformin, and members of the Phytophagy Laboratory at Cornell University (www.herbivory.com) for discussion. This work was supported by German Research Foundation grant PE 2059/1-1 to GP, Swiss National Science Foundation grants P300P3-151191 and PZ00P3-161472 to TZ, and NSF-DEB-1118783 and a Templeton Foundation grant to AAA.
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Züst, T., Petschenka, G., Hastings, A.P. et al. Toxicity of Milkweed Leaves and Latex: Chromatographic Quantification Versus Biological Activity of Cardenolides in 16 Asclepias Species. J Chem Ecol 45, 50–60 (2019). https://doi.org/10.1007/s10886-018-1040-3
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DOI: https://doi.org/10.1007/s10886-018-1040-3