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Toxicity of Milkweed Leaves and Latex: Chromatographic Quantification Versus Biological Activity of Cardenolides in 16 Asclepias Species

  • Tobias Züst
  • Georg Petschenka
  • Amy P. Hastings
  • Anurag A. Agrawal
Article

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.

Keywords

Cardiac glycoside Coevolution Macroevolutionary escalation Mode of action Monarch butterfly Na+/K+ ATPase Phylogenetic chemical ecology Plant-insect interactions Structure-activity relationships Target site insensitivity 

Notes

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.

Supplementary material

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References

  1. Agrawal AA (2011) Current trends in the evolutionary ecology of plant defence. Funct Ecol 25:420–432CrossRefGoogle Scholar
  2. Agrawal AA (2017) Monarchs and milkweed: a migrating butterfly, a poisonous plant, and their remarkable story of coevolution. Princeton University Press, PrincetonCrossRefGoogle Scholar
  3. Agrawal AA, Fishbein M (2008) Phylogenetic escalation and decline of plant defense strategies. Proc Natl Acad Sci U S A 105:10057–10060CrossRefPubMedPubMedCentralGoogle Scholar
  4. Agrawal AA, Konno K (2009) Latex: a model for understanding mechanisms, ecology, and evolution of plant defense against herbivory. Annu Rev Ecol Evol Syst 40:311–331CrossRefGoogle Scholar
  5. 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 128:126–138CrossRefGoogle Scholar
  6. 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 106:18067–18072CrossRefPubMedPubMedCentralGoogle Scholar
  7. Agrawal AA, Salminen JP, Fishbein M (2009b) Phylogenetic trends in phenolic metabolism of milkweeds (Asclepias): evidence for escalation. Evolution 63:663–673CrossRefGoogle Scholar
  8. Agrawal AA, Petschenka G, Bingham RA, Weber MG, Rasmann S (2012) Toxic cardenolides: chemical ecology and coevolution of specialized plant-herbivore interactions. New Phytol 194:28–45CrossRefGoogle Scholar
  9. Agrawal AA, Patrick ET, Hastings AP (2014) Tests of the coupled expression of latex and cardenolide plant defense in common milkweed (Asclepias syriaca). Ecosphere 5:1–11CrossRefGoogle Scholar
  10. 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 47–59Google Scholar
  11. 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 17:155–165CrossRefPubMedGoogle Scholar
  12. Berenbaum M (1978) Toxicity of furanocoumarin to armyworms: a case of biosynthetic escape from insect herbivores. Science 201:532–534CrossRefPubMedGoogle Scholar
  13. Berenbaum MR (1995) The chemistry of defense: theory and practice. Proc Natl Acad Sci U S A 92:2–8CrossRefPubMedPubMedCentralGoogle Scholar
  14. 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 553–571Google Scholar
  15. Berenbaum MR, Zangerl AR (1993) Furanocoumarin metabolism in Papilio polyxenes: biochemistry, genetic variability, and ecological significance. Oecologia 95:370–375CrossRefGoogle Scholar
  16. 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 8:579–633CrossRefGoogle Scholar
  17. Cacho NI, Kliebenstein DJ, Strauss SY (2015) Macroevolutionary patterns of glucosinolate defense and tests of defense-escalation and resource availability hypotheses. New Phytol 208:915–927CrossRefGoogle Scholar
  18. 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 97:14406–14411CrossRefPubMedPubMedCentralGoogle Scholar
  19. Despres L, David JP, Gallet C (2007) The evolutionary ecology of insect resistance to plant chemicals. Trends Ecol Evol 22:298–307CrossRefGoogle Scholar
  20. 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 109:13040–13045CrossRefPubMedPubMedCentralGoogle Scholar
  21. 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 157:30–39CrossRefGoogle Scholar
  22. 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 91:31–38CrossRefPubMedPubMedCentralGoogle Scholar
  23. Ehrlich PR, Raven PH (1964) Butterflies and plants: a study in coevolution. Evolution 18:586–608CrossRefGoogle Scholar
  24. Eisner T, Meinwald J (1995) The chemistry of sexual selection. Proc Natl Acad Sci U S A 92:50–55CrossRefPubMedPubMedCentralGoogle Scholar
  25. 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 63:553–577Google Scholar
  26. Firn RD, Jones CG (2003) Natural products - a simple model to explain chemical diversity. Nat Prod Rep 20:382–391CrossRefPubMedGoogle Scholar
  27. 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 36:1008–1023CrossRefGoogle Scholar
  28. 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 39:465–480CrossRefPubMedGoogle Scholar
  29. Fraenkel GS (1959) The raison d'être of secondary plant substances. Science 129:1466–1470CrossRefPubMedGoogle Scholar
  30. Futuyma DJ, Agrawal AA (2009) Macroevolution and the biological diversity of plants and herbivores. Proc Natl Acad Sci U S A 106:18054–18061CrossRefPubMedPubMedCentralGoogle Scholar
  31. 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 163–194Google Scholar
  32. 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 71:37–48CrossRefGoogle Scholar
  33. Klauck D, Luckner M (1995) In vitro measurement of digitalis-like compounds by inhibition of Na+/K+-ATPase: determination of the inhibitory effect. Pharmazie 50:395–399Google Scholar
  34. Livshultz T et al (2018) Evolution of pyrrolizidine alkaloid biosynthesis in Apocynaceae: revisiting the defence de-escalation hypothesis. New Phytol 218:762–773CrossRefPubMedPubMedCentralGoogle Scholar
  35. 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 251–296CrossRefGoogle Scholar
  36. 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 26:1100–1110CrossRefGoogle Scholar
  37. Marty MA, Krieger RI (1984) Metabolism of uscharidin, a milkweed cardenolide, by tissue homogenates of monarch butterfly larvae, Danaus plexippus L. J Chem Ecol 10:945–956CrossRefGoogle Scholar
  38. 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 7:981–1010CrossRefGoogle Scholar
  39. Nishida R (2002) Sequestration of defensive substances from plants by Lepidoptera. Annu Rev Entomol 47:57–92CrossRefPubMedPubMedCentralGoogle Scholar
  40. Petschenka G, Agrawal AA (2016) How herbivores coopt plant defenses: natural selection, specialization, and sequestration. Curr Opin Insect Sci 14:17–24CrossRefGoogle Scholar
  41. 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 67:2753–2761Google Scholar
  42. 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–S43CrossRefGoogle Scholar
  43. 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 9:1424CrossRefPubMedPubMedCentralGoogle Scholar
  44. Rasmann S, Agrawal AA (2009) Plant defense against herbivory: progress in identifying synergism, redundancy, and antagonism between resistance traits. Curr Opin Plant Biol 12:473–478CrossRefGoogle Scholar
  45. Rasmann S, Agrawal AA (2011) Latitudinal patterns in plant defense: evolution of cardenolides, their toxicity and induction following herbivory. Ecol Lett 14:476–483CrossRefGoogle Scholar
  46. Rasmann S, Agrawal AA, Cook SC, Erwin AC (2009a) Cardenolides, induced responses, and interactions between above- and belowground herbivores of milkweed (Asclepias spp.). Ecology 90:2393–2404CrossRefGoogle Scholar
  47. Rasmann S, Johnson MD, Agrawal AA (2009b) Induced responses to herbivory and jasmonate in three milkweed species. J Chem Ecol 35:1326–1334CrossRefGoogle Scholar
  48. 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 161:861–866CrossRefGoogle Scholar
  49. Richards LA, Glassmire AE, Ochsenrider KM, Smilanich AM, Dodson CD, Jeffrey CS, Dyer LA (2016) Phytochemical diversity and synergistic effects on herbivores. Phytochem Rev 15:1153–1166CrossRefGoogle Scholar
  50. 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 93–167CrossRefGoogle Scholar
  51. Romeo JT, Saunders JA, Barbosa P (eds) (1996) Phytochemical diversity and redundancy in ecological interactions. Plenum Press, New YorkGoogle Scholar
  52. Rosenthal GA, Hughes CG, Janzen DH (1982) L-Canavanine, a dietary nitrogen source for the seed predator Caryedes brasiliensis (Bruchidae). Science 217:353–355CrossRefPubMedGoogle Scholar
  53. 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 6:321–339CrossRefGoogle Scholar
  54. Seiber JN, Nelson CJ, Lee SM (1982) Cardenolides in the latex and leaves of seven Asclepias species and Calotropis procera. Phytochemistry 21:2343–2348CrossRefGoogle Scholar
  55. 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 43–83Google Scholar
  56. Taussky HH, Shorr E (1953) A microcolorimetric method for the determination of inorganic phosphorus. J Biol Chem 202:675–685PubMedGoogle Scholar
  57. Von Euw J, Fishelson L, Parsons JA, Reichstein T, Rothschild M (1967) Cardenolides (heart poisons) in a grasshopper feeding on milkweeds. Nature 214:35–39CrossRefGoogle Scholar
  58. 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 26:212–224CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of Plant SciencesUniversity of BernBernSwitzerland
  2. 2.Institut für InsektenbiotechnologieJustus-Liebig-Universität GiessenGiessenGermany
  3. 3.Department of Ecology and Evolutionary BiologyCornell UniversityIthacaUSA
  4. 4.Department of EntomologyCornell UniversityIthacaUSA

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