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Milkweeds, monarch butterflies and the ecological significance of cardenolides
Abstract
The contribution of Miriam Rothschild to the monarch cardenolide story is reviewed in the light of the 1914 challenge by the evolutionary biologist, E.B. Poulton for North American chemists to explain the chemical basis of unpalatability in monarch butterflies and their milkweed host plants. This challenge had lain unaccepted for nearly 50 years until Miriam Rothschild took up the gauntlet and showed with the help of many able colleagues that monarchs are aposematically coloured because they sequester toxic cardenolides from milkweed host plants for use as a defence against predators. By virtue of Dr Rothschild's inspiration and industry, and subsequently that of Lincoln Brower and his colleagues, this tritrophic interaction has become a familiar paradigm for the evolution of chemical defences and warning colouration. We now know that the cardenolide contents of different milkweeds vary quantitatively, qualitatively and spatially, both within and among species and we are starting to appreciate the implications of such variation. However, as Dr Rothschild has pointed out in her publications, cardenolides have sometimes blinded us to reality and it is curious how little evidence there is for a defensive function to cardenolides in plants — especially against adapted specialists such as the monarch. Thus the review will conclude with a discussion of the significance of temporal variation and induction of cardenolide production in plants, the lethal plant defence paradox and an emphasis on the dynamics of the cardenolide-mediated interaction between milkweeds and monarch larvae.
... Detoxification of pyrrolizidine alkaloids by diverse sequestering herbivores involves modifications for stable storage, only to be reactivated during predation (Hartmann & Ober, 2000;Wang et al., 2012). For monarchs on milkweed, we have long known of their tolerance mechanisms to cardenolides (Holzinger et al., 1992;Karageorgi et al., 2019;Vaughan & Jungreis, 1977), as well as selective storage of these compounds (Malcolm, 1995;Roeske et al., 1976). Nonetheless, our recent demonstration of the breakdown of particular cardenolides and their differential potency (Agrawal et al., 2021, coupled with the reduced toxicity of sequestered compounds in monarch wings compared to leaves shown here, represent novel insights ( Figure 2). ...
... Indeed, across four host plant species, monarch butterflies broke down and sequestered a subset of cardenolide toxins, and these were less toxic to monarchs than the cardenolides extracted from the leaves they eat. It was known that monarchs concentrate some toxins while not sequestering others, and this often results in a pattern of increased polarity of sequestered cardenolides compared to those in leaves (Brower et al., 1982;Malcolm, 1995;Roeske et al., 1976;Seiber et al., 1980) In particular, the three dominant compounds stored by monarchs feeding on A. syriaca are one-sugar glycosides converted from two sugar compounds more abundant in leaves (Figure 2). The mechanistic basis, generality, and implications of removing one sugar before sequestration remain to be discovered. ...
... Two unusual nitrogen-containing cardenolides, voruscharin and labriformin, found in A. curassavica and A. syriaca, respectively, are not sequestered by monarchs (Agrawal et al., 2021Malcolm, 1995;Roeske et al., 1976;Seiber et al., 1980). Both compounds are detoxified by chemical conversion, apparently at some cost to the caterpillars, and when tested on the monarch's sodium pump are among the most toxic cardenolides (Agrawal et al., 2021. ...
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.
... Despite large differences in the cardenolide profiles of milkweed plant tissue and seeds, cardenolides sequestered by bugs converged on an intermediate polarity and diversity relative to their two food sources, supporting previous findings that sequestration of cardenolides by O. fasciatus is selective and involves chemical modification [36]. This result is consistent with work on other milkweed herbivores; when fed on the leaves of multiple species of milkweeds, monarch butterflies (Danaus plexippus) converged in terms of the polarities of sequestered cardenolides [35,43]. Differences in the composition of sequestered toxins between an insect and its food source likely occur for three reasons: (1) insects avoid or break down compounds that are more toxic to them [44], (2) physiological constraints (as opposed to toxicity) prevent storage of certain toxins [43], and (3) sequestration of certain toxins may be adaptive (e.g. ...
... This result is consistent with work on other milkweed herbivores; when fed on the leaves of multiple species of milkweeds, monarch butterflies (Danaus plexippus) converged in terms of the polarities of sequestered cardenolides [35,43]. Differences in the composition of sequestered toxins between an insect and its food source likely occur for three reasons: (1) insects avoid or break down compounds that are more toxic to them [44], (2) physiological constraints (as opposed to toxicity) prevent storage of certain toxins [43], and (3) sequestration of certain toxins may be adaptive (e.g. specific toxins may be more effective deterrents of natural enemies, or may be modified as pheromones) [45,46]. ...
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.
... Monarch butterflies (Nymphalidae: Danainae: Danaini) have an intimate relationship with plant species of the family Apocynaceae, mainly from the genus Asclepias Linnaeus, 1735 (Oberhauser et al. 2015). These plants are known for containing cardenolides in their leaves and latex, which are toxic to animals (Malcolm 1994). Butterflies' caterpillars accumulate these compounds in their bodies, using them as a chemical defense against predators and parasites (Tan et al. 2018;Stenoien et al. 2019). ...
Monarch butterflies have a close relationship with plants of the Apocynaceae family, especially with the genus Asclepias Linnaeus, 1753, using their toxic cardenolides as a defense against predators. Calotropis procera (Aiton) W.T. Aiton, 1811, native from Africa and Asia and introduced in Brazil as an ornamental plant, is a food alternative for monarchs but contains fewer cardenolides than Asclepias, which may make the butterflies more vulnerable to parasitoids. The interaction between wasps of the genus Brachymeria Westwood, 1829 and butterflies of the genus Danaus Kluk, 1802 is seldom reported. This study reports the first case of parasitism by Brachymeria pandora (Crawford, 1914) in pupae of Danaus erippus (Cramer, 1775) in Brazil, collected in the city of Cuiabá, Mato Grosso. Five butterfly pupae were collected on C. procera; three were parasitized, with 34 emergences of parasitoids. We suggest that the relationship between D. erippus and B. pandora may be facilitated by the lower toxicity of C. procera compared to Asclepias, possibly increasing susceptibility to parasitoidism. The high rate of parasitoidism observed suggests that this possible new interaction could be detrimental to the conservation of D. erippus. Further studies are needed to confirm whether this parasitoid-host interaction also occurs with native Asclepias plants and to investigate the impacts of exotic plants on these types of interactions and on butterfly conservation.
... The diversity of defence compounds that we report in the defensive secretion and bodies of large milkweed bugs is characteristic of many aposematic animals including poison frogs, Lepidoptera [51,[57][58][59], nudibranchs [10,60], Coleoptera [61,62] and Orthoptera [63]. That specialist herbivores concentrate some toxins while not sequestering others has long been known [64,65]. A common question about defensive variability is whether it represents 'ecological noise', variation caused by the stochastic nature of prey environments, or is of no adaptive evolutionary significance [2,66]. ...
Aposematic animals rely on diverse secondary metabolites for defence. Various hypotheses, such as competition, life history and multifunctionality, have been posited to explain defence variability and diversity. We investigate the compound selectivity hypothesis using large milkweed bugs, Oncopeltus fasciatus, to determine if distinct cardenolides vary in toxicity to different predators. We quantify cardenolides in the bug’s defensive secretions and body tissues and test the individual compounds against predator target sites, the Na⁺/K⁺-ATPases, that are predicted to differ in sensitivity. Frugoside, gofruside, glucopyranosyl frugoside and glucopyranosyl gofruside were the dominant cardenolides in the body tissues of the insects, whereas the two monoglycosidic cardenolides—frugoside and gofruside—were the most abundant in the defensive fluid. These monoglycosidic cardenolides were highly toxic (IC50 < 1 μM) to an invertebrate and a sensitive vertebrate enzyme, in comparison to the glucosylated compounds. Gofruside was the weakest inhibitor for a putatively resistant vertebrate predator. Glucopyranosyl calotropin, found in only 60% of bugs, was also an effective inhibitor of sensitive vertebrate enzymes. Our results suggest that the compounds sequestered by O. fasciatus probably provide consistency in protection against a range of predators and underscore the need to consider predator communities in prey defence evolution.
... Milkweeds are so named because of the thick white latex laden with toxic cardenolides that they exude when damaged as a chemical defense against herbivory (Malcolm, 1994;Agrawal et al., 2008). Although mammalian herbivores generally avoid them, there is a community of milkweed specialists that have evolved means of either avoiding or tolerating the toxic effects of the plants. ...
... On Asclepias humistrata, a milkweed species with high production of latex and cardenolides, monarch caterpillars have been observed to reduce latex ingestion by removing emerging latex droplets from sabotaged petioles [10]. Since the sequestration of cardenolides in monarchs has been shown to have an upper limit [44], monarch caterpillars may only drink latex until cardenolide saturation is reached and then avoid latex, as has been shown for A. humistrata. ...
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.
... Some notable examples that co-occur with our study species include the smallest instars of black and white striped caterpillars (e.g. monarchs: [82]), treehopper nymphs (e.g. Platycotis vittata and Umbonia crassicornis [83,84], cucumber beetles [85] and the boldly striped eggs and nymphs of harlequin bugs (Murgantia histrionica [86]). ...
Many animals avoid predation using aposematic displays that pair toxic/dangerous defences with conspicuous achromatic warning patterns, such as high-contrast stripes. To understand how these prey defences work, we need to understand the decision-making of visual predators. Here we gave two species of jumping spiders (Phidippus regius and Habronattus trimaculatus) choice tests using live termites that had their back patterns manipulated using paper capes (solid white, solid black, striped). For P. regius, black and striped termites were quicker to capture attention. Yet despite this increased attention, striped termites were attacked at lower rates than either white or black. This suggests that the termite's contrast with the background elicits attention, but the internal striped body patterning reduces attacks. Results from tests with H. trimaculatus were qualitatively similar but did not meet the threshold for statistical significance. Additional exploratory analyses suggest that attention to and aversion to stripes is at least partially innate and provide further insight into how decision-making played out during trials. Because of their rich diversity (over 6500 species) that includes variation in natural history, toxin susceptibility and degree of colour vision, jumping spiders are well suited to test broad generalizations about how and why aposematic displays work.
... The diversity of defence compounds that we report in the defensive secretion and bodies of large milkweed bugs are characteristic of many aposematic animals including poison frogs, lepidoptera (Rothschildet al. 1979;Trigo 2000;Pentzold et al. 2016;Rojaset al. 2017), nudibranchs (Faulkner et al. 1990; Winterset al. 2019), coleoptera (Vogler & Kelley 1998;Triponezet al. 2007), and orthoptera (Jones et al. 1986). That specialist herbivores concentrate some toxins while not sequestering others has long been known (Seiber et al. 1980;Malcolm 1994). A common question about defensive variability is whether it represents 'ecological noise', variation caused by the stochastic nature of prey environments, or is of no adaptive evolutionary significance (Speedet al. 2012;Whitehead et al. 2022). ...
The defences of aposematic animals are characterised by diversity and variability of secondary metabolites. Here we examine the nature and function of chemical defence diversity in large milkweed bugs, Oncopeltus fasciatus, testing the hypothesis that different chemical defence compounds have evolved in response to different enemies. We profiled and quantified the cardenolides sequestered by large milkweed bugs in their defensive secretions and their bodies, and measured the inhibitory properties of a subset of isolated milkweed cardenolides in the insect’s defence against the Na+/K+—ATPase target site of vertebrate and invertebrate predators, using porcine Na+/K+—ATPase data as a reference. We show that highly concentrated coroglaucigenin cardenolides in the insect’s defence (glucopyranosyl frugoside and frugoside) are toxic for both resistant and sensitive predators, whereas corotoxigenin and calotropagenin cardenolides have varying degrees of enzyme inhibition among various predators. Overall, O. fasciatus is well defended against a range of enemies due to the differential effect of these compounds´ target sites. Our results suggest that the compounds the insect sequester have evolved in response to predation pressure.
... For example, alkaloids in Datura stramonium effectively limit herbivory by the specialist beetle, Epitrix parvula, while a triterpenoid in the same plant reduces damage by a second specialist beetle, Lema daturaphila (De-la-Cruz et al., 2020). Other well studied examples include glucosinolates, cardenolides, alkaloids, and iridoid glycosides that often limit generalist but not specialist herbivory (Bowers and Puttick, 1988;Malcolm, 1994;van Dam et al., 1995;Agrawal et al., 2012;Jeschke et al., 2017). In addition, synergistic effects of co-occurring compounds often result in mixtures of defenses that are more effective than isolated compounds (Berenbaum and Neal, 1985;Berenbaum and Zangerl, 1993;Dyer et al., 2003;Richards et al., 2010Richards et al., , 2012. ...
Phytochemical diversity is an effective plant defensive attribute, but much more research has focused on genetic and environmental controls of specific defensive compounds than phytochemical diversity per se. Documenting plasticity in phytochemical richness and plant chemical composition as opposed to individual compounds is important for understanding plant defense. This study outlines a multi-site transplant experiment in Cerrado gallery forests in central Brazil, utilizing Piper arboreum (Piperaceae), a prevalent and widespread neotropical shrub. Clones from four distinct populations were planted either at their origin site or in a different forest. Secondary metabolite composition varied between populations initially and then changed after transplanting. Interestingly, clones with chemical profiles that were distinct from the populations where they were introduced experienced reduced specialist chrysomelid herbivory compared to clones that were more chemically similar to the existing P. arboreum populations where they were planted. Specialist Lepidoptera herbivory also declined in clones transplanted to a new forest, but this change could not be ascribed to chemical profiles. In contrast, generalist herbivory was unaffected by chemical dissimilarity and transplanting. This research adds to the expanding body of evidence suggesting that phytochemical diversity is a dynamic trait exerting unique effects on different herbivore guilds.
Plants have evolved multiple defensive traits in response to herbivory; in turn, herbivore specialists evolved adaptive behaviours to avoid or tolerate such defences. Here, we employ milkweeds ( Asclepias spp.) to test two defences, latex and trichomes, for their independent and interactive effects on behaviour and performance of monarch caterpillars ( Danaus plexippus ).
Latex exuded upon damage and the density of leaf trichomes positively correlate across milkweed species, suggesting they may have evolved together as synergistic defences. Nonetheless, the complementary roles of these two traits have been little‐studied. We focus on two behaviours: shaving, or the removal of trichomes, and chewing, which encompasses both deactivation of latex and leaf consumption.
In an experiment with seven milkweed species, with and without manipulated latex flow, we found latex to be the primary determinant of reducing chewing, while both defences positively predicted shaving behaviour in the first instar. Next, we conducted a factorial experiment throughout the first three instars, manipulating latex and trichomes on a high‐latex, high‐trichome species, the woolly milkweed Asclepias vestita . On plants with latex and trichomes intact, caterpillars spent the most time shaving and least time chewing of all treatment groups, suggesting a possible synergism. These defence‐driven behavioural effects decreased later in larval development.
Latex and trichomes both impacted monarch performance, additively increasing mortality and reducing growth of survivors. Thus, latex and trichomes represent two important plant defences with effects on specialist herbivore behaviour and implications for insect fitness.
There was a strong correlation between the two birds' predation rates on Danaus plexippus over each season, which indicates that they feed synchronously in mixed flocks. In 1985, grosbeaks Pheucticus melanocephalus killed more monarchs than did orioles Icterus galbula abeillei, whereas in 1986 the opposite suggested a reversal in the proportion of the two bird species in the mixed-species flocks. Grosbeaks killed more males than females; orioles killed both sexes equally. Daily variation in predation rate was large: 11.4-fold for orioles and 15.8-fold for grosbeaks. This variation, 14 and 25%, respectively, could be attributed to temperature: more butterflies were eaten on cold days. -from Authors
By suspending nets within and adjacent to a 2.25 hectare overwintering colony of monarch butterflies in Mexico, we estimated that black-backed orioles and black-headed grosbeaks killed 4,550 to 34,300 and an average of 15,067 butterflies per day. A conservative calculation of mortality through the 135 day overwintering season was 2.034 million butterflies, or about 9% of the colony. The birds preyed selectively upon male butterflies, possibly because of a difference in fat content, or possibly because females contain higher concentrations and larger amounts of cardenolide or other defensive chemicals. The risk to individual monarchs of being killed was much greater on the colony periphery and in thinned areas of the forest. Bird predation thus is sufficient to have played a major role in shaping the evolution of the monarch's overwintering and aggregation behavior. Substantial daily variation in predation intensity occurred, 26% of which was attributable to the birds eating more butterflies on colder days, and 30% of which was attributable to a 7.85 day predation cycle. The hypothesis is put forward that the birds feed cyclically because they build up toxic levels of cardenolides or other defensive chemicals contained in the butterflies. The cyclic predation may reduce total predation on the colony by as much as 50%. Such chemical-based group protection is interpreted as a fortuitous by-product of the evolution of unpalatability through selective processes acting on other phases of the monarch's life history.
Many Danaine species feed as larvae on milkweeds and sequester their bitter-tasting and toxic cardiac glycosides, to render them unpalatable for predators. Danaine body odors are characterized by the presence of pyrazines, which may also function as warning signals to predators. A third group of allelochemicals, the bitter-tasting and toxic pyrrolizidine alkaloids (PAs), occur in many plant groups and are actively searched for and ingested by adult butterflies, especially males. The striking odor of many hairbrush signals is due to a wide spectrum of substances and is to some extent species-specific. These odors may facilitate species and sex discrimination. This would be important in those genera, or groups of genera (African Amauris, Asian Euploea, Asian Tirumala/Ideopsis/Parantica), that look similar and live in close sympatry. -from Author
Distributions of the host plants of mobile herbivorous insects form complex mosaics in space and time. Thus it is difficult to track large scale patterns of herbivorous insect movement without time-and labour-intensive trapping, tracking, and marking techniques that are subject to misinterpretation. Here we summarize the use of a chemical fingerprinting technique to describe the continental scale pattern of migration by the monarch butterfly (Danaus plexippus (L.)) in relation to the spatial and temporal distributions of its milkweed larval host plants (Asclepias spp.) in North America.
