Cardenolide toxin diversity impacts monarch butterfly growth and sequestration.

In coevolutionary interactions, host plants accrue novel chemical defenses that specialist herbivores counter by detoxification and sometimes sequestration. We recently found unusual nitrogen- and sulfur-containing (N,S-) cardenolides in some milkweeds—highly toxic compounds that monarch butterflies ( Danaus plexippus ) detoxify during sequestration. We hypothesized that the N,S-cardenolides in Asclepias curassavica (uscharin and voruscharin) would reduce caterpillar performance and sequestration more than other abundant related cardenolides (15-hydroxy-calotropin, frugoside, calactin). Cardenolides generally increased feeding relative to controls, but voruscharin was not stimulatory and substantially reduced growth efficiency. Exposure to either N,S-cardenolide produced the lowest sequestration and reduced sequestration efficiency, consistent with detoxification limiting toxin retention. We next tested whether toxin mixtures impose additional costs relative to individual compounds. We prepared two mixtures, one with equal concentrations of five cardenolides and a ‘realistic mixture’ reflecting natural proportions. Relative to the average of single compounds, mixtures reduced feeding, growth, sequestration, and sequestration efficiency, indicating phytochemical diversity effects exceeded expectations from an additive model. The two mixtures similarly reduced growth, but feeding on the realistic mixture yielded the lowest sequestration. We conclude that coevolution can produce highly specialized defense metabolites such as N,S-cardenolides that thwart even sequestering herbivores, and that phytochemical mixtures strengthen plant defense.

In coevolutionary interactions, host plants accrue novel chemical defenses that specialist herbivores counter by detoxification and sometimes sequestration. We recently found unusual nitrogen- and sulfur-containing (N,S-) cardenolides in some milkweeds—highly toxic compounds that monarch butterflies (Danaus plexippus) detoxify during sequestration. We hypothesized that the N,S-cardenolides in Asclepias curassavica (uscharin and voruscharin) would reduce caterpillar performance and sequestration more than other abundant related cardenolides (15-hydroxy-calotropin, frugoside, calactin). Cardenolides generally increased feeding relative to controls, but voruscharin was not stimulatory and substantially reduced growth efficiency. Exposure to either N,S-cardenolide produced the lowest sequestration and reduced sequestration efficiency, consistent with detoxification limiting toxin retention. We next tested whether toxin mixtures impose additional costs relative to individual compounds. We prepared two mixtures, one with equal concentrations of five cardenolides and a ‘realistic mixture’ reflecting natural proportions. Relative to the average of single compounds, mixtures reduced feeding, growth, sequestration, and sequestration efficiency, indicating phytochemical diversity effects exceeded expectations from an additive model. The two mixtures similarly reduced growth, but feeding on the realistic mixture yielded the lowest sequestration. We conclude that coevolution can produce highly specialized defense metabolites such as N,S-cardenolides that thwart even sequestering herbivores, and that phytochemical mixtures strengthen plant defense.

Plants produce defensive chemicals for protection against insect herbivores that may also alter plant and insect associated microbial communities. However, it is unclear how expression of plant defenses impacts the assembly of insect and plant microbiomes, for example by enhancing communities for microbes that can metabolize defensive chemicals. Monarch butterflies (Danaus plexippus) feed on milkweed species (Asclepias spp.) that vary in production of toxic cardiac glycosides, which could alter associated microbiomes. We therefore sought to understand how different milkweed species, with varying defensive chemical profiles, influence the diversity and composition of monarch and milkweed (root and leaf) bacterial communities. Using a metabarcoding approach, we compared rhizosphere, phyllosphere and monarch microbiomes across two milkweed species (Asclepias curassavica, Asclepias syriaca) and investigated top-down effects of monarch feeding on milkweed microbiomes. Overall, monarch feeding had little effect on host plant microbial communities, but each milkweed species harbored distinct rhizosphere and phyllosphere microbiomes, as did the monarchs feeding on them. There was no difference in diversity between plants species for any of the microbial communities. Taxonomic composition significantly varied between plant species for rhizospheres, phyllospheres, and monarch microbiomes and no dispersion were detected between samples. Interestingly, phyllosphere and monarch microbiomes shared a high proportion of bacterial taxa with the rhizosphere (88.78 and 95.63%, respectively), while phyllosphere and monarch microbiomes had fewer taxa in common. Overall, our results suggest milkweed species select for unique sets of microbial taxa, but to what extent differences in expression of defensive chemicals directly influences microbiome assembly remains to be tested. Host plant species also appears to drive differences in monarch caterpillar microbiomes. Further work is needed to understand how monarchs acquire microbes, for example through horizontal transfer during feeding on leaves or encountering soil when moving on or between host plants.

For highly specialized insect herbivores, plant chemical defenses are often co-opted as cues for oviposition and sequestration. In such interactions, can plants evolve novel defenses, pushing herbivores to trade off benefits of specialization with costs of coping with toxins? We tested how variation in milkweed toxins (cardenolides) impacted monarch butterfly (Danaus plexippus) growth, sequestration, and oviposition when consuming tropical milkweed (Asclepias curassavica), one of two critical host plants worldwide. The most abundant leaf toxin, highly apolar and thiazolidine ring-containing voruscharin, accounted for 40% of leaf cardenolides, negatively predicted caterpillar growth, and was not sequestered. Using whole plants and purified voruscharin, we show that monarch caterpillars convert voruscharin to calotropin and calactin in vivo, imposing a burden on growth. As shown by in vitro experiments, this conversion is facilitated by temperature and alkaline pH. We next employed toxin-target site experiments with isolated cardenolides and the monarch's neural Na+/K+-ATPase, revealing that voruscharin is highly inhibitory compared with several standards and sequestered cardenolides. The monarch's typical >50-fold enhanced resistance to cardenolides compared with sensitive animals was absent for voruscharin, suggesting highly specific plant defense. Finally, oviposition was greatest on intermediate cardenolide plants, supporting the notion of a trade-off between benefits and costs of sequestration for this highly specialized herbivore. There is apparently ample opportunity for continued coevolution between monarchs and milkweeds, although the diffuse nature of the interaction, due to migration and interaction with multiple milkweeds, may limit the ability of monarchs to counteradapt.

Cardenolide sequestration by a hemimetabolous aphid and a holometabolous butterfly from the neotropical milkweed,Asclepias curassavica L., is compared. The oleander aphid,Aphis nerii B. de F., sequestered a similarly narrow range of cardenolide concentrations to the monarch butterfly,Danaus plexippus (L.), from the wide range of concentrations available in leaves of A.curassavica. However, A.nerii sequestered significantly less cardenolide (269 µg/0.1 g) thanD. plexippus (528 µg/0.1 g). The honeydew excreted by A.nerii was comprised of 46% cardenolide. The complete polarity range of 25 cardenolides detected by thin layer chromatography in A.curassavica was represented in the 17 whole aphid cardenolides and the 20 aphid honeydew cardenolides detected. D.plexippus sequestered a narrower polarity range of 11 cardenolides, having eliminated low polarity cardenolide genins and glycosides. It is suggested that these chemical differences may be related to interactions among the broad feeding tactics of sucking or chewing milkweed leaves, life history constraints of holometabolyversus hemimetaboly, the distribution of milkweed food resources in space and time, and the dynamics of natural enemies.

Monarch butterflies are often considered a pinnacle of adaptation, feeding exclusively on milkweed plants and having adapted to the milkweed’s poisonous defenses. This relationship is a textbook example of an evolutionary arms race: milkweeds produce potent toxins called cardenolides, and monarchs have evolved not only to tolerate these poisons but also to accumulate them in their bodies. This accumulation, known as sequestration, protects monarchs from predators. While cardenolides represent a classic case of a defense overcome by a specialist herbivore, ongoing coevolution suggests that plants may continue to evolve increasingly potent strategies. Indeed, plants rarely produce just one toxin, and previous research has shown that some milkweeds produce highly toxic nitrogen- and sulfur-containing cardenolides. Agrawal et al. investigated whether these chemicals impose greater costs on monarch caterpillars compared to other common cardenolides. The researchers isolated and purified five dominant cardenolide toxins from the tropical milkweed, Asclepias curassavica. To test whether consuming a realistic mixture of toxins impairs caterpillar growth and defense sequestration more than consuming individual toxins in isolation, the researchers fed different combinations of the compounds to caterpillars. This revealed that in isolation, nitrogen- and sulfur-containing cardenolides were not stored in their original molecular composition. Instead, caterpillars metabolized them into less toxic forms, reducing their ability to accumulate defensive chemicals compared to when fed other toxins (without nitrogen and sulfur). Caterpillars fed a mixture of all five toxins grew more slowly and sequestered fewer toxins than those fed equal amounts of single compounds. These results suggest that chemical diversity itself is a powerful plant defense, likely by overwhelming the detoxification systems of specialist herbivores. The study of Agrawal et al. provides key insights into how plants defend themselves against pests that have adapted to their toxins. These findings could inform the development of more effective, multi-component pest management strategies in agriculture, mimicking nature’s “cocktail approach” rather than relying on single chemicals. Future research should extend beyond laboratory assays to test these effects on living plants and across a diversity of insect herbivores to validate these ecological theories further.

Monarch butterflies are often considered a pinnacle of adaptation, feeding exclusively on milkweed plants and having adapted to the milkweed’s poisonous defenses. This relationship is a textbook example of an evolutionary arms race: milkweeds produce potent toxins called cardenolides, and monarchs have evolved not only to tolerate these poisons but also to accumulate them in their bodies. This accumulation, known as sequestration, protects monarchs from predators. While cardenolides represent a classic case of a defense overcome by a specialist herbivore, ongoing coevolution suggests that plants may continue to evolve increasingly potent strategies. Indeed, plants rarely produce just one toxin, and previous research has shown that some milkweeds produce highly toxic nitrogen- and sulfur-containing cardenolides. Agrawal et al. investigated whether these chemicals impose greater costs on monarch caterpillars compared to other common cardenolides. The researchers isolated and purified five dominant cardenolide toxins from the tropical milkweed, Asclepias curassavica. To test whether consuming a realistic mixture of toxins impairs caterpillar growth and defense sequestration more than consuming individual toxins in isolation, the researchers fed different combinations of the compounds to caterpillars. This revealed that in isolation, nitrogen- and sulfur-containing cardenolides were not stored in their original molecular composition. Instead, caterpillars metabolized them into less toxic forms, reducing their ability to accumulate defensive chemicals compared to when fed other toxins (without nitrogen and sulfur). Caterpillars fed a mixture of all five toxins grew more slowly and sequestered fewer toxins than those fed equal amounts of single compounds. These results suggest that chemical diversity itself is a powerful plant defense, likely by overwhelming the detoxification systems of specialist herbivores. The study of Agrawal et al. provides key insights into how plants defend themselves against pests that have adapted to their toxins. These findings could inform the development of more effective, multi-component pest management strategies in agriculture, mimicking nature’s “cocktail approach” rather than relying on single chemicals. Future research should extend beyond laboratory assays to test these effects on living plants and across a diversity of insect herbivores to validate these ecological theories further.

Monarch butterflies are often considered a pinnacle of adaptation, feeding exclusively on milkweed plants and having adapted to the milkweed’s poisonous defenses. This relationship is a textbook example of an evolutionary arms race: milkweeds produce potent toxins called cardenolides, and monarchs have evolved not only to tolerate these poisons but also to accumulate them in their bodies. This accumulation, known as sequestration, protects monarchs from predators. While cardenolides represent a classic case of a defense overcome by a specialist herbivore, ongoing coevolution suggests that plants may continue to evolve increasingly potent strategies. Indeed, plants rarely produce just one toxin, and previous research has shown that some milkweeds produce highly toxic nitrogen- and sulfur-containing cardenolides. Agrawal et al. investigated whether these chemicals impose greater costs on monarch caterpillars compared to other common cardenolides. The researchers isolated and purified five dominant cardenolide toxins from the tropical milkweed, Asclepias curassavica. To test whether consuming a realistic mixture of toxins impairs caterpillar growth and defense sequestration more than consuming individual toxins in isolation, the researchers fed different combinations of the compounds to caterpillars. This revealed that in isolation, nitrogen- and sulfur-containing cardenolides were not stored in their original molecular composition. Instead, caterpillars metabolized them into less toxic forms, reducing their ability to accumulate defensive chemicals compared to when fed other toxins (without nitrogen and sulfur). Caterpillars fed a mixture of all five toxins grew more slowly and sequestered fewer toxins than those fed equal amounts of single compounds. These results suggest that chemical diversity itself is a powerful plant defense, likely by overwhelming the detoxification systems of specialist herbivores. The study of Agrawal et al. provides key insights into how plants defend themselves against pests that have adapted to their toxins. These findings could inform the development of more effective, multi-component pest management strategies in agriculture, mimicking nature’s “cocktail approach” rather than relying on single chemicals. Future research should extend beyond laboratory assays to test these effects on living plants and across a diversity of insect herbivores to validate these ecological theories further.

ABSTRACTTropical milkweed (Asclepias curassavica) serves as a host plant for monarch butterflies (Danaus plexippus) and other insect herbivores that can tolerate the abundant cardiac glycosides that are characteristic of this species. Cardiac glycosides, along with additional specialized metabolites, also contribute to the ethnobotanical uses of A. curassavica. To facilitate further research on milkweed metabolism, we assembled the 197‐Mbp genome of a fifth‐generation inbred line of A. curassavica into 619 contigs, with an N50 of 10 Mbp. Scaffolding resulted in 98% of the assembly being anchored to 11 chromosomes, which are mostly colinear with the previously assembled common milkweed (A. syriaca) genome. Assembly completeness evaluations showed that 98% of the BUSCO gene set is present in the A. curassavica genome assembly. The transcriptomes of six tissue types (young leaves, mature leaves, stems, flowers, buds, and roots), with and without defense elicitation by methyl jasmonate treatment, showed both tissue‐specific gene expression and induced expression of genes that may be involved in cardiac glycoside biosynthesis. Expression of a CYP87A gene, the predicted first gene in the cardiac glycoside biosynthesis pathway, was observed only in the stems and roots and was induced by methyl jasmonate. Together, this genome sequence and transcriptome analysis provide important resources for further investigation of the ecological and medicinal uses of A. curassavica.

Anthropogenic disturbance is driving global biodiversity loss, including the monarch butterfly (Danaus plexippus), a dietary specialist of milkweed. In response, ornamental milkweed plantings are increasingly common in urbanized landscapes, and recent evidence indicates they have conservation value for monarch butterflies. Unfortunately, sap-feeding insect herbivores, including the oleander aphid (Aphis nerii), frequently reach high densities on plants in nursery settings and urbanized landscapes. Aphid-infested milkweed may inhibit monarch conservation efforts by reducing host plant quality and inducing plant defenses. To test this, we evaluated the effects of oleander aphid infestation on monarch oviposition, larval performance, and plant traits using tropical milkweed (Asclepias curassavica), the most common commercially available milkweed species in the southern U.S. We quantified monarch oviposition preference, larval herbivory, larval weight, and plant characteristics on aphid-free and aphid-infested milkweed. Monarch butterflies deposited three times more eggs on aphid-free versus aphid-infested milkweed. Similarly, larvae fed aphid-free milkweed consumed and weighed twice as much as larvae fed aphid-infested milkweed. Aphid-free milkweed had higher total dry leaf biomass and nitrogen content than aphid-infested milkweed. Our results indicate that oleander aphid infestations can have indirect negative impacts on urban monarch conservation efforts and highlight the need for effective Lepidoptera-friendly integrated pest management tactics for ornamental plants.

Native plants provide the best habitat for pollinators, but non-native plants can supply resources to native pollinators.The non-native tropical milkweed (bloodflower or scarlet milkweed), Asclepias curassavica L., is a larval food source for the native monarch butterfly (Danaus plexippus).Asclepias curassavica has been widely planted in the southern U.S. where it blooms until late fall, retains healthy vegetation until frost, and does not die back until a hard freeze.In contrast, native Asclepias species senesce and are usually not suitable for monarch larvae consumption in the fall.The late availability of the non-native milkweed may trigger monarchs, normally migrating to Mexico, to break reproductive diapause and lay eggs on their host plant.To determine if non-native A. curassavica was more likely than native Asclepias species to attract egg-laying monarchs, we grew native Asclepias viridis Walter and Asclepias speciosa Torr.along with A. curassavica in Oklahoma and recorded the number of monarch eggs and caterpillars on each plant.From August 2019 until the first freeze, we observed 145 eggs and 39 caterpillars on 40 of 48 A. curassavica plants and one egg on one of 19 native Asclepias plants.First freeze occurred on 12 October.A majority of eggs were laid after 12 September resulting in most eggs having insufficient time to mature.This freeze date was nearly 3 weeks earlier than the average for this area.Our evidence suggests that the monarchs are differentially reacting to the availability of non-native and native Asclepias during late summer and fall.

Abstract. Behavioural events during host selection by ovipositing monarch butterflies (Danaus plexippus (L.), Danainae, Nymphalidae) include tapping the leaf surface with fore‐tarsi and touching this surface with mid‐tarsi (‘drumming’) and antennae. Flavonoids identified from host plant extracts are known to stimulate oviposition. Scanning electron microscopy revealed the presence of contact‐chemoreceptor sensilla on all appendages that contact the leaf surface. This electrophysiological study was conducted to identify the contact chemoreceptors that are sensitive to the known oviposition stimuli and are therefore probably involved in host recognition.Receptor cells of conspicuous sensilla grouped in clusters on fore‐tarsi of females were sensitive to the behaviourally active butanol fraction of host plant (Asclepias curassavica) extract. However, these receptors generally had low sensitivity to three oviposition‐stimulating flavonoids identified from this fraction, but they were also sensitive to the butanol fraction of a non‐host (Brassica oleracea).Chemoreceptors in sensilla of the tarsomers 2–4 of the mid‐legs also responded to the behaviourally active fraction of host plant extract and showed some sensitivity to two of the flavonoids that stimulate oviposition. Similar results were obtained from receptor cells in sensilla on the tip of the antennae. Most of these sensilla had cells responding to the butanol fraction of A. curassavica extract but only 25% of them were also sensitive to one of the behaviourally active flavonoids.These electrophysiological results, in combination with behavioural observations, suggest that host selection in monarch butterflies relies on a complex pattern of peripheral sensory information from several types of tarsal and antennal contact chemoreceptors.

Varroa mites (Varroa destructor) are parasitic mites that, combined with other factors, are contributing to high levels of honey bee (Apis mellifera) colony losses. A Varroa-active dsRNA was recently developed to control Varroa mites within honey bee brood cells. This dsRNA has 372 base pairs that are homologous to a sequence region within the Varroa mite calmodulin gene (cam). The Varroa-active dsRNA also shares a 21-base pair match with monarch butterfly (Danaus plexippus) calmodulin mRNA, raising the possibility of non-target effects if there is environmental exposure. We chronically exposed the entire monarch larval stage to common (Asclepias syriaca) and tropical (Asclepias curassavica) milkweed leaves treated with concentrations of Varroa-active dsRNA that are one- and ten-fold higher than those used to treat honey bee hives. This corresponded to concentrations of 0.025–0.041 and 0.211–0.282 mg/g leaf, respectively. Potassium arsenate and a previously designed monarch-active dsRNA with a 100% base pair match to the monarch v-ATPase A mRNA (leaf concentration was 0.020–0.034 mg/g) were used as positive controls. The Varroa mite and monarch-active dsRNA’s did not cause significant differences in larval mortality, larval or pupal development, pupal weights, or adult eclosion rates when compared to negative controls. Irrespective of control or dsRNA treatment, larvae that consumed approximately 7500 to 10,500-mg milkweed leaf within 10 to 12 days had the highest pupal weights. The lack of mortality and sublethal effects following dietary exposure to dsRNA with 21-base pair and 100% base pair match to mRNAs that correspond to regulatory genes suggest monarch mRNA may be refractory to silencing by dsRNA or monarch dsRNase may degrade dsRNA to a concentration that is insufficient to silence mRNA signaling.

The effect of cardiac glycosides (cardenolides) and nitrogennconcentrations on host selection by Danaas plexippus L. wereninvestigated from two standpoints : (a) presence of eggs andn(b) detailed observation on oviposition behaviour in relation tonthe host plants, Asclepias curassavica L. and A. fruticosa. L..nTwo experiments were carried out, one for each approach.nnnnnnnn The first experiment involved counting the number of eggsnand larvae (immatures) on the two species of milkweed andnrelating these counts to the plant biomass and cardenolidenconcentrations. A total of 323 plants (96 A. curassavica and 227nA. fruticosa) were searched from three randomly selected fieldnplots. The 30 leaves and two flowers bearing immatures and 40nrandomly selected leaves from plants without immatures werenanalysed for cardenolide content.nnnnnnnn The second experiment observed the actual ovipositionnbehaviour and choice of oviposition site by D. plexippus in thenfield. Twelve females were followed and their behaviours recordednonto cassette tapes. Samples of plants on which they : (a) landedn(n=54); (b) landed, tapped their antennae and/or dabbed theirnovipositor (n=20); (c) all of (b) and laid an egg (n=31); and (d)ndid not alight but which were along their flight paths and/ornnearest neighbour to those visited (n=16) were collected forncardenolide and nitrogen analysis. nnnnn Cardenolides concentration (in mg/0.1 gm.) varied widelynbetween and within species and also between seasons. For A.ncurassavica, it varied between 104 and 804, mean 370p156 (n=34)nin autumn and between 204 and 591, mean 382p.207 (n=9) in summernwhile for A. fruticosa it varied between 134 and 889, meann450p193 (n=28) in autumn but between 119 and 719, mean 345p138n(n=120) in summer.nnnnnnn An intermediate concentration level of between 200 and 300nmg/0.1 gm. seemed to be selected by D. plexippus for oviposition;n71% of eggs were laid in this concentration range. Monarchs seemnto avoid plants with very low (l 200 mg/0.1 gm) and very high (gn600 mg/0.1 gm.) cardenolide concentrations; no egg was laid onnplants with cardenolide content below and above thesenconcentrations respectively. It is therefore postulated thatnconcentrations below 200 mg/0.1 gm. are too low to stimulatenoviposition while those above 600 mg/0.1 are to high (i.endeterrent).nnnnnn Nitrogen content seems to have no effect on host selection bynD. plexippus. This is probabaly due to the fact that nitrogen isnnot limiting to the development of its larvae.nnnnnn Key words : Cardiac glycosides (cardenolides), nitrogen,nconcentration (content), milkweeds, Asclepias, A. curassavica. A,nfruticosa. Monarch (Danaus plexippus), emetic potency, sequester,noviposition behaviour, egg laying, tapping of antennae, dabbingnof ovipositor, alight, host selection, cues, visual stimuli,nchemical stimuli.n

Species interactions, specifically plant-insect interactions, are ubiquitous worldwide. Climate change will alter species interactions by affecting abiotic conditions, affecting species phenologies, interaction strengths, and physiological development. However, climate change impacts are often studied using individual species, with limited consideration quantifying the direct and indirect impacts of climate change species interactions. Using lab, field, and greenhouse experiments, I investigated how climate change will directly and indirectly affect species interactions while also fostering undergraduate research experiences using the monarch butterfly (Danaus plexippus)- milkweed (Asclepias sp.) system. In North America, a widely planted, invasive milkweed species, Asclepias curassavica, negatively impacts monarch butterflies. I conducted a fully-factorial field experiment quantifying the indirect impacts of climate change on monarchs, as mediated through the invasive A. curassavica and native A. incarnata. Here, an ecological trap may be developing, driven by lethal increases in milkweed toxicity. Monarchs reared on the invasive A. curassavica at ambient conditions experienced improved performance, but under increased temperatures, monarchs fared much worse. Additionally, I conducted lab and field experiments to quantify the direct impacts of climate change on monarch butterflies and their protozoan parasite, Ophryocystis elektroscirrha (OE). OE threatens monarch populations by decreasing monarch performance, and empirical support is lacking on assessing the impacts of climate change on the interaction between parasites and hosts. Here, simultaneous parasite infection and increased temperatures act as a one-two punch for monarchs, decreasing development time, weights, melanism, and size. I also designed a course-based undergraduate research experience (CURE) for early-division undergraduate students. Here, enrolled students conducted a fully-factorial, greenhouse competition experiment between invasive A. curassavica and two native milkweed species, A. incarnata and A. tuberosa. CURE student performance to that of upper-division students enrolled in a traditional ecology laboratory was also assessed. We found that A. curassavica is a commensal competitor, and that CURE participation can effectively educate and engage early division students in conducting scientific research. In summary, my dissertation highlights the importance of empirically testing the direct and indirect impacts of climate change on species and their interactions, while reinforcing that novel course structures can foster scientific inroads for early division undergraduate students.