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

   https://doi.org/10.1073/pnas.2302251120  

   Agrawal, Anurag A.; Hastings, Amy P. (May 2023, Proceedings of the National Academy of Sciences)

   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. 

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

   https://doi.org/10.1111/ele.14340  

   Agrawal, Anurag A.; Hastings, Amy P.; Duplais, Christophe (November 2023, Ecology Letters)

   Abstract 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 transgenicDrosophilathat 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. 

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3. Evidence for tissue‐specific defence‐offence interactions between milkweed and its community of specialized herbivores

   https://doi.org/10.1111/mec.16450  

   López‐Goldar, Xosé; Hastings, Amy; Züst, Tobias; Agrawal, Anurag (April 2022, Molecular Ecology)

   Abstract Coevolution between plants and herbivores often involves escalation of defence‐offence strategies, but attack by multiple herbivores may obscure the match of plant defence to any one attacker. As herbivores often specialize on distinct plant parts, we hypothesized that defence‐offence interactions in coevolved systems may become physiologically and evolutionarily compartmentalized between plant tissues. We report that roots, leaves, flower buds and seeds of the tropical milkweed (Asclepias curassavica) show increasing concentrations of cardenolide toxins acropetally, with latex showing the highest concentration. In vitro assays of the physiological target of cardenolides, the Na⁺/K⁺–ATPase (hereafter “sodium pump”), of three specialized milkweed herbivores (root‐feedingTetraopes tetrophthalmus, leaf‐feedingDanaus plexippus, and seed‐feedingOncopeltus fasciatus) show that they are proportionally tolerant to the cardenolide concentrations of the tissues they eat. Indeed, molecular substitutions in the insects’ sodium pumps predicted their tolerance to toxins from their target tissues. Nonetheless, the relative inhibition of the sodium pumps of these specialists by the concentration versus composition (inhibition controlled for concentration, what we term “potency”) of cardenolides from their target versus nontarget plant tissues revealed different degrees of insect adaptation to tissue‐specific toxins. In addition, a trade‐off between toxin concentration and potency emerged across plant tissues, potentially reflecting coevolutionary history or plant physiological constraints. Our findings suggest that tissue‐specific coevolutionary dynamics may be proceeding between the plant and its specialized community of herbivores. This novel finding may be common in nature, contributing to ways in which coevolution proceeds in multispecies communities. 

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

   https://doi.org/10.1098/rspb.2023.0987  

   Carlson, Nathaniel J.; Agrawal, Anurag A. (August 2023, Proceedings of the Royal Society B: Biological Sciences)

   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. 

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5. Functional evidence supports adaptive plant chemical defense along a geographical cline

   https://doi.org/10.1073/pnas.2205073119  

   Agrawal, Anurag A.; Espinosa del Alba, Laura; López-Goldar, Xosé; Hastings, Amy P.; White, Ronald A.; Halitschke, Rayko; Dobler, Susanne; Petschenka, Georg; Duplais, Christophe (June 2022, Proceedings of the National Academy of Sciences)

   Environmental clines in organismal defensive traits are usually attributed to stronger selection by enemies at lower latitudes or near the host’s range center. Nonetheless, little functional evidence has supported this hypothesis, especially for coevolving plants and herbivores. We quantified cardenolide toxins in seeds of 24 populations of common milkweed ( Asclepias syriaca ) across 13 degrees of latitude, revealing a pattern of increasing cardenolide concentrations toward the host's range center. The unusual nitrogen-containing cardenolide labriformin was an exception and peaked at higher latitudes. Milkweed seeds are eaten by specialist lygaeid bugs that are even more tolerant of cardenolides than the monarch butterfly, concentrating most cardenolides (but not labriformin) from seeds into their bodies. Accordingly, whether cardenolides defend seeds against these specialist bugs is unclear. We demonstrate that Oncopeltus fasciatus (Lygaeidae) metabolized two major compounds (glycosylated aspecioside and labriformin) into distinct products that were sequestered without impairing growth. We next tested several isolated cardenolides in vitro on the physiological target of cardenolides (Na + /K + -ATPase); there was little variation among compounds in inhibition of an unadapted Na + /K + -ATPase, but tremendous variation in impacts on that of monarchs and Oncopeltu s. Labriformin was the most inhibitive compound tested for both insects, but Oncopeltus had the greater advantage over monarchs in tolerating labriformin compared to other compounds. Three metabolized (and stored) cardenolides were less toxic than their parent compounds found in seeds. Our results suggest that a potent plant defense is evolving by natural selection along a geographical cline and targets specialist herbivores, but is met by insect tolerance, detoxification, and sequestration. 

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