Cardenolide toxin diversity impacts monarch butterfly growth and sequestration
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eLife Assessment
This important study shows that different forms and mixtures of cardenolide toxins in tropical milkweed, especially nitrogen- and sulfur-containing types, change how monarch caterpillars eat, grow, and store these chemicals under laboratory conditions. It solidly demonstrates that chemical diversity within a single group of plant toxins (cardenolides) can have combined effects on even highly specialized herbivores that are different from what one would expect from each toxin alone. However, as all experiments used leaf-disc assays with fixed "natural" toxin ratios and only one adapted herbivore species, tests on living plants, other mixture designs, and non-adapted herbivores would make the broader conclusions stronger.
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Abstract
In classic coevolutionary interactions, host plants are thought to accrue novel chemical defenses which are later countered by detoxification strategies and sometimes sequestration in specialist herbivores. We recently discovered that unusual nitrogen- and sulfur-containing (N,S-) cardenolides in some milkweed species are highly toxic, and broken down to less toxic forms which are sequestered by monarch butterflies ( Danaus plexippus ). Here we isolated and purified five dominant cardenolide toxins from the tropical milkweed, Asclepias curassavica , a globally abundant host plant of monarchs, and fed them to caterpillars individually or in mixture. We hypothesized that the two N,S-cardenolides in A. curassavica (uscharin and voruscharin) would reduce caterpillar growth and sequestration more than other abundant related cardenolides (15-Hydroxy calotropin, frugoside, calactin). Overall, cardenolide treatments caused monarchs to feed more and grow more compared to controls; nonetheless, one N,S-cardenolide (voruscharin) was not stimulatory and caused substantial reductions in growth efficiency. Consuming N,S-cardenolides caused caterpillars to sequester the lowest total amounts of cardenolides, and also reduced their efficiency of sequestration. We next tested the phytochemical diversity hypothesis, that toxin mixtures pose a substantial burden for caterpillars compared to individual compounds provided in equimolar concentrations. We prepared two types of mixtures, one containing equal concentrations of the five compounds and another “realistic mixture” where toxin concentrations reflect their natural proportions in leaves. Mixtures had a negative impact on caterpillar feeding, growth, sequestration, and sequestration efficiency compared to the average of single compounds. The equal and realistic mixtures had similar impacts on feeding and growth, but feeding on the realistic mixture resulted in the lowest sequestration. We conclude that as a result of coevolutionary interactions, even sequestering herbivores may be thwarted by highly specialized plant metabolites such as N,S-cardenolides, and that phytochemical mixtures strengthen plant defense. Toxin mixtures likely challenge detoxification and transport of plant defenses, reducing the herbivore’s growth and sequestration.
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eLife Assessment
This important study shows that different forms and mixtures of cardenolide toxins in tropical milkweed, especially nitrogen- and sulfur-containing types, change how monarch caterpillars eat, grow, and store these chemicals under laboratory conditions. It solidly demonstrates that chemical diversity within a single group of plant toxins (cardenolides) can have combined effects on even highly specialized herbivores that are different from what one would expect from each toxin alone. However, as all experiments used leaf-disc assays with fixed "natural" toxin ratios and only one adapted herbivore species, tests on living plants, other mixture designs, and non-adapted herbivores would make the broader conclusions stronger.
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Reviewer #1 (Public review):
Summary:
In the ecological interactions between wild plants and specialized herbivorous insects, structural innovation-based diversification of secondary metabolites often occurs. In this study, Agrawal et al. utilized two milkweed species (Asclepias curassavica and Asclepias incarnata) and the specialist Monarch butterfly (Danaus plexippus) as a model system to investigate the effects of two N,S-cardenolides - formed through structural diversification and innovation in A. curassavica-on the growth, feeding, and chemical sequestration of D. plexippus, compared to other conventional cardenolides. Additionally, the study examined how cardenolide diversification resulting from the formation of N,S-cardenolides influences the growth and sequestration of D. plexippus. On this basis, the research elucidates the …
Reviewer #1 (Public review):
Summary:
In the ecological interactions between wild plants and specialized herbivorous insects, structural innovation-based diversification of secondary metabolites often occurs. In this study, Agrawal et al. utilized two milkweed species (Asclepias curassavica and Asclepias incarnata) and the specialist Monarch butterfly (Danaus plexippus) as a model system to investigate the effects of two N,S-cardenolides - formed through structural diversification and innovation in A. curassavica-on the growth, feeding, and chemical sequestration of D. plexippus, compared to other conventional cardenolides. Additionally, the study examined how cardenolide diversification resulting from the formation of N,S-cardenolides influences the growth and sequestration of D. plexippus. On this basis, the research elucidates the ecophysiological impact of toxin diversity in wild plants on the detoxification and transport mechanisms of highly adapted herbivores.
Strengths:
The study is characterized by the use of milkweed plants and the specialist Monarch butterfly, which represent a well-established model in chemical ecology research. On one hand, these two organisms have undergone extensive co-evolutionary interactions; on the other hand, the butterfly has developed a remarkable capacity for toxin sequestration. The authors, building upon their substantial prior research in this field and earlier observations of structural evolutionary innovation in cardenolides in A. curassavica, proposed two novel ecological hypotheses. While experimentally validating these hypotheses, they introduced the intriguing concept of a "non-additive diversity effect" of trace plant secondary metabolites when mixed, contrasting with traditional synergistic perspectives, in their impact on herbivores.
Weaknesses:
The manuscript has two main weaknesses. First, as a study reliant on the control of compound concentrations, the authors did not provide sufficient or persuasive justification for their selection of the natural proportions (and concentrations) of cardenolides. The ratios of these compounds likely vary significantly across different environmental conditions, developmental stages, pre- and post-herbivory, and different plant tissues. The ecological relevance of the "natural proportions" emphasized by the authors remains questionable. Furthermore, the same compound may even exert different effects on herbivorous insects at different concentrations. The authors should address this issue in detail within the Introduction, Methods, or Discussion sections.
Second, the study was conducted using leaf discs in an in vitro setting, which may not accurately reflect the responses of Monarch butterflies on living plants. This limitation undermines the foundation for the novel ecological theory proposed by the authors. If the observed phenomena could be validated using specifically engineered plant lines-such as those created through gene editing, knockdown, or overexpression of key enzymes involved in the synthesis of specific N,S-cardenolides - the findings would be substantially more compelling.
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Reviewer #2 (Public review):
This study examined the effects of several cardenolides, including N,S-ring containing variants, on sequestration and performance metrics in monarch larvae. The authors confirm that some cardenolides, which are toxic to non-adapted herbivores, are sequestered by monarchs and enhance performance. Interestingly, N,S-ring-containing cardenolides did not have the same effects and were poorly sequestered, with minimal recovery in frass, suggesting an alternate detoxification or metabolic strategy. These N,S-containing compounds are also known to be less potent defences against non-adapted herbivores. The authors further report that mixtures of cardenolides reduce herbivore performance and sequestration compared to single compounds, highlighting the important role of phytochemical diversity in shaping …
Reviewer #2 (Public review):
This study examined the effects of several cardenolides, including N,S-ring containing variants, on sequestration and performance metrics in monarch larvae. The authors confirm that some cardenolides, which are toxic to non-adapted herbivores, are sequestered by monarchs and enhance performance. Interestingly, N,S-ring-containing cardenolides did not have the same effects and were poorly sequestered, with minimal recovery in frass, suggesting an alternate detoxification or metabolic strategy. These N,S-containing compounds are also known to be less potent defences against non-adapted herbivores. The authors further report that mixtures of cardenolides reduce herbivore performance and sequestration compared to single compounds, highlighting the important role of phytochemical diversity in shaping plant-herbivore interactions.
Overall, this study is clearly written, well-conducted and has the potential to make a valuable contribution to the field. However, I have one major concern regarding the interpretations of the mixture results. From what I understand of the methods, all tested mixtures contain all five compounds. As such, it is not possible to determine whether reduced performance and sequestration result from the complete mixture or from the presence of a single compound, such as voruscharin for performance and uscharin for sequestration. For instance, if all compounds except voruscharin (or uscharin) were combined, would the same pattern emerge? I suspect not, since the effects of the individual N,S-containing compounds alone are generally similar to those of the full mixture (Figure S3). By taking the average of all single compounds, the individual effects of the N,S-containing ones are being inflated by the non-N,S-containing ones (in the main text, Figure 4). In the mix, of course, they are not being 'diluted', as they are always present. This interpretation is further supported by the fact that in the equimolar mix, the relative proportion of voruscharin decreases (from 50% in the 'real mix'), and the target measurements of performance and sequestration tend to increase in the equimolar mix compared to the real mix.
Despite this issue, the discussion of mixtures in the context of plant defence against both adapted and non-adapted herbivores is fascinating and convincing. The rationale that mixtures may serve as a chemical tool-kit that targets different sets of herbivores is compelling. The non-N,S cardenolides are effective against non-adapted herbivores and the N,S-containing cardenolides are effective against adapted herbivores. However, the current experiments focus exclusively on an adapted species. It would be especially interesting to test whether such mixtures reduce overall herbivory when both adapted and non-adapted species are present.
It remains possible that mixtures, even in the absence of voruscharin or uscharin, genuinely reduce sequestration or performance; however, this would need to be tested directly to address the abovementioned concern.
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Author response:
Thanks for these insightful reviews and your summary assessment. We certainly agree that ours was a laboratory study with a single specialized insect, and both mixtures types had all five compounds (controlling for total toxin concentration). Thus, our conclusion that combined effects of naturally occurring toxins (within the cardenolide class) have non-additive effects for the specialized sequestering monarch are constrained by our experimental conditions. In our assay we used two mixture types, equimolar and “natural” proportions. We acknowledge that the natural proportions will vary with plant age, damage history, etc. of the host plant, Asclepias curassavica. Our proportions were based on growing the plants a few different times under variable conditions. Although we did not conduct these experiments on non-adapted …
Author response:
Thanks for these insightful reviews and your summary assessment. We certainly agree that ours was a laboratory study with a single specialized insect, and both mixtures types had all five compounds (controlling for total toxin concentration). Thus, our conclusion that combined effects of naturally occurring toxins (within the cardenolide class) have non-additive effects for the specialized sequestering monarch are constrained by our experimental conditions. In our assay we used two mixture types, equimolar and “natural” proportions. We acknowledge that the natural proportions will vary with plant age, damage history, etc. of the host plant, Asclepias curassavica. Our proportions were based on growing the plants a few different times under variable conditions. Although we did not conduct these experiments on non-adapted insects, we discuss a related experiment that was conducted with wild-type and genetically engineered Drosophila (Lopez-Goldar et al. 2024, PNAS). In sum, we appreciate the reviewers’ comments.
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