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The presence of secondary metabolites in flower nectar can mediate interactions between plants, pollinators, herbivores, and microbes. Milkweeds range in concentrations of cardenolides in flower nectar from ~ 1 to 100 ng μl–1. Using three different behavioral assays with bumblebees Bombus impatiens, we examined the impacts of the commercially available cardenolide ouabain at the range of concentrations at which cardenolides naturally occur in milkweeds. We show that after four days of exposure bees in consumption assays drank more of a nectar solution with a low ouabain concentration of 10 ng μl–1 than the control sucrose nectar, and over the course of the experiment bees consumed less of the 100 ng μl–1 ouabain solution than the control. Bee activity levels in Petri dish arena assays were not impacted by ouabain consumption, even at the highest concentrations; however, in free‐flying choice assays, bees preferentially visited artificial flowers containing 10 ng μl–1 ouabain more than flowers with sucrose control, or flowers with 100 ng μl–1 of ouabain. We therefore conclude that cardenolides may provide plants advantages to pollination at the low end of the naturally occurring range of concentrations for cardenolides, but may be costly to plants at the high end of the range. This research highlights that secondary metabolites in nectar may be under selection to be maintained at low concentrations, and species with high concentrations of secondary metabolites may be under conflicting selection pressures to maintain high circulating levels to deter herbivores at the cost of reduced pollination. [ABSTRACT FROM AUTHOR]

As a general rule, plants defend against herbivores with multiple traits. The defense synergy hypothesis posits that some traits are more effective when co‐expressed with others compared to their independent efficacy. However, this hypothesis has rarely been tested outside of phytochemical mixtures, and seldom under field conditions. We tested for synergies between multiple defense traits of common milkweed (Asclepias syriaca) by assaying the performance of two specialist chewing herbivores on plants in natural populations. We employed regression and a novel application of random forests to identify synergies and antagonisms between defense traits. We found the first direct empirical evidence for two previously hypothesized defense synergies in milkweed (latex by secondary metabolites, latex by trichomes) and identified numerous other potential synergies and antagonisms. Our strongest evidence for a defense synergy was between leaf mass per area and low nitrogen content; given that these "leaf economic" traits typically covary in milkweed, a defense synergy could reinforce their co‐expression. We report that each of the plant defense traits showed context‐dependent effects on herbivores, and increased trait expression could well be beneficial to herbivores for some ranges of observed expression. The novel methods and findings presented here complement more mechanistic approaches to the study of plant defense diversity and provide some of the best evidence to date that multiple classes of plant defense synergize in their impact on insects. Plant defense synergies against highly specialized herbivores, as shown here, are consistent with ongoing reciprocal evolution between these antagonists. [ABSTRACT FROM AUTHOR]

Abstract: Antipredator defense of herbivorous insects often relies on the potential toxicity of defensive chemicals sequestered from their host plants. The colorful Lygaeinae (Heteroptera: Lygaeidae) store a concentrated mixture of toxic cardenolides (cardiac glycosides) in specialized storage compartments of the bugs' integument, from which they are released upon attack. Larvae and adults of the large milkweed bug Oncopeltus fasciatus (Heteroptera: Lygaeinae) are specialized to feed on cardenolide‐containing milkweeds in the plant genus Asclepias and display a conspicuous red and black colorations. To investigate whether O. fasciatus gained improved protection by feeding on a toxic host plant (Asclepias syriaca), compared to a nontoxic alternative (sunflower seeds), we fed nymphs and adults of O. fasciatus to the golden orb‐weaver Nephila senegalensis. While visually oriented vertebrates, such as avian predators, have been intensively investigated for their reaction to defensive compounds and aposematic coloration, less attention has been paid to invertebrate predators. Their different perceptual abilities can provide important opportunities for testing hypotheses on warning coloration and chemical defenses. The predation trials showed that the bugs fed on Asclepias were significantly less likely to be killed than the bugs reared on a cardenolide‐free diet. This suggests that sequestered cardenolides in O. fasciatus nymphs and adults represent a significant fitness advantage on an individual level against this invertebrate predator. Yet, when testing for avoidance learning in the spiders, negative experience did not change the way how similar prey was attacked at the next encounter. In this case, visual or chemical aposematism thus does not seem to matter for predator learning. [ABSTRACT FROM AUTHOR]

Abstract: While it is well established that climate change affects species distributions and abundances, the impacts of climate change on species interactions has not been extensively studied. This is particularly important for specialists whose interactions are tightly linked, such as between the monarch butterfly (Danaus plexippus) and the plant genus Asclepias, on which it depends. We used open‐top chambers (OTCs) to increase temperatures in experimental plots and placed either nonnative Asclepias curassavica or native A. incarnata in each plot along with monarch larvae. We found, under current climatic conditions, adult monarchs had higher survival and mass when feeding on A. curassavica. However, under future conditions, monarchs fared much worse on A. curassavica. The decrease in adult survival and mass was associated with increasing cardenolide concentrations under warmer temperatures. Increased temperatures alone reduced monarch forewing length. Cardenolide concentrations in A. curassavica may have transitioned from beneficial to detrimental as temperature increased. Thus, the increasing cardenolide concentrations may have pushed the larvae over a tipping point into an ecological trap; whereby past environmental cues associated with increased fitness give misleading information. Given the ubiquity of specialist plant–herbivore interactions, the potential for such ecological traps to emerge as temperatures increase may have far‐reaching consequences. [ABSTRACT FROM AUTHOR]

Premise of the Study: Pachypodium (Apocynaceae) is a genus of iconic stem‐succulent and poisonous plants endemic to Madagascar and southern Africa. We tested hypotheses about the mode of action and macroevolution of toxicity in this group. We further hypothesized that while monarch butterflies are highly resistant to cardenolide toxins (a type of cardiac glycoside) from American Asclepias, they may be negatively affected by Pachypodium defenses, which evolved independently. Methods: We grew 16 of 21 known Pachypodium spp. and quantified putative cardenolides by HPLC and also by inhibition of animal Na+/K+‐ATPase (the physiological target of cardiac glycosides) using an in vitro assay. Pachypodium extracts were tested against monarch caterpillars in a feeding bioassay. We also tested four Asclepias spp. and five Pachypodium spp. extracts, contrasting inhibition of the cardenolide‐sensitive porcine Na+/K+‐ATPase to the monarch's resistant form. Key Results: We found evidence for low cardenolides by HPLC, but substantial toxicity when extracts were assayed on Na+/K+‐ATPases. Toxicity showed phylogenetic signal, and taller species showed greater toxicity (this was marginal after phylogenetic correction). Application of Pachypodium extracts to milkweed leaves reduced monarch growth, and this was predicted by inhibition of the sensitive Na+/K+‐ATPase in phylogenetic analyses. Asclepias extracts were 100‐fold less potent against the monarch compared to the porcine Na+/K+‐ATPase, but this difference was absent for Pachypodium extracts. Conclusions: Pachypodium contains potent toxicity capable of inhibiting sensitive and cardenolide‐adapted Na+/K+‐ATPases. Given the monarch's sensitivity to Pachypodium, we suggest that these plants contain novel cardiac glycosides or other compounds that facilitate toxicity by binding to Na+/K+‐ATPases. [ABSTRACT FROM AUTHOR]

1. Although anthropogenic nitrogen (N) enrichment has significantly changed the growth, survival and reproduction of herbivorous insects, its effects on the defensive sequestration of secondary chemicals by insect herbivores are less well understood. Previous studies have shown that soil nutrient availability can affect sequestration directly through changing concentrations of plant defence chemicals, or indirectly through altering growth rates of herbivores. There has been less exploration of how nutrient deposition affects the consumption of secondary chemicals and subsequent sequestration efficiency. In the current study, the overall effect of soil N availability on cardenolide sequestration by the monarch caterpillar Danaus plexippus was examined. Specifically, the effects of soil nutrient availability on growth, consumption, excretion and sequestration efficiency of cardenolides by D. plexippus larvae fed on the tropical milkweed Asclepias curassavica were measured. 2. The results showed that soil N and phosphorus (P) fertilisation significantly reduced caterpillar growth rate and the sequestration efficiency of cardenolides by monarch caterpillars feeding on A. curassavica. The lowered sequestration efficiency was accompanied by higher concentrations of cardenolides in frass. Although the total cardenolide contents of caterpillars were lower under high N or P fertilisation levels, caterpillar cardenolide concentrations were constant across fertilisation treatments because of lower growth rates (and therefore lower body mass) under high fertilisation. It is concluded that anthropogenic N deposition may have multiple effects on insect herbivores, including their ability to defend themselves from predators with sequestered plant defences. [ABSTRACT FROM AUTHOR]

1. Initial herbivory and induced plant responses can influence subsequent above- and below-ground herbivore attack. When two life stages of the same herbivore damage different plant parts sequentially, there is strong potential for plants to respond with induced plant defence against the later attacker. Alternatively, the earlier attacker could manipulate the host plant to facilitate the later-feeding life stage. 2. We studied herbivory by foliage-feeding adults and root-feeding larvae of the red milkweed beetle (Tetraopes tetraophthalmus) on native common milkweed (Asclepias syriaca) in laboratory and field experiments. We applied factorial above- and below-ground herbivory treatments to test for induced responses, effects on later-feeding conspecific larvae, and damage by naturally colonizing herbivores, including adult T. tetraophthalmus. 3. We found that the inducibility of toxic cardenolides was systemic across the root-shoot barrier, with the highest concentrations in plants damaged both above- and below-ground. Initial aboveground herbivory increased root damage and larval survival, suggesting an increase in root quality following leaf herbivory. Initial below-ground herbivory did not affect the performance of later-feeding larvae, indicating limited importance of induced root cardenolides and competition between clutches of T. tetraophthalmus. 4. In a natural milkweed population, initial above-ground herbivory attracted conspecific adults and milkweed leaf beetles (Labidomera clivicollis) and ultimately reduced fruit production by 33%. Nonetheless, the probability of damage by monarch caterpillars (Danaus plexippus) was reduced on plants initially damaged by T. tetraophthalmus above-ground, likely due to reduced oviposition following induced plant responses. 5. Synthesis. Induced plant responses of common milkweed to above-ground damage by adult T. tetraophthalmus both facilitate further damage by adults and enhance the performance of their root-feeding larvae, most likely as a result of host plant manipulation. Although the same induction reduced monarch herbivory, the net effect of these interactions was negative for the plant as fruit production was substantially reduced. These results imply that host plant manipulation may be especially common by specialist herbivores that have sequential above- and below-ground life stages. [ABSTRACT FROM AUTHOR]

1. The plant-herbivore arms race has been postulated to be a major driver for generating biological and biochemical diversity on Earth. Herbivore feeding is reduced by the production of chemical and physical barriers, but increases plant resistance against subsequent attack. Accordingly, specialisation is predicted to be an outcome of herbivores being able to circumvent plant-induced defences. 2. Using a specialised plant-herbivore system, in which adult chrysomelid beetles ( Chrysochus auratus) feed on leaves and larvae feed on roots of dogbane ( Apocynum spp.), this study investigated whether root latex and cardenolides are effective against the soil-dwelling larvae, and whether such defences could be circumvented by the herbivore. 3. Across two Apocynum species, C. auratus larvae were not affected by latex production or cardenolide amounts and diversity. By contrast, cardenolide apolarity was detrimental to larval growth. Yet larval feeding decreased average root cardenolide apolarity in A. cannabinum and larvae performed better on those plants. Finally, above-ground induction rendered the plants more toxic by increasing root cardenolide apolarity and maintaining it, even during subsequent larval herbivory. 4. Therefore, the intimate relationship and interaction between Chrysochus and Apocynum are maintained by a delicate balance of herbivore manipulation and plant chemical induction. [ABSTRACT FROM AUTHOR]

The primary function of secondary plant metabolites is thought to be defence against herbivores. The frequent occurrence of these same noxious compounds in floral nectar, which functions primarily to attract pollinators, has been seen as paradoxical., Although these compounds may have an adaptive purpose in nectar, they may also occur as a nonadaptive consequence of chemical defence in other plant parts. If nectar chemistry reflects physiological constraints or passive leakage from other tissues, we expect that the identity and relative concentration of nectar cardenolides to be correlated with those of other plant parts; in contrast, discordant distributions of compounds in nectar and other tissues may suggest adaptive roles in nectar., We compared the concentrations and identities of cardenolides in the nectar, leaves and flowers of 12 species from a monophyletic clade of Asclepias. To measure putative toxicity of nectar cardenolides, we then examined the effects of a standard cardenolide (digoxin) on the behaviour of bumblebees, a common generalist pollinator of Asclepias., We found that the average cardenolide concentrations in nectar, leaves and flowers of the 12 Asclepias species were positively correlated as predicted by nonadaptive hypotheses. However, significant differences in the identities and concentrations of individual cardenolides between nectar and leaves suggest that the production or allocation of cardenolides may be independently regulated at each plant part. In addition, cardenolide concentrations in leaves and nectar exhibited no phylogenetic signal., Surprisingly, bumblebees did not demonstrate an aversion to digoxin-rich nectar, which may indicate that nectar cardenolides have little effect on pollination., Although the idea that discordant patterns of secondary metabolites across tissue types may signal adaptive functions is attractive, there is evidence to suggest constraint contributes to nectar secondary chemistry. Further work testing the ecological impacts of such patterns will be critical in determining the functional significance of nectar cardenolides. [ABSTRACT FROM AUTHOR]

A tremendous diversity of plants exude sticky and toxic latex upon tissue damage, and its production has been widely studied as a defensive adaptation against insect herbivores. Here, we address variation in latex production and its constituent chemical properties (cardenolides and cysteine proteases) in 53 milkweeds [ Asclepias spp. (Apocynaceae)], employing a phylogenetic approach to test macroevolutionary hypotheses of defense evolution. Species were highly variable for all three traits, and they showed little evidence for strong phylogenetic conservatism. Latex production and the constituent chemical defenses are thus evolutionarily labile and may evolve rapidly. Nonetheless, in phylogenetically independent analyses, we show that the three traits show some correlations (and thus share a correlated evolutionary history), including a positive correlation between latex exudation and cysteine protease activity. Conversely, latex exudation and cysteine protease activity both showed a trade-off with cardenolide concentrations in latex. We also tested whether these traits have increased in their phenotypic values as the milkweeds diversified, as predicted by plant defense escalation theory. Alternative methods of testing this prediction gave conflicting results – there was an overall negative correlation between amount of evolutionary change and amount of latex exudation; however, ancestral state reconstructions indicated that most speciation events were associated with increases in latex. We conclude by (i) summarizing the evidence of milkweed latex itself as a multivariate defense including the amount exuded and toxin concentrations within, (ii) assessing the coordinated evolution of latex traits and how this fits with our previous notion of ‘plant defense syndromes’, and finally, (iii) proposing a novel hypothesis that includes an ‘evolving community of herbivores’ that may promote the escalation or decline of particular defensive strategies as plant lineages diversify. [ABSTRACT FROM AUTHOR]

AbstractMortality of first instars is generally very high, but variable, and is caused by many factors, including physical and chemical plant characters, weather and natural enemies. Here, a summary of detailed field-based studies of the early-stage survival of a specialist lepidopteran herbivore is presented. First-instar larvae of the monarch butterfly, Danaus plexippus, a milkweed specialist, generally grew faster and survived better on leaves when latex flow was reduced by partial severance of the leaf petiole. The outcome depended on milkweed species, and was related to the amount of latex produced, as well as other plant characters, such as leaf hairs, microclimate and concentration of secondary metabolites. Even for a so-called ‘milkweed specialist’, larval performance and survival appears to be related to the concentration of cardenolides produced by the plants (a potential chemical defence against herbivory). This case study of monarchs and milkweeds highlights the need for field-based experiments to assess the effect of plant characters on the usually poor survival of early instar phytophagous insects. Few similar studies concerning the performance and survival of first-instar, eucalypt-specific herbivores have been conducted, but this type of study is considered essential based on the findings obtained using D. plexippus. [ABSTRACT FROM AUTHOR]