Toxic Cardenolides Research Articles

First record of parasitoidism in Danaus erippus (Cramer, 1775) pupae (Lepidoptera, Nymphalidae): A possible mediation by an exotic plant species

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.

Heat waves induce milkweed resistance to a specialist herbivore via increased toxicity and reduced nutrient content.

Over the last decade, a large effort has been made to understand how extreme climate events disrupt species interactions. Yet, it is unclear how these events affect plants and herbivores directly, via metabolic changes, and indirectly, via their subsequent altered interaction. We exposed common milkweed (Asclepias syriaca) and monarch caterpillars (Danaus plexippus) to control (26:14°C, day:night) or heat wave (HW) conditions (36:24°C, day:night) for 4 days and then moved each organism to a new control or HW partner to disentangle the direct and indirect effects of heat exposure on each organism. We found that the HW directly benefited plants in terms of growth and defence expression (increased latex exudation and total cardenolides) and insect her'bivores through faster larval development. Conversely, indirect HW effects caused both plant latex and total cardenolides to decrease after subsequent herbivory. Nonetheless, increasing trends of more toxic cardenolides and lower leaf nutritional quality after herbivory by HW caterpillars likely led to reduced plant damage compared to controls. Our findings reveal that indirect impacts of HWs may play a greater role in shaping plant-herbivore interactions via changes in key physiological traits, providing valuable understanding of how ecological interactions may proceed in a changing world.

Toxic cardenolides are tolerated by wasps but keep bees at bay

Toxic cardenolides are tolerated by wasps but keep bees at bay

Genome sequence of Ophryocystis elektroscirrha, an apicomplexan parasite of monarch butterflies: cryptic diversity and response to host-sequestered plant chemicals

Apicomplexa are ancient and diverse organisms which have been poorly characterized by modern genomics. To better understand the evolution and diversity of these single-celled eukaryotes, we sequenced the genome of Ophryocystis elektroscirrha, a parasite of monarch butterflies, Danaus plexippus. We contextualize our newly generated resources within apicomplexan genomics before answering longstanding questions specific to this host-parasite system. To start, the genome is miniscule, totaling only 9 million bases and containing fewer than 3,000 genes, half the gene content of two other sequenced invertebrate-infecting apicomplexans, Porospora gigantea and Gregarina niphandrodes. We found that O. elektroscirrha shares different orthologs with each sequenced relative, suggesting the true set of universally conserved apicomplexan genes is very small indeed. Next, we show that sequencing data from other potential host butterflies can be used to diagnose infection status as well as to study diversity of parasite sequences. We recovered a similarly sized parasite genome from another butterfly, Danaus chrysippus, that was highly diverged from the O. elektroscirrha reference, possibly representing a distinct species. Using these two new genomes, we investigated potential evolutionary response by parasites to toxic phytochemicals their hosts ingest and sequester. Monarch butterflies are well-known to tolerate toxic cardenolides thanks to changes in the sequence of their Type II ATPase sodium pumps. We show that Ophryocystis completely lacks Type II or Type 4 sodium pumps, and related proteins PMCA calcium pumps show extreme sequence divergence compared to other Apicomplexa, demonstrating new avenues of research opened by genome sequencing of non-model Apicomplexa.

Compound-Specific Behavioral and Enzymatic Resistance to Toxic Milkweed Cardenolides in a Generalist Bumblebee Pollinator.

Plant secondary metabolites that defend leaves from herbivores also occur in floral nectar. While specialist herbivores often have adaptations providing resistance to these compounds in leaves, many social insect pollinators are generalists, and therefore are not expected to be as resistant to such compounds. The milkweeds, Asclepias spp., contain toxic cardenolides in all tissues including floral nectar. We compared the concentrations and identities of cardenolides between tissues of the North American common milkweed Asclepias syriaca, and then studied the effect of the predominant cardenolide in nectar, glycosylated aspecioside, on an abundant pollinator. We show that a generalist bumblebee, Bombus impatiens, a common pollinator in eastern North America, consumes less nectar with experimental addition of ouabain (a standard cardenolide derived from Apocynacid plants native to east Africa) but not with addition of glycosylated aspecioside from milkweeds. At a concentration matching that of the maximum in the natural range, both cardenolides reduced activity levels of bees after four days of consumption, demonstrating toxicity despite variation in behavioral deterrence (i.e., consumption). In vitro enzymatic assays of Na+/K+-ATPase, the target site of cardenolides, showed lower toxicity of the milkweed cardenolide than ouabain for B. impatiens, indicating that the lower deterrence may be due to greater tolerance to glycosylated aspecioside. In contrast, there was no difference between the two cardenolides in toxicity to the Na+/K+-ATPase from a control insect, the fruit fly Drosophila melanogaster. Accordingly, this work reveals that even generalist pollinators such as B. impatiens may have adaptations to reduce the toxicity of specific plant secondary metabolites that occur in nectar, despite visiting flowers from a wide variety of plants over the colony's lifespan.

Gut-specific cardenolide-resistant sodium pump primed an omnivore to feed on toxic oleander.

Apocynaceae plants produce toxic cardenolides to defend against their herbivores. Cardenolides could inhibit the α subunit of Na+⁄K+-ATPase (ATPα), which plays critical roles in biological processes such as muscle contraction, neural function, and osmoregulation. Numerous herbivores that specialized on cardenolides-producing milkweeds carry parallel molecular changes in ATPα that confer resistance. We found that Pacific Ducetia (Ducetia japonica), an omnivorous katydid, could feed on cardenolides-rich oleander leaves. ATPα in D.japonica has undergone parallel molecular changes just like milkweed specialists. These changes evolved at the common ancestors of the leaf katydids before the diversification of Apocynaceae and may prime species in this lineage to feed on cardenolides-rich food. In summary, we reported the first case of cardenolide resistance in katydid, with convergent molecular evolution in ATPα, also an unusual case of cardenolidesresistance in nonspecialist species that evolved earlier than the currently known cardenolide-producing plants.

Reflections on the Predictability of Evolution: Toward a Conceptual Framework.

SummaryEvolution is generally considered to be unpredictable because genetic variations are known to occur randomly. However, remarkable patterns of repeated convergent evolution are observed, for instance, loss of pigments by organisms living in caves. Analogous phenotypes appear in similar environments, sometimes in response to similar constraints. Alongside randomness, a certain evolutionary determinism also exists, for instance, the selection of particular phenotypes subjected to particular environmental constraints in the “evolutionary funnel.” We pursue the idea that eco-evolutionary specialization is in some way determinist. The conceptual framework of phenotypic changes entailing specialization presented in this essay explains how evolution can be predicted. We also discuss how the predictability of evolution could be tested using the case of metabolic specialization through gene losses. We also put forward that microorganisms could be key models to test and possibly make headway evolutionary predictions and knowledge about evolution.

The role of toxic nectar secondary compounds in driving differential bumble bee preferences for milkweed flowers.

While morphological differences such as tongue length are often featured as drivers of pollinator floral preferences, differences in chemical detection and tolerance to secondary compounds may also play a role. We sought to better understand the role of secondary compounds in floral preference by examining visitation of milkweed flowers, which can contain toxic cardenolides in their nectar, by bumble bees (Bombus spp.), some of their most abundant and important pollinators. We examine bumble bee species visitation of common milkweed (Asclepias syriaca) compared to other flowers in the field and test whether observed preferences may be influenced by avoidance and tolerance of cardenolides, as measured by the cardenolide ouabain, in the lab. We reveal that common milkweed is visited predominantly by one bumble bee species, Bombus griseocollis, in a ratio much higher than the abundance of this species in the community. We confirmed the presence and toxicity of cardenolides in A. syriaca nectar. Lab experiments revealed that B. griseocollis, compared to the common bumble bees B. impatiens and B. bimaculatus, exhibit greater avoidance of cardenolides, but only at levels that start to induce illness, whereas the other species exhibit either no or reduced avoidance of cardenolides, resulting in illness and mortality in these bees. Toxicity experiments reveal that B. griseocollis also has a substantially higher tolerance for cardenolides than B. impatiens. Together, these results support a potential evolutionary association between B. griseocollis and milkweed that may involve increased ability to both detect and tolerate milkweed cardenolides.

Transcriptomics of monarch butterflies (Danaus plexippus) reveals that toxic host plants alter expression of detoxification genes and down-regulate a small number of immune genes.

Herbivorous insects have evolved many mechanisms to overcome plant chemical defences, including detoxification and sequestration. Herbivores may also use toxic plants to reduce parasite infection. Plant toxins could directly interfere with parasites or could enhance endogenous immunity. Alternatively, plant toxins could favour down-regulation of endogenous immunity by providing an alternative (exogenous) defence against parasitism. However, studies on genomewide transcriptomic responses to plant defences and the interplay between plant toxicity and parasite infection remain rare. Monarch butterflies (Danaus plexippus) are specialist herbivores of milkweeds (Asclepias spp.), which contain toxic cardenolides. Monarchs have adapted to cardenolides through multiple resistance mechanisms and can sequester cardenolides to defend against bird predators. In addition, high-cardenolide milkweeds confer monarch resistance to a specialist protozoan parasite (Ophryocystis elektroscirrha). We used this system to study the interplay between the effects of plant toxicity and parasite infection on global gene expression. We compared transcriptional profiles between parasite-infected and uninfected monarch larvae reared on two milkweed species. Our results demonstrate that monarch differentially express several hundred genes when feeding on A.curassavica and A.incarnata, two species that differ substantially in cardenolide concentrations. These differentially expressed genes include genes within multiple families of canonical insect detoxification genes, suggesting that they play a role in monarch toxin resistance and sequestration. Interestingly, we found little transcriptional response to infection. However, parasite growth was reduced in monarchs reared on A.curassavica, and in these monarchs, several immune genes were down-regulated, consistent with the hypothesis that medicinal plants can reduce reliance on endogenous immunity.

Cardenolide Intake, Sequestration, and Excretion by the Monarch Butterfly along Gradients of Plant Toxicity and Larval Ontogeny.

Monarch butterflies, Danaus plexippus, migrate long distances over which they encounter host plants that vary broadly in toxic cardenolides. Remarkably little is understood about the mechanisms of sequestration in Lepidoptera that lay eggs on host plants ranging in such toxins. Using closely-related milkweed host plants that differ more than ten-fold in cardenolide concentrations, we mechanistically address the intake, sequestration, and excretion of cardenolides by monarchs. We show that on high cardenolide plant species, adult butterflies saturate in cardenolides, resulting in lower concentrations than in leaves, while on low cardenolide plants, butterflies concentrate toxins. Butterflies appear to focus their sequestration on particular compounds, as the diversity of cardenolides is highest in plant leaves, lower in frass, and least in adult butterflies. Among the variety of cardenolides produced by the plant, sequestered compounds may be less toxic to the butterflies themselves, as they are more polar on average than those in leaves. In accordance with this, results from an in vitro assay based on inhibition of Na+/K+ ATPase (the physiological target of cardenolides) showed that on two milkweed species, including the high cardenolide A. perennis, extracts from butterflies have lower inhibitory effects than leaves when standardized by cardenolide concentration, indicating selective sequestration of less toxic compounds from these host plants. To understand how ontogeny shapes sequestration, we examined cardenolide concentrations in caterpillar body tissues and hemolymph over the course of development. Caterpillars sequestered higher concentrations of cardenolides as early instars than as late instars, but within the fifth instar, concentration increased with body mass. Although it appears that large amounts of sequestration occurs in early instars, a host switching experiment revealed that caterpillars can compensate for feeding on low cardenolide host plants with substantial sequestration in the fifth instar. We highlight commonalities and striking differences in the mechanisms of sequestration depending on host plant chemistry and developmental stage, which have important implications for monarch defense.

Toxicity of Milkweed Leaves and Latex: Chromatographic Quantification Versus Biological Activity of Cardenolides in 16 Asclepias Species.

Cardenolides are classically studied steroidal defenses in chemical ecology and plant-herbivore coevolution. Although milkweed plants (Asclepias spp.) produce up to 200 structurally different cardenolides, all compounds seemingly share the same well-characterized mode of action, inhibition of the ubiquitous Na+/K+ ATPase in animal cells. Over their evolutionary radiation, milkweeds show a quantitative decline of cardenolide production and diversity. This reduction is contrary to coevolutionary predictions and could represent a cost-saving strategy, i.e. production of fewer but more toxic cardenolides. Here we test this hypothesis by tandem cardenolide quantification using HPLC (UV absorption of the unsaturated lactone) and a pharmacological assay (in vitro inhibition of a sensitive Na+/K+ ATPase) in a comparative study of 16 species of Asclepias. We contrast cardenolide concentrations in leaf tissue to the subset of cardenolides present in exuding latex. Results from the two quantification methods were strongly correlated, but the enzymatic assay revealed that milkweed cardenolide mixtures often cause stronger inhibition than equal amounts of a non-milkweed reference cardenolide, ouabain. Cardenolide concentrations in latex and leaves were positively correlated across species, yet latex caused 27% stronger enzyme inhibition than equimolar amounts of leaf cardenolides. Using a novel multiple regression approach, we found three highly potent cardenolides (identified as calactin, calotropin, and voruscharin) to be primarily responsible for the increased pharmacological activity of milkweed cardenolide mixtures. However, contrary to an expected trade-off between concentration and toxicity, later-diverging milkweeds had the lowest amounts of these potent cardenolides, perhaps indicating an evolutionary response to milkweed's diverse community of specialist cardenolide-sequestering insect herbivores.

What doesn’t kill you makes you stronger: The burdens and benefits of toxin sequestration in a milkweed aphid

Abstract Specialized insect herbivores commonly co‐opt plant defences for protection against predators, but the costs, benefits and mechanisms of sequestration are poorly understood. We quantified sequestration of toxic cardenolides by the specialist aphid Aphis nerii when reared on four closely related milkweed (Asclepias) species with >20‐fold variation in cardenolide content, and in the presence or absence of generalist ladybug predators. Increasing concentrations of apolar plant cardenolides increased sequestered amounts in aphids. High concentrations in plants impaired aphid population growth, but also reduced the top‐down effects of predators. An in vitro enzymatic assay of total cardenolides in aphid bodies using the cardenolides’ target (animal Na+/K+‐ATPase) revealed that the subset of sequestered cardenolides is disproportionally more toxic than cardenolides in leaves. All aphids accumulated two cardenolides not present in their host plant, even on plants with very low foliar cardenolide concentrations. Sequestration of potent cardenolides by A. nerii thus involves passive, concentration‐dependent uptake from the host plant, as well as a presumably more active mechanism of modification and up‐concentration of plant cardenolides. The concentration of toxins in the host plant thus not only determines the negative impacts on growth and performance of an aphid, but also the ease and efficiency by which toxins are sequestered for the aphid’s defence, making the costs and benefits of plant toxins highly context‐dependent for both the plant and the herbivore. Therefore, variation in plant toxins is of central importance for co‐evolutionary plant–insect interactions, particularly in environments with variable predator pressure. A plain language summary is available for this article.

Cardenolide‐defended milkweed bugs do not evoke learning in Nephila senegalensis spiders

AbstractAntipredator 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.

Substitutions in the cardenolide binding site and interaction of subunits affect kinetics besides cardenolide sensitivity of insect Na,K-ATPase

Substitutions within the cardenolide target site of several insects' Na,K-ATPase α-subunits may confer resistance against toxic cardenolides. However, to which extent these substitutions alter the Na,K-ATPase's kinetic properties and how they interact with different β-subunits is not clear. The cardenolide-adapted milkweed bug Oncopeltus fasciatus possesses three paralogs of the α-subunit (A, B, and C) that differ in number and identity of resistance-conferring substitutions. We introduced these substitutions into the α-subunit of Drosophila melanogaster and combined them with the β-subunits Nrv2.2 and Nrv3. The substitutions Q111T-N122H-F786N-T797A (A-copy mimic) and Q111T-N122H-F786N (B-copy mimic) mediated high insensitivity to ouabain, yet they drastically lowered ATPase activity. Remarkably, the identity of the β-subunit was decisive and all α-subunits were less active when combined with Nrv3 than when combined with Nrv2.2. Both the substitutions and the co-expressed β-subunit strongly affected the enyzme's affinity for Na+ and K+. Na+ affinity was considerably higher for all enzymes expressed with nrv3 while expression with nrv2.2 mostly increased K+ affinity. Our results provide the first evidence that resistance against cardenolides comes at the cost of significantly altered kinetic properties of the Na,K-ATPase. The β-subunit can strongly modulate these properties but cannot fully compensate for the effect of the substitutions.

Feeding on toxic prey. The praying mantis (Mantodea) as predator of poisonous butterfly and moth (Lepidoptera) caterpillars

Caterpillars of the monarch butterfly, Danaus plexippus, feed on milkweed plants, Asclepias spp. (Apocynaceae), and sequester their toxic cardenolides aimed at deterring predators. Nevertheless, Chinese praying mantids, Tenodera sinensis, consume these caterpillars after removing the midgut (“gutting”) including its plant content. In the present study, monarch caterpillars raised on A. curassavica, and those of the death's-head hawkmoth, Acherontia atropos, raised on Atropa belladonna containing atropine, were fed to mantids, Hierodula membranacea, which removed the gut of both species discarding about 59% of cardenolides and more than 90% of atropine, respectively. The ingestion of these compounds produced no apparent ill effects in the mantids and both were excreted with faeces. On the other hand, when mantids were fed with larvae of two moth species, Amata mogadorensis and Brahmaea certia, raised on non-poisonous host plants, the mantids showed the same gutting behaviour, thereby discarding indigestible plant material. As polar compounds, e.g. cardenolides and atropine, are not absorbed from the mantids midgut and do not pass the gut membrane, this enables the mantids to feed on toxic prey.

Poor sequestration of toxic host plant cardenolides and their rapid loss in the milkweed butterfly Danaus chrysippus (Lepidoptera: Nymphalidae: Danainae: Danaini)

Butterflies of the genus Danaus are known to sequester toxic cardenolides from milkweed host plants (Apocynaceae). In particular, Danaus plexippus efficiently sequesters and stores these compounds, whereas D. chrysippus, is considered to poorly sequester cardenolides. To estimate its sequestration capability compared with that of D. plexippus, larvae of both species were jointly reared on Asclepias curassavica and the major cardenolides of the host plant, calotropin and calactin, were analyzed in adults sampled at different time intervals after eclosion. Both cardenolides were detected in body and wings of D. plexippus. Whereas the calotropin-concentration remained constant over a period of 24 days, that of calactin steadily decreased. In the body, but not in the wings of D. chrysippus, calactin only was detected in low amounts, which was then almost completely lost during the following 8 days after eclosion, suggesting that in contrast to D. plexippus, cardenolides seem to be less important for that butterfly's defence against predators.

Consequences of toxic secondary compounds in nectar for mutualistbees and antagonist butterflies.

Attraction of mutualists and defense against antagonists are critical challenges for most organisms and can be especially acute for plants with pollinating and non-pollinating flower visitors. Secondary compounds in flowers have been hypothesized to adaptively mediate attraction of mutualists and defense against antagonists, but this hypothesis has rarely been tested. The tissues of milkweeds (Asclepias spp.) contain toxic cardenolides that have long been studied as chemical defenses against herbivores. Milkweed nectar also contains cardenolides, and we have examined the impact of manipulating cardenolides in nectar on the foraging choices of two flower visitors: generalist bumble bees, Bombus impatiens, which are mutualistic pollinators, and specialist monarch butterflies, Danaus plexippus, which are herbivores as larvae and ineffective pollinators as adults. Although individual bumble bees in single foraging bouts showed no avoidance of cardenolides at the highest natural concentrations reported for milkweeds, a pattern of deterrence did arise when entire colonies were allowed to forage for several days. Monarch butterflies were not deterred by the presence of cardenolides in nectar when foraging from flowers, but laid fewer eggs on plants paired with cardenolide-laced flowers compared to controls. Thus, although deterrence of bumble bees by cardenolides may only occur after extensive foraging, a primary effect of nectar cardenolides appears to be reduction of monarch butterfly oviposition.

Trade‐offs and tritrophic consequences of host shifts in specialized root herbivores

Summary Trade‐offs in an herbivore's ability to feed, avoid predation and succeed on alternative hosts are thought to be major driving factors in host specialization. In this study, we compared how two closely related milkweed beetles (Tetraopes spp.) that have specialized on separate Asclepias species respond to host switching to alternative milkweed plants. By additionally examining effects on the beetles’ entomopathogenic natural enemies, we test whether host plant specialization is driven by plant–herbivore interactions alone or whether there is a role for the tritrophic interactions. Tetraopes beetles feed on milkweed roots as larvae and on milkweed shoots as adults, and they sequester toxic cardenolides from their host plants. We predicted that growth and sequestration would be compromised on alternative hosts as a consequence of specialization. Larvae of each Tetraopes species were reared on their native host plant, the host plant of the other species and two closely related Asclepias species that do not typically host Tetraopes. Regardless of beetle specialization, survival of both beetles was highest on A. syriaca, which has the second‐to‐lowest levels of root cardenolides and is the native host for T. tetraophthalmus. Larval growth was not affected by host plant for T. texanus, but T. tetraophthalmus grew best on A. exaltata, a close relative of A. syriaca that does not typically experience beetle feeding. We found that larval sequestration of cardenolides largely mirrored host plant values in the roots, with the exception of T. texanus on A. asperula, which sequestered a low proportion of the cardenolides from this host. Although all larvae were susceptible to entomopathogenic nematodes (EPNs), the number of EPNs emerging from cadavers depended on the beetle–plant combination, with more EPNs produced when beetles were feeding on native compared to non‐native hosts. The observed consequences for tritrophic interactions did not fit expectations for specialized herbivores (i.e. EPN predation was highest when beetles were on their native hosts), suggesting that specialization may be driven primarily by a plant–herbivore interaction in this system, not by a tritrophic effect. A Lay Summary is available for this article.

Population growth and sequestration of plant toxins along a gradient of specialization in four aphid species on the common milkweed Asclepias syriaca

Summary Dietary specialization in insect herbivores has long been hypothesized to predict tolerance of plant defences, with more specialized herbivores being highly tolerant of and sometimes sequestering plant secondary compounds. Plant variation in secondary compounds should thus play an important and predictable role in shaping the performance and distribution of insect communities. We compared the performance of four naturally co‐occurring aphid species on twenty genotypes of the common milkweed Asclepias syriaca. Genotypes of milkweed consistently differed in functional traits, including concentrations of toxic cardenolides, while the diet breadths of the four aphids ranged from broadly generalized to monophagous. The two more generalized species had the highest population growth rate overall, while growth rates decreased with increasing specialization. In contrast, honeydew exudation as a measure of phloem consumption increased with specialization; thus, resource‐use efficiency was lower in specialist aphids. The two more generalized aphids grew best on genotypes with the highest plant growth rate (as an approximation for resource availability), while specialist aphids were not affected by plant growth. All four species contained apolar cardenolides in their bodies and excreted polar cardenolides, but only the most specialized aphid Myzocallis asclepiadis was negatively affected by increasing cardenolide concentrations of the host plant. Sequestration of cardenolides increased with diet specialization, with M. asclepiadis accumulating twice as much as any other species, perhaps explaining its susceptibility to plant cardenolides. Heritable plant traits differentially impacted co‐occurring insect herbivores within the same guild. Generalist aphids were susceptible to variation in plant vigour but not defensive compounds. Increased host specialization resulted in lower resource‐use efficiency, increased phloem throughput and ultimately higher cardenolide sequestration. Variation in these traits is thus likely to determine the relative distribution of generalist and specialist herbivores on plants in natural communities.

Na+/K+-ATPase resistance and cardenolide sequestration: basal adaptations to host plant toxins in the milkweed bugs (Hemiptera: Lygaeidae: Lygaeinae).

Despite sequestration of toxins being a common coevolutionary response to plant defence in phytophagous insects, the macroevolution of the traits involved is largely unaddressed. Using a phylogenetic approach comprising species from four continents, we analysed the ability to sequester toxic cardenolides in the hemipteran subfamily Lygaeinae, which is widely associated with cardenolide-producing Apocynaceae. In addition, we analysed cardenolide resistance of their Na(+)/K(+)-ATPases, the molecular target of cardenolides. Our data indicate that cardenolide sequestration and cardenolide-resistant Na(+)/K(+)-ATPase are basal adaptations in the Lygaeinae. In two species that shifted to non-apocynaceous hosts, the ability to sequester was secondarily reduced, yet Na(+)/K(+)-ATPase resistance was maintained. We suggest that both traits evolved together and represent major coevolutionary adaptations responsible for the evolutionary success of lygaeine bugs. Moreover, specialization on cardenolides was not an evolutionary dead end, but enabled this insect lineage to host shift to cardenolide-producing plants from distantly related families.