STEPWISE EVOLUTION OF RESISTANCE TO TOXIC CARDENOLIDES VIA GENETIC SUBSTITUTIONS IN THE NA+/K+‐ATPASE OF MILKWEED BUTTERFLIES (LEPIDOPTERA: DANAINI)

Despite the monarch butterfly (Danaus plexippus) being famous for its adaptations to the defensive traits of its milkweed host plants, little is known about the macroevolution of these traits. Unlike most other animal species, monarchs are largely insensitive to cardenolides, because their target site, the sodium pump (Na⁺/K⁺‐ATPase), has evolved amino acid substitutions that reduce cardenolide binding (so‐called target site insensitivity, TSI). Because many, but not all, species of milkweed butterflies (Danaini) are associated with cardenolide‐containing host plants, we analyzed 16 species, representing all phylogenetic lineages of milkweed butterflies, for the occurrence of TSI by sequence analyses of the Na⁺/K⁺‐ATPase gene and by enzymatic assays with extracted Na⁺/K⁺‐ATPase. Here we report that sensitivity to cardenolides was reduced in a stepwise manner during the macroevolution of milkweed butterflies. Strikingly, not all Danaini typically consuming cardenolides showed TSI, but rather TSI was more strongly associated with sequestration of toxic cardenolides. Thus, the interplay between bottom‐up selection by plant compounds and top‐down selection by natural enemies can explain the evolutionary sequence of adaptations to these toxins.     

Chinese mantids gut toxic monarch caterpillars: avoidance of prey defence?

1. Monarch caterpillars, Danaus plexippus (Linnaeus), feed on milkweed plants in the genus Asclepias and sequester cardenolides as an anti–predator defence. However, some predators are able to consume this otherwise unpalatable prey. 2. Chinese mantids, Tenodera sinensis (Saussure), were observed consuming monarch caterpillars by ‘gutting’ them (i.e. removing the gut and associated internal organs). They then feed on the body of this herbivore without any apparent ill effects. 3. How adult T. sinensis handle and consume toxic (D. plexippus) and non–toxic [Ostrinia nubilalis (Hübner) and Galleria mellonella (Linnaeus)] caterpillars was explored. The differences in the carbon/nitrogen (C:N) ratio and cardenolide content of monarch tissue consumed or discarded by mantids were analysed. 4. Mantids gutted monarchs while wholly consuming non–toxic species. Monarch gut tissue had a higher C:N ratio than non–gut tissue, confirming the presence of plant material. Although there were more cardenolide peaks in the monarch body compared with gut tissue, the total cardenolide concentration and polarity index did not differ. 5. Although T. sinensis treated toxic prey differently than non–toxic prey, gutting did not decrease the mantid's total cardenolide intake. As other predators consume monarch caterpillars whole, this behaviour may be rooted in species–specific vulnerability to particular cardenolides or simply reflect a preference for high–N tissues.     

Effects of soil nutrients on the sequestration of plant defence chemicals by the specialist insect herbivore,Danaus plexippus

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.     

Dietary influence of cardenolides on larval growth and development of the milkweed bug Oncopeltus fasciatus

Larvae of the milkweed bug Oncopeltus fasciatus were reared on the seeds of eight different species of milkweed (Asclepias), representing a wide range of cardenolide concentrations in the diet. There were few significant differences in larval developmental period, wet body weight of teneral adults, dry weight of adults, and pronotal width of adults reared on the different diets. However, the data indicate no significant correlations between cardenolide content, and body weight or size of the adult insects.There was no evidence in this study of a physiological cost or adverse effect on the larval growth and development of insects which sequestered and stored differing quantities of cardenolides. Instead, the data support a recently-proposed model of cardenolide sequestration which may be energy-independent.The validity of evidence supporting a physiological cost hypothesis for sequestration of cardenolides by the monarch butterfly is discussed in the light of these findings.     

Milkweed butterfly resistance to plant toxins is linked to sequestration,not coping with a toxic diet

Insect resistance to plant toxins is widely assumed to have evolved in response to using defended plants as a dietary resource. We tested this hypothesis in the milkweed butterflies (Danaini) which have progressively evolved higher levels of resistance to cardenolide toxins based on amino acid substitutions of their cellular sodium–potassium pump (Na⁺/K⁺-ATPase). Using chemical, physiological and caterpillar growth assays on diverse milkweeds (Asclepias spp.) and isolated cardenolides, we show that resistant Na⁺/K⁺-ATPases are not necessary to cope with dietary cardenolides. By contrast, sequestration of cardenolides in the body (as a defence against predators) is associated with the three levels of Na⁺/K⁺-ATPase resistance. To estimate the potential physiological burden of cardenolide sequestration without Na⁺/K⁺-ATPase adaptations, we applied haemolymph of sequestering species on isolated Na⁺/K⁺-ATPase of sequestering and non-sequestering species. Haemolymph cardenolides dramatically impair non-adapted Na⁺/K⁺-ATPase, but had systematically reduced effects on Na⁺/K⁺-ATPase of sequestering species. Our data indicate that major adaptations to plant toxins may be evolutionarily linked to sequestration, and may not necessarily be a means to eat toxic plants. Na⁺/K⁺-ATPase adaptations thus were a potential mechanism through which predators spurred the coevolutionary arms race between plants and insects.     

The evolution of cardenolide-resistant forms of Na⁺,K⁺ -ATPase in Danainae butterflies

Cardenolides are a class of plant secondary compounds that inhibit the proper functioning of the Na⁺, K⁺‐ATPase enzyme in susceptible animals. Nonetheless, many insect species are able to sequester cardenolides for their own defence. These include butterflies in the subfamily Danainae (Family: Nymphalidae) such as the monarch (Danaus plexippus). Previous studies demonstrated that monarchs harbour an asparagine (N) to histidine (H) substitution (N122H) in the α subunit of Na⁺, K⁺‐ATPase (ATPα) that reduces this enzyme’s sensitivity to cardenolides. More recently, it has been suggested that at ATPα position 111, monarchs may also harbour a leucine (L)/glutamine (Q) polymorphism. This later amino acid could also contribute to cardenolide insensitivity. However, here we find that incorrect annotation of the initially reported DNA sequence for ATPα has led to several erroneous conclusions. Using a population genetic and phylogenetic analysis of monarchs and their close relatives, we show that an ancient Q111L substitution occurred prior to the radiation of all Danainae, followed by a second substitution at the same site to valine (V), which arose before the diversification of the Danaus genus. In contrast, N122H appears to be a recent substitution specific to monarchs. Surprisingly, examination of a broader insect phylogeny reveals that the same progression of amino acid substitutions (Q111L → L111V + N122H) has also occurred in Chyrsochus beetles (Family: Chrysomelidae, Subfamily: Eumolpinae) that feed on cardenolide‐containing host plants. The parallel pattern of amino acid substitution in these two distantly related lineages is consistent with an adaptive role for these substitutions in reducing cardenolide sensitivity and suggests that their temporal order may be limited by epistatic interactions.     

It’s the first bites that count: Survival of first‐instar monarchs on milkweeds

Mortality 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.     

Dietary cardenolides enhance growth and change the direction of the fecundity‐longevity trade‐off in milkweed bugs (Heteroptera: Lygaeinae)

Sequestration, that is, the accumulation of plant toxins into body tissues for defense, was predicted to incur physiological costs and may require resistance traits different from those of non‐sequestering insects. Alternatively, sequestering species could experience a cost in the absence of toxins due to selection on physiological homeostasis under permanent exposure of sequestered toxins in body tissues. Milkweed bugs (Heteroptera: Lygaeinae) sequester high amounts of plant‐derived cardenolides. Although being potent inhibitors of the ubiquitous animal enzyme Na⁺/K⁺‐ATPase, milkweed bugs can tolerate cardenolides by means of resistant Na⁺/K⁺‐ATPases. Both adaptations, resistance and sequestration, are ancestral traits of the Lygaeinae. Using four milkweed bug species (Heteroptera: Lygaeidae: Lygaeinae) and the related European firebug (Heteroptera: Pyrrhocoridae: Pyrrhocoris apterus) showing different combinations of the traits “cardenolide resistance” and “cardenolide sequestration,” we tested how the two traits affect larval growth upon exposure to dietary cardenolides in an artificial diet system. While cardenolides impaired the growth of P. apterus nymphs neither possessing a resistant Na⁺/K⁺‐ATPase nor sequestering cardenolides, growth was not affected in the non‐sequestering milkweed bug Arocatus longiceps, which possesses a resistant Na⁺/K⁺‐ATPase. Remarkably, cardenolides increased growth in the sequestering dietary specialists Caenocoris nerii and Oncopeltus fasciatus but not in the sequestering dietary generalist Spilostethus pandurus, which all possess a resistant Na⁺/K⁺‐ATPase. We furthermore assessed the effect of dietary cardenolides on additional life history parameters, including developmental speed, longevity of adults, and reproductive success in O. fasciatus. Unexpectedly, nymphs under cardenolide exposure developed substantially faster and lived longer as adults. However, fecundity of adults was reduced when maintained on cardenolide‐containing diet for their entire lifetime but not when adults were transferred to non‐toxic sunflower seeds. We speculate that the resistant Na⁺/K⁺‐ATPase of milkweed bugs is selected for working optimally in a “toxic environment,” that is, when sequestered cardenolides are stored in the body.     

Disease ecology across soil boundaries: effects of below-ground fungi on above-ground host–parasite interactions

Host–parasite interactions are subject to strong trait-mediated indirect effects from other species. However, it remains unexplored whether such indirect effects may occur across soil boundaries and connect spatially isolated organisms. Here, we demonstrate that, by changing plant (milkweed Asclepias sp.) traits, arbuscular mycorrhizal fungi (AMF) significantly affect interactions between a herbivore (the monarch butterfly Danaus plexippus) and its protozoan parasite (Ophryocystis elektroscirrha), which represents an interaction across four biological kingdoms. In our experiment, AMF affected parasite virulence, host resistance and host tolerance to the parasite. These effects were dependent on both the density of AMF and the identity of milkweed species: AMF indirectly increased disease in monarchs reared on some species, while alleviating disease in monarchs reared on other species. The species-specificity was driven largely by the effects of AMF on both plant primary (phosphorus) and secondary (cardenolides; toxins in milkweeds) traits. Our study demonstrates that trait-mediated indirect effects in disease ecology are extensive, such that below-ground interactions between AMF and plant roots can alter host–parasite interactions above ground. In general, soil biota may play an underappreciated role in the ecology of many terrestrial host–parasite systems.     

Monarchs in decline: a collateral landscape‐level effect of modern agriculture

We review the postulated threatening processes that may have affected the decline in the eastern population of the monarch butterfly, Danaus plexippus L. (Lepidoptera: Nymphalidae), in North America. Although there are likely multiple contributing factors, such as climate and resource‐related effects on breeding, migrating, and overwintering populations, the key landscape‐level change appears to be associated with the widespread use of genetically modified herbicide resistant crops that have rapidly come to dominate the extensive core summer breeding range. We dismiss misinterpretations of the apparent lack of population change in summer adult count data as logically flawed. Glyphosate‐tolerant soybean and maize have enabled the extensive use of this herbicide, generating widespread losses of milkweed (Asclepias spp.), the only host plants for monarch larvae. Modeling studies that simulate lifetime realized fecundity at a landscape scale, direct counts of milkweeds, and extensive citizen science data across the breeding range suggest that a herbicide‐induced, landscape‐level reduction in milkweed has precipitated the decline in monarchs. A recovery will likely require a monumental effort for the re‐establishment of milkweed resources at a commensurate landscape scale.     

Maintenance of narrow diet breadth in the monarch butterfly caterpillar: response to various plant species and chemicals

In order to better understand the maintenance of a fairly narrow diet breadth in monarch butterfly larvae, Danaus plexippus L. (Lepidoptera: Nymphalidae: Danainae), we measured feeding preference and survival on host and non-host plant species, and sensitivity to host and non-host plant chemicals. For the plant species tested, a hierarchy of feeding preferences was observed; only plants from the Asclepiadaceae were more or equally preferred to Asclepias curassavica, the common control. The feeding preferences among plant species within the Asclepiadaceae are similar to published mean cardenolide concentrations. However, since cardenolide data were not collected from individual plants tested, definitive conclusions regarding cardenolide concentrations and plant acceptability cannot be made. Although several non-Asclepiadaceae were eaten in small quantities, all were less preferred to A. curassavica. Additionally, these non-Asclepiadaceae do not support continued feeding, development, and survival of first and fifth-instar larvae. Preference for a host versus a non-host (A. curassavica versus Vinca rosea) increased for A. curassavica reared larvae as compared to diet-reared larvae suggesting plasticity in larval food preferences. Furthermore, host species were significantly preferred over non-host plant species in bioassays using a host plant or sucrose as a common control. Larval responses to pure chemicals were examined in order to determine if host and non-host chemicals stimulate or deter feeding in monarch larvae. We found that larvae were stimulated to feed by some ubiquitous plant chemicals, such as sucrose, inositol, and rutin. In contrast, several non-host plant chemicals deterred feeding: caffeine, apocynin, gossypol, tomatine, atropine, quercitrin, and sinigrin. Additionally the cardenolides digitoxin and ouabain, which are not in milkweed plants, were neutral in their influence on feeding. Another non-milkweed cardenolide, cymarin, significantly deterred feeding. Extracts of A. curassavica leaves were tested in bioassays to determine which components of the leaf stimulate feeding. Both an ethanol extract of whole leaves and a hexane leaf-surface extract are phagostimulatory, suggesting the involvement of both polar and non-polar plant compounds. These data suggest that the host range of D. plexippus larvae is maintained by both feeding stimulatory and deterrent chemicals in host and non-host plants.     

Stable isotopes (δD and δ13C) are geographic indicators of natal origins of monarch butterflies in eastern North America

Wing membranes of laboratory and field-reared monarch butterflies (Danaus plexippus) were analyzed for their stable-hydrogen (δD) and carbon (δ¹³C) isotope ratios to determine whether this technique could be used to identify their natal origins. We hypothesized that the hydrogen isotopic composition of monarch butterfly wing keratin would reflect the hydrogen isotope patterns of rainfall in areas of natal origin where wings were formed. Monarchs were reared in the laboratory on milkweed plants (Asclepias sp.) grown with water of known deuterium content, and, with the assistance of volunteers, on native milkweeds throughout eastern North America. The results show that the stable hydrogen isotopic composition of monarch butterflies is highly correlated with the isotopic composition of the milkweed host plants, which in turn corresponds closely with the long-term geographic patterns of deuterium in rainfall. Stable-carbon isotope values in milkweed host plants were similarly correlated with those values in monarch butterflies and showed a general pattern of enrichment along a southwest to northeast gradient bisecting the Great Lakes. These findings indicate that natal origins of migratory and wintering monarchs in Mexico can be inferred from the combined δD and δ¹³C isotopic signatures in their wings. This relationship establishes that analysis of hydrogen and carbon isotopes can be used to answer questions concerning the biology of migratory monarch butterflies and provides a new approach to tracking similar migratory movements of other organisms. Received: 1 July 1998 / Accepted: 11 November 1998     

A comparison of the host preference of monarch butterflies (Danaus plexippus) for milkweed (Asclepias syriaca) over dog-strangler vine (Vincetoxicum rossicum)

Observations in the field indicate that monarch butterflies will oviposit on dog‐strangler vine, an invasive introduced species in the same family as milkweed (Asclepias spp.), the principal larval host of monarchs. The potential impact of this behaviour depends on the strength of the preference of monarch adults to oviposit on these two hosts and the relative ability of larvae to survive on each. We determined the preference for milkweed vs. dog‐strangler vine of ovipositing adults and first instar larvae in choice and no‐choice tests. We also compared the ability of larvae to consume, develop, and survive on either host. In the presence of both hosts, adults exhibited a strong preference to oviposit on milkweed over dog‐strangler vine (mean 80.7 eggs compared to 0.4 eggs over 48 h, respectively). In the absence of milkweed, adults ceased oviposition (mean 0.9 eggs in 48 h), but resumed oviposition when the dog‐strangler vine was replaced with milkweed (mean 99.1 eggs in 48 h). Given a choice between hosts over 24 h, 92% of larvae moved to milkweed leaves and consumed 3.94 cm² of milkweed leaves compared to 2% of larvae that moved to dog‐strangler vine and consumed negligible amounts of leaf material (0.01 cm²). Without a choice, larvae on dog‐strangler vine never consumed more than mean 0.02 cm² larva^?1 in a 24‐h period, did not develop beyond the first instar, and died within 96 h. We obtained no data in support of an effect of the presence of dog‐strangler vine on monarch butterfly populations.     

Elevated atmospheric concentrations of carbon dioxide reduce monarch tolerance and increase parasite virulence by altering the medicinal properties of milkweeds

Hosts combat their parasites using mechanisms of resistance and tolerance, which together determine parasite virulence. Environmental factors, including diet, mediate the impact of parasites on hosts, with diet providing nutritional and medicinal properties. Here, we present the first evidence that ongoing environmental change decreases host tolerance and increases parasite virulence through a loss of dietary medicinal quality. Monarch butterflies use dietary toxins (cardenolides) to reduce the deleterious impacts of a protozoan parasite. We fed monarch larvae foliage from four milkweed species grown under either elevated or ambient CO₂, and measured changes in resistance, tolerance, and virulence. The most high‐cardenolide milkweed species lost its medicinal properties under elevated CO₂; monarch tolerance to infection decreased, and parasite virulence increased. Declines in medicinal quality were associated with declines in foliar concentrations of lipophilic cardenolides. Our results emphasize that global environmental change may influence parasite–host interactions through changes in the medicinal properties of plants.     

Can roadside habitat lead monarchs on a route to recovery?

The Eastern North American monarch butterfly population has severely declined over the past decade. The decreasing availability of larval host plants (milkweeds) due to the use of herbicide-tolerant crops has been implicated in this decline. Roadsides could provide additional habitat for monarchs. In this study we document the occurrence of milkweed and monarchs on roadsides, and discuss whether roadsides are appropriate targets for monarch habitat restoration. We sampled roadside rights-of-way in the Upper Midwestern U.S. during the summer of 2015 to estimate the abundance, distribution, and diversity of milkweeds and the extent to which monarchs use these milkweeds. We then compared monarch densities in roadsides to other habitat types and modelled immature monarch densities based on several site characteristics. Our findings suggest that roadsides have conservation potential for monarchs, especially when other habitat is scarce and if wildlife-friendly management practices are enacted. Milkweeds were found on ~60% of roadside transects. Asclepias syriaca was the most common of the seven species encountered, occurring on 97% of transects with milkweed. Immature monarchs were observed in roadsides, but in lower densities than other habitats during the same time period. At lower milkweed densities, immature monarch density per unit area is positively correlated with milkweed density. However, milkweed density weakens as a predictor of immature monarch density over ~0.6 plants per m², possibly indicating a saturation effect.     

Colonization and usage of eight milkweed (<Emphasis Type="Italic">Asclepias</Emphasis>) species by monarch butterflies and bees in urban garden settings

Planting milkweeds on public and private lands has emerged as a central conservation strategy for restoring declining North American migratory populations of the monarch butterfly (Danaus plexippus). Nearly all actionable science on this issue has focused on restoring common milkweed (Asclepias syriaca L.) in rural land types. The aim of this study was to develop recommendations for the best milkweeds for managed gardens intended to support both monarch butterflies and bees. Eight milkweed (Asclepias) species varying in height, form, and leaf shape were grown in a common-garden experiment at a public arboretum. We measured milkweed growth, tillering, and bloom periods, conducted bi-weekly counts of eggs and larvae to assess colonization by wild monarchs, and evaluated suitability for growth of monarch larvae. We also quantified bee visitation and compared the bee assemblages associated with six of the eight species, augmented with additional collections from other sites. Monarchs rapidly colonized the gardens, but did not equally use all of the milkweed species. More eggs and larvae were found on taller, broad-leaved milkweeds, but there was relatively little difference in larval performance, suggesting ovipositional preference for more apparent plants. Asclepias tuberosa and A. fascicularis attracted the greatest number of bees, whereas bee genus diversity was greatest on A. verticillata, A. fascicularis, and A. tuberosa. Milkweeds that do not spread extensively by tillering may be best suited for managed gardens. Combining milkweeds that are preferred by ovipositing monarchs with ones that are particularly attractive to bees may enhance conservation value of small urban gardens.     

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.