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

In coevolution between plants and insects, reciprocal selection often leads to phenotype matching between chemical defense and herbivore offense. Nonetheless, it is not well understood whether distinct plant parts are differentially defended and how herbivores adapted to those parts cope with tissue-specific defense. Milkweed plants produce a diversity of cardenolide toxins and specialist herbivores have substitutions in their target enzyme (Na+/K+-ATPase), each playing a central role in milkweed-insect coevolution. The four-eyed milkweed beetle (Tetraopes tetrophthalmus) is an abundant toxin-sequestering herbivore that feeds exclusively on milkweed roots as larvae and less so on milkweed leaves as adults. Accordingly, we tested the tolerance of this beetle's Na+/K+-ATPase to cardenolide extracts from roots versus leaves of its main host (Asclepias syriaca), along with sequestered cardenolides from beetle tissues. We additionally purified and tested the inhibitory activity of dominant cardenolides from roots (syrioside) and leaves (glycosylated aspecioside). Tetraopes' enzyme was threefold more tolerant of root extracts and syrioside than leaf cardenolides. Nonetheless, beetle-sequestered cardenolides were more potent than those in roots, suggesting selective uptake or dependence on compartmentalization of toxins away from the beetle's enzymatic target. Because Tetraopes has two functionally validated amino acid substitutions in its Na+/K+-ATPase compared to the ancestral form in other insects, we compared its cardenolide tolerance to that of wild-type Drosophila and CRISPR-edited Drosophila with Tetraopes' Na+/K+-ATPase genotype. Those two amino acid substitutions accounted for >50% of Tetraopes' enhanced enzymatic tolerance of cardenolides. Thus, milkweed's tissue-specific expression of root toxins is matched by physiological adaptations in its specialist root herbivore.

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

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

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

Herbivores that sequester toxins are thought to have cracked the code of plant defences. Nonetheless, coevolutionary theory predicts that plants should evolve toxic variants that also negatively impact specialists. We propose and test the selective sequestration hypothesis, that specialists preferentially sequester compounds that are less toxic to themselves while maintaining toxicity to enemies. Using chemically distinct plants, we show that monarch butterflies sequester only a subset of cardenolides from milkweed leaves that are less potent against their target enzyme (Na+ /K+ -ATPase) compared to several dominant cardenolides from leaves. However, sequestered compounds remain highly potent against sensitive Na+ /K+ -ATPases found in most predators. We confirmed this differential toxicity with mixtures of purified cardenolides from leaves and butterflies. The genetic basis of monarch adaptation to sequestered cardenolides was also confirmed with transgenic Drosophila that were CRISPR-edited with the monarch's Na+ /K+ -ATPase. Thus, the monarch's selective sequestration appears to reduce self-harm while maintaining protection from enemies.

Late-instar monarch caterpillars sabotage milkweed to acquire toxins, not to disarm plant defence.

Sabotaging milkweed by monarch caterpillars (Danaus plexippus) is a famous textbook example of disarming plant defence. By severing leaf veins, monarchs are thought to prevent the flow of toxic latex to their feeding site. Here, we show that sabotaging by monarch caterpillars is not only an avoidance strategy. While young caterpillars appear to avoid latex, late-instar caterpillars actively ingest exuding latex, presumably to increase sequestration of cardenolides used for defence against predators. Comparisons with caterpillars of the related but non-sequestering common crow butterfly (Euploea core) revealed three lines of evidence supporting our hypothesis. First, monarch caterpillars sabotage inconsistently and therefore the behaviour is not obligatory to feed on milkweed, whereas sabotaging precedes each feeding event in Euploea caterpillars. Second, monarch caterpillars shift their behaviour from latex avoidance in younger to eager drinking in later stages, whereas Euploea caterpillars consistently avoid latex and spit it out during sabotaging. Third, monarchs reared on detached leaves without latex sequestered more cardenolides when caterpillars imbibed latex offered with a pipette. Thus, we conclude that monarch caterpillars have transformed the ancestral 'sabotage to avoid' strategy into a 'sabotage to consume' strategy, implying a novel behavioural adaptation to increase sequestration of cardenolides for defence.

Mixtures of Milkweed Cardenolides Protect Monarch Butterflies against Parasites.

Plants have evolved a diverse arsenal of defensive secondary metabolites in their evolutionary arms race with insect herbivores. In addition to the bottom-up forces created by plant chemicals, herbivores face top-down pressure from natural enemies, such as predators, parasitoids and parasites. This has led to the evolution of specialist herbivores that do not only tolerate plant secondary metabolites but even use them to fight natural enemies. Monarch butterflies (Danaus plexippus) are known for their use of milkweed chemicals (cardenolides) as protection against vertebrate predators. Recent studies have shown that milkweeds with high cardenolide concentrations can also provide protection against a virulent protozoan parasite. However, whether cardenolides are directly responsible for these effects, and whether individual cardenolides or mixtures of these chemicals are needed to reduce infection, remains unknown. We fed monarch larvae the four most abundant cardenolides found in the anti-parasitic-milkweed Asclepias curassavica at varying concentrations and compositions to determine which provided the highest resistance to parasite infection. Measuring infection rates and infection intensities, we found that resistance is dependent on both concentration and composition of cardenolides, with mixtures of cardenolides performing significantly better than individual compounds, even when mixtures included lower concentrations of individual compounds. These results suggest that cardenolides function synergistically to provide resistance against parasite infection and help explain why only milkweed species that produce diverse cardenolide compounds provide measurable parasite resistance. More broadly, our results suggest that herbivores can benefit from consuming plants with diverse defensive chemical compounds through release from parasitism.

Cardenolides, toxicity, and the costs of sequestration in the coevolutionary interaction between monarchs and milkweeds.

For highly specialized insect herbivores, plant chemical defenses are often co-opted as cues for oviposition and sequestration. In such interactions, can plants evolve novel defenses, pushing herbivores to trade off benefits of specialization with costs of coping with toxins? We tested how variation in milkweed toxins (cardenolides) impacted monarch butterfly (Danaus plexippus) growth, sequestration, and oviposition when consuming tropical milkweed (Asclepias curassavica), one of two critical host plants worldwide. The most abundant leaf toxin, highly apolar and thiazolidine ring-containing voruscharin, accounted for 40% of leaf cardenolides, negatively predicted caterpillar growth, and was not sequestered. Using whole plants and purified voruscharin, we show that monarch caterpillars convert voruscharin to calotropin and calactin in vivo, imposing a burden on growth. As shown by in vitro experiments, this conversion is facilitated by temperature and alkaline pH. We next employed toxin-target site experiments with isolated cardenolides and the monarch's neural Na+/K+-ATPase, revealing that voruscharin is highly inhibitory compared with several standards and sequestered cardenolides. The monarch's typical >50-fold enhanced resistance to cardenolides compared with sensitive animals was absent for voruscharin, suggesting highly specific plant defense. Finally, oviposition was greatest on intermediate cardenolide plants, supporting the notion of a trade-off between benefits and costs of sequestration for this highly specialized herbivore. There is apparently ample opportunity for continued coevolution between monarchs and milkweeds, although the diffuse nature of the interaction, due to migration and interaction with multiple milkweeds, may limit the ability of monarchs to counteradapt.

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

Plant toxicity shapes the dietary choices of herbivores. Especially when herbivores sequester plant toxins, they may experience a trade-off between gaining protection from natural enemies and avoiding toxicity. The availability of toxins for sequestration may additionally trade off with the nutritional quality of a potential food source for sequestering herbivores. We hypothesized that diet mixing might allow a sequestering herbivore to balance nutrition and defence (via sequestration of plant toxins). Accordingly, here we address diet mixing and sequestration of large milkweed bugs (Oncopeltus fasciatus) when they have differential access to toxins (cardenolides) in their diet. In the absence of toxins from a preferred food (milkweed seeds), large milkweed bugs fed on nutritionally adequate non-toxic seeds, but supplemented their diet by feeding on nutritionally poor, but cardenolide-rich milkweed leaf and stem tissues. This dietary shift corresponded to reduced insect growth but facilitated sequestration of defensive toxins. Plant production of cardenolides was also substantially induced by bug feeding on leaf and stem tissues, perhaps benefitting this cardenolide-resistant herbivore. Thus, sequestration appears to drive diet mixing in this toxic plant generalist, even at the cost of feeding on nutritionally poor plant tissue.

The price of defence: toxins, visual signals and oxidative state in an aposematic butterfly.

In a variety of aposematic species, the conspicuousness of an individual's warning signal and the quantity of its chemical defence are positively correlated. This apparent honest signalling is predicted by resource competition models which assume that the production and maintenance of aposematic defences compete for access to antioxidant molecules that have dual functions as pigments and in protecting against oxidative damage. To test for such trade-offs, we raised monarch butterflies (Danaus plexippus) on different species of their milkweed host plants (Apocynaceae) that vary in quantities of cardenolides to test whether (i) the sequestration of cardenolides as a secondary defence is associated with costs in the form of oxidative lipid damage and reduced antioxidant defences; and (ii) lower oxidative state is associated with a reduced capacity to produce aposematic displays. In male monarchs conspicuousness was explained by an interaction between oxidative damage and sequestration: males with high levels of oxidative damage became less conspicuous with increased sequestration of cardenolides, whereas those with low oxidative damage became more conspicuous with increased levels of cardenolides. There was no significant effect of oxidative damage or concentration of sequestered cardenolides on female conspicuousness. Our results demonstrate a physiological linkage between the production of coloration and oxidative state, and differential costs of sequestration and signalling in monarch butterflies.

Herbivory, plant traits and nectar chemistry interact to affect the community of insect visitors and pollination in common milkweed, Asclepias syriaca.

Herbivory can alter plant fitness directly through changing reproductive allocation and indirectly through changing pollinator identity or behavior. Common milkweed is a plant of conservation concern with an inducible chemical defense that is also an important nectar resource. In this study, we aim to understand how herbivory severity and plant traits, including morphology and nectar chemistry, interact to affect insect visitation and pod production in common milkweed. We conducted pollinator watches on plants with experimentally varied herbivory severity and quantified insect frequency and visit length as a response to nectar chemistry, ramet height, number of inflorescences, number of flowers per inflorescence and percent tissue removed. We also quantified pollinator effectiveness and importance. Increased herbivory severity reduced floral displays, including fewer inflorescences and fewer flowers per inflorescence. A reduced floral display was correlated with reduced sucrose, fructose and glucose and resulted in a reduced number and species richness of insect visitors. Fewer flowers per inflorescence reduced the frequency of bumble bee and fly visitors, which were two important pollinators. Although honeybees, flies, small bees, soldier beetles and bumble bees were equally effective pollinators, only bumble bee frequency was positively correlated with pod production. The differences in pollinator visitation have the potential to create diversifying selection on plant floral traits, many of which are also affected by herbivores. This research demonstrates potentially conflicting selection pressures between native and non-native pollinators as well as non-native herbivores.

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.

Plant water limitation and its impact on the oviposition preferences of the monarch butterfly (Lepidoptera: Nymphalidae).

Intensifying drought conditions across the western United States due to global climate change are altering plant-insect interactions. Specialist herbivores must find their host plants within a matrix of nonhosts, and thus often rely upon specific plant secondary chemistry for host location and oviposition cues. Climate-induced alterations to plant chemistry could thus affect female selection of larval food plants. Here, we investigated whether host-plant water limitation influenced oviposition preference in a threatened invertebrate: the monarch butterfly (Danaus plexippus). We found that females deposited more eggs on reduced-water than on well-watered narrowleaf milkweed plants (Asclepias fascicularis), but we could not attribute this change to any specific change in plant chemistry. Specialist herbivores, such as the monarch butterfly, which are tightly linked to specific plant cues, may experience shift in preferences under global-change conditions. Understanding oviposition preferences will be important to directing ongoing habitat restoration activities for this declining insect.

Sequestration of Defenses against Predators Drives Specialized Host Plant Associations in Preadapted Milkweed Bugs (Heteroptera: Lygaeinae).

AbstractHost plant specialization across herbivorous insects varies dramatically, but while the molecular mechanisms of host plant adaptations are increasingly known, we often lack a comprehensive understanding of the selective forces that favor specialization. The milkweed bugs (Heteroptera: Lygaeinae) are ancestrally associated with plants of the Apocynaceae from which they commonly sequester cardiac glycosides for defense, facilitated by resistant Na+/K+-ATPases and adaptations for transport, storage, and discharge of toxins. Here, we show that three Lygaeinae species independently colonized four novel nonapocynaceous hosts that convergently produce cardiac glycosides. A fourth species shifted to a new source of toxins by tolerating and sequestering alkaloids from meadow saffron (Colchicum autumnale, Colchicaceae). Across three milkweed bug species tested, feeding on seeds containing toxins did not improve growth or speed of development and even impaired growth and development in two species, but sequestration mediated protection of milkweed bugs against two natural predators: lacewing larvae and passerine birds. We conclude that physiological preadaptations and convergent phytochemistry facilitated novel specialized host associations. Since toxic seeds did not improve growth but either impaired growth or, at most, had neutral effects, selection by predators on sequestration of defenses, rather than the exploitation of additional profitable dietary resources, can lead to obligatory specialized host associations in otherwise generalist insects.

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

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

Monarch butterfly and milkweed declines substantially predate the use of genetically modified crops.

Monarch butterfly (Danaus plexippus) decline over the past 25 years has received considerable public and scientific attention, in large part because its decline, and that of its milkweed (Asclepias spp.) host plant, have been linked to genetically modified (GM) crops and associated herbicide use. Here, we use museum and herbaria specimens to extend our knowledge of the dynamics of both monarchs and milkweeds in the United States to more than a century, from 1900 to 2016. We show that both monarchs and milkweeds increased during the early 20th century and that recent declines are actually part of a much longer-term decline in both monarchs and milkweed beginning around 1950. Herbicide-resistant crops, therefore, are clearly not the only culprit and, likely, not even the primary culprit: Not only did monarch and milkweed declines begin decades before GM crops were introduced, but other variables, particularly a decline in the number of farms, predict common milkweed trends more strongly over the period studied here.

Pyrethroid insecticide and milkweed cardenolide interactions on detoxification enzyme activity and expression in monarch caterpillars.

Declines of the monarch butterfly population have prompted large-scale plantings of milkweed to restore the population. In North America, there are >73 species of milkweed to choose from for these nationwide plantings. However, it is unclear how different milkweed species affect monarch caterpillar physiology, particularly detoxification enzyme activity and gene expression, given the highly variable cardenolide composition across milkweed species. Here, we investigate the effects of a high cardenolide, tropical milkweed species and a low cardenolide, swamp milkweed species on pyrethroid sensitivity as well as detoxification enzyme activity and expression in monarch caterpillars. Caterpillars fed on each species through the fifth-instar stage and were topically treated with bifenthrin after reaching this final-instar stage. Esterase, glutathione S-transferase, and cytochrome P450 monooxygenase activities were quantified as well as the expression of selected esterase, glutathione S-transferase, ABC transporter, and cytochrome P450 monooxygenase transcripts. There were no significant differences in survival 24 h after treatment with bifenthrin. However, bifenthrin significantly increased glutathione S-transferase activity in caterpillars feeding on tropical milkweed and significantly decreased esterase activity in caterpillars feeding on tropical and swamp milkweed. Significant differential expression of ABC transporter, glutathione S-transferase, and esterase genes was observed for caterpillars feeding on tropical and swamp milkweed and not receiving bifenthrin treatment. Furthermore, significant differential expression of glutathione S-transferase and esterase genes was observed for bifenthrin-treated and -untreated caterpillars feeding on tropical milkweed relative to swamp milkweed. These results suggest that feeding on different milkweed species can affect detoxification and development mechanisms with which monarch caterpillars rely on to cope with their environment.

New Structures, Spectrometric Quantification, and Inhibitory Properties of Cardenolides from Asclepias curassavica Seeds.

Cardiac glycosides are a large class of secondary metabolites found in plants. In the genus Asclepias, cardenolides in milkweed plants have an established role in plant-herbivore and predator-prey interactions, based on their ability to inhibit the membrane-bound Na+/K+-ATPase enzyme. Milkweed seeds are eaten by specialist lygaeid bugs, which are the most cardenolide-tolerant insects known. These insects likely impose natural selection for the repeated derivatisation of cardenolides. A first step in investigating this hypothesis is to conduct a phytochemical profiling of the cardenolides in the seeds. Here, we report the concentrations of 10 purified cardenolides from the seeds of Asclepias curassavica. We report the structures of new compounds: 3-O-ß-allopyranosyl coroglaucigenin (1), 3-[4'-O-ß-glucopyranosyl-ß-allopyranosyl] coroglaucigenin (2), 3'-O-ß-glucopyranosyl-15-ß-hydroxycalotropin (3), and 3-O-ß-glucopyranosyl-12-ß-hydroxyl coroglaucigenin (4), as well as six previously reported cardenolides (5-10). We test the in vitro inhibition of these compounds on the sensitive porcine Na+/K+-ATPase. The least inhibitory compound was also the most abundant in the seeds-4'-O-ß-glucopyranosyl frugoside (5). Gofruside (9) was the most inhibitory. We found no direct correlation between the number of glycosides/sugar moieties in a cardenolide and its inhibitory effect. Our results enhance the literature on cardenolide diversity and concentration among tissues eaten by insects and provide an opportunity to uncover potential evolutionary relationships between tissue-specific defense expression and insect adaptations in plant-herbivore interactions.

Pro-Apoptotic and Pro-Autophagic Properties of Cardenolides from Aerial Parts of Pergularia tomentosa

Pergularia tomentosa L., a milkweed tropical plant belonging to the family Asclepiadaceae, is a rich source of unusual cardiac glycosides, characterised by transfused A/B rings and a sugar moiety linked by a double link, generating a dioxanoid structure. In the present report, five cardenolides isolated from the aerial parts of the plant (calactin, calotropin, 12ß-hydroxycalactin, 12ß,6'-dihydroxycalotropin, and 16α-hydroxycalotropin) were investigated for their biological effects on a human hepatocarcinoma cell line. Cell viability was monitored by an MTT assay. The occurrence of apoptosis was evaluated by detecting caspase-3 activation and chromatin fragmentation. The ability of these compounds to induce autophagy was analysed by monitoring two markers of the autophagic process, LC3 and p62. Our results indicated that all cardenolides had cytotoxic effects, with IC50 ranging from 0.127 to 6.285 µM. All compounds were able to induce apoptosis and autophagy, calactin being the most active one. Some of them also caused a reduction in cell migration and a partial block of the cell cycle into the S-phase. The present study suggests that selected cardenolides from aerial parts of P. tomentosa, particularly calactin, possess potentially desirable properties for further investigation as anticancer agents.

Effects of early-life exposure to sublethal levels of a common neonicotinoid insecticide on the orientation and migration of monarch butterflies (Danaus plexippus).

Migratory insects use a variety of innate mechanisms to determine their orientation and maintain correct bearing. For long-distance migrants, such as the monarch butterfly (Danaus plexippus), these journeys could be affected by exposure to environmental contaminants. Neonicotinoids are synthetic insecticides that work by affecting the nervous system of insects, resulting in impairment of their mobility, cognitive performance, and other physiological and behavioural functions. To examine how neonicotinoids might affect the ability of monarch butterflies to maintain a proper directional orientation on their ∼4000 km migration, we grew swamp milkweed (Asclepias incarnata) in soil that was either untreated (0 ng g-1: control) or mixed with low (15 ng g-1 of soil) or high (25 ng g-1 of soil) levels of the neonicotinoid clothianidin. Monarch caterpillars were raised on control or clothianidin-treated milkweed and, after pupation, either tested for orientation in a static flight simulator or radio-tracked in the wild during the autumn migration period. Despite clothianidin being detectable in milkweed tissue consumed by caterpillars, there was no evidence that clothianidin influenced the orientation, vector strength (i.e. concentration of direction data around the mean) or rate of travel of adult butterflies, nor was there evidence that morphological traits (i.e. mass and forewing length), testing time, wind speed or temperature impacted directionality. Although sample sizes for both flight simulator and radio-tracking tests were limited, our preliminary results suggest that clothianidin exposure during early caterpillar development does not affect the directed flight of adult migratory monarch butterflies or influence their orientation at the beginning of migration.

Elevated atmospheric concentrations of CO2 increase endogenous immune function in a specialist herbivore.

Animals rely on a balance of endogenous and exogenous sources of immunity to mitigate parasite attack. Understanding how environmental context affects that balance is increasingly urgent under rapid environmental change. In herbivores, immunity is determined, in part, by phytochemistry which is plastic in response to environmental conditions. Monarch butterflies Danaus plexippus, consistently experience infection by a virulent parasite Ophryocystis elektroscirrha, and some medicinal milkweed (Asclepias) species, with high concentrations of toxic steroids (cardenolides), provide a potent source of exogenous immunity. We investigated plant-mediated influences of elevated CO2 (eCO2 ) on endogenous immune responses of monarch larvae to infection by O. elektroscirrha. Recently, transcriptomics have revealed that infection by O. elektroscirrha does not alter monarch immune gene regulation in larvae, corroborating that monarchs rely more on exogenous than endogenous immunity. However, monarchs feeding on medicinal milkweed grown under eCO2 lose tolerance to the parasite, associated with changes in phytochemistry. Whether changes in milkweed phytochemistry induced by eCO2 alter the balance between exogenous and endogenous sources of immunity remains unknown. We fed monarchs two species of milkweed; A. curassavica (medicinal) and A. incarnata (non-medicinal) grown under ambient CO2 (aCO2 ) or eCO2 . We then measured endogenous immune responses (phenoloxidase activity, haemocyte concentration and melanization strength), along with foliar chemistry, to assess mechanisms of monarch immunity under future atmospheric conditions. The melanization response of late-instar larvae was reduced on medicinal milkweed in comparison to non-medicinal milkweed. Moreover, the endogenous immune responses of early-instar larvae to infection by O. elektroscirrha were generally lower in larvae reared on foliage from aCO2 plants and higher in larvae reared on foliage from eCO2 plants. When grown under eCO2 , milkweed plants exhibited lower cardenolide concentrations, lower phytochemical diversity and lower nutritional quality (higher C:N ratios). Together, these results suggest that the loss of exogenous immunity from foliage under eCO2 results in increased endogenous immune function. Animal populations face multiple threats induced by anthropogenic environmental change. Our results suggest that shifts in the balance between exogenous and endogenous sources of immunity to parasite attack may represent an underappreciated consequence of environmental change.

Variable effects of mycorrhizal fungi on predator-prey dynamics under field conditions.

Interactions between herbivores and their predators are shaped, in part, by plant phenotype. Consequently, ubiquitous symbionts of plants below-ground, such as arbuscular mycorrhizal fungi (AMF), may influence interactions above-ground between predators and their prey by altering plant phenotype. However, the ecological relevance of below-ground organisms on predator-prey interactions under field conditions remains unclear. We assessed how AMF influence herbivore-predator interactions through a field experiment. We planted two milkweed species (Asclepias curassavica and Asclepias incarnata) provided with different amounts of AMF inoculum (zero, medium, and high) in a randomized block design. We added aphids to plants and reduced predator pressure weekly for 5 weeks to evaluate effects of AMF on predator recruitment. We then allowed herbivore-predator interactions to re-establish naturally for the remainder of the season to examine whether AMF-mediated variation in predator recruitment influenced the suppression of aphid populations. Arbuscular mycorrhizal fungi availability in soils mediated interactions between predaceous aphid midge flies Aphidoletes aphidimyza and their aphid prey Aphis nerii, but the effects were plant species-specific. On A. curassavica, by mid-season, midges were recruited most strongly on plants under medium AMF availability and least on plants under high AMF availability. In contrast, each midge killed fewer aphids with increasing aphid density on medium AMF plants, but killed more aphids with increasing aphid density on high AMF plants. In combination, aphid mortality rates imposed by midges were greatest on medium AMF plants, followed by high and zero AMF plants. By comparison, on A. incarnata, the recruitment of midges was strongest on high AMF plants and weakest on medium AMF plants. AMF had no effect on the number of aphids killed per midge, relative to aphid density, so mortality rates of aphids imposed by midges mirrored recruitment. Rates of decline in aphid populations following predator recolonization were associated with midge densities, as well as lacewing and syrphid densities, which were unaffected by AMF availability. Therefore, the effects of AMF on aphid population decline were not a simple function of AMF-midge interactions. Our findings demonstrate that the availability of AMF in soils has pervasive, but complex, effects on predator-herbivore dynamics in the field.