Milkweed in agricultural field margins - A neonicotinoid exposure route for pollinators at multiple life stages.

Neonicotinoid insecticides move from targeted crops to wildflowers located in adjacent field margins, acting as a potential exposure source for wild pollinators and insect species of conservation concern, including monarch butterflies. Monarchs rely on milkweed over multiple life stages, including as a host plant for eggs and a food source for both larvae (leaves) and adults (flowers). Milkweeds, which are closely associated with field margins, can contain neonicotinoid residues, but previous assessments are constrained to a single plant tissue type. In 2017 and 2018, we sampled milkweeds from 95 field margins adjacent to crop fields (corn, soybean, hay, wheat, and barley) in agricultural landscapes of eastern Ontario, Canada. Milkweeds were sampled during the flower blooming period and leaves and flower tissues were analysed. The neonicotinoids acetamiprid, clothianidin, thiamethoxam, and thiacloprid were detected. Maximum concentrations in leaf samples included 10.30 ng/g of clothianidin in 2017, and 24.4 ng/g of thiamethoxam in 2018. Clothianidin and thiamethoxam percent detections in flowers (72 % and 61 %, respectively) were significantly higher than detections in leaves (24 % and 31 %, respectively). Thiamethoxam concentrations were significantly higher in paired flower samples than leaf samples (median 0.33 ng/g vs <0.07 ng/g) while clothianidin concentrations also trended higher in flowers (median 0.18-0.55 ng/g vs <0.18 ng/g). Only thiamethoxam showed significant differences between years, and we found no effect of crop type, with hay, soybean and corn fields all yielding 50-56 % detections in leaves. We found significantly higher concentrations in older milkweed flowers than young flowers or leaves (medians 0.87 ng/g vs <0.18 ng/g and 0.45 ng/g vs <0.07 ng/g for clothianidin and thiamethoxam, respectively). Our results highlight the importance of considering variation in milkweed tissue type and age of flowers in neonicotinoid exposure risk assessments. Efforts to increase milkweed availability in agricultural landscapes should consider how exposure to neonicotinoids can be mitigated.

Low-Level Dietary Clothianidin Exposure Preferentially Causes Prepupal Mortality of Monarch Butterflies (Danaus plexippus).

Data from prior research indicate the prepupal stage of the monarch butterfly life cycle is more sensitive to clothianidin exposure than the larval stage. A set of experiments was conducted to determine if the dietary clothianidin exposures that cause prepupal mortality are environmentally relevant. Monarch larvae were raised from egg to pupae on clothianidin-contaminated swamp milkweed plants (Asclepias incarnata). Larval growth as well as larval and prepupal survival were monitored throughout the experiments, in which the exposures ranged from 1.4 to 2793.1 ng/g leaf. Exposures of 5.4 to 46.9 ng/g leaf resulted primarily in prepupal mortality, whereas higher exposures of 1042.4 to 2793.1 ng/g leaf resulted exclusively in larval mortality, indicating the prepupal stage is more sensitive to clothianidin exposure than the larval stage. A median lethal concentration and a 10% lethal concentration of 37 and 6 ng/g leaf, respectively, were estimated for prepupal mortality. Both effect concentrations are within the range of clothianidin concentrations reported in leaves collected from wild milkweed plants, indicating prepupal mortality is an environmentally relevant effect. Environ Toxicol Chem 2024;43:2039-2044. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Escalation by duplication: Milkweed bug trumps Monarch butterfly.

The iconic Monarch butterfly is probably the best-known example of chemical defence against predation, as pictures of vomiting naive blue jays in countless textbooks vividly illustrate. Larvae of the butterfly take up toxic cardiac glycosides from their milkweed hostplants and carry them over to the adult stage. These compounds (cardiotonic steroids, including cardenolides and bufadienolides) inhibit the animal transmembrane sodium-potassium ATPase (Na,K-ATPase), but the Monarch enzyme resists this inhibition thanks to amino acid substitutions in its catalytic alpha-subunit. Some birds also have substitutions and can feast on cardiac glycoside-sequestering insects with impunity. A flurry of recent work has shown how the alpha-subunit gene has been duplicated multiple times in separate insect lineages specializing in cardiac glycoside-producing plants. In this issue of Molecular Ecology, Herbertz et al. toss the beta-subunit into the mix, by expressing all nine combinations of three alpha- and three beta-subunits of the milkweed bug Na,K-ATPase and testing their response to a cardenolide from the hostplant. The findings suggest that the diversification and subfunctionalization of genes allow milkweed bugs to balance trade-offs between resistance towards sequestered host plant toxins that protect the bugs from predators, and physiological costs in terms of Na,K-ATPase activity.

Target and non-target effects of insecticide use during ornamental milkweed production.

There are widespread public efforts to conserve wildlife in urbanized landscapes via the installation of nursery-grown plants that support Lepidoptera taxa. Insecticides are commonly used during nursery production to suppress key plant pests, and many products have extended periods of toxicity and affect a wide range of herbivore taxa. While there are plentiful toxicological data on bee species, predominantly the Western honey bee (Apis mellifera L.), little is known about how insecticides affect nonpest lepidopterans. Lepidoptera has different modes of exposure (e.g., leaf-feeding) and differences in susceptibility to insecticide target sites compared to bees. Consequently, many products compatible with bee conservation pose an uncertain risk to nonpest lepidopterans and thus may represent an under-recognized conflict with conservation efforts. Using the monarch butterfly (Danaus plexippus, L.), tropical milkweed (Asclepias curassavica, L.), and oleander aphid (Aphis nerii, Fonscolombe, 1841) system, we conducted leaf and whole-plant feeding assays to evaluate effects of acute and chronic monarch exposure to industry standard and alternative reduced-risk insecticides used during nursery production. We also evaluated the efficacy of these insecticides against their target pest, the oleander aphid. Our results indicate that insecticides used to control pests on ornamental milkweed can cause monarch larval mortality up to 4 wk after treatment application. Furthermore, the duration of aphid suppression is often shorter than the duration of adverse effects on monarchs. This study demonstrates a conflict between insect pest management and Lepidoptera conservation during ornamental plant production and has implications for the conservation value of ornamentals after retail sale.

How do phytophagous insects affect phyllosphere fungi? Tracking fungi from milkweed to monarch caterpillar frass reveals communities dominated by fungal yeast.

Since a significant proportion of plant matter is consumed by herbivores, a necessary adaptation for many phyllosphere microbes could be to survive through the guts of herbivores. While many studies explore the gut microbiome of herbivores by surveying the microbiome in their frass, few studies compare the phyllosphere microbiome to the gut microbiome of herbivores. High-throughput metabarcode sequencing was used to track the fungal community from milkweed (Asclepias spp.) leaves to monarch caterpillar frass. The most commonly identified fungal taxa that dominated the caterpillar frass after the consumption of leaves were yeasts, mostly belonging to the Basidiomycota phylum. While most fungal communities underwent significant bottlenecks and some yeast taxa increased in relative abundance, a consistent directional change in community structure was not identified from leaf to caterpillar frass. These results suggest that some phyllosphere fungi, especially diverse yeasts, can survive herbivory, but whether herbivory is a key stage of their life cycle remains uncertain. For exploring phyllosphere fungi and the potential coprophilous lifestyles of endophytic and epiphytic fungi, methods that target yeast and Basidiomycota fungi are recommended.

Changes in landscape and climate in Mexico and Texas reveal small effects on migratory habitat of monarch butterflies (Danaus plexippus).

The decline of the iconic monarch butterfly (Danaus plexippus) in North America has motivated research on the impacts of land use and land cover (LULC) change and climate variability on monarch habitat and population dynamics. We investigated spring and fall trends in LULC, milkweed and nectar resources over a 20-year period, and ~ 30 years of climate variables in Mexico and Texas, U.S. This region supports spring breeding, and spring and fall migration during the annual life cycle of the monarch. We estimated a - 2.9% decline in milkweed in Texas, but little to no change in Mexico. Fall and spring nectar resources declined < 1% in both study extents. Vegetation greenness increased in the fall and spring in Mexico while the other climate variables did not change in both Mexico and Texas. Monarch habitat in Mexico and Texas appears relatively more intact than in the midwestern, agricultural landscapes of the U.S. Given the relatively modest observed changes in nectar and milkweed, the relatively stable climate conditions, and increased vegetation greenness in Mexico, it seems unlikely that habitat loss (quantity or quality) in Mexico and Texas has caused large declines in population size or survival during migration.

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.

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.

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.

Urbanization and a green corridor do not impact genetic divergence in common milkweed (Asclepias syriaca L.).

Urbanization is altering landscapes globally at an unprecedented rate. While ecological differences between urban and rural environments often promote phenotypic divergence among populations, it is unclear to what degree these trait differences arise from genetic divergence as opposed to phenotypic plasticity. Furthermore, little is known about how specific landscape elements, such as green corridors, impact genetic divergence in urban environments. We tested the hypotheses that: (1) urbanization, and (2) proximity to an urban green corridor influence genetic divergence in common milkweed (Asclepias syriaca) populations for phenotypic traits. Using seeds from 52 populations along three urban-to-rural subtransects in the Greater Toronto Area, Canada, one of which followed a green corridor, we grew ~ 1000 plants in a common garden setup and measured > 20 ecologically-important traits associated with plant defense/damage, reproduction, and growth over four years. We found significant heritable variation for nine traits within common milkweed populations and weak phenotypic divergence among populations. However, neither urbanization nor an urban green corridor influenced genetic divergence in individual traits or multivariate phenotype. These findings contrast with the expanding literature demonstrating that urbanization promotes rapid evolutionary change and offer preliminary insights into the eco-evolutionary role of green corridors in urban environments.

Tissue and toxin-specific divergent evolution in plant defense Evolución divergente específica de tejido y toxina en defensa de plantas.

A major predicted constraint on the evolution of anti-herbivore defense in plants is the nonindependent expression of traits mediating resistance. Since herbivore attack can be highly variable across plant tissues, we hypothesized that correlations in toxin expression within and between plant tissues may limit population differentiation and, thus, plant adaptation. Using full-sib families from two nearby (<1 km) common milkweed (Asclepias syriaca) populations, we investigated genetic correlations among 28 distinct cardenolide toxins within and between roots, leaves, and seeds and examined signatures of tissue-specific divergent selection between populations by QST-FST comparisons. The prevalence, direction, and strength of genetic correlations among cardenolides were tissue specific, and concentrations of individual cardenolides were moderately correlated between tissues; nonetheless, the direction and strength of correlations were population specific. Population divergence in the cardenolide chemistry was stronger in roots than in leaves and seeds. Divergent selection on individual cardenolides was tissue and toxin specific, except for a single highly toxic cardenolide (labriformin), that showed divergent selection across all plant tissues. Heterogeneous evolution of cardenolides within and between tissues across populations appears possible due to their highly independent expression. This independence may be common in nature, especially in specialized interactions in which distinct herbivores feed on different plant tissues.

Differences in δ13C and δ15N values for monarch butterflies raised on milkweeds from within and outside of crop fields: implications for inferences about natal origins.

Stable isotope (δ2H, δ13C) measurements of wing tissue have been used to determine the natal geographic origin of migrant monarch butterflies that overwinter in Mexico. This study examines the possibility of using δ13C and δ15N to identify the milkweed habitat used by monarchs in their natal region. Milkweeds were common in corn and soybean fields before herbicide use led to their extirpation around 2006, and the loss of those milkweeds has been proposed as a reason for the monarch population decline. If crop-field monarchs can be identified, then historical samples of monarchs could be examined to test that hypothesis. The δ15N and δ13C values of leaves from milkweeds growing in corn fields, soybean fields and non-agricultural habitats were examined as well as monarchs that were raised on those leaves. There were no δ15N values for leaves or monarchs that were distinctive for crop fields. Milkweeds in corn fields, and monarchs that were raised on those milkweeds, were found to have δ13C values distinctly lower than those of other habitats and unlike those of locations within the summer breeding range. Thus, it should be possible to identify monarchs that came from cornfields in samples of overwintering monarchs made before ca. 2006.

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.

Temporal matches between monarch butterfly and milkweed population changes over the past 25,000 years.

In intimate ecological interactions, the interdependency of species may result in correlated demographic histories. For species of conservation concern, understanding the long-term dynamics of such interactions may shed light on the drivers of population decline. Here, we address the demographic history of the monarch butterfly, Danaus plexippus, and its dominant host plant, the common milkweed Asclepias syriaca (A. syriaca), using broad-scale sampling and genomic inference. Because genetic resources for milkweed have lagged behind those for monarchs, we first release a chromosome-level genome assembly and annotation for common milkweed. Next, we show that despite its enormous geographic range across eastern North America, A. syriaca is best characterized as a single, roughly panmictic population. Using approximate Bayesian computation with random forests (ABC-RF), a machine learning method for reconstructing demographic histories, we show that both monarchs and milkweed experienced population expansion during the most recent recession of North American glaciers 10,000-20,000 years ago. Our data also identify concurrent population expansions in both species during the large-scale clearing of eastern forests (∼200 years ago). Finally, we find no evidence that either species experienced a reduction in effective population size over the past 75 years. Thus, the well-documented decline of monarch abundance over the past 40 years is not visible in our genomic dataset, reflecting a possible mismatch of the overwintering census population to effective population size in this species.

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.

Aphid infestations reduce monarch butterfly colonization, herbivory, and growth on ornamental milkweed.

Anthropogenic disturbance is driving global biodiversity loss, including the monarch butterfly (Danaus plexippus), a dietary specialist of milkweed. In response, ornamental milkweed plantings are increasingly common in urbanized landscapes, and recent evidence indicates they have conservation value for monarch butterflies. Unfortunately, sap-feeding insect herbivores, including the oleander aphid (Aphis nerii), frequently reach high densities on plants in nursery settings and urbanized landscapes. Aphid-infested milkweed may inhibit monarch conservation efforts by reducing host plant quality and inducing plant defenses. To test this, we evaluated the effects of oleander aphid infestation on monarch oviposition, larval performance, and plant traits using tropical milkweed (Asclepias curassavica), the most common commercially available milkweed species in the southern U.S. We quantified monarch oviposition preference, larval herbivory, larval weight, and plant characteristics on aphid-free and aphid-infested milkweed. Monarch butterflies deposited three times more eggs on aphid-free versus aphid-infested milkweed. Similarly, larvae fed aphid-free milkweed consumed and weighed twice as much as larvae fed aphid-infested milkweed. Aphid-free milkweed had higher total dry leaf biomass and nitrogen content than aphid-infested milkweed. Our results indicate that oleander aphid infestations can have indirect negative impacts on urban monarch conservation efforts and highlight the need for effective Lepidoptera-friendly integrated pest management tactics for ornamental plants.

Extent and impacts of winter breeding in the North American monarch butterfly.

Since the 1960s, scientists have observed the North American monarch butterfly (Danaus plexippus) continuing reproductive activities past the fall migration and into the winter months when the climate is mild. Recent work suggests that small populations of winter breeding monarchs are present in western and southeastern USA, as well as northwestern Mexico, with new winter breeding populations forming in areas where non-native milkweeds are planted. The year-round presence of milkweed plants and temperatures suitable for immature monarch development are vital factors allowing for winter breeding. Non-native milkweeds, in conjunction with novel barriers to migration, are likely contributing to the rise in winter breeding behavior. Warmer climates are already impacting milkweed phenology and range, possibly favoring winter breeding behavior. Similar pressures but different implications are expected for eastern and western winter breeding monarchs given the differences in the migration ecology, milkweed species, and climate changes in the two regions.

Overwintering and breeding patterns of monarch butterflies (Danaus plexippus) in coastal plain habitats of the southeastern USA.

Understanding variability in species' traits can inform our understanding of their ecology and aid in the development of management and conservation strategies. Monarch butterflies (Danaus plexippus) are native to the western hemisphere and are well-known for their long-distance migrations but have experienced significant population declines in recent decades. Here we use a 5-year capture-mark-recapture dataset to compare monarch distributions, mating activity, and larval host plant use between two coastal plain habitats in South Carolina, USA. We observed seasonally specific habitat use, with maritime habitats serving as overwintering areas while nearby inland swamps support significant breeding in spring, summer, and fall seasons due to an abundance of aquatic milkweed (Asclepias perennis). We also observed mating activity by fall migrating monarchs and their use of swallow-wort (Pattalias palustre) in the spring as an important larval host plant in maritime habitats. This phenology and habitat use of monarchs diverges from established paradigms and suggest that a distinct population segment of monarchs may exist, with significance for understanding the conservation status of monarch butterflies and associated habitats in eastern North America. Further research should explore how monarchs along the Atlantic coast of North America relate to other eastern monarch populations.

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.

No physiological costs of dual sequestration of chemically different plant toxins in the milkweed bug Spilostethus saxatilis (Heteroptera: Lygaeidae).

Many herbivorous insects not only cope with plant toxins but also sequester them as a defense against predators and parasitoids. Sequestration is a product of the evolutionary arms race between plants and herbivorous insects and has been hypothesized to incur physiological costs due to specific adaptations required. Contradictory evidence about these costs exists for insects sequestering only one class of toxin, but very little is known about the physiological implications for species sequestering structurally different classes of compounds. Spilostethus saxatilis is a milkweed bug belonging to the cardenolide-sequestering heteropteran subfamily Lygaeinae (Heteroptera: Lygaeidae) that has shifted to the colchicine-containing plant Colchicum autumnale, a resource of chemically unrelated alkaloids. Using feeding-assays on artificial diet and chemical analysis, we assessed whether S. saxatilis is still able to sequester cardenolides apart from colchicine and related metabolites (colchicoids), and tested the effect of (1) either a natural cardenolide concentration (using ouabain as a model compound) or a natural colchicine concentration, (2) an increased concentration of both toxins, and (3) seeds of either Asclepias syriaca (cardenolides) or C. autumnale (colchicoids) on a set of life-history traits. For comparison, we assessed the same life-history traits in the milkweed bug Oncopeltus fasciatus exposed to cardenolides only. Although cardenolides and colchicoids have different physiological targets (Na+/K+-ATPase vs tubulin) and thus require different resistance traits, chronic exposure and sequestration of both isolated toxins caused no physiological costs such as reduced growth, increased mortality, lower fertility, or shorter adult life span in S. saxatilis. Indeed, an increased performance was observed in O. fasciatus and an according trend was found in S. saxatilis when feeding on isolated ouabain and isolated colchicine, respectively. Positive effects were even more pronounced when insects were provided with natural toxic seeds (i.e. C. autumnale for S. saxatilis and A. syriaca for O. fasciatus), especially in O. fasciatus. Our findings suggest, that S. saxatilis can sequester two chemically unrelated classes of plant compounds at a cost-free level, and that colchicoids may even play a beneficial role in terms of fertility.