Faster-growing parasites threaten host populations via patch-level population dynamics and higher virulence; a case study in Varroa mites (Mesostigmata: Varroidae) and honey bees (Hymenoptera: Apidae).

Honey bee parasites remain a critical challenge to management and conservation. Because managed honey bees are maintained in colonies kept in apiaries across landscapes, the study of honey bee parasites allows the investigation of spatial principles in parasite ecology and evolution. We used a controlled field experiment to study the relationship between population growth rate and virulence (colony survival) of the parasite Varroa destructor (Anderson and Trueman). We used a nested design of 10 patches (apiaries) of 14 colonies to examine the spatial scale at which Varroa population growth matters for colony survival. We tracked Varroa population size and colony survival across a full year and found that Varroa populations that grow faster in their host colonies during the spring and summer led to larger Varroa populations across the whole apiary (patch) and higher rates of neighboring colony loss. Crucially, this increased colony loss risk manifested at the patch scale, with mortality risk being related to spatial adjacency to colonies with fast-growing Varroa strains rather than with Varroa growth rate in the colony itself. Thus, within-colony population growth predicts whole-apiary virulence, demonstrating the need to consider multiple scales when investigating parasite growth-virulence relationships.

Insects' essential role in understanding and broadening animal medication.

Like humans, animals use plants and other materials as medication against parasites. Recent decades have shown that the study of insects can greatly advance our understanding of medication behaviors. The ease of rearing insects under laboratory conditions has enabled controlled experiments to test critical hypotheses, while their spectrum of reproductive strategies and living arrangements - ranging from solitary to eusocial communities - has revealed that medication behaviors can evolve to maximize inclusive fitness through both direct and indirect fitness benefits. Studying insects has also demonstrated in some cases that medication can act through modulation of the host's innate immune system and microbiome. We highlight outstanding questions, focusing on costs and benefits in the context of inclusive host fitness.

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.

Interactions between parasitism and migration in monarch butterflies.

In many species, migration can increase parasite burdens or diversity as hosts move between diverse habitats with different parasite assemblages. On the other hand, migration can reduce parasite prevalence by letting animals escape infested habitats, or by exacerbating the costs of parasitism, leading to culling or dropout. How the balance between these negative and positive interactions is maintained or how they will change under anthropogenic pressure remains poorly understood. Here, we summarize the relationship between migration and infectious disease in monarch butterflies, finding that migration can reduce parasite prevalence through a combination of migratory culling and dropout. Because parasite prevalence has risen in recent decades, these processes are now resulting in the loss of tens of millions of monarchs. We highlight the remaining questions, asking how migration influences population genetics and virulence, how the establishment of resident populations interferes with migration, and whether infection can interfere with migratory cognition.

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

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

EXPERIMENTAL INFECTION WITH A NATURALLY OCCURRING PROTOZOAN PARASITE REDUCES MONARCH BUTTERFLY (DANAUS PLEXIPPUS) MATING SUCCESS.

Parasitic infection is known to drive sexual selection in persuasive mating systems, where parasites influence the secondary sexual characteristics that underlie mate choice. However, comparatively little is known about their effects on animals that use coercive mating behavior. We use a tractable system consisting of monarch butterflies and their naturally occurring parasite Ophryocystis elektroscirrha to test how parasites influence host mating dynamics when males force females to copulate. Monarchs were placed in mating cages where all, half, or no individuals were experimentally infected with O. elektroscirrha. We found that parasites reduce a male's mating success such that infected males were not only less likely to copulate but obtained fewer lifetime copulations as well. This reduction in mating success was due primarily to the fact that infected males attempt to mate significantly less than uninfected males. However, we found that O. elektroscirrha did not influence male mate choice. Males chose to mate with both infected and uninfected females at similar rates, regardless of their infection status. Overall, our data highlight how mating dynamics in coercive systems are particularly vulnerable to parasites.

Flying on empty: reduced mitochondrial function and flight capacity in food-deprived monarch butterflies.

Mitochondrial function is fundamental to organismal performance, health and fitness - especially during energetically challenging events, such as migration. With this investigation, we evaluated mitochondrial sensitivity to ecologically relevant stressors. We focused on an iconic migrant, the North American monarch butterfly (Danaus plexippus), and examined the effects of two stressors: 7 days of food deprivation and infection by the protozoan parasite Ophryocystis elektroscirrha (known to reduce survival and flight performance). We measured whole-animal resting metabolic rate (RMR) and peak flight metabolic rate, and mitochondrial respiration of isolated mitochondria from the flight muscles. Food deprivation reduced mass-independent RMR and peak flight metabolic rate, whereas infection did not. Fed monarchs used mainly lipids in flight (respiratory quotient 0.73), but the respiratory quotient dropped in food-deprived individuals, possibly indicating switching to alternative energy sources, such as ketone bodies. Food deprivation decreased mitochondrial maximum oxygen consumption but not basal respiration, resulting in lower respiratory control ratio (RCR). Furthermore, food deprivation decreased mitochondrial complex III activity, but increased complex IV activity. Infection did not result in any changes in these mitochondrial variables. Mitochondrial maximum respiration rate correlated positively with mass-independent RMR and flight metabolic rate, suggesting a link between mitochondria and whole-animal performance. In conclusion, low food availability negatively affects mitochondrial function and flight performance, with potential implications for migration, fitness and population dynamics. Although previous studies have reported poor flight performance in infected monarchs, we found no differences in physiological performance, suggesting that reduced flight capacity may be due to structural differences or low energy stores.

Lack of inbreeding avoidance during mate selection in migratory monarch butterflies.

Inbreeding is generally thought to have negative consequences for organismal health. However, despite the potential fitness effects, it remains surprisingly common among wild populations. In many cases, the complex factors that underlie mating dynamics make predicting whether individuals should or do avoid inbreeding quite challenging. One reason inbreeding may persist among species is that the likelihood of encountering relatives can be rare. Thus, even if inbreeding has severe consequences, selection to avoid mating with kin will be weak in species that are highly dispersed. Here we investigated if migratory monarch butterflies (Danaus plexippus), which are famous for their dispersal ability, actively avoid inbreeding. We found that neither female nor male monarchs choose mates based on relatedness. These results support the hypothesis that movement ecology can mask the deleterious effects of inbreeding and relax selection for active inbreeding avoidance behaviors. Overall, our data add to the growing list of studies showing that inbreeding avoidance is not the behavioral "default" for most species. We also highlight the implications that inbreeding may have on the declining populations of this iconic butterfly.

Crowding does not affect monarch butterflies' resistance to a protozoan parasite.

Host density is an important factor when it comes to parasite transmission and host resistance. Increased host density can increase contact rate between individuals and thus parasite transmission. Host density can also cause physiological changes in the host, which can affect host resistance. Yet, the direction in which host density affects host resistance remains unresolved. It is also unclear whether food limitation plays a role in this effect. We investigated the effect of larval density in monarch butterflies, Danaus plexippus, on the resistance to their natural protozoan parasite Ophryocystis elektroscirrha under both unlimited and limited food conditions. We exposed monarchs to various density treatments as larvae to mimic high densities observed in sedentary populations. Data on infection and parasite spore load were collected as well as development time, survival, wing size, and melanization. Disease susceptibility under either food condition or across density treatments was similar. However, we found high larval density impacted development time, adult survival, and wing morphology when food was limited. This study aids our understanding of the dynamics of environmental parasite transmission in monarch populations, which can help explain the increased prevalence of parasites in sedentary monarch populations compared to migratory populations.

Parasite dynamics in North American monarchs predicted by host density and seasonal migratory culling.

Insect-pathogen dynamics can show seasonal and inter-annual variations that covary with fluctuations in insect abundance and climate. Long-term analyses are especially needed to track parasite dynamics in migratory insects, in part because their vast habitat ranges and high mobility might dampen local effects of density and climate on infection prevalence. Monarch butterflies Danaus plexippus are commonly infected with the protozoan Ophryocystis elektroscirrha (OE). Because this parasite lowers monarch survival and flight performance, and because migratory monarchs have experienced declines in recent decades, it is important to understand the patterns and drivers of infection. Here we compiled data on OE infection spanning 50 years, from wild monarchs sampled in the United States, Canada and Mexico during summer breeding, fall migrating and overwintering periods. We examined eastern versus western North American monarchs separately, to ask how abundance estimates, resource availability, climate and breeding season length impact infection trends. We further assessed the intensity of migratory culling, which occurs when infected individuals are removed from the population during migration. Average infection prevalence was four times higher in western compared to eastern subpopulations. In eastern North America, the proportion of infected monarchs increased threefold since the mid-2000s. In the western region, the proportion of infected monarchs declined sharply from 2000 to 2015, and increased thereafter. For both eastern and western subpopulations, years with greater summer adult abundance predicted greater infection prevalence, indicating that transmission increases with host breeding density. Environmental variables (temperature and NDVI) were not associated with changes in the proportion of infected adults. We found evidence for migratory culling of infected butterflies, based on declines in parasitism during fall migration. We estimated that tens of millions fewer monarchs reach overwintering sites in Mexico as a result of OE, highlighting the need to consider the parasite as a potential threat to the monarch population. Increases in infection among eastern North American monarchs post-2002 suggest that changes to the host's ecology or environment have intensified parasite transmission. Further work is needed to examine the degree to which human practices, such as mass caterpillar rearing and the widespread planting of exotic milkweed, have contributed to this trend.

Population genomics reveals variable patterns of immune gene evolution in monarch butterflies (Danaus plexippus).

Humoral and cellular immune responses provide animals with major defences against harmful pathogens. While it is often assumed that immune genes undergo rapid diversifying selection, this assumption has not been tested in many species. Moreover, it is likely that different classes of immune genes experience different levels of evolutionary constraint, resulting in varying selection patterns. We examined the evolutionary patterns for a set of 91 canonical immune genes of North American monarch butterflies (Danaus plexippus), using as an outgroup the closely related soldier butterfly (Danaus eresimus). As a comparison to these immune genes, we selected a set of control genes that were paired with each immune for approximate size and genomic location. As a whole, these immune genes had a significant but modest reduction in Tajima's D relative to paired-control genes, but otherwise did not show distinct patterns of population genetic variation or evolutionary rates. When further partitioning these immune genes into four functional classes (recognition, signalling, modulation, and effector), we found distinct differences among these groups. Relative to control genes, recognition genes exhibit increased nonsynonymous diversity and divergence, suggesting reduced constraints on evolution, and supporting the notion that coevolution with pathogens results in diversifying selection. In contrast, signalling genes showed an opposite pattern of reduced diversity and divergence, suggesting evolutionary constraints and conservation. Modulator and effector genes showed no statistical differences from controls. These results are consistent with patterns found in immune genes in fruit flies and Pieris butterflies, suggesting that consistent selective pressures on different classes of immune genes broadly govern the evolution of innate immunity among insects.

Thermal tolerance and environmental persistence of a protozoan parasite in monarch butterflies.

Many parasites have external transmission stages that persist in the environment prior to infecting a new host. Understanding how long these stages can persist, and how abiotic conditions such as temperature affect parasite persistence, is important for predicting infection dynamics and parasite responses to future environmental change. In this study, we explored environmental persistence and thermal tolerance of a debilitating protozoan parasite that infects monarch butterflies. Parasite transmission occurs when dormant spores, shed by adult butterflies onto host plants and other surfaces, are later consumed by caterpillars. We exposed parasite spores to a gradient of ecologically-relevant temperatures for 2, 35, or 93 weeks. We tested spore viability by feeding controlled spore doses to susceptible monarch larvae, and examined relationships between temperature, time, and resulting infection metrics. We also examined whether distinct parasite genotypes derived from replicate migratory and resident monarch populations differed in their thermal tolerance. Finally, we examined evidence for a trade-off between short-term within-host replication and long-term persistence ability. Parasite viability decreased in response to warmer temperatures over moderate-to-long time scales. Individual parasite genotypes showed high heterogeneity in viability, but differences did not cluster by migratory vs. resident monarch populations. We found no support for a negative relationship between environmental persistence and within-host replication, as might be expected if parasites invest in short-term reproduction at the cost of longer-term survival. Findings here indicate that dormant spores can survive for many months under cooler conditions, and that heat dramatically shortens the window of transmission for this widespread and virulent butterfly parasite.

Constant Light and Frequent Schedule Changes Do Not Impact Resistance to Parasites in Monarch Butterflies.

Organisms have evolved internal biological clocks to regulate their activities based on external environmental cues, such as light, temperature, and food. Environmental disruption of these rhythms, such as caused by constant light or frequent light schedule changes, has been shown to impair development, reduce survival, and increase infection susceptibility and disease progression in numerous organisms. However, the precise role of the biological clock in host-parasite interactions is understudied and has focused on unnatural host-parasite combinations in lab-adapted inbred models. Here, we use the natural interaction between monarch butterflies (Danaus plexippus) and their virulent protozoan parasite, Ophryocystis elektroscirrha, to investigate the effects of constant light and frequent light schedule changes on development, survival, and parasite susceptibility. We show that constant light exposure slows the monarchs' rate of development but does not increase susceptibility to parasitic infection. Furthermore, frequent schedule changes decrease parasite growth, but have no effect on egg-to-adult survival of infected monarchs. Interestingly, these conditions are usually disruptive to the biological clock, but do not significantly impact the clock of monarch larvae. These unexpected findings show that constant light and frequent schedule changes can uncouple host and parasite performance and highlight how natural relationships are needed to expand our understanding of clocks in host-parasite interactions.

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.

Persistent effects of management history on honeybee colony virus abundances.

Infectious diseases are a major threat to both managed and wild pollinators. One key question is how the movement or transplantation of honeybee colonies under different management regimes affects honeybee disease epidemiology. We opportunistically examined any persistent effect of colony management history following relocation by characterising the virus abundances of honeybee colonies from three management histories, representing different management histories: feral, low-intensity management, and high-intensity "industrial" management. The colonies had been maintained for one year under the same approximate 'common garden' condition. Colonies in this observational study differed in their virus abundances according to management history, with the feral population history showing qualitatively different viral abundance patterns compared to colonies from the two managed population management histories; for example, higher abundance of sacbrood virus but lower abundances of various paralysis viruses. Colonies from the high-intensity management history exhibited higher viral abundances for all viruses than colonies from the low-intensity management history. Our results provide evidence that management history has persistent impacts on honeybee disease epidemiology, suggesting that apicultural intensification could be majorly impacting on pollinator health, justifying much more substantial investigation.

Genomic evidence for gene flow between monarchs with divergent migratory phenotypes and flight performance.

Monarch butterflies are known for their spectacular annual migration in eastern North America, with millions of monarchs flying up to 4,500 km to overwintering sites in central Mexico. Monarchs also live west of the Rocky Mountains, where they travel shorter distances to overwinter along the Pacific Coast. It is often assumed that eastern and western monarchs form distinct evolutionary units, but genomic studies to support this notion are lacking. We used a tethered flight mill to show that migratory eastern monarchs have greater flight performance than western monarchs, consistent with their greater migratory distances. However, analysing more than 20 million SNPs in 43 monarch genomes, we found no evidence for genomic differentiation between eastern and western monarchs. Genomic analysis also showed identical and low levels of genetic diversity, and demographic analyses indicated similar effective population sizes and ongoing gene flow between eastern and western monarchs. Gene expression analysis of a subset of candidate genes during active flight revealed differential gene expression related to nonmuscular motor activity. Our results demonstrate that eastern and western monarchs maintain migratory differences despite ongoing gene flow, and suggest that migratory differences between eastern and western monarchs are not driven by select major-effects alleles. Instead, variation in migratory distance and destination may be driven by environmentally induced differential gene expression or by many alleles of small effect.

Assessing virulence of Varroa destructor mites from different honey bee management regimes.

The mite Varroa destructor is an important honey bee parasite that causes substantial losses of honey bee colonies worldwide. Evolutionary theory suggests that the high densities at which honey bees are managed in large-scale beekeeping settings will likely select for mites with greater growth and virulence, thereby potentially explaining the major damage done by these mites. We tested this hypothesis by collecting mites from feral bee colonies, "lightly" managed colonies (those from small-scale sedentary operations), and "heavily" managed colonies (those from large-scale operations that move thousands of colonies across the US on a yearly basis). We established 8 apiaries, each consisting of 11 colonies from a standardized lightly managed bee background that were cleared of mites, and artificially infested each apiary with controlled numbers of mites from feral, lightly managed, or heavily managed bees or left uninoculated as negative control. We monitored the colonies for more than 2 years for mite levels, colony strength (adult bee population, brood coverage, and honey storage), and survival. As predicted by evolutionary theory, we found that colonies inoculated with mites from managed backgrounds had increased V. destructor mite levels relative to those with mites from feral colonies or negative controls. However, we did not see a difference between heavily and lightly managed colonies, and these higher mite burdens did not translate into greater virulence, as measured by reductions in colony strength and survival. Our results suggest that human management of honey bee colonies may favor the increased population growth rate of V. destructor, but that a range of potential confounders (including viral infections and genotype-by-genotype interactions) likely contribute to the relationship between mite reproduction and virulence.

Diet-microbiome-disease: Investigating diet's influence on infectious disease resistance through alteration of the gut microbiome.

Abiotic and biotic factors can affect host resistance to parasites. Host diet and host gut microbiomes are two increasingly recognized factors influencing disease resistance. In particular, recent studies demonstrate that (1) particular diets can reduce parasitism; (2) diets can alter the gut microbiome; and (3) the gut microbiome can decrease parasitism. These three separate relationships suggest the existence of indirect links through which diets reduce parasitism through an alteration of the gut microbiome. However, such links are rarely considered and even more rarely experimentally validated. This is surprising because there is increasing discussion of the therapeutic potential of diets and gut microbiomes to control infectious disease. To elucidate these potential indirect links, we review and examine studies on a wide range of animal systems commonly used in diet, microbiome, and disease research. We also examine the relative benefits and disadvantages of particular systems for the study of these indirect links and conclude that mice and insects are currently the best animal systems to test for the effect of diet-altered protective gut microbiomes on infectious disease. Focusing on these systems, we provide experimental guidelines and highlight challenges that must be overcome. Although previous studies have recommended these systems for microbiome research, here we specifically recommend these systems because of their proven relationships between diet and parasitism, between diet and the microbiome, and between the microbiome and parasite resistance. Thus, they provide a sound foundation to explore the three-way interaction between diet, the microbiome, and infectious disease.

Industrial bees: The impact of apicultural intensification on local disease prevalence.

It is generally thought that the intensification of farming will result in higher disease prevalences, although there is little specific modelling testing this idea. Focussing on honeybees, we build multi-colony models to inform how "apicultural intensification" is predicted to impact honeybee pathogen epidemiology at the apiary scale.We used both agent-based and analytical models to show that three linked aspects of apicultural intensification (increased population sizes, changes in population network structure and increased between-colony transmission) are unlikely to greatly increase disease prevalence in apiaries. Principally this is because even low-intensity apiculture exhibits high disease prevalence.The greatest impacts of apicultural intensification are found for diseases with relatively low R0 (basic reproduction number), however, such diseases cause little overall disease prevalence and, therefore, the impacts of intensification are minor. Furthermore, the smallest impacts of intensification are for diseases with high R0 values, which we argue are typical of important honeybee diseases. Policy Implications: Our findings contradict the idea that apicultural intensification by crowding honeybee colonies in large, dense apiaries leads to notably higher disease prevalences for established honeybee pathogens. More broadly, our work demonstrates the need for informative models of all agricultural systems and management practices in order to understand the implications of management changes on diseases.

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

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