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.

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.

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.

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.

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.

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.

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.

Within-host competition can delay evolution of drug resistance in malaria.

In the malaria parasite P. falciparum, drug resistance generally evolves first in low-transmission settings, such as Southeast Asia and South America. Resistance takes noticeably longer to appear in the high-transmission settings of sub-Saharan Africa, although it may spread rapidly thereafter. Here, we test the hypothesis that competitive suppression of drug-resistant parasites by drug-sensitive parasites may inhibit evolution of resistance in high-transmission settings, where mixed-strain infections are common. We employ a cross-scale model, which simulates within-host (infection) dynamics and between-host (transmission) dynamics of sensitive and resistant parasites for a population of humans and mosquitoes. Using this model, we examine the effects of transmission intensity, selection pressure, fitness costs of resistance, and cross-reactivity between strains on the establishment and spread of resistant parasites. We find that resistant parasites, introduced into the population at a low frequency, are more likely to go extinct in high-transmission settings, where drug-sensitive competitors and high levels of acquired immunity reduce the absolute fitness of the resistant parasites. Under strong selection from antimalarial drug use, however, resistance spreads faster in high-transmission settings than low-transmission ones. These contrasting results highlight the distinction between establishment and spread of resistance and suggest that the former but not the latter may be inhibited in high-transmission settings. Our results suggest that within-host competition is a key factor shaping the evolution of drug resistance in P. falciparum.

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.

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.

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.

The genetics of monarch butterfly migration and warning colouration.

The monarch butterfly, Danaus plexippus, is famous for its spectacular annual migration across North America, recent worldwide dispersal, and orange warning colouration. Despite decades of study and broad public interest, we know little about the genetic basis of these hallmark traits. Here we uncover the history of the monarch's evolutionary origin and global dispersal, characterize the genes and pathways associated with migratory behaviour, and identify the discrete genetic basis of warning colouration by sequencing 101 Danaus genomes from around the globe. The results rewrite our understanding of this classic system, showing that D. plexippus was ancestrally migratory and dispersed out of North America to occupy its broad distribution. We find the strongest signatures of selection associated with migration centre on flight muscle function, resulting in greater flight efficiency among migratory monarchs, and that variation in monarch warning colouration is controlled by a single myosin gene not previously implicated in insect pigmentation.

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.

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

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

Migratory monarchs that encounter resident monarchs show life-history differences and higher rates of parasite infection.

Environmental change induces some wildlife populations to shift from migratory to resident behaviours. Newly formed resident populations could influence the health and behaviour of remaining migrants. We investigated migrant-resident interactions among monarch butterflies and consequences for life history and parasitism. Eastern North American monarchs migrate annually to Mexico, but some now breed year-round on exotic milkweed in the southern US and experience high infection prevalence of protozoan parasites. Using stable isotopes (δ2 H, δ13 C) and cardenolide profiles to estimate natal origins, we show that migrant and resident monarchs overlap during fall and spring migration. Migrants at sites with residents were 13 times more likely to have infections and three times more likely to be reproductive (outside normal breeding season) compared to other migrants. Exotic milkweed might either attract migrants that are already infected or reproductive, or alternatively, induce these states. Increased migrant-resident interactions could affect monarch parasitism, migratory success and long-term conservation.

Of poisons and parasites-the defensive role of tetrodotoxin against infections in newts.

Classical research on animal toxicity has focused on the role of toxins in protection against predators, but recent studies suggest these same compounds can offer a powerful defense against parasites and infectious diseases. Newts in the genus Taricha are brightly coloured and contain the potent neurotoxin, tetrodotoxin (TTX), which is hypothesized to have evolved as a defense against vertebrate predators such as garter snakes. However, newt populations often vary dramatically in toxicity, which is only partially explained by predation pressure. The primary aim of this study was to evaluate the relationships between TTX concentration and infection by parasites. By systematically assessing micro- and macroparasite infections among 345 adult newts (sympatric populations of Taricha granulosa and T. torosa), we detected 18 unique taxa of helminths, fungi, viruses and protozoans. For both newt species, per-host concentrations of TTX, which varied from undetectable to >60 µg/cm2 skin, negatively predicted overall parasite richness as well as the likelihood of infection by the chytrid fungus, Batrachochytrium dendrobatidis, and ranavirus. No such effect was found on infection load among infected hosts. Despite commonly occurring at the same wetlands, T. torosa supported higher parasite richness and average infection load than T. granulosa. Host body size and sex (females > males) tended to positively predict infection levels in both species. For hosts in which we quantified leucocyte profiles, total white blood cell count correlated positively with both parasite richness and total infection load. By coupling data on host toxicity and infection by a broad range of micro- and macroparasites, these results suggest that-alongside its effects on predators-tetrodotoxin may help protect newts against parasitic infections, highlighting the importance of integrative research on animal chemistry, immunological defenses and natural enemy ecology.

The Effects of Milkweed Induced Defense on Parasite Resistance in Monarch Butterflies, Danaus plexippus.

Many plants express induced defenses against herbivores through increasing the production of toxic secondary chemicals following damage. Phytochemical induction can directly or indirectly affect other organisms within the community. In tri-trophic systems, increased concentrations of plant toxins could be detrimental to plants if herbivores can sequester these toxins as protective chemicals for themselves. Thus, through trophic interactions, induction can lead to either positive or negative effects on plant fitness. We examined the effects of milkweed (Asclepias spp.) induced defenses on the resistance of monarch caterpillars (Danaus plexippus) to a protozoan parasite (Ophryocystis elektroscirrha). Milkweeds contain toxic secondary chemicals called cardenolides, higher concentrations of which are associated with reduced parasite growth. Previous work showed that declines in foliar cardenolides caused by aphid attack render monarch caterpillars more susceptible to infection. Here, we ask whether cardenolide induction by monarchs increases monarch resistance to disease. We subjected the high-cardenolide milkweed A. curassavica and the low-cardenolide A. syriaca to caterpillar grazing, and reared infected and uninfected caterpillars on these plants. As expected, monarchs suffered less parasite growth and disease when reared on A. curassavica than on A. syriaca. We also found that herbivory increased cardenolide concentrations in A. curassavica, but not A. syriaca. However, cardenolide induction in A. curassavica was insufficient to influence monarch resistance to the parasite. Our results suggest that interspecific variation in cardenolide concentration is a more important driver of parasite defense than plasticity via induced defenses in this tri-trophic system.

Within-host competition and drug resistance in the human malaria parasite Plasmodium falciparum.

Infections with the malaria parasite Plasmodium falciparum typically comprise multiple strains, especially in high-transmission areas where infectious mosquito bites occur frequently. However, little is known about the dynamics of mixed-strain infections, particularly whether strains sharing a host compete or grow independently. Competition between drug-sensitive and drug-resistant strains, if it occurs, could be a crucial determinant of the spread of resistance. We analysed 1341 P. falciparum infections in children from Angola, Ghana and Tanzania and found compelling evidence for competition in mixed-strain infections: overall parasite density did not increase with additional strains, and densities of individual chloroquine-sensitive (CQS) and chloroquine-resistant (CQR) strains were reduced in the presence of competitors. We also found that CQR strains exhibited low densities compared with CQS strains (in the absence of chloroquine), which may underlie observed declines of chloroquine resistance in many countries following retirement of chloroquine as a first-line therapy. Our observations support a key role for within-host competition in the evolution of drug-resistant malaria. Malaria control and resistance-management efforts in high-transmission regions may be significantly aided or hindered by the effects of competition in mixed-strain infections. Consideration of within-host dynamics may spur development of novel strategies to minimize resistance while maximizing the benefits of control measures.

Fitness costs of animal medication: antiparasitic plant chemicals reduce fitness of monarch butterfly hosts.

The emerging field of ecological immunology demonstrates that allocation by hosts to immune defence against parasites is constrained by the costs of those defences. However, the costs of non-immunological defences, which are important alternatives to canonical immune systems, are less well characterized. Estimating such costs is essential for our understanding of the ecology and evolution of alternative host defence strategies. Many animals have evolved medication behaviours, whereby they use antiparasitic compounds from their environment to protect themselves or their kin from parasitism. Documenting the costs of medication behaviours is complicated by natural variation in the medicinal components of diets and their covariance with other dietary components, such as macronutrients. In the current study, we explore the costs of the usage of antiparasitic compounds in monarch butterflies (Danaus plexippus), using natural variation in concentrations of antiparasitic compounds among plants. Upon infection by their specialist protozoan parasite Ophryocystis elektroscirrha, monarch butterflies can selectively oviposit on milkweed with high foliar concentrations of cardenolides, secondary chemicals that reduce parasite growth. Here, we show that these antiparasitic cardenolides can also impose significant costs on both uninfected and infected butterflies. Among eight milkweed species that vary substantially in their foliar cardenolide concentration and composition, we observed the opposing effects of cardenolides on monarch fitness traits. While high foliar cardenolide concentrations increased the tolerance of monarch butterflies to infection, they reduced the survival rate of caterpillars to adulthood. Additionally, although non-polar cardenolide compounds decreased the spore load of infected butterflies, they also reduced the life span of uninfected butterflies, resulting in a hump-shaped curve between cardenolide non-polarity and the life span of infected butterflies. Overall, our results suggest that the use of antiparasitic compounds carries substantial costs, which could constrain host investment in medication behaviours.