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Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to declare.

Insects are attacked by a diverse range of microbial pathogens in the wild. In herbivorous species, larval host plants frequently play a critical role in mediating susceptibility to infection. Characterizing such plant-mediated effects on herbivore-pathogen interactions can provide insight into patterns of infection across wild populations. In this study, we investigated the effects of host plant use by two North American butterflies, Euphydryas phaeton (Nymphalidae) and Anartia jatrophae (Nymphalidae), on entomopathogen infection across a range of three doses. Both of these herbivores recently incorporated the same exotic plant, Plantago lanceolata (Plantaginaceae), into their host range and are naturally infected by the same entomopathogen, Junonia coenia densovirus (Parvoviridae), in wild populations. We performed two factorial experiments in which E. phaeton and A. jatrophae were reared on either P. lanceolata or a native host plant [Chelone glabra (Plantaginaceae) for E. phaeton; Bacopa monnieri (Plantaginaceae) for A. jatrophae] and inoculated with either a low, medium, or high dose of the virus. In E. phaeton, the outcomes of infection were highly dose-dependent, with inoculation with higher viral doses resulting in faster time to death and greater mortality. However, neither survival nor postmortem viral burdens varied depending upon the host plant that was consumed. In contrast, host plant use had a strong effect on viral burdens in A. jatrophae, with consumption of the exotic plant appearing to enhance host resistance to infection. Together, these results illustrate the variable influences of host plant use on herbivore resistance to infection, highlighting the importance of investigating plant-herbivore relationships within a tritrophic framework., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Declines in biodiversity generated by anthropogenic stressors at both species and population levels can alter emergent processes instrumental to ecosystem function and resilience. As such, understanding the role of biodiversity in ecosystem function and its response to climate perturbation is increasingly important, especially in tropical systems where responses to changes in biodiversity are less predictable and more challenging to assess experimentally. Using large-scale transplant experiments conducted at five neotropical sites, we documented the impacts of changes in intraspecific and interspecific plant richness in the genus Piper on insect herbivory, insect richness, and ecosystem resilience to perturbations in water availability. We found that reductions of both intraspecific and interspecific Piper diversity had measurable and site-specific effects on herbivory, herbivorous insect richness, and plant mortality. The responses of these ecosystem-relevant processes to reduced intraspecific Piper richness were often similar in magnitude to the effects of reduced interspecific richness. Increased water availability reduced herbivory by 4.2% overall, and the response of herbivorous insect richness and herbivory to water availability were altered by both intra- and interspecific richness in a site-dependent manner. Our results underscore the role of intraspecific and interspecific richness as foundations of ecosystem function and the importance of community and location-specific contingencies in controlling function in complex tropical systems., Competing Interests: AG, TM, KU, LD, YA, LB, MF, LS, MK, HL, AN, TP, WS, AS, JS, ET, TW, LR No competing interests declared, (© 2023, Grele et al.)

Host plant consumption and pathogen infection commonly influence insect traits related to development and immunity, which are ultimately reflected in the behavior and physiology of the insect. Herein, we explored changes in the metabolome of a generalist insect herbivore, Vanessa cardui (Lepidoptera: Nymphalidae), in response to both dietary variation and pathogen infection in order to gain insight into tritrophic interactions for insect metabolism and immunity. Caterpillars were reared on two different host plants, Plantago lanceolata (Plantaginaceae) and Taraxacum officinale (Asteraceae) and subjected to a viral infection by Junonia coenia densovirus (JcDV), along with assays to determine the insect immune response and development. Richness and diversity of plant and caterpillar metabolites were evaluated using a liquid chromatography-mass spectrometry approach and showed that viral infection induced changes to the chemical content of V. cardui hemolymph and frass dependent upon host plant consumption. Overall, the immune response as measured by phenoloxidase (PO) enzymatic activity was higher in individuals feeding on P. lanceolata compared with those feeding on T. officinale. Additionally, infection with JcDV caused suppression of PO activity, which was not host plant dependent. We conclude that viral infection combined with host plant consumption creates a unique chemical environment, particularly within the insect hemolymph. Whether and how these metabolites contribute to defense against viral infection is an open question in chemical ecology., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Pathogens play a key role in insect population dynamics, contributing to short-term fluctuations in abundance as well as long-term demographic trends. Two key factors that influence the effects of entomopathogens on herbivorous insect populations are modes of pathogen transmission and larval host plants. In this study, we examined tritrophic interactions between a sequestering specialist lepidopteran, Euphydryas phaeton, and a viral pathogen, Junonia coenia densovirus, on its native host plant, Chelone glabra, and a novel host plant, Plantago lanceolata, to explore whether host plant mediates viral transmission, survival, and viral loads. A two-factor factorial experiment was conducted in the laboratory with natal larval clusters randomly assigned to either the native or novel host plant and crossed with either uninoculated controls or viral inoculation (20% of individuals in the cluster inoculated). Diapausing clusters were overwintered in the laboratory and checked weekly for mortality. At the end of diapause, all surviving individuals were reared to adulthood to estimate survivorship. All individuals were screened to quantify viral loads, and estimate horizontal transmission postmortem. To test for vertical transmission, adults were mated, and the progeny were screened for viral presence. Within virus-treated groups, we found evidence for both horizontal and vertical transmission. Larval clusters reared on the native host plant had slightly higher horizontal transmission. Survival probability was lower in clusters feeding on the native host plant, with inoculated groups reared on the native host plant experiencing complete mortality. Viral loads did not differ by the host plant, although viral loads decreased with increased sequestration of secondary compounds on both host plants. Our results indicate that the use of a novel host plant may confer fitness benefits in terms of survival and reduced viral transmission when larvae feeding on it are infected with this pathogen, supporting hypotheses of potential evolutionary advantages of a host range expansion in the context of tritrophic interactions., (© 2024 The Ecological Society of America.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Climate change is contributing to declines of insects through rising temperatures, altered precipitation patterns, and an increasing frequency of extreme events. The impacts of both gradual and sudden shifts in weather patterns are realized directly on insect physiology and indirectly through impacts on other trophic levels. Here, we investigated direct effects of seasonal weather on butterfly occurrences and indirect effects mediated by plant productivity using a temporally intensive butterfly monitoring dataset, in combination with high-resolution climate data and a remotely sensed indicator of plant primary productivity. Specifically, we used Bayesian hierarchical path analysis to quantify relationships between weather and weather-driven plant productivity on the occurrence of 94 butterfly species from three localities distributed across an elevational gradient. We found that snow pack exerted a strong direct positive effect on butterfly occurrence and that low snow pack was the primary driver of reductions during drought. Additionally, we found that plant primary productivity had a consistently negative effect on butterfly occurrence. These results highlight mechanisms of weather-driven declines in insect populations and the nuances of climate change effects involving snow melt, which have implications for ecological theories linking topographic complexity to ecological resilience in montane systems., (© 2023 John Wiley & Sons Ltd.)

Secondary plant chemistry mediates a variety of communication signals among species, playing a fundamental role in the evolutionary diversification of communities and ecosystems. Herein, we explored diet-mediated host plant effects on development and immune response of a generalist insect herbivore. Vanessa cardui (Nymphalidae) caterpillars were reared on leaves of three host plants that vary in secondary metabolites, Plantago lanceolata (Plantaginaceae), Taraxacum officinale (Asteraceae) and Tithonia diversifolia (Asteraceae). Insect development was evaluated by larval and pupal viabilities, survivorship, and development rate. Immune response was measured as phenoloxidase (PO) activity. Additionally, chemical profiles of the host plants were obtained by liquid chromatograph-mass spectrometry (LC-MS) and the discriminant metabolites were determined using a metabolomic approach. Caterpillars reared on P. lanceolata exhibited the highest larval and pupal viabilities, as well as PO activity, and P. lanceolata leaves were chemically characterized by the presence of iridoid glycosides, phenylpropanoids and flavonoids. Taraxacum officinale leaves were characterized mainly by the presence of phenylpropanoids, flavones O-glycoside and germacranolide-type sesquiterpene lactones; caterpillars reared on this host plant fully developed to the adult stage, however they exhibited lower larval and pupal viabilities compared to individuals reared on P. lanceolata. Conversely, caterpillars reared on T. diversifolia leaves, which contain phenylpropanoids, flavones and diverse furanoheliangolide-type sesquiterpene lactones, were not able to complete larval development and exhibited the lowest PO activity. These findings suggested that V. cardui have adapted to tolerate potentially toxic metabolites occurring in P. lanceolata (iridoid glycosides), however caterpillars were not able to cope with potentially detrimental metabolites occurring in T. diversifolia (furanoheliangolides). Therefore, we suggest that furanoheliangolide-type sesquiterpene lactones were responsible for the poor development and immune response observed for caterpillars reared on T. diversifolia., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Intraspecific phytochemical variation across a landscape can cascade up trophic levels, potentially mediating the composition of entire insect communities. Surprisingly, we have little understanding of the processes that regulate and maintain phytochemical variation within species, likely because these processes are complex and operate simultaneously both temporally and spatially. To assess how phytochemistry varies within species, we tested the degree to which resource availability, contrasting soil type, and herbivory generate intraspecific chemical variation in growth and defense of the tropical shrub, Piper imperiale (Piperaceae). We quantified changes in both growth (e.g., nutritional protein, above- and below-ground biomass) and defense (e.g., imide chemicals) of individual plants using a well-replicated fully factorial shade-house experiment in Costa Rica. We found that plants grown in high light, nutrient- and richer old alluvial soil had increased biomass. High light was also important for increasing foliar protein. Thus, investment into growth was determined by resource availability and soil composition. Surprisingly, we found that chemical defenses decreased in response to herbivory. We also found that changes in plant protein were more plastic compared to plant defense, indicating that constitutive defenses may be relatively fixed, and thus an adaptation to chronic herbivory that is common in tropical forests. We demonstrate that intraspecific phytochemical variation of P. imperiale is shaped by resource availability from light and soil type. Because environmental heterogeneity occurs over small spatial scales (tens of meters), herbivores may be faced with a complex phytochemical landscape that may regulate how much damage any individual plant sustains., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Exotic plant species represent a novel resource for invertebrates and many herbivorous insects have incorporated exotic plants into their diet. Using a new host plant can have physiological repercussions for these herbivores that may be beneficial or detrimental. In this study, we compared how using an exotic versus native host plant affected the immune system response and feeding efficiency of a specialist lepidopteran, the common buckeye ( Junonia coenia : Nymphalidae, Hübner 1822). Materials and Methods: In a lab experiment, larvae were reared on either the exotic host plant, Plantago lanceolata (Plantaginaceae), or the native host plant, Mimulus guttatus (Phrymaceae). Beginning at second instar feeding efficiency data were collected every 2 days until fifth instar when immune assays were performed. Immune assays consisted of standing phenoloxidase activity, total phenoloxidase activity, and melanization. Results: Interestingly, we found that all three immune system parameters were higher on the exotic host plant compared to the native host plant. The exotic host plant also supported higher pupal weights, faster development time, greater consumption, and more efficient approximate digestibility. In contrast, the native host plant supported higher efficiency of conversion of ingested and digested food. The relationship between immunity and feeding efficiency was more complex but showed a large positive effect of greater host plant consumption on all immune parameters, particularly for the exotic host plant. While not as strong, the efficiency of conversion of digested food tended to show a negative effect on the three immune parameters. Conclusion: Overall, the exotic host plant proved to be beneficial for this specialist insect with regard to immunity and many of the feeding efficiency parameters and continued use of this host plant is predicted for populations already using it., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mo and Smilanich.)

Defense against natural enemies constitutes an important driver of herbivore host range evolution in the wild. Populations of the Baltimore checkerspot butterfly, Euphydryas phaeton (Nymphalidae), have recently incorporated an exotic plant, Plantago lanceolata (Plantaginaceae), into their dietary range. To understand the tritrophic consequences of utilizing this exotic host plant, we examined immune performance, chemical defense, and interactions with a natural entomopathogen (Junonia coenia densovirus, Parvoviridae ) across wild populations of this specialist herbivore. We measured three immune parameters, sequestration of defensive iridoid glycosides (IGs), and viral infection load in field-collected caterpillars using either P . lanceolata or a native plant, Chelone glabra (Plantaginaceae). We found that larvae using the exotic plant exhibited reduced immunocompetence, compositional differences in IG sequestration, and higher in situ viral burdens compared to those using the native plant. On both host plants, high IG sequestration was associated with reduced hemocyte concentration in the larval hemolymph, providing the first evidence of incompatibility between sequestered chemical defenses and the immune response (i.e., the "vulnerable host" hypothesis) from a field-based study. However, despite this negative relationship between IG sequestration and cellular immunity, caterpillars with greater sequestration harbored lower viral loads. While survival of virus-infected individuals decreased with increasing viral burden, it ultimately did not differ between the exotic and native plants. These results provide evidence that: (1) phytochemical sequestration may contribute to defense against pathogens even when immunity is compromised and (2) herbivore persistence on exotic plant species may be facilitated by sequestration and its role in defense against natural enemies., Competing Interests: The authors declare no competing interests., (© 2022 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Foundational hypotheses addressing plant-insect codiversification and plant defense theory typically assume a macroevolutionary pattern whereby closely related plants have similar chemical profiles. However, numerous studies have documented variation in the degree of phytochemical trait lability, raising the possibility that phytochemical evolution is more nuanced than initially assumed. We utilize proton nuclear magnetic resonance ( 1 H NMR) data, chemical classification, and double digest restriction-site associated DNA sequencing (ddRADseq) to resolve evolutionary relationships and characterize the evolution of secondary chemistry in the Neotropical plant clade Radula (Piper; Piperaceae). Sequencing data substantially improved phylogenetic resolution relative to past studies, and spectroscopic characterization revealed the presence of 35 metabolite classes. Metabolite classes displayed phylogenetic signal, whereas the crude 1 H NMR spectra featured little evidence of phylogenetic signal in multivariate tests of chemical resonances. Evolutionary correlations were detected in two pairs of compound classes (flavonoids with chalcones; p-alkenyl phenols with kavalactones), where the gain or loss of a class was dependent on the other's state. Overall, the evolution of secondary chemistry in Radula is characterized by strong phylogenetic signal of traditional compound classes and weak phylogenetic signal of specialized chemical motifs, consistent with both classic evolutionary hypotheses and recent examinations of phytochemical evolution in young lineages., (© 2021. The Author(s).)

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 CO 2 (eCO 2 ) 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 eCO 2 lose tolerance to the parasite, associated with changes in phytochemistry. Whether changes in milkweed phytochemistry induced by eCO 2 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 CO 2 (aCO 2 ) or eCO 2 . 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 aCO 2 plants and higher in larvae reared on foliage from eCO 2 plants. When grown under eCO 2 , 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 eCO 2 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., (© 2020 British Ecological Society.)

An important goal of disease ecology is to understand trophic interactions influencing the host-pathogen relationship. This study focused on the effects of diet and immunity on the outcome of viral infection for the polyphagous butterfly, Vanessa cardui Linnaeus (Lepidoptera: Nymphalidae) (painted lady). Specifically, we aimed to understand the role that larval host plants play when fighting a viral pathogen. Larvae were orally inoculated with the entomopathogenic virus, Junonia coenia densovirus (JcDV) (Parvovirididae: Densovirinae, Lepidopteran Potoambidensovirus 1) and reared on two different host plants (Lupinus albifrons Bentham (Fabales: Fabaceae) or Plantago lanceolata Linnaeus (Lamiales: Plantaginaceae)). Following viral infection, the immune response (i.e., phenoloxidase [PO] activity), survival to adulthood, and viral load were measured for individuals on each host plant. We found that the interaction between the immune response and survival of the viral infection was host plant dependent. The likelihood of survival was lowest for infected larvae exhibiting suppressed PO activity and feeding on P. lanceolata, providing some evidence that PO activity may be an important defense against viral infection. However, for individuals reared on L. albifrons, the viral infection had a negligible effect on the immune response, and these individuals also had higher survival and lower viral load when infected with the pathogen compared to the controls. Therefore, we suggest that host plant modifies the effects of JcDV infection and influences caterpillars' response when infected with the virus. Overall, we conclude that the outcome of viral infection is highly dependent upon diet, and that certain host plants can provide protection from pathogens regardless of immunity., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America.)

Herbivorous insects can defend themselves against pathogens via an immune response, which is influenced by the nutritional quality and phytochemistry of the host plant. However, it is unclear how these aspects of diet interact to influence the insect immune response and what role is played by ingested foliar microbes. We examined dietary protein, phytochemistry, and the caterpillar microbiome to understand variation in immune response of the Melissa blue butterfly, Lycaeides melissa. We also asked if these factors have host plant-specific effects by measuring L. melissa immune response when reared on a recently colonized exotic host plant (Medicago sativa) as compared to the immune response on an ancestral, native host (Astragalus canadensis). L. melissa did not experience immunological benefits directly related to consumption of the novel plant M. sativa. However, we did find negative, direct effects of phytochemical diversity and negative, direct effects of diet-derived microbial diversity on constitutive immune response for caterpillars fed M. sativa, as measured by phenoloxidase activity. Foliar protein did not directly influence the immune response, but did do so indirectly by increasing weight gain. Our results highlight the important effects of host diet on caterpillar physiology and raise the possibility that foliar microbiota, despite being rapidly passed through the gut, can affect the caterpillar immune response.

Terrestrial tri-trophic interactions account for a large part of biodiversity, with approximately 75% represented in plant-insect-parasitoid interactions. Herbivore diet breadth is an important factor mediating these tri-trophic interactions, as specialisation can influence how herbivore fitness is affected by plant traits. We investigated how phytochemistry, herbivore immunity, and herbivore diet breadth mediate plant-caterpillar-parasitoid interactions on the tropical plant genus Piper (Piperaceae) at La Selva Biological station in Costa Rica and at Yanayacu Biological Station in Ecuador. We collected larval stages of one Piper generalist species, Quadrus cerealis , (Lepidoptera: Hesperiidae) and 4 specialist species in the genus Eois (Lepidoptera: Geometridae) from 15 different species of Piper , reared them on host leaf material, and assayed phenoloxidase activity as a measure of potential larval immunity. We combined these data with parasitism and caterpillar species diet breadth calculated from a 19-year database, as well as established values of phytochemical diversity calculated for each plant species, in order to test specific hypotheses about how these variables are related. We found that phytochemical diversity was an important predictor for herbivore immunity, herbivore parasitism, and diet breadth for specialist caterpillars, but that the direction and magnitude of these relationships differed between sites. In Costa Rica, specialist herbivore immune function was negatively associated with the phytochemical diversity of the Piper host plants, and rates of parasitism decreased with higher immune function. The same was true for Ecuador with the exception that there was a positive association between immune function and phytochemical diversity. Furthermore, phytochemical diversity did not affect herbivore immunity and parasitism for the more generalised herbivore. Results also indicated that small differences in herbivore diet breadth are an important factor mediating herbivore immunity and parasitism success for Eois at both sites. These patterns contribute to a growing body of literature that demonstrate strong cascading effects of phytochemistry on higher trophic levels that are dependent on herbivore specialisation and that can vary in space and time. Investigating the interface between herbivore immunity, plant chemical defence, and parasitoids is an important facet of tri-trophic interactions that can help to explain the enormous amount of biodiversity found in the tropics.

Understanding the interaction between host plant chemistry, the immune response, and insect pathogens can shed light on host plant use by insect herbivores. In this study, we focused on how interactions between the insect immune response and plant secondary metabolites affect the response to a viral pathogen. Based upon prior research, we asked whether the buckeye caterpillar, Junonia coenia (Nymphalidae), which specializes on plants containing iridoid glycosides (IGs), is less able to resist the pathogenic effects of a densovirus infection when feeding on plants with high concentrations of IGs. In a fully factorial design, individuals were randomly assigned to three treatments, each of which had two levels: (1) exposed to the densovirus versus control, (2) placed on a plant species with high concentrations of IGs (Plantago lanceolata, Plantaginaceae) versus low concentrations of IGs (P. major), and (3) control versus surface sterilized to exclude surface microbes that may contribute to viral resistance. We measured phenoloxidase (PO) activity, hemocyte counts, and gut bacterial diversity (16S ribosomal RNA) during the fourth larval instar, as well as development time, pupal weight, and survival to adult. Individuals infected with the virus were immune-suppressed (as measured by PO response and hemocyte count) and developed significantly faster than virus-free individuals. Contrary to our predictions,mortality was significantly less for virus challengedindividuals reared on the high IG plant compared to the low IG plant.This suggests that plant secondary metabolites can influence survival from viral infection and may be associated with activation of PO. Removing egg microbes did not affect the immune response or survival of the larvae. In summary, these results suggest that plant secondary metabolites are important for survival against a viral pathogen. Even though the PO response was better on the high IG plant, the extent to which this result contributes to survival against the virus needs further investigation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The origins of evolutionary radiations are often traced to the colonization of novel adaptive zones, including unoccupied habitats or unutilized resources. For herbivorous insects, the predominant mechanism of diversification is typically assumed to be a shift onto a novel lineage of host plants. However, other drivers of diversification are important in shaping evolutionary history, especially for groups residing in regions with complex geological histories. We evaluated the contributions of shifts in host plant clade, bioregion, and elevation to diversification in Eois (Lepidoptera: Geometridae), a hyper-diverse genus of moths found throughout the Neotropics. Relationships among 107 taxa were reconstructed using one mitochondrial and two nuclear genes. In addition, we used a genotyping-by-sequencing approach to generate 4641 SNPs for 137 taxa. Both datasets yielded similar phylogenetic histories, with relationships structured by host plant clade, bioregion, and elevation. While diversification of basal lineages often coincided with host clade shifts, more recent speciation events were more typically associated with shifts across bioregions or elevational gradients. Overall, patterns of diversification in Eois are consistent with the perspective that shifts across multiple adaptive zones synergistically drive diversification in hyper-diverse lineages., (© 2017 The Author(s). Evolution © 2017 The Society for the Study of Evolution.)

Chemically mediated plant-herbivore interactions contribute to the diversity of terrestrial communities and the diversification of plants and insects. While our understanding of the processes affecting community structure and evolutionary diversification has grown, few studies have investigated how trait variation shapes genetic and species diversity simultaneously in a tropical ecosystem. We investigated secondary metabolite variation among subpopulations of a single plant species, Piper kelleyi (Piperaceae), using high-performance liquid chromatography (HPLC), to understand associations between plant phytochemistry and host-specialized caterpillars in the genus Eois (Geometridae: Larentiinae) and associated parasitoid wasps and flies. In addition, we used a genotyping-by-sequencing approach to examine the genetic structure of one abundant caterpillar species, Eois encina, in relation to host phytochemical variation. We found substantive concentration differences among three major secondary metabolites, and these differences in chemistry predicted caterpillar and parasitoid community structure among host plant populations. Furthermore, E. encina populations located at high elevations were genetically different from other populations. They fed on plants containing high concentrations of prenylated benzoic acid. Thus, phytochemistry potentially shapes caterpillar and wasp community composition and geographic variation in species interactions, both of which can contribute to diversification of plants and insects., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

According to the plant-apparency hypothesis, apparent plants allocate resources to quantitative defenses that negatively affect generalist and specialist herbivores, while unapparent plants invest more in qualitative defenses that negatively affect nonadapted generalists. Although this hypothesis has provided a useful framework for understanding the evolution of plant chemical defense, there are many inconsistencies surrounding associated predictions, and it has been heavily criticized and deemed obsolete. We used a hierarchical Bayesian meta-analysis model to test whether defenses from apparent and unapparent plants differ in their effects on herbivores. We collected a total of 225 effect sizes from 158 published papers in which the effects of plant chemistry on herbivore performance were reported. As predicted by the plant-apparency hypothesis, we found a prevalence of quantitative defenses in woody plants and qualitative defenses in herbaceous plants. However, the detrimental impacts of qualitative defenses were more effective against specialists than generalists, and the effects of chemical defenses did not significantly differ between specialists and generalists for woody or herbaceous plants. A striking pattern that emerged from our data was a pervasiveness of beneficial effects of secondary metabolites on herbivore performance, especially generalists. This pattern provides evidence that herbivores are evolving effective counteradaptations to putative plant defenses., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

What are the ecological causes and consequences of variation in phytochemical diversity within and between plant taxa? Despite decades of natural products discovery by organic chemists and research by chemical ecologists, our understanding of phytochemically mediated ecological processes in natural communities has been restricted to studies of either broad classes of compounds or a small number of well-characterized molecules. Until now, no studies have assessed the ecological causes or consequences of rigorously quantified phytochemical diversity across taxa in natural systems. Consequently, hypotheses that attempt to explain variation in phytochemical diversity among plants remain largely untested. We use spectral data from crude plant extracts to characterize phytochemical diversity in a suite of co-occurring plants in the tropical genus Piper (Piperaceae). In combination with 20 years of data focused on Piper-associated insects, we find that phytochemical diversity has a direct and positive effect on the diversity of herbivores but also reduces overall herbivore damage. Elevated chemical diversity is associated with more specialized assemblages of herbivores, and the cascading positive effect of phytochemistry on herbivore enemies is stronger as herbivore diet breadth narrows. These results are consistent with traditional hypotheses that predict positive associations between plant chemical diversity, insect herbivore diversity, and trophic specialization. It is clear from these results that high phytochemical diversity not only enhances the diversity of plant-associated insects but also contributes to the ecological predominance of specialized insect herbivores.

Understanding variation in resource specialization is important for progress on issues that include coevolution, community assembly, ecosystem processes, and the latitudinal gradient of species richness. Herbivorous insects are useful models for studying resource specialization, and the interaction between plants and herbivorous insects is one of the most common and consequential ecological associations on the planet. However, uncertainty persists regarding fundamental features of herbivore diet breadth, including its relationship to latitude and plant species richness. Here, we use a global dataset to investigate host range for over 7,500 insect herbivore species covering a wide taxonomic breadth and interacting with more than 2,000 species of plants in 165 families. We ask whether relatively specialized and generalized herbivores represent a dichotomy rather than a continuum from few to many host families and species attacked and whether diet breadth changes with increasing plant species richness toward the tropics. Across geographic regions and taxonomic subsets of the data, we find that the distribution of diet breadth is fit well by a discrete, truncated Pareto power law characterized by the predominance of specialized herbivores and a long, thin tail of more generalized species. Both the taxonomic and phylogenetic distributions of diet breadth shift globally with latitude, consistent with a higher frequency of specialized insects in tropical regions. We also find that more diverse lineages of plants support assemblages of relatively more specialized herbivores and that the global distribution of plant diversity contributes to but does not fully explain the latitudinal gradient in insect herbivore specialization.

A rapidly advancing area of ecological immunology concerns the effects of diet on animals' immunological responses to parasites and pathogens. Here, we focus on diet-mediated ecological immunology in herbivorous insects, in part because these organisms commonly experience nutritional limitations from their diets of plants. Nutritional immunology highlights nutrient-based trade-offs between immunological and other physiological processes as well as trade-offs among distinct immunological processes. This field reveals that nutrition influences the quality and quantity of immunological defense in herbivorous insects, and conversely that nutritional intake by herbivorous insects can be an adaptive response to the specific types of immune-challenge they face in the context of other physiological processes. Because the diets of herbivores challenge them physiologically with plants' secondary metabolites, another area of study analyzes constraints on immunological defense imposed by secondary metabolites of plants in the diets of herbivorous insects. Alternatively, some herbivores can use secondary metabolites as medicine against parasites or pathogens. Animal-medication theory makes an important contribution to ecological immunology by distinguishing prophylactic and therapeutic mechanisms of anti-parasite defense. Integrating ideas from animal-medication and nutritional immunology, we outline a conceptual framework in which the immunological role of the diet consists of mechanisms of prophylaxis, therapy, compensation, and combinations thereof. Then, we use this framework to organize findings from our own research on diet-mediated ecological immunology of woolly bear caterpillars. We show evidence that the woolly bear caterpillar, Grammia incorrupta (Hy. Edwards) (Lepidoptera, Erebidae, and Arctiinae), can employ both diet-mediated prophylaxis and therapy. First, increased consumption of carbohydrate-biased food prior to immune-challenge increased its melanization-response. Second, increased consumption of pyrrolizidine alkaloids (PAs) more than 24 h after parasitism by tachinid flies resulted in anti-parasite resistance. Caterpillars reduced feeding on protein-biased food within 24 h after immune-challenge, showing evidence of illness-induced anorexia. We synthesize our work to generate the hypothesis that a diet-mediated defense by the host against parasites acts as a temporally explicit, multi-stage process., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.)

Advances in ecological immunity have illustrated that, like vertebrates, insects exhibit adaptive immunity, including induced changes in feeding behavior that aid the immune system. In particular, recent studies have pointed to the importance of protein intake in mounting an immune response. In this study, we tested the hypothesis that the polyphagous caterpillar Grammia incorrupta (H. Edwards) (Family: Erebidae) would adaptively change its feeding behavior in response to immune challenge, predicting that caterpillars would increase their intake of dietary protein. We further predicted that this response would enhance the melanization response, a component of the immune system that acts against parasitoids. We challenged the immune system using either tachinid fly parasitoids or a bead injection technique that has been used in studies to simulate parasitism, and measured feeding before and after immune challenge on diets varying in their macronutrient content. To evaluate the effects of diet on melanization, we quantified melanization of beads following feeding assays. Contrary to our prediction, we found that parasitized or injected caterpillars given a choice between high- and low-protein foods reduced their intake of the high-protein food. Furthermore, in a no-choice experiment, caterpillars offered food with a protein concentration that is optimal for growth reduced feeding following immune challenge, whereas those offered a low-protein food did not. Although variation in protein intake did not change the caterpillars' melanization response, increased carbohydrate intake did increase melanization, suggesting a prophylactic role for carbohydrates. We discuss alternative mechanisms by which variation in protein intake could negatively or positively affect parasitized caterpillars, including nutritional interactions with the caterpillar's self-medication response., (© 2014. Published by The Company of Biologists Ltd.)

Plants use a diverse mix of defenses against herbivores, including multiple secondary metabolites, which may affect herbivores synergistically. Chemical defenses also can affect natural enemies of herbivores via limiting herbivore populations or by affecting herbivore resistance or susceptibility to these enemies. In this study, we conducted larval feeding experiments to examine the potential synergistic effects of iridoid glycosides (IGs) found in Plantago spp. (Plantaginaceae) on the specialist buckeye caterpillar, Junonia coenia (Nymphalidae). Caterpillars were placed on artificial diets containing different concentrations of single IGs (aucubin or catalpol alone) or combinations of the two IGs. Larval performance and immune response were recorded to test the hypothesis that IGs would have positive synergistic effects on buckeyes, which are specialists on IG plants. The positive synergistic effects that IGs had on buckeyes in our experiments included lower mortality, faster development, and higher total iridoid glycoside sequestration on mixed diets than on aucubin- or catalpol-only diets. Furthermore, we found negative synergistic effects of IGs on the immune response of buckeye caterpillars. These results demonstrate multiple synergistic effects of IGs and indicate a potential trade-off between larval performance and parasitoid resistance.

Basic research on the insect immune response has progressed dramatically within the last two decades, showing that immunity is one of the most effective defenses against foreign invaders. As such, it is important to understand the causes of variation in this response. Here, we investigate the effects of pesticides used in Costa Rican banana plantations on the immune response of the lepidopteran larva, Caligo memnon (Brassolinae). In addition, we performed a parasitism survey of the banana plantations and surrounding forests to provide a broader assessment of pesticide effects on parasitoid populations. All caterpillars for the immune assay were collected from two banana plantations and brought to La Selva Biology Station for immune challenge. Individuals were fed leaves from the plantations (pesticide) or leaves from La Selva (pesticide-free), then immune challenged with injected sephadex beads. We found that individuals feeding on pesticide leaves had significantly lower bead melanization compared to individuals feeding on pesticide-free leaves. Nonetheless, the parasitism survey showed that caterpillars from the banana plantations had lower parasitism rates compared to caterpillars from the La Selva forest. This study adds to the growing body of evidence documenting negative effects of pesticides on the insect immune response and on adult parasitoids, and underscores the need for more research at the intersection between ecological entomology and immunology.

This study investigates complex effects of parasitoid infection on herbivore diet choice. Specifically, we examine how immunological resistance, parasitoid infection stage, and parasitoid taxonomic identity affect the pharmacophagous behavior of the polyphagous caterpillar, Grammia incorrupta (Arctiidae). Using a combination of lab and field experiments, we test the caterpillar's pharmacophagous response to pyrrolizidine alkaloids (PAs) over the course of parasitoid infection, as well as the effect of dietary PAs on the caterpillar's immunological response. Previous work from other systems gave the prediction that dietary PAs would be detrimental to the immune response and thus less acceptable to feeding early in the infection, when encapsulation of the parasitoid is most crucial. We found that the feeding acceptability of PAs was indeed low for caterpillars with early-stage parasitoid infections; however, this was not explained by PA interference with immune function. When allowed to choose among three host plant species, individuals harboring early-stage parasitoids increased their consumption of a nutritious plant containing antioxidants. This result was driven by wasp-parasitized caterpillars, whereas fly-parasitized caterpillars increased their consumption of plants containing iridoid glycosides. Individuals in the later time phase of infection exhibited an increase in PA intake that was consistent with previously reported self-medication behavior during late-stage parasitoid infection. This study reveals the depth of complexity and the dynamic nature of herbivore host plant choice, and underscores the importance of considering multitrophic interactions when studying insect diet choice.

We considered the effects of plant secondary metabolites on the immune response, a key physiological defense of herbivores against pathogens and parasitoids. We tested the effect of host plant species and ingested iridoid glycosides on the immune response of the grazing, polyphagous caterpillar, Grammia incorrupta (Arctiidae). Individuals of G. incorrupta were fed either one of three plant diets with varying secondary metabolites, or an artificial diet with high or low concentrations of iridoid glycosides. An immune challenge was presented, followed by measurement of the encapsulation response. We failed to detect a significant difference in the immune response of G. incorrupta feeding on diets with varying concentrations of iridoid glycosides, or feeding on different host plants. However, the immune response was lower in caterpillars consuming the artificial diet compared to those consuming the plant diets. When caterpillar performance was measured, pupal weights were lower when caterpillars ingested high concentrations of iridoid glycosides due to a decrease in feeding efficiency. Overall, individuals of G. incorrupta that consumed different plant diets exhibited a high immune response with low variation. We conclude that the immune response of G. incorrupta is adapted to feeding on a variety of plants, which may contribute to the maintenance of this caterpillar's polyphagous habit.

Plants use a diverse mix of defenses against herbivores, including multiple secondary metabolites, which often affect herbivores synergistically. Chemical defenses also can affect natural enemies of herbivores via limiting herbivore populations or by affecting herbivore resistance to parasitoids. In this study, we performed feeding experiments to examine the synergistic effects of imides and amides (hereafter "amides") from Piper cenocladum and P. imperiale on specialist (Eois nympha, Geometridae) and generalist (Spodoptera frugiperda, Noctuidae) lepidopteran larvae. Each Piper species has three unique amides, and in each experiment, larvae were fed diets containing different concentrations of single amides or combinations of the three. The amides from P. imperiale had negative synergistic effects on generalist survival and specialist pupal mass, but had no effect on specialist survival. Piper cenocladum amides also acted synergistically to increase mortality caused by parasitoids, and the direct negative effects of mixtures on parasitoid resistance and pupal mass were stronger than indirect effects via changes in growth rate and approximate digestibility. Our results are consistent with plant defense theory that predicts different effects of plant chemistry on generalist versus adapted specialist herbivores. The toxicity of Piper amide mixtures to generalist herbivores are standard bottom-up effects, while specialists experienced the top-down mediated effect of mixtures causing reduced parasitoid resistance and associated decreases in pupal mass.

Selective pressures from host plant chemistry and natural enemies may contribute independently to driving insect herbivores towards narrow diet breadths. We used the specialist caterpillar, Junonia coenia (Nymphalidae), which sequesters defensive compounds, iridoid glycosides, from its host plants to assess the effects of plant chemistry and sequestration on the larval immune response. A series of experiments using implanted glass beads to challenge immune function showed that larvae feeding on diets with high concentrations of iridoid glycosides are more likely to have their immune response compromised than those feeding on diets low in these compounds. These results indicate that larvae feeding on plants with high concentrations of toxins might be more poorly defended against parasitoids, while at the same time being better defended against predators, suggesting that predators and parasitoids can exert different selective pressures on the evolution of herbivore diet breadth.

Parasitic wasps and flies (parasitoids) exert high mortality on caterpillars, and previous studies have demonstrated that most primary and secondary defenses do not protect caterpillars against parasitoids. We investigated the efficacy of tertiary defenses (i.e., immune responses) against parasitoids. Using a bead injection technique to measure the immune response and a 15-year database to measure parasitism, we compared the immune response for 16 species of caterpillars in nine different families. We found that caterpillar species with a strong immune response had the lowest incidence of parasitism, and when statistically compared to other defensive traits, the immune response was the best predictor of parasitism. Parasitoids either avoid attacking caterpillar species with a capacity for high levels of melanization or are killed once they have parasitized. In either case, the immune response is clearly one of the most effective defenses that caterpillars have against parasitism, and elucidating consistent predictors of variation in encapsulation could improve understanding of parasitism patterns in time and space and could enhance biological control efforts.

The tropical rainforest shrub Piper cenocladum, which is normally defended against herbivores by a mutualistic ant, contains three amides that have various defensive functions. While the ants are effective primarily against specialist herbivores, we hypothesized that these secondary compounds would be effective against a wider range of insects, thus providing a broad array of defenses against herbivores. We also tested whether a mixture of amides would be more effective against herbivores than individual amides. Diets spiked with amides were offered to five herbivores: a naïve generalist caterpillar (Spodoptera frugiperda), two caterpillar species that are monophagous on P. cenocladum (Eois spp.), leaf-cutting ants (Atta cephalotes), and an omnivorous ant (Paraponera clavata). Amides had negative effects on all insects, whether they were naïve, experienced, generalized, or specialized feeders. For Spodoptera, amide mixtures caused decreased pupal weights and survivorship and increased development times. Eois pupal weights, larval mass gain, and development times were affected by additions of individual amides, but increased parasitism and lower survivorship were caused only by the amide mixture. Amide mixtures also deterred feeding by the two ant species, and crude plant extracts were strongly deterrent to P. clavata. The mixture of all three amides had the most dramatic deterrent and toxic effects across experiments, with the effects usually surpassing expected additive responses, indicating that these compounds can act synergistically against a wide array of herbivores.