Additive genetic effects in interacting species jointly determine the outcome of caterpillar herbivory.

Plant-insect interactions are common and important in basic and applied biology. Trait and genetic variation can affect the outcome and evolution of these interactions, but the relative contributions of plant and insect genetic variation and how these interact remain unclear and are rarely subject to assessment in the same experimental context. Here, we address this knowledge gap using a recent host-range expansion onto alfalfa by the Melissa blue butterfly. Common garden rearing experiments and genomic data show that caterpillar performance depends on plant and insect genetic variation, with insect genetics contributing to performance earlier in development and plant genetics later. Our models of performance based on caterpillar genetics retained predictive power when applied to a second common garden. Much of the plant genetic effect could be explained by heritable variation in plant phytochemicals, especially saponins, peptides, and phosphatidyl cholines, providing a possible mechanistic understanding of variation in the species interaction. We find evidence of polygenic, mostly additive effects within and between species, with consistent effects of plant genotype on growth and development across multiple butterfly species. Our results inform theories of plant-insect coevolution and the evolution of diet breadth in herbivorous insects and other host-specific parasites.

Multiple and contrasting pressures determine intraspecific phytochemical variation in a tropical shrub.

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.

Host plant-dependent effects of microbes and phytochemistry on the insect immune response.

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.

Phytochemical diversity drives plant-insect community diversity.

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.

Intraspecific phytochemical variation shapes community and population structure for specialist caterpillars.

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.

Antiherbivore prenylated benzoic acid derivatives from Piper kelleyi.

The known prenylated benzoic acid derivative 3-geranyl-4-hydroxy-5-(3″,3″-dimethylallyl)benzoic acid (1) and two new chromane natural products were isolated from the methanolic extract of the leaves of Piper kelleyi Tepe (Piperaceae), a midcanopy tropical shrub that grows in lower montane rain forests in Ecuador and Peru. Structure determination using 1D and 2D NMR analysis led to the structure of the chromene 2 and to the reassignment of the structure of cumanensic acid as 4, an isomeric chromene previously isolated from Piper gaudichaudianum. The structure and relative configuration of new chromane 3 was determined using 1D and 2D NMR spectroscopic analysis and was found to be racemic by ECD spectropolarimetry. The biological activity of 1-3 was evaluated against a lab colony of the generalist caterpillar Spodoptera exigua (Noctuidae), and low concentrations of 2 and 3 were found to significantly reduce fitness. Further consideration of the biosynthetic relationship of the three compounds led to the proposal that 1 is converted to 2 via an oxidative process, whereas 3 is produced through hetero-[4+2] dimerization of a quinone methide derived from the chromene 2.

Synergistic effects of iridoid glycosides on the survival, development and immune response of a specialist caterpillar, Junonia coenia (Nymphalidae).

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.

Phytochemistry reflects different evolutionary history in traditional classes versus specialized structural motifs.

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 (1H 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 1H 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.

Synergistic effects of amides from two piper species on generalist and specialist herbivores.

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.

Secondary metabolites in a neotropical shrub: spatiotemporal allocation and role in fruit defense and dispersal.

Deciphering the ecological roles of plant secondary metabolites requires integrative studies that assess both the allocation patterns of compounds and their bioactivity in ecological interactions. Secondary metabolites have been primarily studied in leaves, but many are unique to fruits and can have numerous potential roles in interactions with both mutualists (seed dispersers) and antagonists (pathogens and predators). We described 10 alkenylphenol compounds from the plant species Piper sancti-felicis (Piperaceae), quantified their patterns of intraplant allocation across tissues and fruit development, and examined their ecological role in fruit interactions. We found that unripe and ripe fruit pulp had the highest concentrations and diversity of alkenylphenols, followed by flowers; leaves and seeds had only a few compounds at detectable concentrations. We observed a nonlinear pattern of alkenylphenol allocation across fruit development, increasing as flowers developed into unripe pulp then decreasing as pulp ripened. This pattern is consistent with the hypothesis that alkenylphenols function to defend fruits from pre-dispersal antagonists and are allocated based on the contribution of the tissue to the plant's fitness, but could also be explained by non-adaptive constraints. To assess the impacts of alkenylphenols in interactions with antagonists and mutualists, we performed fungal bioassays, field observations, and vertebrate feeding experiments. In fungal bioassays, we found that alkenylphenols had a negative effect on the growth of most fungal taxa. In field observations, nocturnal dispersers (bats) removed the majority of infructescences, and diurnal dispersers (birds) removed a larger proportion of unripe infructescences. In feeding experiments, bats exhibited an aversion to alkenylphenols, but birds did not. This observed behavior in bats, combined with our results showing a decrease in alkenylphenols during ripening, suggests that alkenylphenols in fruits represent a trade-off (defending against pathogens but reducing disperser preference). These results provide insight into the ecological significance of a little studied class of secondary metabolites in seed dispersal and fruit defense. More generally, documenting intraplant spatiotemporal allocation patterns in angiosperms and examining mechanisms behind these patterns with ecological experiments is likely to further our understanding of the evolutionary ecology of plant chemical traits.

Caterpillars on a phytochemical landscape: The case of alfalfa and the Melissa blue butterfly.

Modern metabolomic approaches that generate more comprehensive phytochemical profiles than were previously available are providing new opportunities for understanding plant-animal interactions. Specifically, we can characterize the phytochemical landscape by asking how a larger number of individual compounds affect herbivores and how compounds covary among plants. Here we use the recent colonization of alfalfa (Medicago sativa) by the Melissa blue butterfly (Lycaeides melissa) to investigate the effects of indivdiual compounds and suites of covarying phytochemicals on caterpillar performance. We find that survival, development time, and adult weight are all associated with variation in nutrition and toxicity, including biomolecules associated with plant cell function as well as putative anti-herbivore action. The plant-insect interface is complex, with clusters of covarying compounds in many cases encompassing divergent effects on different aspects of caterpillar performance. Individual compounds with the strongest associations are largely specialized metabolites, including alkaloids, phenolic glycosides, and saponins. The saponins are represented in our data by more than 25 individual compounds with beneficial and detrimental effects on L. melissa caterpillars, which highlights the value of metabolomic data as opposed to approaches that rely on total concentrations within broad defensive classes.

Shedding Light on Chemically Mediated Tri-Trophic Interactions: A 1H-NMR Network Approach to Identify Compound Structural Features and Associated Biological Activity.

Diverse mixtures of plant natural products play an important role in plant-herbivore-parasitoid interactions. In the pursuit of understanding these chemically-mediated interactions, we are often faced with the challenge of determining ecologically and biologically relevant compounds present in complex phytochemical mixtures. Using a network-based approach, we analyzed binned 1H-NMR data from 196 prepared mixtures of commonly studied secondary metabolites including alkaloids, amides, terpenes, iridoid glycosides, saponins, phenylpropanoids, flavonoids and phytosterols. The mixtures included multiple dimensions of chemical diversity, including molecular complexity, mixture complexity and differences in relative compound concentrations. This approach yielded modules of co-occurring chemical shifts that were correlated with specific compounds or common structural features shared across compounds. This approach was then applied to crude phytochemical extracts of 31 species in the phytochemically diverse tropical plant genus Piper (Piperaceae). Combining the activity of crude plant extracts in an array of bioassays with our 1H-NMR network approach, we identified a potential prenylated benzoic acid from these mixtures that exhibits antifungal properties and identified small structural differences that were potentially responsible for antifungal activity. In an intraspecific analysis of individual Piper kelleyi plants, we also found ontogenetic differences in chemistry that may affect natural plant enemies. In sum, this approach allowed us to characterize mixtures as useful network modules and to combine chemical and ecological datasets to identify biologically important compounds from crude extracts.

Effects of CO2 and temperature on tritrophic interactions.

There has been a significant increase in studies of how global change parameters affect interacting species or entire communities, yet the combined or interactive effects of increased atmospheric CO2 and associated increases in global mean temperatures on chemically mediated trophic interactions are mostly unknown. Thus, predictions of climate-induced changes on plant-insect interactions are still based primarily on studies of individual species, individual global change parameters, pairwise interactions, or parameters that summarize communities. A clear understanding of community response to global change will only emerge from studies that examine effects of multiple variables on biotic interactions. We examined the effects of increased CO2 and temperature on simple laboratory communities of interacting alfalfa, chemical defense, armyworm caterpillars, and parasitoid wasps. Higher temperatures and CO2 caused decreased plant quality, decreased caterpillar development times, developmental asynchrony between caterpillars and wasps, and complete wasp mortality. The effects measured here, along with other effects of global change on natural enemies suggest that biological control and other top-down effects of insect predators will decline over the coming decades.

The roots of defense: plant resistance and tolerance to belowground herbivory.

BACKGROUND: There is conclusive evidence that there are fitness costs of plant defense and that herbivores can drive selection for defense. However, most work has focused on above-ground interactions, even though belowground herbivory may have greater impacts on individual plants than above-ground herbivory. Given the role of belowground plant structures in resource acquisition and storage, research on belowground herbivores has much to contribute to theories on the evolution of plant defense. Pocket gophers (Geomyidae) provide an excellent opportunity to study root herbivory. These subterranean rodents spend their entire lives belowground and specialize on consuming belowground plant parts. METHODOLOGY AND PRINCIPAL FINDINGS: We compared the root defenses of native forbs from mainland populations (with a history of gopher herbivory) to island populations (free from gophers for up to 500,000 years). Defense includes both resistance against herbivores and tolerance of herbivore damage. We used three approaches to compare these traits in island and mainland populations of two native California forbs: 1) Eschscholzia californica populations were assayed to compare alkaloid deterrents, 2) captive gophers were used to test the palatability of E. californica roots and 3) simulated root herbivory assessed tolerance to root damage in Deinandra fasciculata and E. californica. Mainland forms of E. californica contained 2.5 times greater concentration of alkaloids and were less palatable to gophers than island forms. Mainland forms of D. fasciculata and, to a lesser extent, E. californica were also more tolerant of root damage than island conspecifics. Interestingly, undamaged island individuals of D. fasciculata produced significantly more fruit than either damaged or undamaged mainland individuals. CONCLUSIONS AND SIGNIFICANCE: These results suggest that mainland plants are effective at deterring and tolerating pocket gopher herbivory. Results also suggest that both forms of defense are costly to fitness and thus reduced in the absence of the putative target herbivore.

A bioassay for insect deterrent compounds found in plant and animal tissues.

A general field bioassay for detecting biologically active compounds in plants and insects has been developed and tested for efficacy and sensitivity. Methanolic extracts, in sucrose solution, of 20 plant and six caterpillar species were offered to the ponerine ant Paraponera clavata and the feeding preferences observed. The bioassay resulted in the detection of nine plant and three caterpillar species with ant-deterrent extracts, and 11 plant and three caterpillar species with neutral or attractant extracts. All of the plants showing ant-deterrent characteristics which had been chemically investigated in our laboratory, or for which chemical literature was available, contained secondary metabolites of known deterrence. Both naturally occurring and artificial differences in chemical concentrations could be detected using the bioassay. The method provides a means of screening plants and insects for compounds that are insect anti-feedants or that can modify insect behaviour.