Craig D. Dodson

Synergistic Effects of Three Piper Amides on Generalist and Specialist Herbivores

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.

Trade-offs in Antiherbivore Defenses in Piper cenocladum: Ant Mutualists Versus Plant Secondary Metabolites

Ant–plant mutualisms may provide indirect evidence for costs of antiherbivore defenses when plants demonstrate trade-offs between allocating resources and energy into ant attractants versus chemical defenses. We tested the hypothesis that ecological trade-offs in defenses are present in Piper cenocladum. This plant possesses two distinct defenses: food bodies that attract predatory ants that destory herbivore eggs and amides that deter herbivores. Previous studies have demonstrated that the food bodies in P. cenocladum are an effective defense because the ants deter herbivory by specialist herbivores. Amides in other Piper species have been shown to have toxic qualities, but we tested the additional hypothesis that these amides have an actual defensive function in P. cenocladum. To test for ecological trade-offs between the two putative defenses, fragments of P. cenocladum were examined for the presence of amides both when the plant was producing food bodies and when it was not producing food bodies. Plants with active ant colonies had redundant defenses, producing food bodies and high levels of amides at the same time, but we detected a trade-off in that they had significantly lower levels of amides than did plants with no ants. To test for the defensive value of P. cenocladum amides, we used an ant bioassay and we examined herbivory results from previous experiments with plants that had variable levels of amides. These tests demonstrated that amides are deterrent to omnivorous ants, leaf cutting ants, and orthopterans. In contrast, the resident Pheidole bicornis ants are effective at deterring herbivory by specialist herbivores that oviposit eggs on the plant but not at deterring herbivory by nonresident omnivores. We concluded that although both amides and food body production appear to be costly, redundancy in defenses is necessary to avoid damage by a complex suit of herbivores.

ECOLOGICAL CAUSES AND CONSEQUENCES OF VARIATION IN DEFENSIVE CHEMISTRY OF A NEOTROPICAL SHRUB

Prominent models of herbivore regulation focus on predators, low plant biomass, or poor resource quality as factors that limit herbivore populations. We examined predictions of these tritrophic models for herbivores on the understory shrub, Piper cen- ocladum, which is defended by mutualistic ants (Pheidole bicornis) and three amide sec- ondary metabolites. To examine sources of variation in P. cenocladum amide content and to compare the effects of amides vs. ants on herbivores, we used three linked experiments in the field and in shadehouses. We manipulated light, nutrient availability, and presence of symbionts for experimental plant fragments and shrubs and then quantified leaf amide concentration. We also examined relationships between amide content and damage by the three most common groups of folivores on P. cenocladum: generalist orthopterans, specialist coleopterans, and specialist lepidopterans. For all experiments, enhanced resources and absence of symbionts caused higher levels of amides. These increased chemical defenses had strong effects on generalist herbivores in this system, while the ant predators were more effective at depressing herbivory by specialists. The negative effects of amides on specialist lepidopterans were small, suggesting that these herbivores are adapted to chemical defenses in their host plant. It is possible that our results are part of a more general trend where top-down effects are stronger against specialist herbivores, while chemical defenses are more effective against generalists. We concluded that different models of herbivore regulation were supported by components of the P. cenocladum arthropod community, depending on resource availability and on the portion of the web examined.

Phytochemical diversity drives plant–insect community diversity

Significance Phytochemical diversity is a key component of functional diversity. Challenges in quantifying phytochemical diversity have limited our understanding of the causes and consequences of variation in phytochemical diversity across plant species and families. Here we show that phytochemical diversity across dozens of plant species predicts herbivore diversity, herbivore specialization, phototoxicity, herbivory, and attack by natural enemies. Our approach and findings provide a framework for future investigations focused on uncovering chemical underpinnings of trophic interactions at realistic ecological, geographic, and taxonomic scales, and have implications for the conservation of functional and taxonomic 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.

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.

Isolation, Synthesis, and Evolutionary Ecology of Piper Amides

The phytochemistry of the genus Piper is rich in terms of numbers of compounds discovered, but given the diversity of this genus and the intraspecific diversity of secondary metabolites in those species that have been examined, Piper chemistry has not been adequately investigated. The natural products chemistry that has been elucidated is well documented and has been the subject of extensive review (Sengupta and Ray 1987, Parmar et al. 1997). Since those reviews were published, 28 new species have been investigated (Benevides et al. 1999, Chen et al. 2002, Ciccio 1997, de Abreu et al. 2002, Dodson et al. 2000, dos Santos et al. 2001, Facundo and Morais 2003, Jacobs et al. 1999, Joshi et al. 2001, Martins et al. 1998, Masuoka et al. 2002, McFerren and Rodriquez 1998, Moreira et al. 1998, Mundina et al. 1998, Srivastava et al. 2000a, Stohr et al. 2001, Terreaux et al. 1998, Torquilho et al. 2000, Vila et al. 2001, 2003, Wu et al. 1997), and 69 compounds new to Piper have been discovered (Adesina et al. 2002, Alecio et al. 1998, Baldoqui et al. 1999, Banerji et al. 1993, 2002b, Boll et al. 1994, Chen et al. 2002, Ciccio 1997, Da Cunha and Chaves 2001, Das and Kashinatham 1998, daSilva et al. 2002, de Araujo-Junior et al. 1997, Dodson et al. 2000, Gupta et al. 1999, Jacobs et al. 1999, Joshi et al. 2001, Martins et al. 1998, Masuoka et al. 2002, Menon et al. 2000, 2002, Moreira et al. 1998, 2000, Mundina et al. 1998, Navickiene et al. 2000, Pande et al. 1997, Parmar et al. 1998, Santos and Chaves 1999a,b, Santos et al. 1998, Seeram et al. 1996, Siddiqui et al. 2002, Srivastava et al. 2000a,b, Stohr et al. 1999, Terreaux et al. 1998, Wu et al. 2002a,b, Zeng et al. 1997).

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.

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.

Inter- and intraspecific comparisons of antiherbivore defenses in three species of rainforest understory shrubs.

Plants defend themselves against herbivores and pathogens with a suite of morphological, phenological, biochemical, and biotic defenses, each of which is presumably costly. The best studied are allocation costs that involve trade-offs in investment of resources to defense versus other plant functions. Decreases in growth or reproductive effort are the costs most often associated with antiherbivore defenses, but trade-offs among different defenses may also occur within a single plant species. We examined trade-offs among defenses in closely related tropical rain forest shrubs (Piper cenocladum, P. imperiale, and P. melanocladum) that possess different combinations of three types of defense: ant mutualists, secondary compounds, and leaf toughness. We also examined the effectiveness of different defenses and suites of defenses against the most abundant generalist and specialist Piper herbivores. For all species examined, leaf toughness was the most effective defense, with the toughest species, P. melanocladum, receiving the lowest incidence of total herbivory, and the least tough species, P. imperiale, receiving the highest incidence. Although variation in toughness within each species was substantial, there were no intraspecific relationships between toughness and herbivory. In other Piper studies, chemical and biotic defenses had strong intraspecific negative correlations with herbivory. A wide variety of defensive mechanisms was quantified in the three Piper species studied, ranging from low concentrations of chemical defenses in P. imperiale to a complex suite of defenses in P. cenocladum that includes ant mutualists, secondary metabolites, and moderate toughness. Ecological costs were evident for the array of defensive mechanisms within these Piper species, and the differences in defensive strategies among species may represent evolutionary trade-offs between costly defenses.

Intraspecific phytochemical variation shapes community and population structure for specialist caterpillars

Summary 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.