Quantity versus quality: Effects of diet protein-carbohydrate ratios and amounts on insect herbivore gene expression.

Dietary protein and digestible carbohydrates are two key macronutrients for insect herbivores, but the amounts and ratios of these two macronutrients in plant vegetative tissues can be highly variable. Typically, insect herbivores regulate their protein-carbohydrate intake by feeding selectively on nutritionally complementary plant tissues, but this may not always be possible. Interestingly, lab experiments consistently demonstrate that performance - especially growth and survival - does not vary greatly when caterpillars and nymphal grasshoppers are reared on diets that differ in their protein-carbohydrate content. This suggests insect herbivores employ post-ingestive physiological mechanisms to compensate for variation in diet protein-carbohydrate profile. However, the molecular mechanisms that underlie this compensation are not well understood. Here we explore, for the first time in an insect herbivore, the transcriptional effects of two dietary factors: protein-to-carbohydrate ratio (p:c) and total macronutrient (p + c) content. Specifically, we reared Helicoverpa zea caterpillars on three diets that varied in diet p:c ratio and one diet that varied in total p + c concentration, all within an ecologically-relevant range. We observed two key findings. Caterpillars reared on diets with elevated total p + c content showed large differences in gene expression. In contrast, only small differences in gene expression were observed when caterpillars were reared on diets with different p:c ratios (spanning from protein-biased to carbohydrate-biased). The invariable expression of many metabolic genes across these variable diets suggests that H. zea caterpillars employ a strategy of constitutive expression to deal with protein-carbohydrate imbalances rather than diet-specific changes. This is further supported by two findings. First, few genes were uniquely associated with feeding on a protein- and carbohydrate-biased diet. Second, many differentially-expressed genes were shared across protein-biased, carbohydrate-biased, and concentrated diet treatments. Our study provides insights into the post-ingestive physiological mechanisms insect herbivores employ to regulate protein-carbohydrate intake. Most notably, it suggests that H. zea, and perhaps other generalist species, use similar post-ingestive mechanisms to deal with protein-carbohydrate imbalances - regardless of the direction of the imbalance.

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar.

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of nitrogen-containing plant defensive compounds, and variation in species-specific correlations between nitrogen and plant protein content all limit the accuracy of these inferences. Additionally, research focused on plant and/or herbivore physiology require a level of accuracy that is not achieved using generalized correlations. The methods presented here offer researchers a clear and rapid protocol for directly measuring plant soluble proteins and digestible carbohydrates, the two plant macronutrients most closely tied to animal physiological performance. The protocols combine well characterized colorimetric assays with optimized plant-specific digestion steps to provide precise and reproducible results. Our analyses of different sweet corn tissues show that these assays have the sensitivity to detect variation in plant soluble protein and digestible carbohydrate content across multiple spatial scales. These include between-plant differences across growing regions and plant species or varieties, as well as within-plant differences in tissue type and even positional differences within the same tissue. Combining soluble protein and digestible carbohydrate content with elemental data also has the potential to provide new opportunities in plant biology to connect plant mineral nutrition with plant physiological processes. These analyses also help generate the soluble protein and digestible carbohydrate data needed to study nutritional ecology, plant-herbivore interactions and food-web dynamics, which will in turn enhance physiology and ecological research.

Nutrition affects insect susceptibility to Bt toxins.

Pesticide resistance represents a major challenge to global food production. The spread of resistance alleles is the primary explanation for observations of reduced pesticide efficacy over time, but the potential for gene-by-environment interactions (plasticity) to mediate susceptibility has largely been overlooked. Here we show that nutrition is an environmental factor that affects susceptibility to Bt toxins. Protein and carbohydrates are two key macronutrients for insect herbivores, and the polyphagous pest Helicoverpa zea self-selects and performs best on diets that are protein-biased relative to carbohydrates. Despite this, most Bt bioassays employ carbohydrate-biased rearing diets. This study explored the effect of diet protein-carbohydrate content on H. zea susceptibility to Cry1Ac, a common Bt endotoxin. We detected a 100-fold increase in LC50 for larvae on optimal versus carbohydrate-biased diets, and significant diet-mediated variation in survival and performance when challenged with Cry1Ac. Our results suggest that Bt resistance bioassays that use ecologically- and physiologically-mismatched diets over-estimate susceptibility and under-estimate resistance.

Spatio-Temporal, Genotypic, and Environmental Effects on Plant Soluble Protein and Digestible Carbohydrate Content: Implications for Insect Herbivores with Cotton as an Exemplar.

Plant soluble protein and digestible carbohydrate content significantly affect insect herbivore fitness, but studies reporting plant protein and carbohydrate content are rare. Instead, the elements nitrogen and carbon often are used as surrogates for plant protein and digestible carbohydrate content, respectively. However, this is problematic for two reasons. First, carbon is found in all organic molecules, which precludes strong correlations with ecologically important dietary macronutrients (e.g., digestible carbohydrates, the primary energy source for most insect herbivores). Second, some elements (e.g., nitrogen) are present in both macronutrients (e.g., protein) and non-nutritive secondary compounds (e.g., alkaloids, protease inhibitors); in these cases N values would greatly overestimate protein available for an insect herbivore. Thus, the objective of this study was to explicitly document plant protein-carbohydrate content and assess its variation in cotton (Gossypium hirsutum and G. barbadense), which is a nutritional resource for a number of insect herbivores. We did this by measuring plant soluble protein (P) and digestible carbohydrate (C) content across seven plant tissues, five varieties, and two growing environments. Significant differences in P and C concentration, total macronutrient content (P + C), and P:C ratio were observed across plant tissues, plant age and environment; smaller differences were seen across plant genotype. Foliar tissues had higher total P + C content compared to reproductive tissues, except for developing seeds and developing flowers, which contained twice the total P + C content; these two tissues also had the highest P content. Our data show that even agricultural monocultures offer a highly heterogeneous protein-carbohydrate landscape for insect herbivores. Characterizing plant resources using nutritional currencies (e.g., protein and carbohydrates) that are ecologically and physiologically-relevant to insect herbivores can be used to enhance our understanding of plant-insect interactions.

Revisiting macronutrient regulation in the polyphagous herbivore Helicoverpa zea (Lepidoptera: Noctuidae): New insights via nutritional geometry.

Insect herbivores that ingest protein and carbohydrates in physiologically-optimal proportions and concentrations show superior performance and fitness. The first-ever study of protein-carbohydrate regulation in an insect herbivore was performed using the polyphagous agricultural pest Helicoverpa zea. In that study, experimental final instar caterpillars were presented two diets - one containing protein but no carbohydrates, the other containing carbohydrates but no protein - and allowed to self-select their protein-carbohydrate intake. The results showed that H. zea selected a diet with a protein-to-carbohydrate (p:c) ratio of 4:1. At about this same time, the geometric framework (GF) for the study of nutrition was introduced. The GF is now established as the most rigorous means to study nutrient regulation (in any animal). It has been used to study protein-carbohydrate regulation in several lepidopteran species, which exhibit a range of self-selected p:c ratios between 0.8 and 1.5. Given the economic importance of H. zea, and it is extremely protein-biased p:c ratio of 4:1 relative to those reported for other lepidopterans, we decided to revisit its protein-carbohydrate regulation. Our results, using the experimental approach of the GF, show that H. zea larvae self-select a p:c ratio of 1.6:1. This p:c ratio strongly matches that of its close relative, Heliothis virescens, and is more consistent with self-selected p:c ratios reported for other lepidopterans. Having accurate protein and carbohydrate regulation information for an insect herbivore pest such as H. zea is valuable for two reasons. First, it can be used to better understand feeding patterns in the field, which might lead to enhanced management. Second, it will allow researchers to develop rearing diets that more accurately reflect larval nutritional needs, which has important implications for resistance bioassays and other measures of physiological stress.