Predator Performance and Fitness Is Dictated by Herbivore Prey Type Plus Indirect Effects of their Host Plant.

Animals, including herbivores and predators, use diet-mixing to balance their macro- and micronutrient intake. Recent work demonstrated that lady beetles fed only pea aphids from fava beans had reduced fitness caused by a deficiency of dietary sterols. However, beetles redressed this deficit by eating fava bean leaves. In the current study we used Coccinella septempunctata as a model to test the hypotheses that pea aphids are a poor sterol resource independent of their host plant, and that fava beans produce low quality prey regardless of aphid species. Additionally, we tested the reproductive rescue capacity of alfalfa and barley foliage compared to fava, and profiled the sterols of phloem exudates, foliage, and aphids reared on these different hosts. Beetle fecundity and egg viability was significantly better when provided pea aphids reared on alfalfa (compared to fava beans) and green peach aphids reared on fava plants. Alfalfa and barley leaves were not consumed by beetles and did not support beetle reproduction. The sterol profile of aphids largely reflected their host plant phloem. However, green peach aphids from fava acquired 125-times more sterol than pea aphids from fava. Our findings show how the sterol content of different host-plants can affect the third trophic level. Our results suggest that 1) prey quality varies depending on prey species, even when they occur on the same plant, 2) plant species can mediate prey quality, 3) host plant-mediated effects on prey quality partially drive omnivory, and 4) diet-mixing benefits growth and reproduction by redressing micronutrient deficits.

Insect Sterol Nutrition: Physiological Mechanisms, Ecology, and Applications.

Insects, like all eukaryotes, require sterols for structural and metabolic purposes. However, insects, like all arthropods, cannot make sterols. Cholesterol is the dominant tissue sterol for most insects; insect herbivores produce cholesterol by metabolizing phytosterols, but not always with high efficiency. Many insects grow on a mixed-sterol diet, but this ability varies depending on the types and ratio of dietary sterols. Dietary sterol uptake, transport, and metabolism are regulated by several proteins and processes that are relatively conserved across eukaryotes. Sterol requirements also impact insect ecology and behavior. There is potential to exploit insect sterol requirements to (a) control insect pests in agricultural systems and (b) better understand sterol biology, including in humans. We suggest that future studies focus on the genetic mechanism of sterol metabolism and reverse transportation, characterizing sterol distribution and function at the cellular level, the role of bacterial symbionts in sterol metabolism, and interrupting sterol trafficking for pest control.

Herbivory improves the fitness of predatory beetles.

While many predatory arthropods consume non-prey foods from lower trophic levels, little is known about what drives the shift from predator to omnivore. Predatory lady beetles often consume non-prey foods like plant foliage and pollen. One species, Coccinella septempunctata, eats foliage to redress sterol deficits caused by eating sterol-deficient prey. Here we explore how omnivory benefits lady beetle fitness. We reared seven species of lady beetles-from five genera distributed across the tribe Coccinellini-on pea aphids in the presence or absence of fava bean foliage; pea aphids have very low sterol content. Foliage supplements lengthened the development times of four species and decreased survival to adulthood of two species; it had no effect on adult mass. We mated beetles in a 2 × 2 factorial design (males with or without foliage paired with females with or without foliage). For each species, we observed a profound paternal effect of foliage supplements on fitness. Females mated to foliage-supplemented males laid more eggs and more viable eggs compared to females mated to non-supplemented males. Foliage-supplemented males produced 2.9-4.6 times more sperm compared to non-supplemented males for the three species we examined. We analysed the sterol profile of four beetle species reared on pea aphids-with or without foliage-and compared their sterol profile to field-collected adults. For two laboratory-reared species, sterols were not detected in adult male beetles, and overall levels were generally low (total ng of sterol/beetle range: 3-33 ng); the exception being Propylea quatuordecimpunctata females (total ng of sterol/beetle range: 50-157 ng). Laboratory-reared lady beetle sterol content was not significantly affected by the presence of foliage. Field-collected beetles had higher levels of sterols compared to laboratory-reared beetles (2,452-145,348 ng per beetle); cholesterol and sitosterol were the dominant sterols in both field-collected and laboratory-reared beetles. Our findings indicate that herbivory benefits lady beetle fitness across the Coccinellini, and that this was entirely a paternal effect. Our data provide a rare example of a nutritional constraint impacting fitness in a sex-specific manner. It also shows, more broadly, how a nutritional constraint can drive predators towards omnivory.

Prey nutrient content creates omnivores out of predators.

The proximate forces that create omnivores out of herbivores and predators have long fascinated ecologists, but the causal reasons for a shift to omnivory are poorly understood. Determining what factors influence changes in trophic position are essential as omnivory plays a central role in theoretical and applied ecology. We used sevenspotted lady beetles (Coccinella septempunctata) to test how prey nutrient content affects beetles' propensity to engage in herbivory. We show that beetles consuming an all-prey diet demonstrate normal growth and development, but suffer a complete loss of fitness (spermatogenic failure) that is restored via herbivory and supplementation with phytosterols and cholesterol. Furthermore, we show that lady beetles possess a state-dependent sterol-specific appetite and redressed their sterol deficit by feeding on foliage. These results demonstrate that predators balance their nutrient intake via herbivory when prey quality is low, and reveal a selective force (sterol nutrition) that drives predatory taxa to omnivory.

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.

Nutritional physiology of life-history trade-offs: how food protein-carbohydrate content influences life-history traits in the wing-polymorphic cricket Gryllus firmus.

Although life-history trade-offs result from the differential acquisition and allocation of nutritional resources to competing physiological functions, many aspects of this topic remain poorly understood. Wing-polymorphic insects, which possess alternative morphs that trade off allocation to flight capability versus early reproduction, provide a good model system for exploring this topic. In this study, we used the wing-polymorphic cricket Gryllus firmus to test how expression of the flight capability versus reproduction trade-off was modified across a heterogeneous protein-carbohydrate nutritional landscape. Newly molted adult female long- and short-winged crickets were given one of 13 diets with different concentrations and ratios of protein and digestible carbohydrate; for each cricket, we measured consumption patterns, growth and allocation to reproduction (ovary mass) versus flight muscle maintenance (flight muscle mass and somatic lipid stores). Feeding responses in both morphs were influenced more by total macronutrient concentration than by protein-carbohydrate ratio, except at high-macronutrient concentration, where protein-carbohydrate balance was important. Mass gain tended to be greatest on protein-biased diets for both morphs, but was consistently lower across all diets for long-winged females. When long-winged females were fed high-carbohydrate foods, they accumulated greater somatic lipid stores; on high-protein foods, they accumulated greater somatic protein stores. Food protein-carbohydrate content also affected short-winged females (selected for early reproductive onset), which showed dramatic increases in ovary size, including ovarian stores of lipid and protein, on protein-biased foods. This is the first study to show how the concentration and ratio of dietary protein and carbohydrate affects consumption and allocation to key physiological features associated with the reproduction-dispersal life-history trade-off.

Microbial symbionts shape the sterol profile of the xylem-feeding woodwasp, Sirex noctilio.

The symbiotic fungus Amylostereum areolatum is essential for growth and development of larvae of the invasive woodwasp, Sirex noctilio. In the nutrient poor xylem of pine trees, upon which Sirex feeds, it is unknown whether Amylostereum facilitates survival directly through consumption (mycetophagy) and/or indirectly through digestion of recalcitrant plant polymers (external rumen hypothesis). We tested these alternative hypotheses for Amylostereum involvement in Sirex foraging using the innate dependency of all insects on dietary sources of sterol and the unique sterols indicative of fungi and plants. We tested alternative hypotheses by using GC-MS to quantify concentrations of free and bound sterol pools from multiple life-stages of Sirex, food sources, and waste products in red pine (Pinus resinosa). Cholesterol was the primary sterol found in all life-stages of Sirex. However, cholesterol was not found in significant quantities in either plant or fungal resources. Ergosterol was the most prevalent sterol in Amylostereum but was not detectable in either wood or insect tissue (<0.001 µg/g). Phytosterols were ubiquitous in both pine xylem and Sirex. Therefore, dealkylation of phytosterols (sitosterol and campesterol) is the most likely pathway to meet dietary demand for cholesterol in Sirex. Ergosterol concentrations from fungal-infested wood demonstrated low fungal biomass, which suggests mycetophagy is not the primary source of sterol or bulk nutrition for Sirex. Our findings suggest there is a potentially greater importance for fungal enzymes, including the external digestion of recalcitrant plant polymers (e.g., lignin and cellulose), shaping this insect-fungal symbiosis.

Not just the usual suspects: insect herbivore populations and communities are associated with multiple plant nutrients.

The relationship between plant nutrient content and insect herbivore populations and community structure has long interested ecologists. Insect herbivores require multiple nutrients, but ecologists have focused mostly on nitrogen (an estimate of plant protein content), and more recently phosphorus (P); other nutrients have received little attention. Here we document nutrient variation in grass and forb samples from grassland habitats in central Nebraska using an elemental approach; in total we measured foliar concentrations of 12 elements (N and P, plus S, B, Ca, Mg, Na, K, Zn, Fe, Mn, and Cu). We detected significant variability among sites for N, P, Mg, Na, K, and Cu. We next used a model selection approach to explore how this nutritional variation and plant biomass correlate with grasshopper densities (collectively and at the feeding-guild level), and principal component analysis to explore nutrient correlations with grasshopper community species composition. When all grasshoppers were pooled, densities varied among sites, but only P was associated with abundance of the elements shown to vary between sites. Different responses occurred at the feeding-guild level. For grass specialists, densities were associated with N, plus P, Mg, and Na. For forb specialists, N and P were often associated with density, but associations with Na and K were also observed. Finally, mixed-feeder abundance was strongly associated with biomass, and to a lesser extent P, Mg, Na, and Cu. At the community level, B, Ca, Zn, and Cu, plus biomass, explained > 30% of species composition variation. Our results confirm the positive association of N and P with insect herbivore populations, while suggesting a potential role for Mg, Na, and K. They also demonstrate the importance of exploring effects at the feeding-guild level. We hope our data motivate ecologists to think beyond N and P when considering plant nutrient effects on insect herbivores, and make a call for studies to examine functional responses of insect herbivores to dietary manipulation of Mg, Na, and K. Finally, our results demonstrate correlations between variation in nutrients and species assemblages, but factors not linked to plant nutrient quality or biomass likely explain most of the observed variation.

Effect of queen number on colony-level nutrient regulation, food collection and performance in two polygynous ant species.

There is growing appreciation for how social interactions influence animal foraging behavior, especially with respect to key nutrients. Ants, given their eusocial nature and ability to be reared and manipulated in the laboratory, offer unique opportunities to explore how social interactions influence nutrient regulation and related processes. At the colony-level, ants simultaneously regulate their protein and carbohydrate intake; a regulation tied to the presence of larvae. However, even though 45% of the approximately 10,000 ant species are polygynous, we know little about the influence of queen number on colony-level foraging behavior and performance. Here we explored the direct effects of queen number on colony-level protein-carbohydrate regulation, food collection, survival, and brood production in two polygynous ant species (Nylanderia fulva and Solenopsis invicta). For both species we conducted choice and no-choice experiments using small experimental colonoids (20 workers) with 0, 1, or 2 queens. Both species regulated their relative intake of protein and carbohydrate around a P1:C2 mark. However, only N. fulva responded to the addition of queens, increasing overall food collection, biasing intake towards carbohydrates, and over-collecting imbalanced foods. N. fulva also exhibited reduced survival and reproduction on protein-biased foods. In contrast, S. invicta showed no response to queen number and reduced food collection on the protein-biased diet while maintaining high survival and reproduction. Our results demonstrate the potential for queens of some ant species to impact colony-level foraging and performance, with interspecific variation likely being shaped by differences in life history traits.

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.

Assessing pollen nutrient content: a unifying approach for the study of bee nutritional ecology.

Poor nutrition and landscape changes are regularly cited as key factors causing the decline of wild and managed bee populations. However, what constitutes 'poor nutrition' for bees currently is inadequately defined. Bees collect and eat pollen: it is their only solid food source and it provides a broad suite of required macro- and micronutrients. Bees are also generalist foragers and thus the different pollen types they collect and eat can be highly nutritionally variable. Therefore, characterizing the multidimensional nutrient content of different pollen types is needed to fully understand pollen as a nutritional resource. Unfortunately, the use of different analytical approaches to assess pollen nutrient content has complicated between-studies comparisons and blurred our understanding of pollen nutrient content. In the current study, we start by reviewing the common methods used to estimate protein and lipids found in pollen. Next, using monofloral Brassica and Rosa pollen, we experimentally reveal biases in results using these methods. Finally, we use our collective data to propose a unifying approach for analysing pollen nutrient content. This will help researchers better study and understand the nutritional ecology-including foraging behaviour, nutrient regulation and health-of bees and other pollen feeders. This article is part of the theme issue 'Natural processes influencing pollinator health: from chemistry to landscapes'.

Coexisting generalist herbivores occupy unique nutritional feeding niches.

A mainstay of ecological theory and practice is that coexisting species use different resources, leading to the local development of biodiversity. However, a problem arises for understanding coexistence of multiple species if they share critical resources too generally. Here, we employ an experimental framework grounded in nutritional physiology to show that closely related, cooccurring and generalist-feeding herbivores (seven grasshopper species in the genus Melanoplus; Orthoptera: Acrididae) eat protein and carbohydrate in different absolute amounts and ratios even if they eat the same plant taxa. The existence of species-specific nutritional niches provides a cryptic mechanism that helps explain how generalist herbivores with broadly overlapping diets might coexist. We also show that performance by grasshoppers allowed to mix their diets and thus regulate their protein-carbohydrate intake matched optimal performance peaks generated from no-choice treatments. These results indicate the active nature of diet selection to achieve balanced diets and provide buffering capacity in the face of variable food quality. Our empirical findings and experimental approach can be extended to generate and test predictions concerning the intensity of biotic interactions between species, the relative abundance of species, yearly fluctuations in population size, and the nature of interactions with natural enemies in tritrophic niche space.

Protein-carbohydrate regulation and nutritionally mediated responses to Bt are affected by caterpillar population history.

BACKGROUND: The widespread adoption of genetically modified crops, including Bacillius thuringensis (Bt) crops that target chewing insects, has transformed agricultural pest management. This increased use of Bt has raised concerns about the onset of resistance amongst target pests. Recent studies have shown that for some caterpillars, nutritional foraging (e.g. the ratio of proteins and carbohydrates consumed) can affect the insect susceptibility to the Bt toxin Cry1Ac. However, studies on both nutritional foraging and Bt susceptibility tend to rely on laboratory colonies without specifically addressing physiological differences that may occur between populations of the same species. Here, we used choice assays, no choice assays and dose response assays to address two overarching questions: Do populations of Spodoptera frugiperda (J.E. Smith) vary in their protein-carbohydrate foraging behavior? and Does protein-carbohydrate intake impact S. frugiperda's susceptibility to the Bt toxin Cry1F? RESULTS: All three of our S. frugiperda populations actively regulated their protein-carbohydrate intake, but we observed significant differences between populations with respect to their self-selected protein-carbohydrate intake. We also found that feeding at the protein-carbohydrate intake target slightly increased Cry1F susceptibility for one S. frugiperda population, but had no effect on the other two populations. CONCLUSIONS: Our findings indicate that inherent differences exist in the nutritional physiology of three S. frugiperda populations, possibly related to the time spent in culture. This suggests that population-level differences are an important consideration when drawing parallels between field-collected and laboratory-reared insects.

Evaluation of a microbial inhibitor in artificial diets of a generalist caterpillar, Heliothis virescens.

Controlling microbial growth in artificial diets is a key component in the rearing of laboratory insects. In this study an antimicrobial agent, Diet Antimicrobial Agent (DAA), was tested for its ability to suppress microbial growth on a range of different diets, and for its effect on larval and pupal performance of individuals from two different strains of Heliothis virescens Fabricus (Lepidoptera: Noctuidae). In the first experiment, it was found that the presence of DAA in a pinto bean-based diet was highly effective at suppressing microbial growth relative to other methods, and that survival of caterpillars on diets with DAA was superior to other treatments. Caterpillars also performed best on diets with DAA, although this may have been the result of laboratory selection pressure as these caterpillars had been reared on pinto bean-based diets with DAA for several hundred generations. A second experiment was conducted, using different diets and a different strain of H. virescens to more fully evaluate DAA. Here it was found that DAA significantly suppressed microbial growth and development, particularly in synthetic diets. There was no significant effect of DAA on pupal development time or mass gain. There was a statistically significant effect of DAA on eclosion time for two of the diets, although the effect did not seem to be biologically meaningful. The findings suggest that DAA is an effective suppressor of microbial growth on artificial diets, and that its net effect on developing diet-reared insects is neutral.

Aphid growth and reproduction on plants with altered sterol profiles: Novel insights using Arabidopsis mutant and overexpression lines.

Sterols are essential membrane components and are critical for many physiological processes in all eukaryotes. Insects and other arthropods are sterol auxotrophs that typically rely on a dietary source of sterols. Herbivorous insects generally obtain sterols from plants and then metabolize them into cholesterol, the dominant sterol in most insects. However, there is significant variation in phytosterol structure, and not all phytosterols are equally suitable for insects. In the current study, we used seven Arabidopsis thaliana lines that display altered sterol profiles due to mutations in the sterol biosynthetic pathway or to overexpression of key enzymes of the pathway, and investigated how plant sterol profiles affected green peach aphid (Myzus persicae) growth and reproduction. We also characterized the sterol profile of aphids reared on these Arabidopsis genotypes. Aphids on two mutant lines (14R/fk and ste1-1) that accumulated biosynthetic sterol intermediates (Δ8,14-sterols, and Δ7-sterols, respectively) all showed significantly reduced growth and reproduction. Aphids on SMT2COSUP plants (which have decreased ß-sitosterol but increased campesterol) also displayed significantly reduced growth and reproduction. However, aphids on SMT2OE plants (which have increased ß-sitosterol but decreased campesterol) performed similarly to aphids on wild-type plants. Finally, Arabidopsis plants that had an overproduction of sterols (CD-HMGROE) or decreased sterol esters (psat1-2) had no impact on aphid performance. Two noteworthy results come from the aphid sterol profile study. First, ß-sitosterol, cholesterol and stigmasterol were recovered in all aphids. Second, we did not detect Δ8,14-sterols in aphids reared on 14R/fk plants. We discuss the implications of our findings, including how aphid sterol content does not appear to reflect plant leaf sterol profiles. We also discuss the potential of modifying plant sterol profiles to control insect herbivore pests, including aphids.

First draft genome assembly of the desert locust, Schistocerca gregaria.

Background: At the time of publication, the most devastating desert locust crisis in decades is affecting East Africa, the Arabian Peninsula and South-West Asia. The situation is extremely alarming in East Africa, where Kenya, Ethiopia and Somalia face an unprecedented threat to food security and livelihoods. Most of the time, however, locusts do not occur in swarms, but live as relatively harmless solitary insects. The phenotypically distinct solitarious and gregarious locust phases differ markedly in many aspects of behaviour, physiology and morphology, making them an excellent model to study how environmental factors shape behaviour and development. A better understanding of the extreme phenotypic plasticity in desert locusts will offer new, more environmentally sustainable ways of fighting devastating swarms. Methods: High molecular weight DNA derived from two adult males was used for Mate Pair and Paired End Illumina sequencing and PacBio sequencing. A reliable reference genome of Schistocerca gregaria was assembled using the ABySS pipeline, scaffolding was improved using LINKS. Results: In total, 1,316 Gb Illumina reads and 112 Gb PacBio reads were produced and assembled. The resulting draft genome consists of 8,817,834,205 bp organised in 955,015 scaffolds with an N50 of 157,705 bp, making the desert locust genome the largest insect genome sequenced and assembled to date. In total, 18,815 protein-encoding genes are predicted in the desert locust genome, of which 13,646 (72.53%) obtained at least one functional assignment based on similarity to known proteins. Conclusions: The desert locust genome data will contribute greatly to studies of phenotypic plasticity, physiology, neurobiology, molecular ecology, evolutionary genetics and comparative genomics, and will promote the desert locust's use as a model system. The data will also facilitate the development of novel, more sustainable strategies for preventing or combating swarms of these infamous insects.

Same host-plant, different sterols: variation in sterol metabolism in an insect herbivore community.

Insects lack the ability to synthesize sterols de novo, which are required as cell membrane inserts and as precursors for steroid hormones. Herbivorous insects typically utilize cholesterol as their primary sterol. However, plants rarely contain cholesterol, and herbivorous insects must, therefore, produce cholesterol by metabolizing plant sterols. Previous studies have shown that insects generally display diversity in phytosterol metabolism. Despite the biological importance of sterols, there has been no investigation of their metabolism in a naturally occurring herbivorous insect community. Therefore, we determined the neutral sterol profile of Solidago altissima L., six taxonomically and ecologically diverse herbivorous insect associates, and the fungal symbiont of one herbivore. Our results demonstrated that S. altissima contained Delta(7)-sterols (spinasterol, 22-dihydrospinasterol, avenasterol, and 24-epifungisterol), and that 85% of the sterol pool existed in a conjugated form. Despite feeding on a shared host plant, we observed significant variation among herbivores in terms of their qualitative tissue sterol profiles and significant variation in the cholesterol content. Cholesterol was absent in two dipteran gall-formers and present at extremely low levels in a beetle. Cholesterol content was highly variable in three hemipteran phloem feeders; even species of the same genus showed substantial differences in their cholesterol contents. The fungal ectosymbiont of a dipteran gall former contained primarily ergosterol and two ergosterol precursors. The larvae and pupae of the symbiotic gall-former lacked phytosterols, phytosterol metabolites, or cholesterol, instead containing an ergosterol metabolite in addition to unmetabolized ergosterol and erogsterol precursors, thus demonstrating the crucial role that a fungal symbiont plays in their nutritional ecology. These data are discussed in the context of sterol physiology and metabolism in insects, and the potential ecological and evolutionary implications.

First evidence of protein-carbohydrate regulation in a plant bug (Lygus hesperus).

Lygus bugs are highly polyphagous piercing/sucking insects found throughout North America. Collectively, they have been reported to feed on over 330 plant species (one of the broadest host range ever documented for a group of insects); they also feed on many economically important crops. Despite its prevalence across North America and status as a common pest in many agroecosystems, very little is known about how Lygus bugs regulate their intake of nutrients. In reality, little is known about nutrient regulation for most hemipterans, specifically non-phloem feeding species in the suborder Heteroptera. This likely reflects difficulties in developing adequate artificial diets for insects with piercing/sucking mouthparts. There is, however, an artificial diet for L. hersperus, and in this study we modified it and performed choice and no-choice experiments to determine how L. hesperus regulates its intake of two macronutrients - protein (p) and carbohydrates (c) - that are tightly linked to survival and performance in other insect herbivores. In choice experiments L. hesperus was allowed to select between two foods with different protein:carbohydrate ratios. We documented strong regulation for protein and carbohydrates, with late instar nymphs selecting a slightly protein-biased intake target (protein-carbohydrate ratio = 1.5:1). We also performed no-choice experiments, where nymphs were restricted to a single food. Here, the protein-carbohydrate ratio of their food had a strong impact on survival, which was highest for nymphs reared on the treatment with a protein-carbohydrate ratio closest to the self-selected intake target (determined by the choice experiments), but no significant impact on developmental time or mass gain. Our data are the first of their kind for a non-phloem feeding hemipteran and provide a starting point for more broadly understanding and further investigating the nutritional ecology/physiology of Lygus bugs. Our study also provides a framework for exploring nutrient regulation in other hemipterans and for optimizing artificial diets for piercing/sucking insects, especially heteropterans.

Protein-carbohydrate regulation in Helicoverpa amigera and H. punctigera and how diet protein-carbohydrate content affects insect susceptibility to Bt toxins.

Many animals, including insects, demonstrate a remarkable ability to regulate their intake of key macronutrients (e.g., soluble protein and digestible carbohydrates), which allows them to optimize fitness and performance. Additionally, regulating the intake of these two macronutrients enhances an animal's ability to defend itself against pathogens, mitigate the effects of secondary plant metabolites, and decrease susceptibility to toxins. In this study, we first compared how Bt-resistant and -susceptible lines of Helicoverpa armigera and Helicoverpa punctigera regulate their intake of protein (p) and digestible carbohydrates (c). We found that there was no difference in the self-selected protein-carbohydrate intake target between resistant and susceptible genotypes of either species. We then explored the extent to which food protein-carbohydrate content altered the susceptibility of these species to three Bt toxins: Cry1Ac, Cry2Ab, and Vip3Aa. We found that H. armigera on diets that had protein-carbohydrate profiles that matched their self-selected protein-carbohydrate intake target were significantly less susceptible to Cry1Ac. In contrast, diet protein-carbohydrate content did not affect H. punctigera susceptibility to Cry1Ac. For both H. armigera and H. punctigera, susceptibility to Cry2Ab and Vip3Aa toxins did not change as a function of diet protein-carbohydrate profile. These results, when combined with earlier work on H. zea, suggest food protein-carbohydrate content can modify susceptibility to some Bt toxins, but not others. An increased understanding of how the nutritional environment can modify susceptibility to different Bt toxins could help improve pest management and resistance management practices.

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.