Phytosterols occur in the phloem of vascular plants as free, acylated, and glycosylated derivatives. The phloem is increasingly recognized as an active site of sterol synthesis and transport used to support plant metabolism, while also providing a potential nutritional source for phloem-feeding insects. This chapter describes a method for the collection and quantitative analysis of phloem sterols using an EDTA-facilitated exudation procedure followed by gas chromatography-mass spectrometry (GC-MS). Excised leaves are incubated in an EDTA solution to induce phloem exudation, and sterols from collected exudate are extracted, hydrolyzed to their free forms, derivatized as trimethylsilyl ethers, and analyzed by GC-MS using internal standards for quantification. This method has been successfully applied to several plant species and can be adapted to others with suitable modifications. It provides a reproducible approach for characterizing sterol composition of phloem sap and assessing its biological relevance in plant-insect interactions.

Sterols are essential for eukaryotic cell structure and metabolism, yet insects cannot synthesize them de novo and must acquire them through their diet. For insect herbivores, plant-derived sterols are typically converted into cholesterol to support development and reproduction. We previously engineered Arabidopsis thaliana lines with silenced HYD1, resulting in altered sterol composition. In this study, we evaluated the performance of the phloem-feeding aphid Myzus persicae on these sterol-modified plants. Aphids reared on the modified lines (HYD1RNAi10, 12, 25) exhibited significantly reduced growth, reproduction, and survival compared to those on wild-type Col-0. However, choice assays and electropenetrography (EPG) revealed no differences in host preference or probing and feeding behaviors. Unlike the chewing insect Plutella xylostella, M. persicae did not accumulate atypical sterols but instead showed a significant reduction of total sterol content. Phloem-sap analysis mirrored aphid sterol profiles, lacking the atypical sterols found in leaf tissue of the modified lines. RNA-seq of HYD1RNAi lines revealed no induction of known plant defense pathways; instead, genes involved in translation and nitrate metabolism were upregulated. These findings show that HYD1 silencing reduces host suitability for aphids by limiting sterol availability for insect development and reproduction. Our results highlight the potential of sterol-modified plants as a promising strategy for managing phloem sap-feeding insect pests.

Abstract Herbivorous insects and their host plants comprise most known species on Earth. Illuminating how herbivory repeatedly evolved in insects from nonherbivorous lineages is critical to understanding how this biodiversity is created and maintained. We characterized the trophic niche of Scaptomyza flava, a representative of a lineage nested within the Drosophila that transitioned to herbivory ~10–15 million years ago. We used natural history studies to determine whether S. flava is a true herbivore or a cryptic microbe‐feeder, given that the ancestral character state for the family Drosophilidae is likely microbe‐feeding. Specifically, we quantified oviposition substrate choice and larval viability across food types, trophic‐related morphological traits, and nitrogen isotope and sterol profiles across putatively herbivorous and nonherbivorous drosophilids. The results of these studies show that S. flava is an obligate herbivore of living plants. Paired with its genetic model host, Arabidopsis thaliana, S. flava is a novel and powerful system for exploring mechanisms underlying the evolution of herbivory, a complex trait that enabled the exceptional diversification of insects.

All eukaryotes use sterols as structural components in cell membranes and as precursors for key hormones. However, arthropods are unique among eukaryotes because they cannot synthesize sterols de novo and must acquire sterols through diet. Cholesterol is the dominant sterol in most insects; however, because plants contain little cholesterol, most insect herbivores convert phytosterols to cholesterol. An additional challenge for plant-feeding insects is that only limited types of phytosterols can be converted to cholesterol. In this study, we used RNA interference to modify the plant sterol profile to generate phytosterols that would negatively impact insect herbivores. Specifically, we knocked down the expression of Arabidopsis thaliana Δ8–Δ7-sterol isomerase gene (HYD1), responsible for the conversion of Δ8-sterols into Δ7-sterols. Silenced lines with > 95

Abstract All eukaryotes use sterols as structural components in cell membranes and as precursors for key hormones. However, arthropods are unique among eukaryotes because they are unable to synthesize sterols de novo and must acquire sterols through their diet. Cholesterol is the dominant sterol in most insects, but because plants contain little cholesterol, most insect herbivores convert phytosterols to cholesterol. However, metabolic constraints limit which phytosterols insect herbivores can convert to cholesterol. In this study we used RNA interference to modify the sterol profile of Arabidopsis thaliana to generate phytosterols that would be problematic for insect herbivores. We did this by silencing the Δ8-Δ7-sterol isomerase gene (HYD1) that is responsible for the conversion of Δ8-sterols into Δ7-sterols. Sterol production decreased in Arabidopsis lines with > 95% transcript reduction and half of the total sterol profile in these plants showed a Δ8 configuration. These sterol-modified lines exhibited normal growth, but caterpillars (Plutella xylostella) reared on these plants showed reduced growth and survival, while adults showed decreased reproductive output. Sterol analysis of the pupae reared on sterol-modified plants suggests these negative effects are a function of reduced cholesterol levels and the accumulation of Δ8-sterols. Finally, we used our collective performance and reproduction data to model the effects of sterol-modified plants at the population level. We show that population numbers are significantly reduced, and generation time extended, after only two generations. Our results suggest that modifying plant sterols has real potential for management of pest caterpillars, especially multivoltine species.

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'.

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.

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 β-sitosterol but increased campesterol) also displayed significantly reduced growth and reproduction. However, aphids on SMT2OE plants (which have increased β-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, β-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.

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.

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.

The aphid Myzus persicae displays high mortality on tobacco plants bearing a transgene which results in the accumulation of the ketosteroids cholestan-3-one and cholest-4-en-3-one in the phloem sap. To test whether the ketosteroids are the basis of the plant resistance to the aphids, M. persicae were reared on chemically-defined diets with different steroid contents at 0.1–10 µg ml−1. Relative to sterol-free diet and dietary supplements of the two ketosteroids and two phytosterols, dietary cholesterol significantly extended aphid lifespan and increased fecundity at one or more dietary concentrations tested. Median lifespan was 50% lower on the diet supplemented with cholest-4-en-3-one than on the cholesterol-supplemented diet. Aphid feeding rate did not vary significantly across the treatments, indicative of no anti-feedant effect of any sterol/steroid. Aphids reared on diets containing equal amounts of cholesterol and cholest-4-en-3-one showed fecundity equivalent to aphids on diets containing only cholesterol. Aphids were reared on diets that reproduced the relative steroid abundance in the phloem sap of the control and modified tobacco plants, and their performance on the two diet formulations was broadly equivalent. We conclude that, at the concentrations tested, plant ketosteroids support weaker aphid performance than cholesterol, but do not cause acute toxicity to the aphids. In plants, the ketosteroids may act synergistically with plant factors absent from artificial diets but are unlikely to be solely responsible for resistance of modified tobacco plants.

Insects lack the ability to synthesize sterols de novo so they acquire this essential nutrient from their food. Cholesterol is the dominant sterol found in most insects, but in plant vegetative tissue it makes up only a small fraction of the total sterol profile. Instead, plants mostly contain phytosterols; plant-feeding insects generate the majority of their cholesterol by metabolizing phytosterols. However, not all phytosterols are readily converted to cholesterol, and some are even deleterious when ingested above a threshold level. In a recent study we showed that caterpillars reared on tobacco accumulating novel sterols/steroids exhibited reduced performance, even when suitable sterols were present. In the current study we examined how the dominant sterols (cholesterol and stigmasterol) and steroids (cholestanol and cholestanone) typical of the modified tobacco plants affected two insect herbivores (Heliothis virescens and Helicoverpa zea). The sterols/steroids were incorporated into synthetic diets singly, as well as in various combinations, ratios and amounts. For each insect species, a range of performance values was recorded for two generations, with the eggs from the 1st-generation adults as the source of neonates for the 2nd-generation. Performance on the novel steroids (cholestanol and cholestanone) was extremely poor compared to suitable sterols (cholesterol and stigmasterol). Additionally, performance tended to decrease as the ratio of the novel dietary steroids increased. We discuss how the balance of different dietary sterols/steroids affected our two caterpillar species, relate this back to recent studies on sterol/steroid metabolism in these two species, and consider the potential application of sterol/steroid modification in crops.

All eukaryotes contain sterols, which serve as structural components in cell membranes, and as precursors for important hormones. Plant vegetative tissues are known to contain mixtures of sterols, but very little is known about the sterol composition of phloem. Plants are food for many animals, but plant-feeding arthropods (including phloem-feeding insets) are unique among animals in that they have lost the ability to synthesize sterols, and must therefore acquire these essential nutrients from their food, or via endosymbionts. Our paper starts by providing a very brief overview of variation in plant sterol content, and how different sterols can affect insect herbivores, including those specializing on phloem. We then describe an experiment, where we bulk collected phloem sap exudate from bean and tobacco, and analyzed its sterol content. This approach revealed two significant observations concerning phloem sterols. First, the phloem exudate from each plant was found to contain sterols in three different fractions - free sterols, sterols conjugated to lipids (acylated), and sterols conjugated to carbohydrates (glycosylated). Second, for both plants, cholesterol was identified as the dominant sterol in each phloem exudate fraction; the remaining sterols in each fraction were a mixture of common phytosterols. We discuss our phloem exudate sterol profiles in a plant physiology/biochemistry context, and how it relates to the nutritional physiology/ecology of phloem-feeding insects. We close by proposing important next steps that will advance our knowledge concerning plant phloem sterol biology, and how phloem-sterol content might affect phloem-feeding insects.

Insects cannot synthesize sterols de novo, so they typically require a dietary source. Cholesterol is the dominant sterol in most insects, but because plants contain only small amounts of cholesterol, plant-feeding insects generate most of their cholesterol by metabolizing plant sterols. Plants almost always contain mixtures of different sterols, but some are not readily metabolized to cholesterol. Here we explore, in two separate experiments, how dietary phytosterols and phytosteroids, in different mixtures, ratios, and amounts, affect insect herbivore sterol/steroid metabolism and absorption; we use two caterpillars species - one a generalist (Heliothis virescens), the other a specialist (Manduca sexta). In our first experiment caterpillars were reared on two tobacco lines one expressing a typical phystosterol profile, the other expressing high amounts/ratios of stanols and 3-ketosteroids. Caterpillars reared on the control tobacco contained mostly cholesterol, but those reared on the modified tobacco had reduced amounts of cholesterol, and lower total sterol/steroid body profiles. In our second experiment, caterpillars were reared on artificial diets containing known amounts of cholesterol, stigmasterol, cholestanol and/or cholestanone, either singly or in various combinations and ratios. Cholesterol and stigmasterol-reared moths were mostly cholesterol, while cholestanol-reared moths were mostly cholestanol. Moth tissue cholesterol concentration tended to decrease as the ratio of dietary cholestanol and/or cholestanone increased. In both moths cholestanone was metabolized into cholestanol and epichblestanol. Interestingly, M. sexta generated much more cholestanol than epicholestanol, while H. virescens did the opposite. Finally, total tissue steroid levels were significantly reduced in moths reared on diets containing very high levels of cholestanol. We discuss how dietary sterol/steroid structural differences are important with respect to sterol/steroid metabolism and uptake, including species-specific differences. (C) 2013 Elsevier Ltd. All rights reserved.

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.

The phloem sap of fava bean (Vicia faba) plants utilized by the pea aphid Acyrthosiphon pisum contains three sterols, cholesterol, stigmasterol and sitosterol, in a 2:2:1 ratio. To investigate the nutritional value of these sterols, pea aphids were reared on chemically-defined diets containing each sterol at 0.1, 1 and 10μgml(-1) with a sterol-free diet as control. Larval growth rate and aphid lifespan did not vary significantly across the diets, indicating that sterol reserves can buffer some performance indices against a shortfall in dietary sterol over at least one generation. However, lifetime reproductive output was depressed in aphids on diets containing stigmasterol or no sterol, relative to diets supplemented with cholesterol or sitosterol. The cholesterol density of embryos in teneral adults was significantly higher than in the total body; and the number and biomass of embryos in aphids on diets with stigmasterol and no sterols were reduced relative to diets with cholesterol or sitosterol, indicating that the reproductive output of the pea aphid can be limited by the amount and composition of dietary sterol. In a complementary RNA-seq analysis of pea aphids reared on plants and diets with different sterol contents, 7.6% of the 17,417 detected gene transcripts were differentially expressed. Transcript abundance of genes with annotated function in sterol utilization did not vary significantly among treatments, suggesting that the metabolic response to dietary sterol may be mediated primarily at the level of enzyme function or metabolite concentration.

Sterols are essential nutrients for insects because, in contrast to mammals, no insect (or arthropod for that matter) can synthesize sterols de novo. Plant-feeding insects typically generate their sterols, commonly cholesterol, by metabolizing phytosterols. However, not all phytosterols are readily converted to cholesterol. In this study we examined, using artificial diets containing single sterols/steroids, how typical (cholesterol and stigmasterol) and atypical (cholestanol and cholestanone) sterols/steroids affect the performance of a generalist caterpillar (Helicoverpa zea). We also performed sterols/steroids analyses, using GC/MS techniques, to explore the metabolic fate of these different dietary sterols/steroids. Finally, we used a microarray approach to measure, and compare, midgut gene expression patterns that arise as a function of dietary sterols/steroids. In general, H. zea performed best on the cholesterol and stigmasterol diets, with cholesterol as the dominant tissue sterol on these two treatments. Compared to the cholesterol and stigmasterol diets, performance was reduced on the cholestanol and cholestanone diets; on these latter treatments stanols were the dominant tissue sterol. Finally, midgut gene expression patterns differed as a function of dietary sterol/steroid; using the cholesterol treatment as a reference, gene expression differences were smallest on stigmasterol, intermediate on cholestanol, and greatest on cholestanone. Inspection of our data revealed two broad insights. First, they identify a number of genes potentially involved in sterol/steroid metabolism and absorption. Second, they provide unique mechanistic insights into how variation in dietary sterol/steroid structure can affect insect herbivores.

Insects, unlike plants and vertebrates, lack the ability to biosynthesize sterols. Cholesterol is typically the most common sterol found in plant-feeding insects, but it is rarely found in plants above trace levels, so plant-feeding insects must produce the cholesterol they need by metabolizing the sterols found in the plants they eat. Plant-feeding insects are, however, often limited in terms of which sterols can be converted to cholesterol. In the current study we used a transgenic tobacco plant line that displays high levels of atypical plant steroids, specifically stanols and ketone-steroids, to explore how novel steroid structural features affect performance in three economically important caterpillars (Heliothis virescens, Spodoptera exigua, and Manduca sexta). For each species we measured pupation success, larval development, pupal mass, pupal development, and eclosion success. For the two generalists species (H. virescens and S. exigua) we also measured egg production and egg viability. We then used these eggs to replicate the experiment, so that we could examine the effect of parental steroid dietary history on survival, growth and reproduction of 2nd-generation individuals. Significant negative effects of novel steroids on larval and pupal performance were observed for each caterpillar in the first generation, although these were often subtle, and were not consistent between the three species. In the second generation, larval survival estimated by ‘pupation number/plant’ on the tobacco plants with novel steroids was significantly reduced, while eclosion success was significantly lower for H. virescens. With respect to adult reproduction (i.e. egg production and egg viability) there were no observed differences in the first generation, but novel steroids significantly negatively impacted reproduction in the second generation. The findings from this study, when integrated into a simple population growth model, demonstrate the potential in using plants with modified steroids as a novel approach to manage populations of economically important caterpillar species.

1. Arthropods, including insects, are unique among animals in that they cannot synthesize sterols, including cholesterol, de novo. Some phytophagous insects (e.g. caterpillars, grasshoppers) generate tissue cholesterol by metabolizing plant phytosterols. Currently, little is known about sterols in plant phloem sap, and their significance to the nutritional physiology and ecology of phloem-feeding insects. 2. The sterol profiles of leaves from two plant species, Chinese cabbage and tobacco, were dominated by the phytosterols sitosterol and stigmasterol, respectively. In contrast, the principle sterol in the phloem sap of both plants was cholesterol, which is traditionally considered an animal sterol. Cholesterol was also the most abundant sterol in the carcass and honeydew of Myzus persicae aphids feeding on these plants. 3. The effect of sterol structure on M. persicae was investigated using modified tobacco plants that contained high levels of atypical steroids, specifically ketone-steroids. Aphids reared on the modified tobacco plants had a high atypical steroid content, severely reduced reproduction and high mortality. 4. Our data indicate that the total sterol composition of plants is not necessarily representative of the sterol profile available to phloem-feeding insects, and that the sterol utilization patterns of phloem-feeding insects may differ from chewing insect herbivores utilizing the same plant. Atypical steroids are naturally at insufficient concentrations for significant deleterious effects on insect herbivores, and possible reasons why plants apparently do not use them as defensive compounds are considered.