Sterols regulate cell membrane fluidity and are precursors for hormone and secondary metabolite production in plants, but plant sterols also have a critical role as nutrients in herbivores. Here we describe the distribution of 78 different sterols from pollen of 295 UK wildflower taxa and use this data to develop an evolutionary rationale for the diversity of sterols in pollen compared to vegetative tissues. The sterolome was a function of plant lineage and conserved in groups as high as subfamily. Insect herbivores are auxotrophic for sterols, and notably bees can not modify them therefore rely on dietary sources, primarily pollen, to meet their metabolic needs. Most pollen in the present study contained high proportions of Δ5 sterols including β-sitosterol, 24-methylenecholesterol and isofucosterol, which are important sterols for bees. The sterols recorded in honey bees occurred in the pollen of only 68% of plant taxa, however, none matched these proportions exactly suggesting they must forage pollen from multiple plant taxa to satisfy their sterol requirements. We conclude that there is evidence for pollen sterol composition being the result of diverse driving forces including plant lineage and pollinator nutritional requirements. ### Competing Interest Statement The authors have declared no competing interest.

Pollen sterols are essential micronutrients for bees as membrane components, hormone precursors and gene regulation. Sterols vary among plant species; therefore, bee development may be influenced by or adapted to specific pollen diets. To better understand the roles of pollen diet and sterol provisioning, we investigated the effect of different pollen types with sterol supplementation (i.e., pollen provided by the bee mother, Castanea sativa pollen, C. sativa pollen supplemented with sterols, polyfloral pollen, and a combination of polyfloral and C. sativa pollen) on the development, and sterolome of the generalist, solitary bee Osmia bicornis . Pollen diet significantly affected bee development (i.e., final weight, feeding period, growth rate) and larvae fed on pollen supplemented with sterols had an increased growth rate compared to those from the other treatments. Despite sterolomes being distinctive among pollen diets, sterolomes were more similar within some bees than between bees and their pollen diets. Moreover, larvae fed on polyfloral pollen had much higher relative concentrations of campesterol (∼10%) than the pollen itself (∼0.2%), indicating Osmia bees are highly efficient in nutrient accumulation or can metabolically produce campesterol. Our results contrast with previous work which suggests bees cannot modify sterols and highlight the complexity of bee nutrition. ### Competing Interest Statement The authors have declared no competing interest.

Plants produce floral nectar as a reward for pollinators, which contains carbohydrates and amino acids (AAs). We designed experiments to test whether pollinators could exert selection pressure on the profiles of AAs in nectar. We used HPLC to measure the free AAs and sugars in the nectar of 102 UK plant species. Six distinct profiles of essential amino acids (EAAs) were defined using the relative proportions of AAs with a clustering algorithm; we then tested bumblebee (Bombus terrestris) preferences for the EAA profiles and proline using a two-choice assay. We found a phylogenetic signal for the proportions of phenylalanine, methionine and proline as well as the total concentrations of essential and nonessential amino acids. However, there was no phylogenetic signal for EAA profile. Bumblebees did not exhibit a preference for any of the six EAA nectar profiles; however, four of the EAA profiles stimulated feeding. By contrast, bumblebees avoided proline in an inverse concentration-dependent manner. Our data indicate that bees are likely to have mechanisms for the postingestive evaluation of free AAs in solution but are unlikely to taste EAAs at nectar-relevant quantities. We predict that EAAs increase nectar value to bumblebees postingestively.

Phytosterols regulate cell membrane fluidity and are precursors for plant hormones and secondary metabolites in plants. Insects are auxotrophic for sterols; therefore, they have to consume phytosterols and dealkylate them to cholesterol. Some insects, including bees, which rely on dietary sources, primarily pollen, for sterols, cannot modify them; therefore, they have adapted to use them unmodified as they occur in pollen. Here, using high resolution chemical analysis, we describe the distribution of 78 different pollen sterols from 295 UK wildflower taxa and use this data to develop an evolutionary rationale for the diversity of sterols in pollen compared to vegetative tissues. The sterol occurrence in pollen was a function of plant lineage and conserved in groups as high as subfamily. Most pollen in the present study contained high proportions of Δ5 sterols including β-sitosterol, 24-methylenecholesterol and isofucosterol, which are important sterols for bees. The sterols recorded in honeybees occurred in the pollen of only 68% of plant taxa; however, none matched these proportions exactly, suggesting they must forage pollen from multiple plant taxa to satisfy their sterol requirements. We conclude that there is evidence for pollen sterol composition being the result of diverse driving forces including plant lineage and potentially pollinator nutritional requirements.

Learning cues such as tastes associated with palatable food is an important mechanism animals have for foraging optimally. Insects can use gustatory receptor neurons (GRNs) in their mouthparts to detect nutrients and toxins, but they also taste compounds using sensilla on peripheral organs such as their antennae. Bees are adept at learning to associate floral traits with the presence of nectar rewards, but few studies have examined how they incorporate gustatory information from their antennae with rewards. Here, we characterize the ability of adult worker bumblebees (Bombus terrestris) to taste sugar, salt, and bitter compounds using their antennae and then tested whether they could use this sensory information to associate it with food. We show that bumblebees have antennal GRNs sensitive to sugars, salts, and bitter compounds and that they can use surface chemistry differences detected by their periphery to learn about the presence or absence of flower rewards in a free-flight assay. Naïve bumblebees showed no instinctual preferences toward or against any surface chemistry tested. Bumblebees performed best when sucrose surface cues were associated with sucrose reward, but they could learn to associate any cue with the presence or absence of sucrose solution. Interestingly, the bees found it more difficult to associate quinine surface chemistry with the presence of reward than its absence. These results indicate that bees have the potential to learn to associate another floral trait - chemicals on the surfaces of petals - with the quality of floral rewards. ### Competing Interest Statement The authors have declared no competing interest.

Learning cues such as tastes associated with palatable food is an important mechanism animals have for foraging optimally. Insects can use gustatory receptor neurons (GRNs) in their mouthparts to detect nutrients and toxins, but they also taste compounds using sensilla on other organs such as their antennae and tarsi. Bees are adept at learning to associate floral traits with the presence of nectar rewards, but few studies have examined how they incorporate gustatory information from their antennae or tarsi detected on flower surfaces. Here, we characterize the ability of bumblebees (Bombus terrestris) to taste sugar, salt and bitter compounds using their antennae and then tested whether they could use this sensory information to associate it with rewarding artificial floral displays. We show that bumblebees have antennal GRNs sensitive to sugars, salts and bitter compounds and can use surface chemistry differences detected by their antennae and/or tarsi to learn about the presence or absence of flower rewards in a free-flight assay. Naïve bumblebees showed no detected spontaneous preferences toward or against any surface chemistry tested. Bumblebees performed best when sucrose surface cues were associated with rewards, but they could learn to associate any cue with the presence or absence of sucrose solution. Interestingly, the bees found it more difficult to associate quinine surface chemistry with the presence of reward than its absence. These results indicate that bees have the potential to learn to associate another floral trait - chemicals on the surfaces of petals - with the quality of floral rewards.

The sugar concentration of floral nectar is a key metric for describing nectar composition and a major factor influencing pollinator visitation to flowers. Across pollination biology research there are multiple approaches in use for describing nectar sugar concentration. With these different approaches there are several potential sources of confusion which, if not accounted for, can lead to errors. Further potential for error arises if researchers wish to make comparisons between the energetic content of nectars containing different ratios of sucrose, fructose and glucose. Regardless of whether concentration is measured per mole or per unit mass, the energetic content differs between the hexose sugars (glucose and fructose) and sucrose. Appropriate conversion is needed for direct comparison. Here we address these two issues with the following aims. We consolidate the literature on this topic with examples of the different methods for reporting nectar sugar concentrations, provide insight into potential sources of error, and derive equations for converting between the different ways of expressing sugar concentration for the three primary nectar sugars: sucrose, glucose and fructose. Second, we discuss the relative energetic content of sucrose, glucose, and fructose, and rationalise adjustment of "energetic value" rather than reporting concentration directly. In this way, we hope to harmonise ongoing work in pollination ecology.

Honeybees are important crop pollinators, but they increasingly face pollen starvation as a result of agricultural intensification and climate change1. Frequent flowering dearth periods and high-density rearing conditions weaken colonies, which often leads to their demise2. Beekeepers provide colonies with pollen substitutes, but these feeds do not sustain brood production because they lack essential sterols found in pollen3,4. Here we describe a technological advance in honeybee nutrition with wide-reaching impacts on global food security. We first measured the quantity and proportion of sterols present in honeybee tissues. Using this information, we genetically engineered a strain of the oleaginous yeast Yarrowia lipolytica to produce a mixture of essential sterols for bees and incorporated this yeast strain into an otherwise nutritionally complete diet. Colonies exclusively fed with this diet reared brood for significantly longer than those fed diets without suitable sterols. The use of this method to incorporate sterol supplements into pollen substitutes will enable honeybee colonies to produce brood in the absence of floral pollen. Optimized diets created using this yeast strain could also reduce competition between bee species for access to natural floral resources and stem the decline in wild bee populations.

Complete pipeline for system-level of biological systems, from indivituals to ecosystems, using only metabolite data.

Nutrition is vital to health, but while the link between diet and body nutritional composition is well explored in humans and other vertebrates, this information is not well understood in insects, despite the vital roles they play in ecosystems, and their increasing use as experimental models. Here we used Nutritional Geometry to explore the rapid physiological response to ingested nutrients in the haemolymph nutritional profile of Spodoptera littoralis caterpillars. We ask whether blood nutrients are maintained homeostatically in the face of variable nutritional intake, or if regulation is more flexible for some nutrients than others (allostasis), which allows animals to adapt to stress by responding in a way that prioritises efficiency of responses in the face of trade-offs. Caterpillars were placed on 1 of 20 diets, systematically varying in their nutrient ratios (protein: carbohydrate) and density (calorie content), and their consumption was measured. After 48 h, caterpillars were bled, and the macronutrient (protein, carbohydrates and lipids) and nutrient metabolite (amino acids and simple sugars) content of the haemolymph was measured. Proteins comprised 93% of the haemolymph macronutrient pool on average and their concentration increased with protein eaten. The amino acid (AA) pool was dominated by five AAs, and the total pool increased with total nutrient intake. However, the ratio of essential to non-essential AAs increased as the proportion of protein consumed increased. Carbohydrates were tightly controlled, increasing only on the most extreme carbohydrate intakes. Simple sugars were dominated by glucose and trehalose, and overall, the simple sugar pool showed high levels of homeostasis. Rather than strict homeostasis of blood nutritional properties, an allostatic model seemed to be a better fit for blood nutrient regulation in this generalist herbivore. This flexibility in response to the nutritional composition of the diet may, in part, explain how this species has evolved to extreme dietary generalism and may play a role in its worldwide pest status. Given the range of fitness-related processes affected by the haemolymph, future studies should examine the physiological impacts of blood nutrient variation on reproduction, growth and response to infection and the trade-offs between them.

Honeybees, the world's most important crop pollinators, are increasingly facing pollen starvation arising from agricultural intensification and climate change. Frequent flowering dearth periods and high-density rearing conditions weaken colonies, often leading to their demise. Beekeepers provide colonies with pollen substitutes, but these feeds cannot sustain brood production because they lack essential sterols found in pollen. Here, we describe a technological breakthrough in honeybee nutrition with wide-reaching impacts on global food security. We first measured the quantity and proportion of sterols found in honeybee tissues. Using this information, we genetically engineered a strain of the oleaginous yeast, Yarrowia lipolytica, to produce a mixture of essential sterols for bees and incorporated it into an otherwise nutritionally complete diet. Colonies fed exclusively with this diet reared brood for significantly longer than those fed diets without suitable sterols. Incorporating sterol supplements into pollen substitutes using this method will enable beekeepers to rear healthier, longer-lived colonies to meet the growing demands for global crop pollination. It could also reduce competition between bee species for access to natural floral resources, stemming the decline of wild bee populations. ### Competing Interest Statement The authors have declared no competing interest.

In insects, nicotine activates nicotinic acetylcholine receptors, which are expressed throughout the central nervous system. However, little work has been done to investigate the effects of chronic nicotine treatment on learning or other behaviors in non-herbivorous insects. To examine the effects of long term nicotine consumption on learning and memory, honey bees were fed nicotine containing solutions over four days. Bees were able to detect nicotine at 0.1 mM in sucrose solutions, and in a no choice assay, bees reduced food intake when nicotine was 1 mM or higher. Treatment with a low dose of nicotine decreased the proportion of bees able to form an associative memory when bees were conditioned with either a massed or spaced appetitive olfactory training paradigm. On the other hand, higher doses of nicotine increased memory retention and the proportion of bees responding to the odor during 10 min and 24 h recall tests. The reduction in nicotine containing food consumed may also impact response levels during learning and recall tests. These data suggest that long term exposure to nicotine has complex effects on learning and memory.

Bees are important pollinators of agricultural crops, but their populations are at risk when pesticides are used. One of the largest risks bees face is poisoning of floral nectar and pollen by insecticides. Studies of bee detection of neonicotinoids have reported contradictory evidence about whether bees can taste these pesticides in sucrose solutions and hence avoid them. Here, we use an assay for the detection of food aversion combined with single-sensillum electrophysiology to test whether the mouthparts of the buff-tailed bumblebee (Bombus terrestris) detect the presence of pesticides in a solution that mimicked the nectar of oilseed rape (Brassica napus). Bees did not avoid consuming solutions containing concentrations of imidacloprid, thiamethoxam, clothianidin, or sulfoxaflor spanning six orders of magnitude, even when these solutions contained lethal doses. Only extremely high concentrations of the pesticides altered spiking in gustatory neurons through a slight reduction in firing rate or change in the rate of adaptation. These data provide strong evidence that bumblebees cannot detect or avoid field-relevant concentrations of pesticides using information from their mouthparts. As bees rarely contact floral nectar with other body parts, we predict that they are at high risk of unwittingly consuming pesticides in the nectar of pesticide-treated crops.

Background Bees provide essential pollination services for many food crops and are critical in supporting wild plant diversity. However, the dietary landscape of pollen food sources for social and solitary bees has changed because of agricultural intensification and habitat loss. For this reason, understanding the basic nutrient metabolism and meeting the nutritional needs of bees is becoming an urgent requirement for agriculture and conservation. We know that pollen is the principal source of dietary fat and sterols for pollinators, but a precise understanding of what the essential nutrients are and how much is needed is not yet clear. Sterols are key for producing the hormones that control development and may be present in cell membranes, where fatty-acid-containing species are important structural and signalling molecules (phospholipids) or to supply, store and distribute energy (glycerides). Aim of the review In this critical review, we examine the current general understanding of sterol and lipid metabolism of social and solitary bees from a variety of literature sources and discuss implications for bee health. Key scientific concepts of review We found that while eusocial bees are resilient to some dietary variation in sterol supply the scope for this is limited. The evidence of both de novo lipogenesis and a dietary need for particular fatty acids (FAs) shows that FA metabolism in insects is analogous to mammals but with distinct features. Bees rely on their dietary intake for essential sterols and lipids in a way that is dependent upon pollen availability.

This file contains the dataset used in the paper "Macronutrient balance has opposing effects on cognition and survival in honey bees"

Insects require dietary sources of B-vitamins, but relatively little is known about whether they regulate B-vitamin intake in the same way they regulate other nutrients. Honey bees meet their B-vitamin requirements mainly from the pollen they collect. Employing the geometric framework for nutrition, we found that honey bees actively regulate their vitamin intake following Bertrand's rule. We fed bees with a diet of essential amino acids (EAAs) and carbohydrate (C) to identify how the addition of B-vitamins affected the regulation of these macronutrients. In our experiments, honey bees preferred vitamins in concentrations comparable to those found in honey bee food (pollen, beebread, and royal jelly). Honey bees actively regulated niacin around an optimal value. Supplementing honey bee diets with B-vitamins influenced the amount of EAAs and carbohydrate ingested differently depending on the type of the vitamin. The impact of these vitamins was observed over the course of seven days where honey bees' mortality increased on diets of low and medium folic acid concentrations. This study provides insights into honey bee food intake regulation and the feeding preferences and sets the basis for future studies considering B-vitamins in honey bees diets.

The sense of taste permits the recognition of valuable nutrients and the avoidance of potential toxins. Previously, we found that bumblebees have a specialized mechanism for sensing sugars whereby two gustatory receptor neurons (GRNs) within the galeal sensilla of the bees’ mouthparts exhibit bursts of spikes. Here, we show that the temporal firing patterns of these GRNs separate sugars into four distinct groups that correlate with sugar nutritional value and palatability. We also identified a third GRN that responded to stimulation with relatively high concentrations of fructose, sucrose, and maltose. Sugars that were nonmetabolizable or toxic suppressed the responses of bursting GRNs to sucrose. These abilities to encode information about sugar value are a refinement to the bumblebee’s sense of sweet taste that could be an adaptation that enables precise calculations of the nature and nutritional value of floral nectar.

Essential macronutrients are critical to the fitness and survival of animals. Many studies have shown that animals regulate the amount of protein and carbohydrate they eat for optimal performance. Regulation of dietary fat is important but less often studied. Honeybees collect and consume floral pollen to obtain protein and fat but how they achieve the optimal balance of these two macronutrients is presently unknown. Here, using chemically defined diets composed of essential amino acids and lipids (lecithin), we show that adult worker honeybees actively regulate their intake of lipids around optimal values relative to the amount of protein in their diet. We found that broodless, nurse-age worker honeybees consume foods to achieve a ratio between 1:2 and 1:3 for essential amino acids to lipid or ∼1.25:1 protein to fat. Bees fed diets relatively high in fat gained abdominal fat and had enlarged hypopharyngeal glands. In most cases, eating diets high in fat did not result in increased mortality. Importantly, we also discovered that the total quantity of food the bees ate increased when they were given a choice of two diets relatively high in fat, implying that dietary fat influences bee nutritional state in a way that, in turn, influences behaviour. We speculate that dietary fat plays a critical role in maintaining workers in the nurse-like behavioural state independently of the influence of queen pheromone.

Summary Phytosterols are primary plant metabolites that have fundamental structural and regulatory functions. They are also essential nutrients for phytophagous insects, including pollinators, that cannot synthesize sterols. Despite the well‐described composition and diversity in vegetative plant tissues, few studies have examined phytosterol diversity in pollen. We quantified 25 pollen phytosterols in 122 plant species (105 genera, 51 families) to determine their composition and diversity across plant taxa. We searched literature and databases for plant phylogeny, environmental conditions, and pollinator guilds of the species to examine the relationships with pollen sterols. 24‐methylenecholesterol, sitosterol and isofucosterol were the most common and abundant pollen sterols. We found phylogenetic clustering of twelve individual sterols, total sterol content and sterol diversity, and of sterol groupings that reflect their underlying biosynthesis pathway (C‐24 alkylation, ring B desaturation). Plants originating in tropical‐like climates (higher mean annual temperature, lower temperature seasonality, higher precipitation in wettest quarter) were more likely to record higher pollen sterol content. However, pollen sterol composition and content showed no clear relationship with pollinator guilds. Our study is the first to show that pollen sterol diversity is phylogenetically clustered and that pollen sterol content may adapt to environmental conditions.