Land-use-associated stressors interact to reduce bumblebee health at the individual and colony level.

In agricultural landscapes, bees face a variety of stressors, including insecticides and poor-quality food. Although both stressors individually have been shown to affect bumblebee health negatively, few studies have focused on stressor interactions, a scenario expected in intensively used agricultural landscapes. Using the bumblebee Bombus terrestris, a key pollinator in agricultural landscapes, we conducted a fully factorial laboratory experiment starting at nest initiation. We assessed the effects of food quality and insecticides, alone and in interaction, on health traits at various levels, some of which have been rarely studied. Pollen with a diluted nutrient content (low quality) reduced ovary size and delayed colony development. Wing asymmetry, indicating developmental stress, was increased during insecticide exposure and interactions with poor food, whereas both stressors reduced body size. Both stressors and their interaction changed the workers' chemical profile and reduced worker interactions and the immune response. Our findings suggest that insecticides combined with nutritional stress reduce bumblebee health at the individual and colony levels, thus possibly affecting colony performance, such as development and reproduction, and the stability of plant-pollinator networks. The synergistic effects highlight the need of combining stressors in risk assessments and when studying the complex effects of anthropogenic stressors on health outcomes.

The ecology of nutrient sensation and perception in insects.

Insects are equipped with neurological, physiological, and behavioral tools to locate potential food sources and assess their nutritional quality based on volatile and chemotactile cues. We summarize current knowledge on insect taste perception and the different modalities of reception and perception. We suggest that the neurophysiological mechanisms of reception and perception are closely linked to the species-specific ecology of different insects. Understanding these links consequently requires a multidisciplinary approach. We also highlight existing knowledge gaps, especially in terms of the exact ligands of receptors, and provide evidence for a perceptional hierarchy suggesting that insects have adapted their reception and perception to preferentially perceive nutrient stimuli that are important for their fitness.

Perception, regulation, and fitness effects of pollen phytosterols in the bumble bee, Bombus terrestris.

PREMISE: Many flowering plants depend on insects for pollination and thus attract pollinators by offering rewards, mostly nectar and pollen. Bee pollinators rely on pollen as their main nutrient source. Pollen provides all essential micro- and macronutrients including substances that cannot be synthesized by bees themselves, such as sterols, which bees need for processes such as hormone production. Variations in sterol concentrations may consequently affect bee health and reproductive fitness. We therefore hypothesized that (1) these variations in pollen sterols affect longevity and reproduction in bumble bees and (2) can thus be perceived via the bees' antennae before consumption. METHODS: We studied the effect of sterols on longevity and reproduction of Bombus terrestris workers in feeding experiments and investigated sterol perception using chemotactile proboscis extension response (PER) conditioning. RESULTS: Workers could perceive several sterols (cholesterol, cholestenone, desmosterol, stigmasterol, ß-sitosterol) via their antennae but not differentiate between them. However, when sterols were presented in pollen, and not as a single compound, the bees were unable to differentiate between pollen differing in sterol content. Additionally, different sterol concentrations in pollen neither affected pollen consumption nor brood development or worker longevity. CONCLUSIONS: Since we used both natural concentrations and concentrations higher than those found in pollen, our results indicate that bumble bees may not need to pay specific attention to pollen sterol content beyond a specific threshold. Naturally encountered concentrations might fully support their sterol requirements and higher concentrations do not seem to have negative effects.

Young bumblebees may rely on both direct pollen cues and early experience when foraging.

An adequate supply of macro- and micronutrients determines health and reproductive success in most animals. Many bee species, for example, collect nectar and pollen to satisfy their demands for carbohydrates, protein and fat, respectively. Bees can assess the quality of pollen by feeding on it, but also pre-digestively by means of chemotactile assessment. Whether they additionally use larval nutritional experience, as has been shown for Drosophila melanogaster and Bombyx mori, is unknown. In this study, we tested whether pollen selection of bumblebee foragers is affected by nutritional experience (acquired before the onset of foraging) or solely by food quality. Bumblebee larvae were fed with one out of three different pollen blends. As adults, they were offered all three blends when they started foraging for the first time. We found all treatment groups to prefer one out of the three blends. This blend provided the highest nutritional quality and increased the bees' lifespan, as shown by feeding studies with microcolonies. Besides, bees also chose the pollen blend fed during their larval stage more often than expected, indicating a significant effect of pre-foraging experience on adult pollen foraging behaviour. The combination of both direct pollen quality assessment and pre-foraging experience (i.e. during the larval phase or as early imagines) seems to allow foraging bumblebees to efficiently select the most suitable pollen for their colony.

Adding Amino Acids to a Sucrose Diet Is Not Sufficient to Support Longevity of Adult Bumble Bees.

Dietary macro-nutrients (i.e., carbohydrates, protein, and fat) are important for bee larval development and, thus, colony health and fitness. To which extent different diets (varying in macro-nutrient composition) affect adult bees and whether they can thrive on nectar as the sole amino acid source has, however, been little investigated. We investigated how diets varying in protein concentration and overall nutrient composition affected consumption, longevity, and breeding behavior of the buff-tailed bumble bee, Bombus terrestris (Hymenoptera: Apidae). Queenless micro-colonies were fed either natural nutrient sources (pollen), nearly pure protein (i.e., the milk protein casein), or sucrose solutions with low and with high essential amino acid content in concentrations as can be found in nectar. We observed micro-colonies for 110 days. We found that longevity was highest for pure pollen and lowest for pure sucrose solution and sucrose solution supplemented with amino acids in concentrations as found in the nectar of several plant species. Adding higher concentrations of amino acids to sucrose solution did only slightly increase longevity compared to sucrose alone. Consequently, sucrose solution with the applied concentrations and proportions of amino acids or other protein sources (e.g., casein) alone did not meet the nutritional needs of healthy adult bumble bees. In fact, longevity was highest and reproduction only successful in micro-colonies fed pollen. These results indicate that, in addition to carbohydrates and protein, adult bumble bees, like larvae, need further nutrients (e.g., lipids and micro-nutrients) for their well-being. An appropriate nutritional composition seemed to be best provided by floral pollen, suggesting that pollen is an essential dietary component not only for larvae but also for adult bees.

Best be(e) on low fat: linking nutrient perception, regulation and fitness.

Preventing malnutrition through consuming nutritionally appropriate resources represents a challenge for foraging animals. This is due to often high variation in the nutritional quality of available resources. Foragers consequently need to evaluate different food sources. However, even the same food source can provide a plethora of nutritional and non-nutritional cues, which could serve for quality assessment. We show that bumblebees, Bombus terrestris, overcome this challenge by relying on lipids as nutritional cue when selecting pollen. The bees 'prioritised' lipid perception in learning experiments and avoided lipid consumption in feeding experiments, which supported survival and reproduction. In contrast, survival and reproduction were severely reduced by increased lipid contents. Our study highlights the importance of fat regulation for pollen foraging bumblebees. It also reveals that nutrient perception, nutrient regulation and reproductive fitness can be linked, which represents an effective strategy enabling quick foraging decisions that prevent malnutrition and maximise fitness.

Pollinator or pedigree: which factors determine the evolution of pollen nutrients?

A prime example of plant-animal interactions is the interaction between plants and pollinators, which typically receive nectar and/or pollen as reward for their pollination service. While nectar provides mostly carbohydrates, pollen represents the main source of protein and lipids for many pollinators. However, the main function of pollen is to carry nutrients for pollen tube growth and thus fertilization. It is unclear whether pollinator attraction exerts a sufficiently strong selective pressure to alter the nutritional profile of pollen, e.g., through increasing its crude protein content or protein-to-lipid ratio, which both strongly affect bee foraging. Pollen nutritional quality may also be merely determined by phylogenetic relatedness, with pollen of closely related plants showing similar nutritional profiles due to shared biosynthetic pathways or floral morphologies. Here, we present a meta-analysis of studies on pollen nutrients to test whether differences in pollen nutrient contents and ratios correlated with plant insect pollinator dependence and/or phylogenetic relatedness. We hypothesized that if pollen nutritional content was affected by pollinator attraction, it should be different (e.g., higher) in highly pollinator-dependent plants, independent of phylogenetic relatedness. We found that crude protein and the protein-to-lipid ratio in pollen strongly correlated with phylogeny. Moreover, pollen protein content was higher in plants depending mostly or exclusively on insect pollination. Pollen nutritional quality thus correlated with both phylogenetic relatedness and pollinator dependency, indicating that, besides producing pollen with sufficient nutrients for reproduction, the nutrient profile of zoophilous plants may have been shaped by their pollinators' nutritional needs.

Bumblebees are able to perceive amino acids via chemotactile antennal stimulation.

Like all animals, bees need to consume essential amino acids to maintain their body's protein synthesis. Perception and discrimination of amino acids are, however, still poorly understood in bees (and insects in general). We used chemotactile conditioning of the proboscis extension response (PER) to examine (1) whether Bombus terrestris workers are able to perceive amino acids by means of their antennae and (if so) which ones, (2) whether they are able to differentiate between different amino acids, and (3) whether they are able to differentiate between different concentrations of the same amino acid. We found that workers perceived asparagine, cysteine, hydroxyproline, glutamic acid, lysine, phenylalanine, and serine, but not alanine, leucine, proline, or valine by means of their antennae. Surprisingly, they were unable to differentiate between different (perceivable) amino acids, but they distinguished between different concentrations of lysine. Consequently, bumblebees seem to possess amino acid receptors at the tip of their antennae, which enable a general perception of those solute amino acids that have an additional functional group (besides the common amino and carboxylic groups). They may thus have the ability to assess the overall amino acid content of pollen and nectar prior to ingestion.

Do honeybees (Apis mellifera) differentiate between different pollen types?

Bees receive nectar and pollen as reward for pollinating plants. Pollen of different plant species varies widely in nutritional composition. In order to select pollen of appropriate nutritional quality, bees would benefit if they could distinguish different pollen types. Whether they rely on visual, olfactory and/or chemotactile cues to distinguish between different pollen types, has however been little studied. In this study, we examined whether and how Apis mellifera workers differentiate between almond and apple pollen. We used differential proboscis extension response conditioning with olfactory and chemotactile stimulation, in light and darkness, and in summer and winter bees. We found that honeybees were only able to differentiate between different pollen types, when they could use both chemotactile and olfactory cues. Visual cues further improved learning performance. Summer bees learned faster than winter bees. Our results thus highlight the importance of multisensory information for pollen discrimination.

Separation of different pollen types by chemotactile sensing in Bombus terrestris.

When tasting food, animals rely on chemical and tactile cues, which determine the animal's decision on whether to eat food. As food nutritional composition has enormous consequences for the survival of animals, food items should generally be tasted before they are eaten or collected for later consumption. Even though recent studies have confirmed the importance of, for example, gustatory cues, compared with olfaction only little is known about the representation of chemotactile stimuli at the receptor level (let alone higher brain centers) in animals other than vertebrates. To better understand how invertebrates may process chemotactile cues, we used bumblebees as a model species and combined electroantennographical (EAG) recordings with a novel technique for chemotactile antennal stimulation in bees. The recorded EAG responses to chemotactile stimulation clearly separated volatile compounds by both compound identity and concentration, and could be successfully applied to test the receptor activity evoked by different types of pollen. We found that two different pollen types (apple and almond; which were readily distinguished by bumblebees in a classical conditioning task) evoked significantly distinct neural activity already at the antennal receptor level. Our novel stimulation technique therefore enables investigation of chemotactile sensing, which is highly important for assessing food nutritional quality while foraging. It can further be applied to test other chemosensory behaviors, such as mate or nest mate recognition, or to investigate whether toxic substances, e.g. in pollen, affect neuronal separation of different food types.

How to know which food is good for you: bumblebees use taste to discriminate between different concentrations of food differing in nutrient content.

In view of the ongoing pollinator decline, the role of nutrition in bee health has received increasing attention. Bees obtain fat, carbohydrates and protein from pollen and nectar. As both excessive and deficient amounts of these macronutrients are detrimental, bees would benefit from assessing food quality to guarantee an optimal nutrient supply. While bees can detect sucrose and use it to assess nectar quality, it is unknown whether they can assess the macronutrient content of pollen. Previous studies have shown that bees preferentially collect pollen of higher protein content, suggesting that differences in pollen quality can be detected either by individual bees or via feedback from larvae. In this study, we examined whether and, if so, how individuals of the buff-tailed bumblebee (Bombus terrestris) discriminate between different concentrations of pollen and casein mixtures and thus nutrients. Bumblebees were trained using absolute and differential conditioning of the proboscis extension response (PER). As cues related to nutrient concentration could theoretically be perceived by either smell or taste, bees were tested on both olfactory and, for the first time, chemotactile perception. Using olfactory cues, bumblebees learned and discriminated between different pollen types and casein, but were unable to discriminate between different concentrations of these substances. However, when they touched the substances with their antennae, using chemotactile cues, they could also discriminate between different concentrations. Bumblebees are therefore able to discriminate between foods of different concentrations using contact chemosensory perception (taste). This ability may enable them to individually regulate the nutrient intake of their colonies.