Cuticular Hydrocarbon Profiles of Himalayan Bumble Bees (Hymenoptera: Bombus Latreille) are Species-Specific and Show Elevational Variation.

Bumble bees are important pollinators in natural environments and agricultural farmlands, and they are in particular adapted to harsh environments like high mountain habitats. In these environments, animals are exposed to low temperature and face the risk of desiccation. The Eastern Himalayas are one of the recognized biodiversity hotspots worldwide. The area covers subtropical rainforest with warm temperature and high precipitation as well as high mountain ranges with peaks reaching up to 7,000 m, shaping a diverse floral and faunal community at the different elevational zones. To identify possible adaptation strategies, we investigated the cuticular hydrocarbon profiles of four bumble bee species occurring at different elevational ranges in Arunachal Pradesh, the northeastern most state in India. At 17 locations along an elevational gradient, we collected workers of two species from lower elevations (B. albopleuralis and B. breviceps; ~ 100 m - 3,000 m asl) and two species from higher elevations (B. prshewalskyi and B. mirus; ~ 2,800 m - 4,500 m asl). The CHC profiles of all four species showed a significant degree of variation in the composition of hydrocarbons, indicating species specificity. We also found clear correlation with elevation. The weighted mean chain length of the hydrocarbons significantly differed between the low and high elevation species, and the proportion of saturated hydrocarbons in CHC profiles significantly increased with the elevational range of the bumble bee species. Our results indicate that bumble bees living at high elevations reduce the risk of water loss by adapting their CHC composition on their cuticle, a phenomenon that has also been found in other insects like ants and fruit flies.

Monarch butterflies memorize the spatial location of a food source.

Spatial memory helps animals to navigate familiar environments. In insects, spatial memory has extensively been studied in central place foragers such as ants and bees. However, if butterflies memorize a spatial location remains unclear. Here, we conducted behavioural experiments to test whether monarch butterflies (Danaus plexippus) can remember and retrieve the spatial location of a food source. We placed several visually identical feeders in a flight cage, with only one feeder providing sucrose solution. Across multiple days, individual butterflies predominantly visited the rewarding feeder. Next, we displaced a salient landmark close to the feeders to test which visual cue the butterflies used to relocate the rewarding feeder. While occasional landmark displacements were ignored by the butterflies and did not affect their decisions, systematic displacement of both the landmark and the rewarding feeder demonstrated that the butterflies associated the salient landmark with the feeder's position. Altogether, we show that butterflies consolidate and retrieve spatial memory in the context of foraging.

Visual accelerated and olfactory decelerated responses during multimodal learning in honeybees.

To obtain accurate information about the outside world and to make appropriate decisions, animals often combine information from different sensory pathways to form a comprehensive representation of their environment. This process of multimodal integration is poorly understood, but it is common view that the single elements of a multimodal stimulus influence each other's perception by enhancing or suppressing their neural representation. The neuronal level of interference might be manifold, for instance, an enhancement might increase, whereas suppression might decrease behavioural response times. In order to investigate this in an insect behavioural model, the Western honeybee, we trained individual bees to associate a sugar reward with an odour, a light, or a combined olfactory-visual stimulus, using the proboscis extension response (PER). We precisely monitored the PER latency (the time between stimulus onset and the first response of the proboscis) by recording the muscle M17, which innervates the proboscis. We found that odours evoked a fast response, whereas visual stimuli elicited a delayed PER. Interestingly, the combined stimulus showed a response time in between the unimodal stimuli, suggesting that olfactory-visual integration accelerates visual responses but decelerates the olfactory response time.

Visual ecology: How glossy colours shine and confuse.

Most colours in nature are matte, but across the tree of life glossiness has evolved numerous times, suggesting that glossiness can be beneficial. Recent research finds that glossiness may confuse observers and protect against predators.

Honesty, reliability, and information content of floral signals.

Plants advertise their presence by displaying attractive flowers, which pollinators use to locate a floral reward. Understanding how floral traits scale with reward status lies at the heart of pollination biology, because it connects the different interests of plants and pollinators. Studies on plant phenotype-reward associations often use different terms and concepts, which limits developing a broader synthesis. Here, we present a framework with definitions of the key aspects of plant phenotype-reward associations and provide measures to quantify them across different species and studies. We first distinguish between cues and signals, which are often used interchangeably, but have different meanings and are subject to different selective pressures. We then define honesty, reliability, and information content of floral cues/signals and provide ways to quantify them. Finally, we discuss the ecological and evolutionary factors that determine flower phenotype-reward associations, how context-dependent and temporally variable they are, and highlight promising research directions.

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.

Colour vision in ants (Formicidae, Hymenoptera).

Ants are ecologically one of the most important groups of insects and exhibit impressive capabilities for visual learning and orientation. Studies on numerous ant species demonstrate that ants can learn to discriminate between different colours irrespective of light intensity and modify their behaviour accordingly. However, the findings across species are variable and inconsistent, suggesting that our understanding of colour vision in ants and what roles ecological and phylogenetic factors play is at an early stage. This review provides a brief synopsis of the critical findings of the past century of research by compiling studies that address molecular, physiological and behavioural aspects of ant colour vision. With this, we aim to improve our understanding of colour vision and to gain deeper insights into the mysterious and colourful world of ants. This article is part of the theme issue 'Understanding colour vision: molecular, physiological, neuronal and behavioural studies in arthropods'.

Caution with colour calculations: spectral purity is a poor descriptor of flower colour visibility.

BACKGROUND: The colours of flowers are of key interest to plant and pollination biologists. An increasing number of studies have investigated the importance of saturation of flower colours (often called 'spectral purity' or 'chroma') for visibility to pollinators, but the conceptual, physiological and behavioural foundations for these metrics as well as the calculations used rest on slender foundations. METHODS: We discuss the caveats of colour attributes that are derived from human perception, and in particular spectral purity and chroma, as variables in flower colour analysis. We re-analysed seven published datasets encompassing 774 measured reflectance spectra to test for correlations between colour contrast, spectral purity and chroma. MAIN FINDINGS AND CONCLUSIONS: We identify several concerns with common calculation procedures in animal colour spaces. Studies on animal colour vision provide no ground to assume that any pollinator perceives (or responds to) saturation, chroma or spectral purity in the way humans do. A re-analysis of published datasets revealed that values for colour contrast between flowers and their background are highly correlated with measures for spectral purity and chroma, which invalidates treating these factors as independent variables as is currently commonplace. Strikingly, spectral purity and chroma - both of which are metrics for saturation and are often used synonymously - are not correlated at all. We conclude that alternative, behaviourally validated metrics for the visibility of flowers to pollinators, such as colour contrast and achromatic contrast, are better in understanding the role of flower colour in plant-pollinator signalling.

Does quantity matter to a stingless bee?

Quantitative information is omnipresent in the world and a wide range of species has been shown to use quantities to optimize their decisions. While most studies have focused on vertebrates, a growing body of research demonstrates that also insects such as honeybees possess basic quantitative abilities that might aid them in finding profitable flower patches. However, it remains unclear if for insects, quantity is a salient feature relative to other stimulus dimensions, or if it is only used as a "last resort" strategy in case other stimulus dimensions are inconclusive. Here, we tested the stingless bee Trigona fuscipennis, a species representative of a vastly understudied group of tropical pollinators, in a quantity discrimination task. In four experiments, we trained wild, free-flying bees on stimuli that depicted either one or four elements. Subsequently, bees were confronted with a choice between stimuli that matched the training stimulus either in terms of quantity or another stimulus dimension. We found that bees were able to discriminate between the two quantities, but performance differed depending on which quantity was rewarded. Furthermore, quantity was more salient than was shape. However, quantity did not measurably influence the bees' decisions when contrasted with color or surface area. Our results demonstrate that just as honeybees, small-brained stingless bees also possess basic quantitative abilities. Moreover, invertebrate pollinators seem to utilize quantity not only as "last resort" but as a salient stimulus dimension. Our study contributes to the growing body of knowledge on quantitative cognition in invertebrate species and adds to our understanding of the evolution of numerical cognition.

Evidence for UV-green dichromacy in the basal hymenopteran Sirex noctilio (Siricidae).

A precondition for colour vision is the presence of at least two spectral types of photoreceptors in the eye. The order Hymenoptera is traditionally divided into the Apocrita (ants, bees, wasps) and the Symphyta (sawflies, woodwasps, horntails). Most apocritan species possess three different photoreceptor types. In contrast, physiological studies in the Symphyta have reported one to four photoreceptor types. To better understand the evolution of photoreceptor diversity in the Hymenoptera, we studied the Symphyta Sirex noctilio, which belongs to the superfamily Siricoidea, a closely related group of the Apocrita suborder. Our aim was to (i) identify the photoreceptor types of the compound eye by electroretinography (ERG), (ii) characterise the visual opsin genes of S. noctilio by genomic comparisons and phylogenetic analyses and (iii) analyse opsin mRNA expression. ERG measurements revealed two photoreceptor types in the compound eye, maximally sensitive to 527 and 364 nm. In addition, we identified three opsins in the genome, homologous to the hymenopteran green or long-wavelength sensitive (LW) LW1, LW2 and ultra-violet sensitive (UV) opsin genes. The LW1 and UV opsins were found to be expressed in the compound eyes, and LW2 and UV opsins in the ocelli. The lack of a blue or short-wavelength sensitive (SW) homologous opsin gene and a corresponding receptor suggests that S. noctilio is a UV-green dichromate.

Effect of Trap Color on Captures of Bark-and Wood-Boring Beetles (Coleoptera; Buprestidae and Scolytinae) and Associated Predators.

Traps baited with attractive lures are increasingly used at entry-points and surrounding natural areas to intercept exotic wood-boring beetles accidentally introduced via international trade. Several trapping variables can affect the efficacy of this activity, including trap color. In this study, we tested whether species richness and abundance of jewel beetles (Buprestidae), bark and ambrosia beetles (Scolytinae), and their common predators (i.e., checkered beetles, Cleridae) can be modified using trap colors different to those currently used for surveillance of jewel beetles and bark and ambrosia beetles (i.e., green or black). We show that green and black traps are generally efficient, but also that many flower-visiting or dark-metallic colored jewel beetles and certain bark beetles are more attracted by other colors. In addition, we show that checkered beetles have color preferences similar to those of their Scolytinae preys, which limits using trap color to minimize their inadvertent removal. Overall, this study confirmed that understanding the color perception mechanisms in wood-boring beetles can lead to important improvements in trapping techniques and thereby increase the efficacy of surveillance programs.

Mechanisms of Nutritional Resource Exploitation by Insects.

Insects have evolved an extraordinary range of nutritional adaptations to exploit other animals, plants, bacteria, fungi and soils as resources in terrestrial and aquatic environments. This special issue provides some new insights into the mechanisms underlying these adaptations. Contributions comprise lab and field studies investigating the chemical, physiological, cognitive and behavioral mechanisms that enable resource exploitation and nutrient intake regulation in insects. The collection of papers highlights the need for more studies on the comparative sensory ecology, underlying nutritional quality assessment, cue perception and decision making to fully understand how insects adjust resource selection and exploitation in response to environmental heterogeneity and variability.

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.

Color preference and spatial distribution of glaphyrid beetles suggest a key role in the maintenance of the color polymorphism in the peacock anemone (Anemone pavonina, Ranunculaceae) in Northern Greece.

In the Mediterranean region, a group of unrelated plant species share an unusual deep-red flower color and are pollinated by glaphyrid beetles. Some of these species possess different color morphs, but the mechanisms maintaining this color polymorphism are unknown. The peacock anemone, Anemone pavonina, is a color polymorphic species with red or purple flowers. We investigated the spatial distribution of its color morphs and its potential glaphyrid pollinators, Pygopleurus spp., along an elevational gradient on the southern slopes of Mount Olympus, Greece. We found a correlation between relative proportions of the two color morphs with both elevation and beetle abundance. At low elevations (< 1000 m a.s.l.), beetles were abundant and anemone populations comprised only red flowers. Above a steep transition zone with mixed-colored populations (c. 1000-1300 m) most flowers were purple and beetles were rare. Color-trapping experiments revealed a strong preference for red over other colors in beetles and colorimetric modeling suggests that a simple chromatic mechanism is sufficient to explain their color choices. We thus hypothesize that beetles select for red flowers and that with increasing elevation and decreasing beetle density, other flower visitors (e.g., bees) gain importance as pollinators and select for a different color.

Species composition and elevational distribution of bumble bees (Hymenoptera, Apidae, Bombus Latreille) in the East Himalaya, Arunachal Pradesh, India.

The East Himalaya is one of the world's most biodiverse ecosystems. However, very little is known about the abundance and distribution of many plant and animal taxa in this region. Bumble bees are a group of cold-adapted and high elevation insects that fulfil an important ecological and economical function as pollinators of wild and agricultural flowering plants and crops. The Himalayan mountain range provides ample suitable habitats for bumble bees. Systematic study of Himalayan bumble bees began a few decades ago and the main focus has centred on the western region, while the eastern part of the mountain range has received little attention and only a few species have been verified. During a three-year survey, more than 700 bumble bee specimens of 21 species were collected in Arunachal Pradesh, the largest of the north-eastern states of India. The material included a range of species that were previously known from a limited number of collected specimens, which highlights the unique character of the East Himalayan ecosystem. Our results are an important first step towards a future assessment of species distribution, threat, and conservation. Clear elevation patterns of species diversity were observed, which raise important questions about the functional adaptations that allow bumble bees to thrive in this particularly moist region in the East Himalaya.

Neuronal Plasticity in the Mushroom-Body Calyx of Bumble Bee Workers During Early Adult Development.

Division of labor among workers is a key feature of social insects and frequently characterized by an age-related transition between tasks, which is accompanied by considerable structural changes in higher brain centers. Bumble bees (Bombus terrestris), in contrast, exhibit a size-related rather than an age-related task allocation, and thus workers may already start foraging at two days of age. We ask how this early behavioral maturation and distinct size variation are represented at the neuronal level and focused our analysis on the mushroom bodies (MBs), brain centers associated with sensory integration, learning and memory. To test for structural neuronal changes related to age, light exposure, and body size, whole-mount brains of age-marked workers were dissected for synapsin immunolabeling. MB calyx volumes, densities, and absolute numbers of olfactory and visual projection neuron (PN) boutons were determined by confocal laser scanning microscopy and three-dimensional image analyses. Dark-reared bumble bee workers showed an early age-related volume increase in olfactory and visual calyx subcompartments together with a decrease in PN-bouton density during the first three days of adult life. A 12:12  h light-dark cycle did not affect structural organization of the MB calyces compared to dark-reared individuals. MB calyx volumes and bouton numbers positively correlated with body size, whereas bouton density was lower in larger workers. We conclude that, in comparison to the closely related honey bees, neuronal maturation in bumble bees is completed at a much earlier stage, suggesting a strong correlation between neuronal maturation time and lifestyle in both species.