Comparative psychophysics of Western honey bee (Apis mellifera) and stingless bee (Tetragonula carbonaria) colour purity and intensity perception.

Bees play a vital role as pollinators worldwide and have influenced how flower colour signals have evolved. The Western honey bee, Apis mellifera (Apini), and the Buff-tailed bumble bee, Bombus terrestris (Bombini) are well-studied model species with regard to their sensory physiology and pollination capacity, although currently far less is known about stingless bees (Meliponini) that are common in pantropical regions. We conducted comparative experiments with two highly eusocial bee species, the Western honey bee, A. mellifera, and the Australian stingless bee, Tetragonula carbonaria, to understand their colour preferences considering fine-scaled stimuli specifically designed for testing bee colour vision. We employed stimuli made of pigment powders to allow manipulation of single colour parameters including spectral purity (saturation) or colour intensity (brightness) of a blue colour (hue) for which both species have previously shown innate preferences. Both A. mellifera and T. carbonaria demonstrated a significant preference for spectrally purer colour stimuli, although this preference is more pronounced in honey bees than in stingless bees. When all other colour cues were tightly controlled, honey bees receiving absolute conditioning demonstrated a capacity to learn a high-intensity stimulus significant from chance expectation demonstrating some capacity of plasticity for this dimension of colour perception. However, honey bees failed to learn low-intensity stimuli, and T. carbonaria was insensitive to stimulus intensity as a cue. These comparative findings suggest that there may be some common roots underpinning colour perception in bee pollinators and how they interact with flowers, although species-specific differences do exist.

A bee's eye view of remarkable floral colour patterns in the south-west Australian biodiversity hotspot revealed by false colour photography.

BACKGROUND AND AIMS: Colour pattern is a key cue of bee attraction selectively driving the appeal of pollinators. It comprises the main colour of the flower with extra fine patterns, indicating a reward focal point such as nectar, nectaries, pollen, stamens and floral guides. Such advertising of floral traits guides visitation by the insects, ensuring precision in pollen gathering and deposition. The study, focused in the Southwest Australian Floristic Region, aimed to spot bee colour patterns that are usual and unusual, missing, accomplished by mimicry of pollen and anthers, and overlapping between mimic-model species in floral mimicry cases. METHODS: Floral colour patterns were examined by false colour photography in 55 flower species of multiple highly diverse natural plant communities in south-west Australia. False colour photography is a method to transform a UV photograph and a colour photograph into a false colour photograph based on the trichromatic vision of bees. This method is particularly effective for rapid screening of large numbers of flowers for the presence of fine-scale bee-sensitive structures and surface roughness that are not detectable using standard spectrophotometry. KEY RESULTS: Bee- and bird-pollinated flowers showed the expected but also some remarkable and unusual previously undetected floral colour pattern syndromes. Typical colour patterns include cases of pollen and flower mimicry and UV-absorbing targets. Among the atypical floral colour patterns are unusual white and UV-reflecting flowers of bee-pollinated plants, bicoloured floral guides, consistently occurring in Fabaceae spp., and flowers displaying a selective attractiveness to birds only. In the orchid genera (Diuris and Thelymitra) that employ floral mimicry of model species, we revealed a surprising mimicry phenomenon of anthers mimicked in turn by model species. CONCLUSION: The study demonstrates the applicability of 'bee view' colour imaging for deciphering pollinator cues in a biodiverse flora with potential to be applied to other eco regions. The technique provides an exciting opportunity for indexing floral traits on a biome scale to establish pollination drivers of ecological and evolutionary relevance.

Changes in floral nectar are unlikely adaptive responses to pollinator flight sound.

Under noiseless experimental conditions, sugar concentration of secreted floral nectar may increase after flower exposure to nearby sounds of pollinator flight (Veits et al. 2019). However, we reject the argument that this represents adaptive plant behaviour, and consider that the appealing analogy between a flower and human ear is unjustified.

How flower colour signals allure bees and hummingbirds: a community-level test of the bee avoidance hypothesis.

Colour signals are the main floral trait for plant-pollinator communication. Owing to visual specificities, flower visitors exert different selective pressures on flower colour signals of plant communities. Although they evolved to attract pollinators, matching their visual sensitivity and colour preferences, floral signals may also evolve to avoid less efficient pollinators and antagonistic flower visitors. We evaluated evidence for the bee avoidance hypothesis in a Neotropical community pollinated mainly by bees and hummingbirds, the campo rupestre. We analysed flower reflectance spectra, compared colour variables of bee-pollinated flowers (bee-flowers; 244 species) and hummingbird-pollinated flowers (hummingbird-flowers; 39 species), and looked for evidence of bee sensorial exclusion in hummingbird-flowers. Flowers were equally contrasting for hummingbirds. Hummingbird-flowers were less conspicuous to bees, reflecting mainly long wavelengths and avoiding red-blind visitors. Bee-flowers reflected more short wavelengths, were more conspicuous to bees (higher contrasts and spectral purity) than hummingbird-flowers and displayed floral guides more frequently, favouring flower attractiveness, discrimination and handling by bees. Along with no phylogenetic signal, the differences in colour signal strategies between bee- and hummingbird-flowers are the first evidence of the bee avoidance hypothesis at a community level and reinforce the role of pollinators as a selective pressure driving flower colour diversity.

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.

Functional significance of the optical properties of flowers for visual signalling.

Background: Flower coloration is a key enabler for pollinator attraction. Floral visual signals comprise several components that are generated by specific anatomical structures and pigmentation, and often have different functions in pollinator attraction. Anatomical studies have advanced our understanding of the optical properties of flowers, and evidence from behavioural experiments has elucidated the biological relevance of different components of floral visual signals, but these two lines of research are often considered independently. Scope: Here, we review current knowledge about different aspects of the floral visual signals, their anatomical and optical properties, and their functional significance in plant-pollinator visual signalling. We discuss common aspects, such as chromatic and achromatic contrast, hue, saturation and brightness, as well as less common types of visual signals, including gloss, fluorescence, polarization and iridescence in the context of salience of floral colour signals and their evolution, and highlight promising avenues for future research.

The yellow specialist: dronefly Eristalis tenax prefers different yellow colours for landing and proboscis extension.

Droneflies, imagoes of the hoverfly Eristalis tenax, are known to possess a preference for yellow flowers, i.e. they prefer to visit yellow flowers and prefer to extend the proboscis to yellow colours. In this study we disentangle these colour preferences by investigating the landing reaction and proboscis reflex with particular reference to intensity, spectral purity and dominant wavelength of colour stimuli and their UV reflection properties. In multiple-choice tests, naïve and non-trained flies prefer to land on yellow colours independent of their UV reflection characteristics, but also accept blue, white and pink colours if they absorb UV and are of sufficient brightness. Flies trained to land on colours other than yellow still prefer yellow colours to some extent. Moreover, the flies prefer bright over dark yellow colours even if trained to dark yellow ones. The flies refuse to land on dark colours of all hues. Naïve flies exhibit the proboscis reflex only to pure yellow pollen. These experiments show for the first time that landing in droneflies is triggered by a yellow colour independent of its UV reflection properties, but proboscis extension is triggered by yellow colours strongly absorbing blue and UV. The ability to discriminate colours is better than predicted by the categorical colour vision model. The colour preferences in E. tenax represent a fine-tuned ability to visit yellow flowers displaying a UV bull's-eye pattern.

Testing the influence of gravity on flower symmetry in five Saxifraga species.

Flower symmetry is considered a species-specific trait and is categorized in asymmetry, actinomorphic symmetry, bisymmetry and zygomorphic symmetry. Here we report on the intra-individual variation of flower symmetry in the genus Saxifraga and the influence of light, gravity and intrinsic factors on the development of flower symmetry. We tested five species-Saxifraga cuneifolia, Saxifraga imparilis, Saxifraga rotundifolia, Saxifraga stolonifera and Saxifraga umbrosa-concerning six flower parameters-angles between petals, petal length, petal pigmentation, angular position of carpels, movement of stamens and (only for S. imparilis and S. stolonifera) the length of the two lower elongated petals in regard to their position towards the stem. Specimens of all species were tested on a vertical clinostat as a gravity compensator, on a horizontal clinostat as a light incidence compensator and on a stationary control. The results show that the angle of incident light has no apparent impact on flower symmetry, whereas gravity affects the angular position of petals in S. cuneifolia and S. umbrosa and the petal colouration in S. rotundifolia. In S. cuneifolia and S. umbrosa, the absence of directional gravity resulted in the development of actinomorphic flowers, whereas the corresponding control flowers were zygomorphic. The development of flowers in S. rotundifolia was not altered by this treatment. The length of the two elongated petals in S. stolonifera and S. imparilis was not affected by gravity, but rather was determined by position of the flower within the inflorescence and resulted in asymmetrical flowers.

Colour is more than hue: preferences for compiled colour traits in the stingless bees Melipona mondury and M. quadrifasciata.

The colour vision of bees has been extensively analysed in honeybees and bumblebees, but few studies consider the visual perception of stingless bees (Meliponini). In a five-stage experiment the preference for colour intensity and purity, and the preference for the dominant wavelength were tested by presenting four colour stimuli in each test to freely flying experienced workers of two stingless bee species, Melipona mondury and Melipona quadrifasciata. The results with bee-blue, bee-UV-blue and bee-green colours offered in four combinations of varying colour intensity and purity suggest a complex interaction between these colour traits for the determination of colour choice. Specifically, M. mondury preferred bee-UV-blue colours over bee-green, bee-blue and bee-blue-green colours while M. quadrifasciata preferred bee-green colour stimuli. Moreover in M. mondury the preferences were different if the background colour was changed from grey to green. There was a significant difference between species where M. mondury preferred UV-reflecting over UV-absorbing bee-blue-green colour stimuli, whereas M. quadrifasciata showed an opposite preference. The different colour preferences of the free flying bees in identical conditions may be caused by the bees' experience with natural flowers precedent to the choice tests, suggesting reward partitioning between species.

Innate colour preferences of the Australian native stingless bee Tetragonula carbonaria Sm.

Innate preferences promote the capacity of pollinators to find flowers. Honeybees and bumblebees have strong preferences for 'blue' stimuli, and flowers of this colour typically present higher nectar rewards. Interestingly, flowers from multiple different locations around the world independently have the same distribution in bee colour space. Currently, however, there is a paucity of data on the innate colour preferences of stingless bees that are often implicated as being key pollinators in many parts of the world. In Australia, the endemic stingless bee Tetragonula carbonaria is widely distributed and known to be an efficient pollinator of both native plants and agricultural crops. In controlled laboratory conditions, we tested the innate colour responses of naïve bees using standard broadband reflectance stimuli representative of common flower colours. Colorimetric analyses considering hymenopteran vision and a hexagon colour space revealed a difference between test colonies, and a significant effect of green contrast and an interaction effect of green contrast with spectral purity on bee choices. We also observed colour preferences for stimuli from the blue and blue-green categorical regions of colour space. Our results are discussed in relation to the similar distribution of flower colours observed from bee pollination around the world.

Visual ecology of flies with particular reference to colour vision and colour preferences.

The visual ecology of flies is outstanding among insects due to a combination of specific attributes. Flies' compound eyes possess an open rhabdom and thus separate rhabdomeres in each ommatidium assigned to two visual pathways. The highly sensitive, monovariant neural superposition system is based on the excitation of the peripheral rhabdomeres of the retinula cells R1-6 and controls optomotor reactions. The two forms of central rhabdomeres of R7/8 retinula cells in each ommatidium build up a system with four photoreceptors sensitive in different wavelength ranges and thought to account for colour vision. Evidence from wavelength discrimination tests suggests that all colour stimuli are assigned to one of just four colour categories, but cooperation of the two pathways is also evident. Flies use colour cues for various behavioural reactions such as flower visitation, proboscis extension, host finding, and egg deposition. Direct evidence for colour vision, the ability to discriminate colours according to spectral shape but independent of intensity, has been demonstrated for few fly species only. Indirect evidence for colour vision provided from electrophysiological recordings of the spectral sensitivity of photoreceptors and opsin genes indicates similar requisites in various flies; the flies' responses to coloured targets, however, are much more diverse.

Bumblebees (Bombus terrestris) and honeybees (Apis mellifera) prefer similar colours of higher spectral purity over trained colours.

Differences in the concentration of pigments as well as their composition and spatial arrangement cause intraspecific variation in the spectral signature of flowers. Known colour preferences and requirements for flower-constant foraging bees predict different responses to colour variability. In experimental settings, we simulated small variations of unicoloured petals and variations in the spatial arrangement of colours within tricoloured petals using artificial flowers and studied their impact on the colour choices of bumblebees and honeybees. Workers were trained to artificial flowers of a given colour and then given the simultaneous choice between three test colours: either the training colour, one colour of lower and one of higher spectral purity, or the training colour, one colour of lower and one of higher dominant wavelength; in all cases the perceptual contrast between the training colour and the additional test colours was similarly small. Bees preferred artificial test flowers which resembled the training colour with the exception that they preferred test colours with higher spectral purity over trained colours. Testing the behaviour of bees at artificial flowers displaying a centripetal or centrifugal arrangement of three equally sized colours with small differences in spectral purity, bees did not prefer any type of artificial flowers, but preferentially choose the most spectrally pure area for the first antenna contact at both types of artificial flowers. Our results indicate that innate preferences for flower colours of high spectral purity in pollinators might exert selective pressure on the evolution of flower colours.

Bees' subtle colour preferences: how bees respond to small changes in pigment concentration.

Variability in flower colour of animal-pollinated plants is common and caused, inter alia, by inter-individual differences in pigment concentrations. If and how pollinators, especially bees, respond to these small differences in pigment concentration is not known, but it is likely that flower colour variability impacts the choice behaviour of all flower visitors that exhibit innate and learned colour preferences. In behavioural experiments, we simulated varying pigment concentrations and studied its impact on the colour choices of bumblebees and honeybees. Individual bees were trained to artificial flowers having a specific concentration of a pigment, i.e. Acridine Orange or Aniline Blue, and then given the simultaneous choice between three test colours including the training colour, one colour of lower and one colour of higher pigment concentration. For each pigment, two set-ups were provided, covering the range of low to middle and the range of middle to high pigment concentrations. Despite the small bee-subjective perceptual contrasts between the tested stimuli and regardless of training towards medium concentrations, bees preferred neither the training stimuli nor the stimuli offering the highest pigment concentration but more often chose those stimuli offering the highest spectral purity and the highest chromatic contrast against the background. Overall, this study suggests that bees choose an intermediate pigment concentration due to its optimal conspicuousness. It is concluded that the spontaneous preferences of bees for flower colours of high spectral purity might exert selective pressure on the evolution of floral colours and of flower pigmentation.

Pollinator-mediated plant coexistence requires high levels of pollinator specialization.

In pollen-limited plant communities, the foraging behavior of pollinators might mediate coexistence and competitive exclusion of plant species by determining which plants receive conspecific pollen. A key question is whether realistic pollinator foraging behavior promotes coexistence or exclusion of plant species. We use a simulation model to understand how pollinator foraging behavior impacts the coexistence dynamics of pollen-limited plants. To determine whether pollinators are likely to provide a biologically important coexistence mechanism, we compare our results to bee foraging data from the literature and from a novel experimental analysis. Model results indicate that strong specialization at the level of individual foraging paths is required to promote coexistence. However, few empirical studies have robustly quantified within-bout specialization. Species-level data suggest that foraging behavior is sufficient to permit pollinator-mediated coexistence in species-poor plant communities and possibly in diverse communities where congeneric plants co-occur. Our experiments using bumblebees show that individual-level specialization does exist, but not at levels sufficient to substantially impact coexistence dynamics. The literature on specialization within natural foraging paths suffers from key limitations, but overall suggests that pollinator-mediated coexistence should be rare in diverse plant communities.

Mimicking orchids lure bees from afar with exaggerated ultraviolet signals.

Flowers have many traits to appeal to pollinators, including ultraviolet (UV) absorbing markings, which are well-known for attracting bees at close proximity (e.g., <1 m). While striking UV signals have been thought to attract pollinators also from far away, if these signals impact the plant pollinia removal over distance remains unknown. Here, we report the case of the Australian orchid Diuris brumalis, a nonrewarding species, pollinated by bees via mimicry of the rewarding pea plant Daviesia decurrens. When distant from the pea plant, Diuris was hypothesized to enhance pollinator attraction by exaggeratedly mimicking the floral ultraviolet (UV) reflecting patterns of its model. By experimentally modulating floral UV reflectance with a UV screening solution, we quantified the orchid pollinia removal at a variable distance from the model pea plants. We demonstrate that the deceptive orchid Diuris attracts bee pollinators by emphasizing the visual stimuli, which mimic the floral UV signaling of the rewarding model Daviesia. Moreover, the exaggerated UV reflectance of Diuris flowers impacted pollinators' visitation at an optimal distance from Da. decurrens, and the effect decreased when orchids were too close or too far away from the model. Our findings support the hypothesis that salient UV flower signaling plays a functional role in visual floral mimicry, likely exploiting perceptual gaps in bee neural coding, and mediates the plant pollinia removal at much greater spatial scales than previously expected. The ruse works most effectively at an optimal distance of several meters revealing the importance of salient visual stimuli when mimicry is imperfect.

Dynamic monitoring of vital functions and tissue re-organization in Saturnia pavonia (Lepidoptera, Saturniidae) during final metamorphosis by non-invasive MRI.

Magnetic resonance imaging (MRI) is the key whole-body imaging technology for observing processes within a living object providing excellent resolution and contrast between soft tissues. In the present work, we exploited the non-destructive properties of MRI to track longitudinally the dynamic changes that take place in developing pupae of the Emperor Moth (Saturnia pavonia) during the last days before eclosion. While in diapause pupae, body fluid was almost homogeneously distributed over the internal compartments, as soon as wings, legs, flight muscles and the head region were fully developed, a significant redistribution of water levels occurred between thoracic and abdominal regions. During the last two days before eclosion, the developing moths transferred substantial amounts of liquid into the gut and the labial gland, and in case of females, into developing eggs. Concomitantly, the volume of the air sacs increased drastically and their expansion/compression became clearly visible in time-resolved MR images. Furthermore, besides ventilation of the tracheal system, air sacs are likely to serve as volume reservoir for liquid transfer during development of the moths inside their pupal case. In parallel, we were able to monitor noninvasively lipid consumption, cardiac activity and haemolymph circulation during final metamorphosis.

Avoidance of achromatic colours by bees provides a private niche for hummingbirds.

That hummingbird-pollinated plants predominantly have red flowers has been known for decades, but well-investigated research studies are still rare. Preference tests have shown that hummingbirds do not have an innate preference for red colours. In addition, hummingbirds do not depend solely upon red flowers, because white-flowered hummingbird-pollinated plants are also common and temporarily abundant. Here we show that both white and red hummingbird-pollinated flowers differ from bee-pollinated flowers in their reflection properties for ultraviolet (UV) light. Hummingbird-pollinated red flowers are on average less UV reflective, and white hummingbird-pollinated flowers are more UV reflective than the same coloured bee-pollinated ones. In preference tests with artificial flowers, neotropical orchid bees prefer red UV-reflecting artificial flowers and white UV-nonreflecting flowers over red and white flowers with the opposite UV properties. By contrast, hummingbirds showed no preference for any colour in the same tests. Plotting floral colours and test stimuli into the honeybees' perceptual colour space suggests that the less attractive colours are achromatic for bees and therefore more difficult to detect against the background. This underlying colour preference in bees might provide hummingbirds with a private niche that is not attractive to bees.

Flower Color Evolution and the Evidence of Pollinator-Mediated Selection.

The evolution of floral traits in animal-pollinated plants involves the interaction between flowers as signal senders and pollinators as signal receivers. Flower colors are very diverse, effect pollinator attraction and flower foraging behavior, and are hypothesized to be shaped through pollinator-mediated selection. However, most of our current understanding of flower color evolution arises from variation between discrete color morphs and completed color shifts accompanying pollinator shifts, while evidence for pollinator-mediated selection on continuous variation in flower colors within populations is still scarce. In this review, we summarize experiments quantifying selection on continuous flower color variation in natural plant populations in the context of pollinator interactions. We found that evidence for significant pollinator-mediated selection is surprisingly limited among existing studies. We propose several possible explanations related to the complexity in the interaction between the colors of flowers and the sensory and cognitive abilities of pollinators as well as pollinator behavioral responses, on the one hand, and the distribution of variation in color phenotypes and fitness, on the other hand. We emphasize currently persisting weaknesses in experimental procedures, and provide some suggestions for how to improve methodology. In conclusion, we encourage future research to bring together plant and animal scientists to jointly forward our understanding of the mechanisms and circumstances of pollinator-mediated selection on flower color.

Visibility and attractiveness of Fritillaria (Liliaceae) flowers to potential pollinators.

Visual floral characters play an important role in shaping plant-pollinator interactions. The genus Fritillaria L. (Liliaceae), comprising approximately 140 species, is described as displaying a remarkable variety of flower colours and sizes. Despite this variation in visual floral traits of fritillaries, little is known about the potential role of these features in shaping plant-pollinator interactions. Here, we seek to clarify the role of visual attraction in species offering a robust food reward for pollinators early in the spring, which is the case for Fritillaria. We also searched for potential tendencies in the evolution of floral traits crucial for plant-pollinator communication. The generality of species with green and purple flowers may indicate an influence of environmental factors other than pollinators. The flowers of the studied species seem to be visible but not very visually attractive to potential pollinators. The food rewards are hidden within the nodding perianth, and both traits are conserved among fritillaries. Additionally, visual floral traits are not good predictors of nectar properties. When in the flowers, pollinators are navigated by nectar guides in the form of contrasting nectary area colouration. Flower colour does not serve as a phenotypic filter against illegitimate pollinators-red and orange bird-pollinated fritillaries are visible to bees.