Why do <Emphasis Type="Italic">Manduca sexta</Emphasis> feed from white flowers? Innate and learnt colour preferences in a hawkmoth

Flower colour is an important signal used by flowering plants to attract pollinators. Many anthophilous insects have an innate colour preference that is displayed during their first foraging bouts and which could help them locate their first nectar reward. Nevertheless, learning capabilities allow insects to switch their colour preferences with experience and thus, to track variation in floral nectar availability. Manduca sexta, a crepuscular hawkmoth widely studied as a model system for sensory physiology and behaviour, visits mostly white, night-blooming flowers lacking UV reflectance throughout its range in the Americas. Nevertheless, the spectral sensitivity of the feeding behaviour of naïve moths shows a narrow peak around 450 nm wavelengths, suggesting an innate preference for the colour blue. Under more natural conditions (i.e. broader wavelength reflectance) than in previous studies, we used dual choice experiments with blue- and white-coloured feeders to investigate the innate preference of naïve moths and trained different groups to each colour to evaluate their learning capabilities. We confirmed the innate preference of M. sexta for blue and found that these moths were able to switch colour preferences after training experience. These results unequivocally demonstrate that M. sexta moths innately prefer blue when presented against white flower models and offer novel experimental evidence supporting the hypothesis that learning capabilities could be involved in their foraging preferences, including their widely observed attraction to white flowers in nature.     

The influence of pigmentation patterning on bumblebee foraging from flowers of Antirrhinum majus

Patterns of pigmentation overlying the petal vasculature are common in flowering plants and have been postulated to play a role in pollinator attraction. Previous studies report that such venation patterning is significantly more attractive to bee foragers in the field than ivory or white flowers without veins. To dissect the ways in which venation patterning of pigment can influence bumblebee behaviour, we investigated the response of flower-naïve individuals of Bombus terrestris to veined, ivory and red near-isogenic lines of Antirrhinum majus. We find that red venation shifts flower colour slightly, although the ivory background is the dominant colour. Bees were readily able to discriminate between ivory and veined flowers under differential conditioning but showed no innate preference when presented with a free choice of rewarding ivory and veined flowers. In contrast, both ivory and veined flowers were selected significantly more often than were red flowers. We conclude that advantages conferred by venation patterning might stem from bees learning of their use as nectar guides, rather than from any innate preference for striped flowers.     

Individual lifetime pollen and nectar foraging preferences in bumble bees

Foraging specialization plays an important role in the ability of social insects to efficiently allocate labor. However, relatively little is known about the degree to which individual bumble bees specialize on collecting nectar or pollen, when such preferences manifest, and if individuals can alter their foraging preferences in response to changes in the colony workforce. Using Bombus impatiens, we monitored all foraging visits made by every bee in multiple colonies and showed that individual foragers exhibit consistent lifetime foraging preferences. Based upon the distribution of foraging preferences, we defined three forager types (pollen specialists, nectar specialists, and generalists). In unmanipulated colonies, 16–36?% of individuals specialized (≥90?% of visits) on nectar or pollen only. On its first day of foraging, an individual’s foraging choices (nectar only, pollen only, or nectar and pollen) significantly predicted its lifetime foraging preferences. Foragers that only collected pollen on their first day of foraging made 1.61- to 1.67-fold more lifetime pollen foraging visits (as a proportion of total trips) than foragers that only collected nectar on their first foraging day. Foragers were significantly larger than bees that stayed only in the nest. We also determined the effect of removing pollen specialists at early (brood present) or later (brood absent) stages in colony life. These results suggest that generalists can alter their foraging preferences in response to the loss of a small subset of foragers. Thus, bumble bees exhibit individual lifetime foraging preferences that are established early in life, but generalists may be able to adapt to colony needs.     

Floral colour change in <Emphasis Type="Italic">Byrsonima variabilis</Emphasis> (Malpighiaceae) as a visual cue for pollen but not oil foraging by oil-collecting bees

Pollinators search for multiple flora resources throughout their life cycle. Most studies, however, only assess how bees discriminate floral cues in the context of nectar foraging. In the present study, we sought to elucidate whether oil-collecting bees discriminate flowers of Byrsonima variabilis (Malpighiaceae) with petals of different colours when foraging for pollen or oil. As the colour of the standard petal changes during anthesis, we characterised the spectral reflectance patterns of flowers throughout anthesis and modelled chromatic perceptual space to determine how these colour patterns are perceived by bees. Through the quantification of flower pollen in the different phases, we found that the colour of the standard petal is an honest cue of the presence of pollen. Centridine bees preferentially visited flowers with a yellow (bee’s green) colour when searching for pollen, but indiscriminately visited flowers with different petal colours when searching for floral oil. We suggest that standard petals, in the species studied and others of the genus, like nectar guides, act as pollen guides, which oil-collecting females use to detect pollen-rich flowers. Moreover, they use different floral clues during foraging for different resources in the same host plant.     

Pollen foraging: learning a complex motor skill by bumblebees (<Emphasis Type="Italic">Bombus terrestris</Emphasis>)

To investigate how bumblebees (Bombus terrestris) learn the complex motor skills involved in pollen foraging, we observed naïve workers foraging on arrays of nectarless poppy flowers (Papaver rhoeas) in a greenhouse. Foraging skills were quantified by measuring the pollen load collected during each foraging bout and relating this to the number of flowers visited and bout duration on two consecutive days. The pollen standing crop (PSC) in each flower decreased drastically from 0530 to 0900 hours. Therefore, we related foraging performance to the changing levels of pollen available (per flower) and found that collection rate increased over the course of four consecutive foraging bouts (comprising between 277 and 354 individual flower visits), suggesting that learning to forage for pollen represents a substantial time investment for individual foragers. The pollen collection rate and size of pollen loads collected at the start of day 2 were markedly lower than at the end of day 1, suggesting that components of pollen foraging behaviour could be subject to imperfect overnight retention. Our results suggest that learning the necessary motor skills to collect pollen effectively from morphologically simple flowers takes three times as many visits as learning how to handle the most morphologically complex flowers to extract nectar, potentially explaining why bees are more specialised in their choice of pollen flowers.     

Visual targeting of components of floral colour patterns in flower-naïve bumblebees (Bombus terrestris; Apidae)

Floral colour patterns are contrasting colour patches on flowers, a part of the signalling apparatus that was considered to display shape and colour signals used by flower-visitors to detect flowers and locate the site of floral reward. Here, we show that flower-naïve bumblebees (Bombus terrestris) spontaneously direct their approach towards the outside margin of artificial flowers, which provides contrast between these dummy flowers and the background. If no floral guides are present, the bumblebees continue to approach the margin and finally touch the marginal area of the dummy flower with the tips of their antennae. Whilst approaching dummy flowers that also have a central floral guide, the bumblebees change their direction of flight: Initially, they approach the margin, later they switch to approaching the colour guide, and finally they precisely touch the floral guide with their antennae. Variation of the shape of equally sized dummy flowers did not alter the bumblebees’ preferential orientation towards the guide. Using reciprocal combinations of guide colour and surrounding colour, we showed that the approach from a distance towards the corolla and the antennal contact with the guide are elicited by the same colour parameter: spectral purity. As a consequence, the dummy flowers eliciting the greatest frequency of antennal reactions at the guide are those that combine a floral guide of high spectral purity with a corolla of less spectral purity. Our results support the hypothesis that floral guides direct bumblebees’ approaches to the site of first contact with the flower, which is achieved by the tips of the antennae.Electronic Supplementary Material  Supplementary material is available for this article at     

The cues have it; nest-based, cue-mediated recruitment to carbohydrate resources in a swarm-founding social wasp

This study explores whether or not foragers of the Neotropical swarm-founding wasp Polybia occidentalis use nest-based recruitment to direct colony mates to carbohydrate resources. Recruitment allows social insect colonies to rapidly exploit ephemeral resources, an ability especially advantageous to species such as P. occidentalis, which store nectar and prey in their nests. Although recruitment is often defined as being strictly signal mediated, it can also occur via cue-mediated information transfer. Previous studies indicated that P. occidentalis employs local enhancement, a type of cue-mediated recruitment in which the presence of conspecifics at a site attracts foragers. This recruitment is resource-based, and as such, is a blunt recruitment tool, which does not exclude non-colony mates. We therefore investigated whether P. occidentalis also employs a form of nest-based recruitment. A scented sucrose solution was applied directly to the nest. This mimicked a scented carbohydrate resource brought back by employed foragers, but, as foragers were not allowed to return to the nest with the resource, there was no possibility for on-nest recruitment behavior. Foragers were offered two dishes—one containing the test scent and the other an alternate scent. Foragers chose the test scent more often, signifying that its presence in the nest induces naïve foragers to search for it off-nest. P. occidentalis, therefore, employs a form of nest-based recruitment to carbohydrate resources that is mediated by a cue, the presence of a scented resource in the nest.     

Recruits of the stingless bee Scaptotrigona pectoralis learn food odors from the nest atmosphere

The ability to learn food odors inside the nest and to associate them with food sources in the field is of essential importance for the recruitment of nestmates in social bees. We investigated odor learning by workers within the hive and the influence of these odors on their food choice in the field in the stingless bee Scaptotrigona pectoralis. During the experiments, recruited bees had to choose between two feeders, one with an odor that was present inside the nest during the recruitment process, and one with an unknown odor. In all experiments with different odor combinations (linalool/phenylacetaldehyde, geraniol/eugenol) a significant majority of bees visited the feeder with the odor they had experienced in their nest (χ ²-tests; p?<?0.05). By contrast, the bees showed no preference for one of two feeders when they were either baited with the same odor (linalool) or contained no odor. Our results clearly show that naïve workers of S. pectoralis can learn the odor of a food source during the recruitment process from the nest atmosphere and that their subsequent food search in the field is influenced by the learned odor.     

How floral odours are learned inside the bumblebee (Bombus terrestris) nest

Recruitment in social insects often involves not only inducing nestmates to leave the nest, but also communicating crucial information about finding profitable food sources. Although bumblebees transmit chemosensory information (floral scent), the transmission mechanism is unknown as mouth-to-mouth fluid transfer (as in honeybees) does not occur. Because recruiting bumblebees release a pheromone in the nest that triggers foraging in previously inactive workers, we tested whether this pheromone helps workers learn currently rewarding floral odours, as found in food social learning in rats. We exposed colonies to artificial recruitment pheromone, paired with anise scent. The pheromone did not facilitate learning of floral scent. However, we found that releasing floral scent in the air of the colony was sufficient to trigger learning and that learning performance was improved when the chemosensory cue was provided in the nectar in honeypots; probably because it guarantees a tighter link between scent and reward, and possibly because gustatory cues are involved in addition to olfaction. Scent learning was maximal when anise-scented nectar was brought into the nest by demonstrator foragers, suggesting that previously unidentified cues provided by successful foragers play an important role in nestmates learning new floral odours.     

Foraging scent marks of bumblebees: footprint cues rather than pheromone signals

In their natural habitat foraging bumblebees refuse to land on and probe flowers that have been recently visited (and depleted) by themselves, conspecifics or other bees, which increases their overall rate of nectar intake. This avoidance is often based on recognition of scent marks deposited by previous visitors. While the term 'scent mark' implies active labelling, it is an open question whether the repellent chemicals are pheromones actively and specifically released during flower visits, or mere footprints deposited unspecifically wherever bees walk. To distinguish between the two possibilities, we presented worker bumblebees (Bombus terrestris) with three types of feeders in a laboratory experiment: unvisited control feeders, passive feeders with a corolla that the bee had walked over on its way from the nest (with unspecific footprints), and active feeders, which the bee had just visited and depleted, but which were immediately refilled with sugar-water (potentially with specific scent marks). Bumblebees rejected both active and passive feeders more frequently than unvisited controls. The rate of rejection of passive feeders was only slightly lower than that of active feeders, and this difference vanished completely when passive corollas were walked over repeatedly on the way from the nest. Thus, mere footprints were sufficient to emulate the repellent effect of an actual feeder visit. In confirmation, glass slides on which bumblebees had walked on near the nest entrance accumulated hydrocarbons (alkanes and alkenes, C23 to C31), which had previously been shown to elicit repellency in flower choice experiments. We conclude that repellent scent marks are mere footprints, which foraging bees avoid when they encounter them in a foraging context.     

Convergent evolution: floral guides, stingless bee nest entrances, and insectivorous pitchers

Several recent hypotheses, including sensory drive and sensory exploitation, suggest that receiver biases may drive selection of biological signals in the context of sexual selection. Here we suggest that a similar mechanism may have led to convergence of patterns in flowers, stingless bee nest entrances, and pitchers of insectivorous plants. A survey of these non-related visual stimuli shows that they share features such as stripes, dark centre, and peripheral dots. Next, we experimentally show that in stingless bees the close-up approach to a flower is guided by dark centre preference. Moreover, in the approach towards their nest entrance, they have a spontaneous preference for entrance patterns containing a dark centre and disrupted ornamentation. Together with existing empirical evidence on the honeybee's and other insects’ orientation to flowers, this suggests that the signal receivers of the natural patterns we examined, mainly Hymenoptera, have spontaneous preferences for radiating stripes, dark centres, and peripheral dots. These receiver biases may have evolved in other behavioural contexts in the ancestors of Hymenoptera, but our findings suggest that they have triggered the convergent evolution of visual stimuli in floral guides, stingless bee nest entrances, and insectivorous pitchers.     

The use of heterospecific scent marks by the sweat bee <Emphasis Type="Italic">Halictus aerarius</Emphasis>

To forage effectively amongst flowers, some bee species utilize olfactory cues left by previous visitors in addition to direct assessment of visual cues to identify rewarding flowers. This ability can be more advantageous if the bees can recognize and use scent marks left by heterospecifics, not just marks left by members of their own species. We conducted field experiments to investigate whether the sweat bee Halictus aerarius avoids visiting flowers of trailing water willow Justicia procumbens emptied by other bee species. We found that H. aerarius rejected the flowers visited by both heterospecifics and conspecifics. They also rejected visited flowers artificially replenished with nectar. Our results demonstrate that social bees outside the Apidae can detect marks left on flowers by heterospecifics but that (on this plant species) they are unable to discriminate against flowers by directly detecting nectar volume. H. aerarius exhibited different rejection rates according to the identity of the previous bee species. We suggest that the frequency of rejection responses may depend on the amount of chemical substances left by the previous bee. In general, the use of scent marks left by previous visitors is almost certainly advantageous, enabling foragers to avoid flowers with depleted nectar levels and thereby improving their foraging efficiency.     

Non-random nectar unloading interactions between foragers and their receivers in the honeybee hive

Nectar acquisition in the honeybee Apis mellifera is a partitioned task in which foragers gather nectar and bring it to the hive, where nest mates unload via trophallaxis (i.e. mouth-to-mouth transfer) the collected food for further storage. Because forager mates exploit different feeding places simultaneously, this study addresses the question of whether nectar unloading interactions between foragers and hive-bees are established randomly, as it is commonly assumed. Two groups of foragers were trained to exploit a different scented food source for 5 days. We recorded their trophallaxes with hive-mates, marking the latter ones according to the forager group they were unloading. We found non-random probabilities for the occurrence of trophallaxes between experimental foragers and hive-bees, instead, we found that trophallactic interactions were more likely to involve groups of individuals which had formerly interacted orally. We propose that olfactory cues present in the transferred nectar promoted the observed bias, and we discuss this bias in the context of the organization of nectar acquisition: a partitioned task carried out in a decentralized insect society.     

Honeybees learn floral odors while receiving nectar from foragers within the hive

Recent studies showed that nectar odors brought back by honeybee foragers can be learned associatively inside the hive. In the present study, we focused on the learning abilities of bees, which directly interact via trophallaxis with the incoming nectar foragers: the workers that perform nectar-receiving tasks inside the hive. Workers that have received food directly from foragers coming back from a feeder offering either unscented or scented sugar solution [phenylacetaldehyde (PHE) or nonanal diluted] were captured from two observational hives, and their olfactory memories were tested using the proboscis extension response paradigm. Bees that have received scented solution from incoming foragers showed significantly increased response frequencies for the corresponding solution odor in comparison with those that have received unscented solution. No differences in the response frequencies were found between food odors and colonies. The results indicate that first-order receivers learn via trophallaxis the association between the scent and the sugar solution transferred by incoming foragers. The implications of these results should be considered at three levels: the operational cohesion of bees involved in foraging-related tasks, the information propagation inside the hive related to the floral type exploited, and the putative effect of these memories on future preferences for resources.     

Floral odor learning within the hive affects honeybees’ foraging decisions

Honeybees learn odor cues quickly and efficiently when visiting rewarding flowers. Memorization of these cues facilitates the localization and recognition of food sources during foraging flights. Bees can also use information gained inside the hive during social interactions with successful foragers. An important information cue that can be learned during these interactions is food odor. However, little is known about how floral odors learned in the hive affect later decisions of foragers in the field. We studied the effect of food scent on foraging preferences when this learning is acquired directly inside the hive. By using in-hive feeders that were removed 24 h before the test, we showed that foragers use the odor information acquired during a 3-day stimulation period with a scented solution during a food-choice situation outside the nest. This bias in food preference is maintained even 24 h after the replacement of all the hive combs. Thus, without being previously collected outside by foragers, food odors learned within the hive can be used during short-range foraging flights. Moreover, correct landings at a dual-choice device after replacing the storing combs suggests that long-term memories formed within the colony can be retrieved while bees search for food in the field.     

Nectar production dynamics and sugar composition in two <Emphasis Type="Italic">Mucuna</Emphasis> species (Leguminosae,Faboideae) with different specialized pollinators

Nectar is secreted in particular rhythms throughout the lifespan of a flower, which allows determining the nectar production dynamics. This paper compares nectar features in Mucuna japira and Mucuna urens describing: dynamics of nectar production, floral response to nectar removal, resorption, nectar sugar composition, and variation in nectar sugar composition. M. japira inflorescence bears 12–21 yellow flowers, which are in anthesis for 7 days, whereas M. urens inflorescence bears 36–54 greenish flowers, but only 1–3 flowers are in anthesis simultaneously that last one night. Nectar volume and sugar concentration were measured, and the amount of sugar was estimated. Qualitative and quantitative nectar sugar composition was determined. Both species had a constant nectar sugar concentration (ca. 10% for M. japira and ca. 16% for M. urens) and secreted high volumes of nectar (ca. 340 μl per flower for M. japira and 310 μl per flower for M. urens), during 5 days for M. japira and 6 h for M. urens, but after the first removal, i.e., when flower opening mechanism is triggered, nectar production stops immediately. Nectar resorption occurred in both species. Nectar sugar composition showed some similarities between the species. Variation in nectar sugar composition occurred in both species. The Mucuna species are dependent on their pollinators to produce fruits and seeds, and they have different strategies to promote the necessary interaction with birds or bats, especially related to nectar and flower characteristics.     

Bumblebees exhibit the memory spacing effect

Associative learning is key to how bees recognize and return to rewarding floral resources. It thus plays a major role in pollinator floral constancy and plant gene flow. Honeybees are the primary model for pollinator associative learning, but bumblebees play an important ecological role in a wider range of habitats, and their associative learning abilities are less well understood. We assayed learning with the proboscis extension reflex (PER), using a novel method for restraining bees (capsules) designed to improve bumblebee learning. We present the first results demonstrating that bumblebees exhibit the memory spacing effect. They improve their associative learning of odor and nectar reward by exhibiting increased memory acquisition, a component of long-term memory formation, when the time interval between rewarding trials is increased. Bombus impatiens forager memory acquisition (average discrimination index values) improved by 129% and 65% at inter-trial intervals (ITI) of 5 and 3 min, respectively, as compared to an ITI of 1 min. Memory acquisition rate also increased with increasing ITI. Encapsulation significantly increases olfactory memory acquisition. Ten times more foragers exhibited at least one PER response during training in capsules as compared to traditional PER harnesses. Thus, a novel conditioning assay, encapsulation, enabled us to improve bumblebee-learning acquisition and demonstrate that spaced learning results in better memory consolidation. Such spaced learning likely plays a role in forming long-term memories of rewarding floral resources.     

Flower Constancy, Insect Psychology, and Plant Evolution

 Individuals of some species of pollinating insects tend to restrict their visits to only a few of the available plant species, in the process bypassing valuable food sources. The question of why this flower constancy exists is a rich and important one with implications for the organization of natural communities of plants, floral evolution, and our understanding of the learning processes involved in finding food. Some scientists have assumed that flower constancy is adaptive per se. Others argued that constancy occurs because memory capacity for floral features in insects is limited, but attempts to identify the limitations often remained rather simplistic. We elucidate now different sensory and motor memories from natural foraging tasks are stored and retrieved, using concepts from modern learning science and visual search, and conclude that flower constancy is likely to have multiple causes. Possible constraints favoring constancy are interference sensitivity of short-term memory, and temporal limitations on retrieving information from long-term memory as rapidly as from short-term memory, but further empirical evidence is needed to substantiate these possibilities. In addition, retrieving memories may be slower and more prone to errors when there are several options than when an insect copes with only a single task. In addition to memory limitations, we also point out alternative explanations for flower constancy. We then consider the way in which floral parameters, such as interplant distances, nectar rewards, flower morphology, and floral color (as seen through bees' eyes) affect constancy. Finally, we discuss the implications of pollinator constancy for plant evolution. To date there is no evidence that flowers have diverged to favor constancy, although the appropriate tests may not have yet been conducted. However, there is good evidence against the notion that pollinator constancy is involved in speciation or maintenance of plant species integrity.     

Bird pollination of Canary Island endemic plants

The Canary Islands are home to a guild of endemic, threatened bird-pollinated plants. Previous work has suggested that these plants evolved floral traits as adaptations to pollination by flower specialist sunbirds, but subsequently, they appear to have co-opted generalist passerine birds as sub-optimal pollinators. To test this idea, we carried out a quantitative study of the pollination biology of three of the bird-pollinated plants, Canarina canariensis (Campanulaceae), Isoplexis canariensis (Veronicaceae) and Lotus berthelotii (Fabaceae), on the island of Tenerife. Using colour vision models, we predicted the detectability of flowers to bird and bee pollinators. We measured pollinator visitation rates, nectar standing crops as well as seed-set and pollen removal and deposition. These data showed that the plants are effectively pollinated by non-flower specialist passerine birds that only occasionally visit flowers. The large nectar standing crops and extended flower longevities (>10 days) of Canarina and Isoplexis suggests that they have evolved a bird pollination system that effectively exploits these low frequency non-specialist pollen vectors and is in no way sub-optimal. Seed set in two of the three species was high and was significantly reduced or zero in flowers where pollinator access was restricted. In L. berthelotii, however, no fruit set was observed, probably because the plants were self-incompatible horticultural clones of a single genet. We also show that, while all three species are easily detectable for birds, the orange Canarina and the red Lotus (but less so the yellow-orange Isoplexis) should be difficult to detect for insect pollinators without specialised red receptors, such as bumblebees. Contrary to expectations if we accept that the flowers are primarily adapted to sunbird pollination, the chiffchaff (Phylloscopus canariensis) was an effective pollinator of these species.     

Pollination syndromes ignored: importance of non-ornithophilous flowers to Neotropical savanna hummingbirds

Generalization prevails in flower–animal interactions, and although animal visitors are not equally effective pollinators, most interactions likely represent an important energy intake for the animal visitor. Hummingbirds are nectar-feeding specialists, and many tropical plants are specialized toward hummingbird-pollination. In spite of this, especially in dry and seasonal tropical habitats, hummingbirds may often rely on non-ornithophilous plants to meet their energy requirements. However, quantitative studies evaluating the relative importance of ornithophilous vs. non-ornithophilous plants for hummingbirds in these areas are scarce. We here studied the availability and use of floral resources by hummingbirds in two different areas of the Cerrado, the seasonal savannas in Central Brazil. Roughly half the hummingbird visited plant species were non-ornithophilous, and these contributed greatly to increase the overall nectar availability. We showed that mean nectar offer, at the transect scale, was the only parameter related to hummingbird visitation frequency, more so than nectar offer at single flowers and at the plant scale, or pollination syndrome. Centrality indices, calculated using hummingbird–plant networks, showed that ornithophilous and non-ornithophilous plants have similar importance for network cohesion. How this foraging behaviour affects reproduction of non-ornithophilous plants remains largely unexplored and is probably case specific, however, we suggest that the additional energy provided by non-ornithophilous plants may facilitate reproduction of truly ornithophilous flowers by attracting and maintaining hummingbirds in the area. This may promote asymmetric hummingbird–plant associations, i.e., pollination depends on floral traits adapted to hummingbird morphology, but hummingbird visitation is determined more by the energetic "reward" than by pollination syndromes.