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.     

Colour preferences influences odour learning in the hawkmoth, Macroglossum stellatarum

The hummingbird hawkmoth, Macroglossum stellatarum, learns colour fast and reliably. It has earlier been shown to spontaneously feed from odourless artificial flowers. Now, we have studied odour learning. The moths were trained to discriminate feeders of the same colour but marked with different odours. They did not learn to discriminate two natural flower odours when they were presented with the innately preferred colour blue, but they did learn this discrimination combined with yellow or green colours that are less attractive to the moth. The yellow colour could be trained to become as attractive as the innately preferred blue colour and the blue colour could be trained to become less attractive. This is the first proof of odour learning in a diurnal moth. The results show that M. stellatarum can use more than one modality in their foraging behaviour and that the system is plastic. By manipulating the preferences for the different colours, their influence on odour learning could be changed.     

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.     

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.     

A comparative analysis of colour preferences in temperate and tropical social bees

The spontaneous occurrence of colour preferences without learning has been demonstrated in several insect species; however, the underlying mechanisms are still not understood. Here, we use a comparative approach to investigate spontaneous and learned colour preferences in foraging bees of two tropical and one temperate species. We hypothesised that tropical bees utilise different sets of plants and therefore might differ in their spontaneous colour preferences. We tested colour-naive bees and foragers from colonies that had been enclosed in large flight cages for a long time. Bees were shortly trained with triplets of neutral, UV-grey stimuli placed randomly at eight locations on a black training disk to induce foraging motivation. During unrewarded tests, the bees’ responses to eight colours were video-recorded. Bees explored all colours and displayed an overall preference for colours dominated by long or short wavelengths, rather than a single colour stimulus. Naive Apis cerana and Bombus terrestris showed similar choices. Both inspected long-wavelength stimuli more than short-wavelength stimuli, whilst responses of the tropical stingless bee Tetragonula iridipennis differed, suggesting that resource partitioning could be a determinant of spontaneous colour preferences. Reward on an unsaturated yellow colour shifted the bees’ preference curves as predicted, which is in line with previous findings that brief colour experience overrides the expression of spontaneous preferences. We conclude that rather than determining foraging behaviour in inflexible ways, spontaneous colour preferences vary depending on experimental settings and reflect potential biases in mechanisms of learning and decision-making in pollinating insects.     

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.     

Biogenic amine levels, reproduction and social dominance in the queenless ant Streblognathus peetersi

Social harmony often relies on ritualised dominance interactions between society members, particularly in queenless ant societies, where colony members do not have developmentally predetermined castes but have to fight for their status in the reproductive and work hierarchy. In this behavioural plasticity, their social organisation resembles more that of vertebrates than that of the “classic” social insects. The present study investigates the neurochemistry of the queenless ant species, Streblognathus peetersi, to better understand the neural basis of the high behavioural plasticity observed in queenless ants. We report measurements of brain biogenic amines [octopamine, dopamine, serotonin] of S. peetersi ants; they reveal a new set of biogenic amine influences on social organisation with no common features with other “primitively organised societies” (bumble bees) and some common features with “highly eusocial” species (honey bees). This similarity to honey bees may either confirm the heritage of queenless species from their probably highly eusocial ancestors or highlight independent patterns of biogenic amine influences on the social organisation of these highly derived species.     

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     

Bees use three-dimensional information to improve target detection

Bumblebee detection of a flat circular disc (two-dimensional (2D) presentation) and a disc which was presented 10 cm in front of a structured background (and thus provided three-dimensional (3D) cues) was compared. A dual choice test using a Y-maze apparatus was conducted to estimate the minimum visual angle at which the bees were able to detect the disc. At large visual angles of 15, 10 and 5° bees’ performance between the 2D and the 3D presentation did not differ. However, when the disc subtended 3° at the bee’s eye, the bees performed significantly better when 3D information was available. Overall, bees were able to detect a target subtending a 40% smaller visual angle when it was presented in front of the structured background compared to a 2D presentation. This suggests that previous reports on the limits of target detection in bees using flat stimuli might have underestimated the bees’ ability to locate small flowers under natural conditions. Bees use motion parallax, i.e. the apparent relative motion of a stationary object against a background, for perceiving the third dimension. Our data suggest that bumblebees can integrate information from at least two types of feature detectors, motion and area, to improve single target detection.     

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.     

Experience,but not distance,influences the recruitment precision in the stingless bee <Emphasis Type="Italic">Scaptotrigona mexicana</Emphasis>

Recruitment precision, i.e. the proportion of recruits that reach an advertised food source, is a crucial adaptation of social bees to their environment. Studies with honeybees showed that recruitment precision is not a fixed feature, but it may be enhanced by factors like experience and distance. However, little is known regarding the recruitment precision of stingless bees. Hence, in this study, we examined the effects of experience and spatial distance on the precision of the food communication system of the stingless bee Scaptotrigona mexicana. We conducted the experiments by training bees to a three-dimensional artificial patch at several distances from the colony. We recorded the choices of individual recruited foragers, either being newcomers (foragers without experience with the advertised food source) or experienced (foragers that had previously visited the feeder). We found that the average precision of newcomers (95.6 ± 2.61%) was significantly higher than that of experienced bees (80.2 ± 1.12%). While this might seem counter-intuitive on first sight, this “loss” of precision can be explained by the tendency of experienced recruits to explore nearby areas to find new rewarding food sources after they had initially learned the exact location of the food source. Increasing the distance from the colony had no significant effect on the precision of the foraging bees. Thus, our data show that experience, but not the distance of the food source, affected the patch precision of S. mexicana foragers.     

When males whistle at females: complex FM acoustic signals in cockroaches

Male cockroaches of the species Elliptorhina chopardi expel air through a pair of modified abdominal spiracles during courtship. This air expulsion simultaneously produces air and substrate-borne vibrations. We described and compared in details these two types of vibrations. Our analysis of the air-borne signals shows that males can produce three categories of signals with distinct temporal and frequency parameters. “Pure whistles” consist of two independent harmonic series fast frequency modulated with independent harmonics that can cross each other. “Noisy whistles” also possess two independent voices but include a noisy broad-band frequency part in the middle. Hiss sounds are more noise-like, being made of a broad-band frequency spectrum. All three call types are unusually high in dominant frequency (>5 kHz) for cockroaches. The substrate-borne signals are categorised similarly. Some harmonics of the substrate-borne signals were filtered out, however, making the acoustic energy centered on fewer frequency bands. Our analysis shows that cockroach signals are complex, with fast frequency modulations and two distinct voices. These results also readdress the question of what system could potentially receive and decode the information contained within such complex sounds.Electronic supplementary material  Supplementary material is available for this article at and is accessible for authorized users.     

Dynamics of sperm transfer in the ant Leptothorax gredleri

Mating tactics differ remarkably between and within species of social Hymenoptera (bees, wasps, ants) concerning, e.g., mating frequencies, sperm competition, and the degree of male sperm limitation. Although social Hymenoptera might, therefore, potentially be ideal model systems for testing sexual selection theory, the dynamics of mating and sperm transfer have rarely been studied in species other than social bees, and basic information needed to draw conclusions about possible sperm competition and female choice is lacking. We investigated sperm transfer in the ant Leptothorax gredleri, a species in which female sexuals attract males by “female calling.” The analysis of 38 female sexuals fixed immediately or up to 7 days after copulation with a single male each revealed that the sperm is transferred into the female bursa copulatrix embedded in a gelatinous mass, presumably a spermatophore. Sperm cells rapidly start to migrate from the tip of the spermatophore towards the spermatheca, but transfer is drastically slowed down by an extreme constriction of the spermathecal duct, through which sperm cells have to pass virtually one by one. This results in the spermatheca being filled only between one and several hours after mating. During this time, the posterior part of the spermatophore seals the junction between bursa copulatrix and spermathecal duct and prevents sperm loss. The prolonged duration of sperm transfer might allow female sexuals to chose between ejaculates and explain previously reported patterns of single paternity of the offspring of multiply mated queens. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Do females pay attention to secondary sexual coloration in vervet monkeys (Chlorocebus aethiops)?

Several primate species show sexual dichromatism with males displaying conspicuous coloration of the pelage or skin. Studies of scrotal coloration in male vervet monkeys (Chlorocebus aethiops) suggest that it is an important intrasexual signal, with relatively dark, colourful males dominating paler males. To date, no studies have examined the influence of male colour on intersexual social interactions in vervet monkeys. The primary goal of the present study was to evaluate whether female vervet monkeys attend to male coloration. We experimentally introduced females, housed with either “pale” or “dark” males, to stimulus males whose scrota were pale, dark, or pale but painted to look dark. Overall, during introductions, females did not differ in time spent directing affiliative behaviour toward pale, dark, and painted males; however, females, permanently housed with dark males, spent significantly more time directing affiliative behaviour toward pale than painted males. When the stimulus male was pale, affiliative exchanges between males and females were longer than when the stimulus male was painted. Home male colour was not related to female-initiated aggression. Home male colour was also not related to male-initiated aggression, although painted stimulus males were more likely to initiate aggression than pale stimulus males. These findings lead us to conclude that females pay attention to male coloration, but do not bias their interactions toward males solely on the basis of natural male coloration.     

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.     

How do water striders, Aquarius paludum, react to brackish water simulated by NaCl solutions?

Several stages, from eggs to adults, of the water strider, Aquarius paludum (Fabricius), inhabiting fresh water are sometimes conveyed by heavy flow in the rainy or typhoon seasons in Japan to lotic brackish water in the mouth of rivers. The water striders might then respond to salinity either by remaining to wait for extensive rainfall to reduce osmotic pressure locally before reproducing (“breed here and later tactic”) or by flying away to reproduce in fresh waters elsewhere (“breed elsewhere and later tactic”). All first instars died before the first molt when they were exposed to 1.75 and 3.5% NaCl solutions in a laboratory experiment. Living on 0.5 and 0.9% solutions through larval and adult stages slowed down larval growth and suppressed female reproduction. When exposed to the 0.5 and 0.9% solutions, 90 and 92% of males, respectively, showed histolysis of their flight muscles. Therefore, in brackish natural habitats, larvae and adults seem to follow the strategy “breed here and later.” When water striders were exposed to 0.9% solution either just after emergence or 20 days later, females showed a higher flight propensity than those kept on fresh waters throughout, and they delayed the deposition of eggs. Therefore, when conveyed to brackish water after emergence by stream flow after heavy rain, adults seem to leave the area by flight, demonstrating the strategy “breed elsewhere and later” tactic. We conclude that water striders use alternative tactics for responding to salinity, depending on the stage of exposure.     

RNAi-mediated silencing of vitellogenin gene function turns honeybee (Apis mellifera) workers into extremely precocious foragers

The switch from within-hive activities to foraging behavior is a major transition in the life cycle of a honeybee (Apis mellifera) worker. A prominent regulatory role in this switch has long been attributed to juvenile hormone (JH), but recent evidence also points to the yolk precursor protein vitellogenin as a major player in behavioral development. In the present study, we injected vitellogenin double-stranded RNA (dsVg) into newly emerged worker bees of Africanized genetic origin and introduced them together with controls into observation hives to record flight behavior. RNA interference-mediated silencing of vitellogenin gene function shifted the onset of long-duration flights (>10 min) to earlier in life (by 3–4 days) when compared with sham and untreated control bees. In fact, dsVg bees were observed conducting such flights extremely precociously, when only 3 days old. Short-duration flights (<10 min), which bees usually perform for orientation and cleaning, were not affected. Additionally, we found that the JH titer in dsVg bees collected after 7 days was not significantly different from the controls. The finding that depletion of the vitellogenin titer can drive young bees to become extremely precocious foragers could imply that vitellogenin is the primary switch signal. At this young age, downregulation of vitellogenin gene activity apparently had little effect on the JH titer. As this unexpected finding stands in contrast with previous results on the vitellogenin/JH interaction at a later age, when bees normally become foragers, we propose a three-step sequence in the constellation of physiological parameters underlying behavioral development. David Santos Marco Antonio and Karina Rosa Guidugli-Lazzarini contributed equally to the present study.     

Fine colour discrimination requires differential conditioning in bumblebees

Accurate recognition requires that visual systems must be able to discriminate between target and distractor stimuli. Flowers are learned and recognised by bees using visual cues including colour and shape. We investigated whether bees were able to learn to discriminate between colours differently depending upon absolute or differential conditioning. For absolute conditioning bees were rewarded with sucrose solution for visits to target flowers. When distractor stimuli were subsequently presented, a high level of discrimination was observed if there was a perceptually large colour distance separating distractors and targets, but for a perceptually small colour distance the bees generalised and did not discriminate between stimuli. When provided with differential conditioning where both target and distractors were present, the bees learnt to discriminate stimuli separated by a perceptually small colour distance. This shows that for bees to learn fine colour discrimination tasks it is important to use differential conditioning. The findings are discussed within the context of the necessity for plants to produce distinctive flower colours.     

Optical properties of the uropygial gland secretion: no evidence for UV cosmetics in birds

Ultraviolet (UV) reflectance of the plumage is common in birds and plays an important role in sexual signalling. Recently, it has been proposed that birds are able to modify plumage UV reflectance by the application of uropygial gland secretion. Based on a survey of the optical properties of this secretion from 51 species belonging to 12 avian orders, we show that two main types of uropygial secretions exist, one predominantly found in passerines and one in non-passerines, both reducing relative UV reflectance of a white background (Teflon™ tape). We quantified how each type of secretion (exemplified by blue tit and mallard) affected feather UV reflectance. Both secretions reduced overall brightness and relative UV reflectance of white mallard feathers but hardly affected the reflectance of UV/blue blue tit crown feathers. According to models of avian colour vision, changes in reflectance due to application of the secretion were at or below the discrimination threshold of most birds. We conclude that the uropygial secretion is unlikely to play a major role in modifying plumage UV reflectance. However, the optical properties of the uropygial secretion may have been selected to interfere as little as possible with visual signaling through plumage reflectance. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Flower-visiting behavior of male bees is triggered by nectar-feeding insects

Bees are important pollinators for many flowering plants. Female bees are thought to be more effective pollinators than male bees because they carry much more pollen than males. Males of some solitary bee species are known to patrol near flowers that females visit. Because patrolling males visit flowers to mate or defend their territories, they may function as pollinators. However, the significance of patrolling males to pollination has not been studied. We studied males of a solitary bee, Heriades fulvohispidus (Megachilidae), patrolling near flowers and visiting flowers that attracted nectar-feeding insects, including conspecifics, on the Ogasawara (Bonin) Islands. To test the hypothesis that patrolling male bees may function as pollen vectors, we compared the frequency of visits by H. fulvohispidus to flowers of an endemic plant, Schima mertensiana (Theaceae); comparisons were made among flowers with a dead H. fulvohispidus, a dead beetle, a piece of plastic, and nothing (control flowers). Patrolling H. fulvohispidus more frequently visited flowers with a dead conspecific, a dead beetle, or a piece of plastic than the control flowers. Our experiment demonstrates that nectar-feeding insects (including conspecifics and other insects) enhance the flower-visiting frequency of patrolling H. fulvohispidus males on S. mertensiana flowers. Furthermore, we observed S. mertensiana pollen on patrolling males as well as females, suggesting that male bees may also function as pollen vectors.