Brood Odor Discrimination Abilities in Hygienic Honey Bees (Apis mellifera L.) Using Proboscis Extension Reflex Conditioning

To understand the effect of abnormal brood odors on the initiation or control of hygienic behavior in honey bees, we employed the associative learning paradigm, proboscis extension reflex conditioning. Bees from two genetic lines(hygienic and non-hygienic) were able to discriminate between high concentrations of two floral odors equally well. Differential discrimination abilities were observed between the two lines when healthy and diseased brood odors were used, with the bees from the hygienic line discriminating between the pair of brood odors better than the non-hygienic bees. These results suggest that hygienic behavior in individual bees is associated with the bees' responses to olfactory stimuli emanating from diseased brood.     

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees (Apis mellifera)

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability^1,2. Because the survival of a honeybee colony depends on the exploitation of food sources, forager bees learn and memorize variable flower sites as well as their profitability. Forager bees can be easily trained in natural settings where they forage at a feeding site and learn the related signals such as odor or color. Appetitive associative learning can also be studied under controlled conditions in the laboratory by conditioning the proboscis extension response (PER) of individually harnessed honeybees^3,4. This learning paradigm enables the study of the neuronal and molecular mechanisms that underlie learning and memory formation in a simple and highly reliable way^5-12. A behavioral pharmacology approach is used to study molecular mechanisms. Drugs are injected systemically to interfere with the function of specific molecules during or after learning and memory formation^13-16.Here we demonstrate how to train harnessed honeybees in PER conditioning and how to apply drugs systemically by injection into the bee flight muscle.     

Winter Active Bumblebees (Bombus terrestris) Achieve High Foraging Rates in Urban Britain

Background

Foraging bumblebees are normally associated with spring and summer in northern Europe. However, there have been sightings of the bumblebee Bombus terrestris during the warmer winters in recent years in southern England. But what floral resources are they relying upon during winter and how much winter forage can they collect?

Methodology/Principal Findings

To test if urban areas in the UK provide a rich foraging niche for bees we set up colonies of B. terrestris in the field during two late winter periods (2005/6 & 2006/7) in London, UK, and measured their foraging performance. Fully automatic radio-frequency identification (RFID) technology was used in 2006/7 to enable us to record the complete foraging activity of individually tagged bees. The number of bumblebees present during winter (October 2007 to March 2008) and the main plants they visited were also recorded during transect walks. Queens and workers were observed throughout the winter, suggesting a second generation of bee colonies active during the winter months. Mass flowering shrubs such as Mahonia spp. were identified as important food resources. The foraging experiments showed that bees active during the winter can attain nectar and pollen foraging rates that match, and even surpass, those recorded during summer.

Conclusions/Significance

B. terrestris in the UK are now able to utilise a rich winter foraging resource in urban parks and gardens that might at present still be under-exploited, opening up the possibility of further changes in pollinator phenology.     

Biotope Associations and the Decline of Bumblebees (Bombus spp.)

Much of the ecology of rare bumblebee species remains poorly understood and in need of further study. It has recently been suggested that differences in the range and rate of decline among bumblebee species may relate to differences in their degree of habitat specialization. We examine biotope use by 17 bumblebee species in the Hebrides, southern UK and South Island, New Zealand. We identify a cluster of widespread and abundant species that occur in almost all biotopes and exploit man-made environments such as gardens and arable margins, this group corresponding to the “mainland ubiquitous” species of previous studies. A second grouping of species includes those associated to varying degrees with heathland. It is notable that some species occupy markedly different biotopes in different parts of their range; for example B. soroeensis is found largely on upland heaths in the Hebrides, but on calcareous grassland in the south. Some species, such as B. subterraneus and B. distinguendus, now survive only in specific rare biotopes and could be mistaken for habitat specialists, but it is clear from their historic distributions that they formerly occupied a broader range of biotopes. Surviving populations of several of the species that have declined most (B. distinguendus, B. sylvarum, B. muscorum sladeni, B. humilis) exhibit a markedly coastal distribution, when once they were widespread inland. We suggest that this is probably simply because some coastal biotopes are less amenable to agricultural improvement, and so more have escaped the detrimental effects of intensive farming. Our results concur with previous suggestions that bumblebees are generally not habitat specialists, so that the conservation of most bumblebee species could be achieved by restoration of flower-rich unimproved meadows.     

Proboscis length and resource utilization in two Uruguayan bumblebees: Bombus atratus Franklin and Bombus bellicosus Smith (Hymenoptera: Apidae)

Bumblebees (Bombus sp.) are eusocial insects with an annual life cycle whose use as pollinator of crops has gained great importance in modern agriculture. Several authors have found that resource use in Bombus species is usually based on the correlation between the proboscis length of the bumblebees and the corolla depth of the flowers. The aim of this study was to determine proboscis length of Bombus atratus and B. bellicosus, two Uruguayan bumblebees, and verify the resource exploitation testing two cultivated species, the red clover and the bird's foot trefoil. Bumblebee foraging activity was recorded in two culture conditions: in a red clover and bird's foot trefoil mixed meadow, and in contiguous plots of these legumes, and the proboscis length of collected foragers was determined. Both species of bumblebees exploited red clover and bird's foot trefoil although they did it in different proportions in all instances tested. The results indicated that the choice of the resources in B. atratus and B. bellicosus was influenced by their proboscis length. Bombus atratus has a longer proboscis and preferably visited red clover, possibly obtaining nectar easier and faster than B. bellicosus, which has a shorter proboscis. Bombus bellicosus used both resources without any clear preference.     

Bumblebees (Bombus terrestris) use social information as an indicator of safety in dangerous environments

Avoiding predation is one of the most important challenges that an animal faces. Several anti-predation behaviours can be employed, yet simply using the presence of conspecifics can be a good signal of safety in an environment with potential predation hazards. Here, we show, for the first time, that past experience of predation causes bumblebees (Bombus terrestris) to aggregate with conspecifics, facilitating the identification of safe foraging patches. Bees were trained to differentiate between flowers that harboured predators and flowers that were predator free. When test subjects were subsequently presented solely with the previously predator-infested flower species, there was a significant preference to only land on flowers occupied by other feeding conspecifics. Yet, when safe flowers were made available to subjects previously entrained to discriminate safe from predator-occupied flowers, subjects ignored other bees and the social information potentially provided by them, demonstrating that attraction towards conspecifics is confined to dangerous situations. Our findings demonstrate a previously unknown social interaction in pollinators which may have important implications for plant–pollinator interactions.     

Daily Changes in Ultraviolet Light Levels Can Synchronize the Circadian Clock of Bumblebees (Bombus terrestris)

Endogenous circadian clocks are synchronized to the 24-h day by external zeitgebers such as daily light and temperature cycles. Bumblebee foragers show diurnal rhythms under daily light:dark cycles and short-period free-running circadian rhythms in constant light conditions in the laboratory. In contrast, during the continuous light conditions of the arctic summer, they show robust 24-h rhythms in their foraging patterns, meaning that some external zeitgeber must entrain their circadian clocks in the presence of constant light. Although the sun stays above the horizon for weeks during the arctic summer, the light quality, especially in the ultraviolet (UV) range, exhibits pronounced daily changes. Since the photoreceptors and photopigments that synchronize the circadian system of bees are not known, we tested if the circadian clocks of bumblebees (Bombus terrestris) can be entrained by daily cycles in UV light levels. Bumblebee colonies were set up in the laboratory and exposed to 12?h:12?h UV?+?:UV? cycles in otherwise continuous lighting conditions by placing UV filters on their foraging arenas for 12?h each day. The activity patterns of individual bees were recorded using fully automatic radiofrequency identification (RFID). We found that colonies manipulated in such a way showed synchronized 24-h rhythms, whereas simultaneously tested control colonies with no variation in UV light levels showed free-running rhythms instead. The results of our study show that bumblebee circadian rhythms can indeed be synchronized by daily cycles in ambient light spectral composition. (Author correspondence: r.stanewsky@qmul.ac.uk)     

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.     

Location and Color Learning in Bumblebees in a Two-Phase Conditioning Experiment

Bees use spatial and visual cues that characterize flowers to make dietary choices. If two such cues always appear together nonambiguously, they provide identical information. In such cases, do bees base dietary choices on one cue and ignore the other, or do they consider both cues? We allowed bumblebees to forage on two patches of artificial flowers that differed in location, color, and reward presence in a two-phase experiment. We switched either the display color, the location, or both the color and the location associated with the rewarding patch between phases. We tested for the effects of the switch on the bees' choices. Immediately following a switch in the location or both the location and the color of the rewarding patch, the bees' performance decreased, as they continued to visit the patch that was previously rewarding. This decrease did not occur when only the color of the rewarding patch was changed or in no-change controls. We suggest that the bees' foraging choices were guided mostly by a location cue when both the location and the color conveyed the same information.     

Effects of Chronic Morphine Treatment on an Odor Conditioning Paradigm,Locomotor Activity and Sucrose Responsiveness in Honeybees (Apis mellifera)

Honeybee is a widely used insect model for learning and memory research. Recently, it has become a potentially good subject for evaluating the effects of addictive drugs on the nervous systems. Our previous study has found that acute morphine injection affected associative memory and locomotor activity in honeybees. In the current study, the effect of chronic morphine treatment and its cessation in honeybees were assessed. The results demonstrated that 1) chronic morphine (0.01, 0.1 and 1 mg/ml) treatment for 7 days severely diminished associative memory in honeybees; 2) 1 mg/ml morphine consumption for 5, 7 and 10 but not 3 days impaired the olfactory memory; 3) Bees withdrawn from morphine for 1 day but not 3 days showed amnesia in the PER conditioning. We also found that bees displayed hyperactivity and tolerance in response to chronic morphine administration. In addition, morphine dose-dependently altered the sucrose responsiveness of bees. The data indicated that chronic morphine has sensory-motor effects and may impair learning and/or memory in honeybees, which were comparative to that in vertebrates.     

A Protocol to Assess Insect Resistance to Heat Waves,Applied to Bumblebees (Bombus Latreille, 1802)

Insect decline results from numerous interacting factors including climate change. One of the major phenomena related to climate change is the increase of the frequency of extreme events such as heat waves. Since heat waves are suspected to dramatically increase insect mortality, there is an urgent need to assess their potential impact. Here, we determined and compared the resistance to heat waves of insects under hyperthermic stress through their time before heat stupor (THS) when they are exposed to an extreme temperature (40°C). For this, we used a new experimental standardised device available in the field or in locations close to the field collecting sites. We applied this approach on different Arctic, Boreo-Alpine and Widespread bumblebee species in order to predict consequences of heat waves. Our results show a heat resistance gradient: the heat stress resistance of species with a centred arctic distribution is weaker than the heat resistance of the Boreo-Alpine species with a larger distribution which is itself lower than the heat stress resistance of the ubiquitous species.     

Proboscis extension response (PER) assay in Drosophila

Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.On the surface of the fly's mouth (labellum), there are hair-like structures called sensilla which houses taste neurons. When an attractive substance makes contact to the labellum, the fly extends its proboscis to consume the material. Proboscis Extension Response (PER) assay measures this taste behavior response, and it is a useful method to learn about food preferences in a single fly. Solutions of various sugars, such as sucrose, glucose and fructose, are very attractive to the fly. The effect of aversive substances can also be tested as reduction of PER when mixed in a sweet solution.Despite the simplicity of the basic procedure, there are many things that can prevent it from working. One of the factors that requires attention is the fly's responsive state. The required starvation time to bring the fly to the proper responsive state varies drastically from 36 to 72 hours. We established a series of controls to evaluate the fly's state and which allows screening out of non-responsive or hyper-responsive individual animals. Another important factor is the impact level and the position of the contact to the labellum, which would be difficult to describe by words. This video presentation demonstrates all these together with several other improvements that would increase the reproducibility of this method.     

Innate Flower Recognition in Bumblebees (Bombus terrestris,B. lucorum; Apidae): Optical Signals from Stamens as Landing Reaction Releasers

Innate behavioural reactions in bumblebees were observed in flower dummy experiments. In a previous paper, it was shown that the approach of flower-naive, untrained bumblebees toward flower dummies is released by colour signals, i.e. a gradient of spectral purity. The bumblebees are directed toward that part of a flower dummy which exhibits the highest spectral purity (Lunau 1990). In the present work it is shown that the bumblebees, while still in flight, make contact with this part using their antennal tips. The antennae reaction is followed by a landing reaction which is released by olfactory and optical signals. The specific optical signals of the guide area which release the landing reaction were investigated. Since in the flower dummies which were used, the guide had the highest spectral purity, most of the antennae reactions were executed at the guide. The antennae reaction allows for a precise orientation and may involve the stimulation of a specialized area of ommatidia. Minute changes in optical guide parameters could thus be detected. The following features of stamens which most effectively released the landing reaction were: a minimum of 2 theca dummies; an oval shape of theca dummies which face each other along the longitudinal axis (both these characteristics are found in genuine stamens); 4 mm distance between theca dummies (this coincides with the distance between the antennal tips of bumblebees); two equally sized theca dummies each with an area of 7 mm² (this size of theca dummies was preferred over larger or smaller ones; the latter parameter was not changed when the corolla size was varied). Colour parameters are not involved in releasing the landing reaction. Clearly, bumblebees possess an innate releasing mechanism (IRM) that responds to optical signals of anthers which release the landing reaction. The experimental results are in accordance with conclusions drawn from the comparative studies of Osche (1979, 1983a, 1983b) that many flower guides can be interpreted as stamen mimics. Therefore stamens as well as stamen mimics represent a highly standardized signal for the insect pollinators and they are well suited for innate orientation at flowers.     

Comparison and examination of Bombus occidentalis and Bombus impatiens (Hymenoptera: Apidae) in tomato greenhouses

Experiments were conducted in commercial tomato, Lycopersicon esculentum Miller (Solanaceae), greenhouses to compare the relative foraging effort of two bumble bee species, Bombus occidentalis Greene and Bombus impatiens Cresson, to examine interspecific competition between B. occidentalis and B. impatiens, and to determine whether bumble bee colonies grew to their full population potential in commercial tomato greenhouses. B. impatiens colonies had more brood and workers and made more foraging trips per hour than B. occidentalis colonies. However, B. impatiens returned to the colony without pollen loads and left their colonies without dropping off their pollen loads more frequently than B. occidentalis greenhouse colonies. Our data also suggest that the presence of B. impatiens had a detrimental effect on B. occidentalis populations. Furthermore, B. occidentalis colonies did not grow to their full population potential in tomato greenhouses, with fewer workers in greenhouse colonies than in colonies placed outside in a natural environment, or in colonies that were physically enclosed and protected from external mortality. Together, this study suggests that B. impatiens is a better pollinator than B. occidentalis. It also shows that unknown factors are limiting the size of B. occidentalis colonies in tomato greenhouses.     

Effects of clothianidin on Bombus impatiens (Hymenoptera: Apidae) colony health and foraging ability

We conducted laboratory experiments to investigate the lethal and sublethal effects of clothianidin on bumble bee, Bombus impatiens Cresson, colony health and foraging ability. Bumble bee colonies were exposed to 6 ppb clothianidin, representing the highest residue levels found in field studies on pollen, and a higher dose of 36 ppb clothianidin in pollen. Clothianidin did not effect pollen consumption, newly emerged worker weights, amount of brood or the number of workers, males, and queens at either dose. The foraging ability of worker bees tested on an artificial array of complex flowers also did not differ among treatments. These results suggest that clothianidin residues found in seed-treated canola and possibly other crops will not adversely affect the health of bumble bee colonies or the foraging ability of workers.     

Effects of Nosema bombi and its treatment fumagillin on bumble bee (Bombus occidentalis) colonies

We examined the effects of Nosema bombi (Microsporidia: Nosematidae) on colonies of bumble bees, Bombus occidentalis Greene (Hymenoptera: Apidae), used to pollinate tomatoes in commercial greenhouses. We assessed methods of detecting N. bombi and tested the effectiveness of fumagillin to control this parasite. N. bombi did not affect adult population size or amount of brood in B. occidentalis colonies. Fumagillin was not effective against N. bombi at the doses we tested, and frass samples did not provide accurate estimates of the intensity of N. bombi infections. The number of N. bombi spores per bee was highly variable among bumble bees within colonies, and accurate estimates could only be obtained by sampling a large proportion of bees in each colony. Therefore, whole bee and frass sampling is useful for determining if N. bombi is present or absent, but not for obtaining accurate estimates of the intensity of N. bombi infections.