Colour learning when foraging for nectar and pollen: bees learn two colours at once

Bees are model organisms for the study of learning and memory, yet nearly all such research to date has used a single reward, nectar. Many bees collect both nectar (carbohydrates) and pollen (protein) on a single foraging bout, sometimes from different plant species. We tested whether individual bumblebees could learn colour associations with nectar and pollen rewards simultaneously in a foraging scenario where one floral type offered only nectar and the other only pollen. We found that bees readily learned multiple reward–colour associations, and when presented with novel floral targets generalized to colours similar to those trained for each reward type. These results expand the ecological significance of work on bee learning and raise new questions regarding the cognitive ecology of pollination.     

An arthropod deterrent attracts specialised bees to their host plants

Many bee species are adapted to just a few specific plants in order to collect pollen (oligolecty). To reproduce successfully, it is important for oligolectic bees to find and recognise the specific host flowers. In this study, we investigated the role of floral volatiles used by an oligolectic bee to recognise its host plants. We compared the attractiveness of natural and synthetic scent samples of host flowers to foraging-naïve and -experienced Hoplitis adunca (Megachilidae) bees that are specialised on Echium and Pontechium (Boraginaceae) plants. The investigations showed that naïve H. adunca females are attracted to 1,4-benzoquinone. During their lifetime, bees learn additional floral cues while foraging on host flowers. In contrast to naïve ones, experienced H. adunca females use, in addition to 1,4-benzoquinone, other compounds to recognise their host plants. 1,4-Benzoquinone is an uncommon floral compound only known from the host plants of H. adunca, and is therefore ideally suited to be used as a plant-specific recognition cue. Several arthropods use this compound to deter insect predators. Therefore, 1,4-benzoquinone as an attractant in Echium flowers may have evolved from a primary function as a defensive compound against insect herbivores.     

The evolution of floral scent: the influence of olfactory learning by insect pollinators on the honest signalling of floral rewards

1.  The evolution of flowering plants has undoubtedly been influenced by a pollinator's ability to learn to associate floral signals with food. Here, we address the question of 'why' flowers produce scent by examining the ways in which olfactory learning by insect pollinators could influence how floral scent emission evolves in plant populations.
2.  Being provided with a floral scent signal allows pollinators to learn to be specific in their foraging habits, which could, in turn, produce a selective advantage for plants if sexual reproduction is limited by the income of compatible gametes. Learning studies with honeybees predict that pollinator-mediated selection for floral scent production should favour signals which are distinctive and exhibit low variation within species because these signals are learned faster. Social bees quickly learn to associate scent with the presence of nectar, and their ability to do this is generally faster and more reliable than their ability to learn visual cues.
3.  Pollinators rely on floral scent as a means of distinguishing honestly signalling flowers from deceptive ones. Furthermore, a pollinator's sensitivity to differences in nectar rewards can bias the way that it responds to floral scent. This mechanism may select for flowers that provide olfactory signals as an honest indicator of the presence of nectar or which select against the production of a detectable scent signal when no nectar is present.
4.  We expect that an important yet commonly overlooked function of floral scent is an improvement in short-term pollinator specificity which provides an advantage to both pollinator and plant over the use of a visual signal alone. This, in turn, impacts the evolution of plant mating systems via its influence on the species-specific patterns of floral visitation by pollinators.     

蜜蜂对复合气味中特征因素的选择性记忆巩固的研究

目的 蜜蜂天生具有丰富的嗅觉辨识能力,觅食、交配、导航以及社交活动均依赖其嗅觉系统,是研究嗅觉感知和学习记忆的行为及神经机制的理想模型。蜜蜂既能够将某个复合气味作为一个整体也可以将复合气味的各组成成分进行辨别和区分,但是在特征依赖的联合记忆中依据何种原则进行加工并存储到长期记忆还不清楚。方法 本文利用特征阳性（feature positive：AB+,B-）和特征阴性（feature negative：AB-,B+）的奖赏性嗅觉条件化,训练蜜蜂对复合气味和成分气味的辨别,并检测蜜蜂对复合气味（AB）、成分气味（B）以及特征气味（A）的中长时记忆（3 h）和长时记忆（24 h）。结果 在特征阳性的奖赏性嗅觉条件化中,蜜蜂对训练过的气味可以形成稳定的中长时和长时记忆,并且对复合气味中的特征气味的记忆与复合气味的记忆呈现高度相似。但在特征阴性的奖赏性嗅觉条件化中,蜜蜂虽能够在3 h和24 h对训练过的两种气味具有显著的伸喙反应差异,且对特征阴性的气味无显著反应,但对复合气味的反应随时间的推移而增加。结论 实验结果表明,蜜蜂选择性地将与奖赏信息联合出现的气味巩固到长时记忆中,但并未依据特征成分加工储存到长时记忆中。奖赏信息预示着食物源,与生存息息相关,表明对环境信息进行选择性的记忆巩固加工并储存可能是低等动物高效地编码生存相关信息的重要策略。     

THE EVOLUTION OF FLORAL SCENT AND OLFACTORY PREFERENCES IN POLLINATORS: COEVOLUTION OR PRE‐EXISTING BIAS?

Coevolution is thought to be a major factor in shaping plant-pollinator interactions. Alternatively, plants may have evolved traits that fitted pre-existing preferences or morphologies in the pollinators. Here, we test these two scenarios in the plant family of Araceae and scarab beetles (Coleoptera, Scarabaeidae) as pollinators. We focused on floral volatile organic compounds (VOCs) and production/detection of VOCs by scarab beetles. We found phylogenetic structure in the production/detection of methoxylated aromatics in scarabs, but not plants. Within the plants, most of the compounds showed a well-supported pattern of correlated evolution with scarab-beetle pollination. In contrast, the scarabs showed no correlation between VOC production/detection and visitation to Araceae flowers, with the exception of the VOC skatole. Moreover, many VOCs were found in nonpollinating beetle groups (e.g., Melolonthinae) that are ancestors of pollinating scarabs. Importantly, none of the tested VOCs were found to have originated in pollinating taxa. Our analysis indicates a Jurassic origin of VOC production/detection in scarabs, but a Cretaceous/Paleocene origin of floral VOCs in plants. Therefore, we argue against coevolution, instead supporting the scenario of sequential evolution of floral VOCs in Araceae driven by pre-existing bias of pollinators.     

Carry-over effects of bumblebee associative learning in changing plant communities leads to increased costs of foraging

Flower visitors learn to avoid food-deceptive plants and to prefer rewarding ones by associating floral cues to rewards. As co-occurring plant species have different phenologies, cue-reward associations vary over time. It is not known how these variations affect flower visitors’ foraging costs and learning. We trained bumblebees of two colonies to forage in a community of deceptive and rewarding artificial inflorescences whose flower colours were either similar or dissimilar. We then modified the community composition by turning the rewarding inflorescences into unrewarding and adding rewarding inflorescences of a novel flower colour. In the short term, bees trained to similar rather than dissimilar inflorescences experienced higher costs of foraging (decreased foraging speed and accuracy) in the novel community. The colonies differed in their speed-accuracy trade-off. In the longer term, bees adapted their foraging behaviour to the novel community composition by increasingly visiting the novel rewarding inflorescences.     

Echium and Pontechium specific floral cues for host–plant recognition by the oligolectic bee Hoplitis adunca

Flowering plants often have specific floral cues, which allow bees and other pollinators to differentiate between them. Many bee species exhibit specialised associations with flowers (oligolecty) and it is important for them to find and recognise their specific host plants. In this study we compared the visual and olfactory floral cues of different Echium and Pontechium (Boraginaceae) species with the closely related Anchusa officinalis (Boraginaceae). We tested whether plant-specific cues occur in Echium and Pontechium which may allow oligolectic Hoplitis adunca (Megachilidae) to recognise its host plants and to distinguish them from Anchusa non-hosts. Our investigations showed that Echium/Pontechium provides a specific scent bouquet. Furthermore, we identified compounds which were not described as floral scent before ((Z)-3-nonenal and 1,4-benzoquinone). These unique volatiles and the specific bouquet could act as a recognition cue for H. adunca. The corolla colours differed between all species, but were grouped together in the bee colour categories blue and UV-blue and can indicate potential host flowers for H. adunca.     

Olfactory conditioning of the proboscis extension in bumble bees

The foraging behaviour of bumble bees is well documented for nectar and/or pollen gathering, but little is known about the learning processes underlying such behaviour. We report olfactory conditioning in worker bumble bees Bombus terrestris L. (Hymenoptera: Apidae) obtained under laboratory conditions on restrained individuals. The protocol was adapted from the proboscis extension conditioning previously described in the honey bee Apis mellifera L. Bumble bees were found to be able to learn a pure odorant when it was presented in paired association with a sugar reward, but not when odour and reward were presented in an explicitly unpaired procedure. This suggests an associative basis for this olfactory learning. Bumble bees showed similar conditioning abilities when stimulated with two different floral odours. An effect of the sugar reward concentration on the learning performances was found.     

Productivity differences between dandelion (Taraxacum officinale; Asteraceae) clones from pollution impacted versus non-impacted soils

Common dandelions (Taraxacum officinale Weber, sensu lato; Asteraceae) introduced to North America form an assemblage of asexual (agamospermous), clonal lineages derived from Eurasian mixed sexual and asexual populations. We investigated whether selection for more pollution tolerant clonal lineages occurs at polluted sites and selection for more pollution intolerant lineages occurs at unpolluted sites. We tested the above hypothesis by performing reciprocal greenhouse productivity experiments in which unique dandelion clones (12 clones, identified by DNA fingerprinting, from each site type) sampled from two unpolluted and two polluted (moderately enhanced Cu, Pb and Zn soil concentrations) sites were grown pairwise in both unpolluted (nutrient solution only) and polluted (nutrient solution + Cu, Pb and Zn) media (n?=?48 paired tests for each media type). Dandelion clones from polluted sites produced fewer and smaller leaves, shorter roots and smaller root diameters, reduced shoot and root dry weights, and reduced total biomass compared to clones from unpolluted sites when clones were grown in unpolluted-media (P?≤?0.05). In contrast, clones taken from unpolluted sites were shown to produce significantly fewer and shorter leaves, shorter roots and smaller root diameters, reduced shoot and root dry weights, reduced total biomass, a reduced shoot : root biomass ratio, and have much lower survival compared to clones from polluted sites when both were grown in polluted-media (P?≤?0.05). These results reveal that there was increased selection against unpolluted-site clonal lineages in polluted-media and against polluted-site clonal lineages in unpolluted-media. Across all treatments, clones from unpolluted sites growing in unpolluted-media had the highest proximate measures of fitness. Overall, these findings provide insight into the relationships among anthropogenic environmental contamination and the consequent effects of selective forces acting on dandelion clones and their population genetic architecture.     

Impact of wastewater discharge on the plant diversity,community structure and heavy metal pollution of range plants in eastern Saudi Arabia

The main objectives of this study were to determine the floristic composition of the vegetation cover and to find the effects of wastewater pollution on the plant community structure in eastern Saudi Arabia. 28 stands which were distributed among polluted and unpolluted sites, were chosen for this study. A total of 42 plant species were recorded (14 in the polluted and 28 in the unpolluted sites). The recorded plants comprised of 13 perennial plant species and 29 annual plant species. Seven vegetation communities were determined using TWINSPAN and DCA classification and ordination techniques. Three in the polluted sites, two in the unpolluted sites and the remaining two were mixed communities. The (Sarcopoterium spinosum – Pistacia len‐tiscus) community in the polluted sites, had the highest values of soil moisture, salinity, sulphate, calcium and potassium. On the other hand, Juniperus phoenicea – Olea europaea community in the unpolluted site, had the lowest value of organic matter, salinity and magnesium. In respect of genetic diversity, the community of Foeniculum vulgare - Nicotiana glauca in the unpolluted site, was the most diverse, while Ricinus communis - Chrysanthemum coronarium in the polluted sites was the least one. Both soil and wastewater heavy metal analysis indicated that Pb, Cd, Cr, Cu and Ni concentrations in the polluted sites were significantly higher than those in the unpolluted ones. The impact of wastewater discharge led to the appearance of new invasive plant species that may significantly affect plant diversity and community structure in eastern Saudi Arabia. Finally wastewater discharge in open rangelands could adversely affect the growth of plant species in the rangelands and thus adversely affect plant community structure and diversity.     

Asymmetrical generalisation between pheromonal and floral odours in appetitive olfactory conditioning of the honey bee (Apis mellifera L.)

The capacity to generalise between similar but not identical olfactory stimuli is crucial for honey bees, allowing them to find rewarding food sources with varying volatile emissions. We studied bees' generalisation behaviour with odours having different biological values: typical floral odours or alarm compounds. Bees' behavioural and peripheral electrophysiological responses were investigated using a combined proboscis extension response conditioning-electroantennogram assay. Bees were conditioned to pure linalool (floral) or to pure isoamyl acetate (alarm) and were tested with different concentrations of both compounds. Electrophysiological responses were not influenced by conditioning, suggesting that the learning of individual compounds does not rely on modulations of peripheral sensitivity. Behaviourally, generalisation responses of bees conditioned to the alarm compound were much higher than those of bees conditioned to the floral odour. We further demonstrated such asymmetrical generalisation between alarm and floral odours by using differential conditioning procedures. Conditioning to alarm compounds (isoamyl acetate or 2-heptanone) consistently induced more generalisation than conditioning to floral compounds (linalool or phenylacetaldehyde). Interestingly, generalisation between the two alarm compounds, which are otherwise chemically different, was extremely high. These results are discussed in relation to the neural representation of compounds with different biological significance for bees.     

Does pollen function as a reward for honeybees in associative learning?

The ability to learn an association between floral characteristics such as its odor, color and shape and a reward such as nectar is key to honeybee foraging success. Here, we tested if also pollen could function as a reward for associative learning in honeybees. We found that large proportions of bees with and without field experience showed an unconditioned response, the extension of the proboscis, after touching their antennae with bee-collected pollen. Furthermore, bees readily learn to associate an odor with pollen in a classical conditioning assay. We suggest that pollen might play an important role as a reward for free-flying bees. Received 12 February 2008; revised 17 June 2008; accepted 15 July 2008. C. Grüter, A. Arenas: Both authors contributed equally to this work.     

Gastropod grazing on a benthic alga leads to liberation of food‐finding infochemicals

Chemical information transfer is a major agent in the regulation of interspecific and intraspecific interactions in natural ecosystems. One important group of such infochemicals both in terrestrial and aquatic ecosystems are so‐called volatile organic compounds (VOCs) that can evoke behavioral or physiological responses like predator avoidance and mate or host location. In previous work, we have demonstrated that freshwater gastropods utilize VOCs released from benthic algae as food finding cues, although the specific nature of the VOC release and perception were not yet clear. Therefore we tested whether gastropod grazing on biofilms leads to algal cell damage and a subsequent liberation of wounding‐associated VOCs. In bioassays we investigated the algal VOC bouquet level which is necessary to elicit a behavioural response of freshwater gastropods. The results of the liberation experiment showed that gastropod grazing leads to VOCs release. We also found that a certain threshold level of volatiles is necessary for snails to recognise the volatile infochemicals and subsequently respond with a directed foraging behaviour towards the odour. Finally, a calculated mass balance model demonstrated that the grazer mediated VOC release produced a signal concentration that is sufficient to be recognized by conspecifics and utilized as foraging infochemicals. The emission of ecologically relevant volatiles through snail grazing with subsequent attraction of other gastropod grazers to algal biofilms indicates an important but so far understudied chemical signaling mechanism of ecological importance.     

Do Insectivorous Birds use Volatile Organic Compounds from Plants as Olfactory Foraging Cues? Three Experimental Tests

Some insectivorous birds orient towards insect‐defoliated trees even when they do not see the foliar damage or the herbivores. There are, however, only a few studies that have examined the mechanisms behind this foraging behaviour. Previous studies suggest that birds can use olfactory foraging cues (e.g. volatile organic compounds (VOCs) emitted by defoliated plants), indirect visual cues or a combination of the two sensory cues. VOCs from insect‐defoliated plants are known to attract natural enemies of herbivores, and researchers have hypothesized that VOCs could also act as olfactory foraging cues for birds. We conducted three experiments across a range of spatial scales to test this hypothesis. In each experiment, birds were presented with olfactory cues and their behavioural responses or foraging outcomes were observed. In the first experiment, two different VOC blends, designed to simulate the volatile emissions of mountain birch (Betula pubescens ssp. czerepanovii) after defoliation by autumnal moth (Epirrita autumnata) larvae, were used in behavioural experiments in aviaries with pied flycatchers (Ficedula hypoleuca). The second experiment was a field‐based trial of bird foraging efficiency; the same VOC blends were applied to mountain birches, silver birches (B. pendula) and European white birches (B. pubescens) with plasticine larvae attached to the trees to serve as artificial prey for birds and provide a means to monitor predation rate. In the third experiment, the attractiveness of silver birch saplings defoliated by autumnal moth larvae versus intact controls was tested with great tits (Parus major) and blue tits (Cyanistes caeruleus) in an aviary. Birds did not orient towards either artificial or real trees with VOC supplements or towards herbivore‐damaged saplings when these saplings and undamaged alternatives were hidden from view. These findings do not support the hypothesis that olfactory foraging cues are necessary in the attraction of birds to herbivore‐damaged trees.     

Emissions of Total Volatile Organic Compounds from Anthropogenic Sources in India

Volatile organic compounds (VOCs) have a direct bearing on the levels of ozone and other reactive chemicals in the atmosphere and play an important role in determining air quality Anthropogenic emission of VOCs has greatly increased due to growing consumption of fossil fuels and related activities. This article presents an emissions inventory for VOCs emitted from anthropogenic soutres in India. VOC emissions factors for important source categories and activities are assembled from the literature and an effort is made to use Indian emission factors as far as possible. Important sources of VOCs include livestock, combustion of firewood and fossil fuels, rice paddy fields, manufacturing. petroleum (production and refining), natural gas (production and distribution), vehicular exhaust, and coal mining. The annual anthropogenic VOC emissions for India have been estimated to be 21 million metric tons (mt). A comparison of VOC emissions inventories for a group of countries varying in their industrial and economic development, in terms of income (gross domestic product, or GDP), population, and land area, reflects the differences among the countries. This VOC emissions inventory provides baseline information for comparisons over time and across countries. In addition, it may serve as an important tool for formulating national VOC control policies.     

Honey Bees Modulate Their Olfactory Learning in the Presence of Hornet Predators and Alarm Component

In Southeast Asia the native honey bee species Apis cerana is often attacked by hornets (Vespa velutina), mainly in the period from April to November. During the co-evolution of these two species honey bees have developed several strategies to defend themselves such as learning the odors of hornets and releasing alarm components to inform other mates. However, so far little is known about whether and how honey bees modulate their olfactory learning in the presence of the hornet predator and alarm components of honey bee itself. In the present study, we test for associative olfactory learning of A. cerana in the presence of predator odors, the alarm pheromone component isopentyl acetate (IPA), or a floral odor (hexanal) as a control. The results show that bees can detect live hornet odors, that there is almost no association between the innately aversive hornet odor and the appetitive stimulus sucrose, and that IPA is less well associated with an appetitive stimulus when compared with a floral odor. In order to imitate natural conditions, e.g. when bees are foraging on flowers and a predator shows up, or alarm pheromone is released by a captured mate, we tested combinations of the hornet odor and floral odor, or IPA and floral odor. Both of these combinations led to reduced learning scores. This study aims to contribute to a better understanding of the prey-predator system between A. cerana and V. velutina.     

Bee visitation rates to cultivated sunflowers increase with the amount and accessibility of nectar sugars

Pollinators make foraging decisions based on numerous floral traits, including nectar and pollen rewards, and associated visual and olfactory cues. For insect‐pollinated crops, identifying and breeding for attractive floral traits may increase yields. In this study, we examined floral trait variation within cultivated sunflowers and its effects on bee foraging behaviours. Over 2 years, we planted different sunflower inbred lines, including male‐fertile and male‐sterile lines, and measured nectar volume, nectar sugar concentration and composition, and corolla length. During bloom, we recorded visits by both managed honey bees and wild bees. We then examined consistency in relative nectar production by comparing field results to those from a greenhouse experiment. Sunflower inbred lines varied significantly in all floral traits, including the amount and composition of nectar sugars, and in corolla length. Both wild bee and honey bee visits significantly increased with nectar sugar amount and decreased with corolla length, but appeared unaffected by nectar sugar composition. While wild bees made more visits to sunflowers providing pollen (male‐fertile), honey bees preferred plants without pollen (male‐sterile). Differences in nectar quantity among greenhouse‐grown sunflower lines were similar to those measured in the field, and bumble bees preferentially visited lines with more nectar in greenhouse observations. Our results show that sunflowers with greater quantities of nectar sugar and shorter corollas receive greater pollination services from both managed and wild bees. Selecting for these traits could thus increase sunflower crop yields and provide greater floral resources for bees.     

Intrafloral patterns of color and scent in Capparis spinosa L. and the ghosts of its selection past

Premise

Capparis spinosa is a widespread charismatic plant, in which the nocturnal floral habit contrasts with the high visitation by diurnal bees and the pronounced scarcity of hawkmoths. To resolve this discrepancy and elucidate floral evolution of C. spinosa, we analyzed the intrafloral patterns of visual and olfactory cues in relation to the known sensory biases of the different visitor guilds (bees, butterflies, and hawkmoths).

Methods

We measured the intrafloral variation of scent, reflectance spectra, and colorimetric properties according to three guilds of known visitors of C. spinosa. Additionally, we sampled visitation rates using a motion-activated camera.

Results

Carpenter bees visited the flowers eight times more frequently than nocturnal hawkmoths, at dusk and in the following morning. Yet, the floral headspace of C. spinosa contained a typical sphingophilous scent with high emission rates of certain monoterpenes and amino-acid derived compounds. Visual cues included a special case of multisensory nectar guide and color patterns conspicuous to the visual systems of both hawkmoths and bees.

Conclusions

The intrafloral patterns of sensory stimuli suggest that hawkmoths have exerted strong historical selection on C. spinosa. Our study revealed two interesting paradoxes: (a) the flowers phenotypically biased towards the more inconsistent pollinator; and (b) floral display demands an abundance of resources that seems maladaptive in the habitats of C. spinosa. The transition to a binary pollination system accommodating large bees has not required phenotypic changes, owing to specific eco-physiological adaptations, unrelated to pollination, which make this plant an unusual case in pollination ecology.     

Proteomics-based method for the assessment of marine pollution using liquid chromatography coupled with two-dimensional electrophoresis

Using a proteomic approach, we have developed a new method for the assessment of marine pollution that generates highly reproducible protein expression patterns and it is simple and scalable. The protocol is based on applying liquid chromatography (LC) coupled with two-dimensional electrophoresis (2-DE) to analyze changes in the protein expression pattern after exposure to marine pollution. The digestive gland of the sentinel "blue mussel" (Mytilus edulis) was batch-processed through a simple cell fractionation followed by ion-exchange chromatography and 2-DE. The selection of ligands, elution method, and small volume design was carefully considered to define a protocol that could be mainly robotized. A pilot study with samples collected from different Gothenburg harbor areas indicated that the clean area could be distinguished from the polluted ones based on a protein expression pattern (PES) composed of 13 proteins. Principal component analysis (PCA) and hierarchical clustering confirmed that the PES was sufficient to discriminate polluted and unpolluted areas and to provide a spatial gradient from the polluted source. Several proteins from the PES were identified by electrospray ionization tandem mass spectrometry (ESI-MS/MS), and they are involved in beta-oxidation, amino acid metabolism, detoxification, protein degradation, organelle biogenesis, and protein folding. In the near future, this methodology could show potential advantages to assess marine pollution and could become a stable platform to elucidate ecotoxicological questions.     

Reward quality influences the development of learned olfactory biases in honeybees

Plants produce flowers with complex visual and olfactory signals, but we know relatively little about the way that signals such as floral scents have evolved. One important factor that may direct the evolution of floral signals is a pollinator''s ability to learn. When animals learn to associate two similar signals with different outcomes, biases in their responses to new signals can be formed. Here, we investigated whether or not pollinators develop learned biases towards floral scents that depend on nectar reward quality by training restrained honeybees to learn to associate two similar odour signals with different outcomes using a classical conditioning assay. Honeybees developed learned biases towards odours as a result of differential conditioning, and the extent to which an olfactory bias could be produced depended upon the difference in the quality of the nectar rewards experienced during conditioning. Our results suggest that differences in reward quality offered by flowers influence odour recognition by pollinators, which in turn could influence the evolution of floral scents in natural populations of co-flowering plants.