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.     

Africanized honeybees are slower learners than their European counterparts

Does cognitive ability always correlate with a positive fitness consequence? Previous research in both vertebrates and invertebrates provides mixed results. Here, we compare the learning and memory abilities of Africanized honeybees (Apis mellifera scutellata hybrid) and European honeybees (Apis mellifera ligustica). The range of the Africanized honeybee continues to expand, superseding the European honeybee, which led us to hypothesize that they might possess greater cognitive capabilities as revealed by a classical conditioning assay. Surprisingly, we found that fewer Africanized honeybees learn to associate an odor with a reward. Additionally, fewer Africanized honeybees remembered the association a day later. While Africanized honeybees are replacing European honeybees, our results show that they do so despite displaying a relatively poorer performance on an associative learning paradigm.     

The proboscis extension reflex to evaluate learning and memory in honeybees (Apis mellifera): some caveats

The proboscis extension reflex (PER) is widely used in a classical conditioning (Pavlovian) context to evaluate learning and memory of a variety of insect species. The literature is particularly prodigious for honeybees (Apis mellifera) with more than a thousand publications. Imagination appears to be the only limit to the types of challenges to which researchers subject honeybees, including all the sensory modalities and a broad diversity of environmental treatments. Accordingly, some remarkable insights have been achieved using PER. However, there are several challenges to evaluating the PER literature that warrant a careful and thorough review. We assess here variation in methods that makes interpretation of studies, even those researching the same question, tenuous. We suggest that the numerous variables that might influence experimental outcomes from PER be thoroughly detailed by researchers. Moreover, the influence of individual variables on results needs to carefully evaluated, as well as among two or more variables. Our intent is to encourage investigation of the influence of numerous variables on PER results.     

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.     

Self body-size perception in an insect

Animals negotiating complex environments encounter a wide range of obstacles of different shapes and sizes. It is greatly beneficial for the animal to react to such obstacles in a precise, context-specific manner, in order to avoid harm or even simply to minimize energy expenditure. An essential key challenge is, therefore, an estimation of the animal’s own physical characteristics, such as body size. A further important aspect of self body-size perception (or SBSP) is the need to update it in accordance with changes in the animal’s size and proportions. Despite the major role of SBSP in functional behavior, little is known about if and how it is mediated. Here, we demonstrate that insects are also capable of self perception of body size and that this is a vital factor in allowing them to adjust their behavior following the sudden and dramatic growth associated with periodic molting. We reveal that locusts’ SBSP is strongly correlated with their body size. However, we show that the dramatic change in size accompanying adult emergence is not sufficient to create a new and updated SBSP. Rather, this is created and then consolidated only following the individuals’ experience and interaction with the physical environment. Behavioral or pharmacological manipulations can both result in maintenance of the old larval SBSP. Our results emphasize the importance of learning and memory-related processes in the development and update of SBSP, and highlight the advantage of insects as good models for a detailed study on the neurobiological and molecular aspects of SBSP.     

Comparison of pollination and defensive buzzes in bumblebees indicates species-specific and context-dependent vibrations

Bees produce vibrations in many contexts, including for defense and while foraging. Buzz pollination is a unique foraging behavior in which bees vibrate the anthers of flowers to eject pollen which is then collected and used as food. The relationships between buzzing properties and pollen release are well understood, but it is less clear to what extent buzzing vibrations vary among species, even though such information is crucial to understanding the functional relationships between bees and buzz-pollinated plants. Our goals in this study were (1) to examine whether pollination buzzes differ from those produced during defense, (2) to evaluate the similarity of buzzes between different species of bumblebees (Bombus spp.), and (3) to determine if body size affects the expression of buzzing properties. We found that relative peak amplitude, peak frequency, and duration were significantly different between species, but only relative peak amplitude differed between pollination and defensive buzzes. There were significant interactions between species and buzz type for peak frequency and duration, revealing that species differed in their patterns of expression in these buzz properties depending on the context. The only parameter affected by body size was duration, with larger bees producing shorter buzzes. Our findings suggest that although pollination and defensive buzzes differ in some properties, variability in buzz structure also exhibits a marked species-specific component. Species differences in pollination buzzes may have important implications for foraging preferences in bumblebees, especially if bees select flowers best matched to release pollen for their specific buzzing characteristics.     

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.     

Behavioral genomics of honeybee foraging and nest defense

The honeybee has been the most important insect species for study of social behavior. The recently released draft genomic sequence for the bee will accelerate honeybee behavioral genetics. Although we lack sufficient tools to manipulate this genome easily, quantitative trait loci (QTLs) that influence natural variation in behavior have been identified and tested for their effects on correlated behavioral traits. We review what is known about the genetics and physiology of two behavioral traits in honeybees, foraging specialization (pollen versus nectar), and defensive behavior, and present evidence that map-based cloning of genes is more feasible in the bee than in other metazoans. We also present bioinformatic analyses of candidate genes within QTL confidence intervals (CIs). The high recombination rate of the bee made it possible to narrow the search to regions containing only 17–61 predicted peptides for each QTL, although CIs covered large genetic distances. Knowledge of correlated behavioral traits, comparative bioinformatics, and expression assays facilitated evaluation of candidate genes. An overrepresentation of genes involved in ovarian development and insulin-like signaling components within pollen foraging QTL regions suggests that an ancestral reproductive gene network was co-opted during the evolution of foraging specialization. The major QTL influencing defensive/aggressive behavior contains orthologs of genes involved in central nervous system activity and neurogenesis. Candidates at the other two defensive-behavior QTLs include modulators of sensory signaling (Am5HT ₇ serotonin receptor, AmArr4 arrestin, and GABA-B-R1 receptor). These studies are the first step in linking natural variation in honeybee social behavior to the identification of underlying genes.     

Chemical compounds of the foraging recruitment pheromone in bumblebees

When the frenzied and irregular food-recruitment dances of bumblebees were first discovered, it was thought that they might represent an evolutionary prototype to the honeybee waggle dance. It later emerged that the primary function of the bumblebee dance was the distribution of an alerting pheromone. Here, we identify the chemical compounds of the bumblebee recruitment pheromone and their behaviour effects. The presence of two monoterpenes and one sesquiterpene (eucalyptol, ocimene and farnesol) in the nest airspace and in the tergal glands increases strongly during foraging. Of these, eucalyptol has the strongest recruitment effect when a bee nest is experimentally exposed to it. Since honeybees use terpenes for marking food sources rather than recruiting foragers inside the nest, this suggests independent evolutionary roots of food recruitment in these two groups of bees.     

Mating flights select for symmetry in honeybee drones (Apis mellifera)

Males of the honeybee (Apis mellifera) fly to specific drone congregation areas (DCAs), which virgin queens visit in order to mate. From the thousands of drones that are reared in a single colony, only very few succeed in copulating with a queen, and therefore, a strong selection is expected to act on adult drones during their mating flights. In consequence, the gathering of drones at DCAs may serve as an indirect mate selection mechanism, assuring that queens only mate with those individuals having a better flight ability and a higher responsiveness to the queen’s visual and chemical cues. Here, we tested this idea relying on wing fluctuating asymmetry (FA) as a measure of phenotypic quality. By recapturing marked drones at a natural DCA and comparing their size and FA with a control sample of drones collected at their maternal hives, we were able to detect any selection on wing size and wing FA occurring during the mating flights. Although we found no solid evidence for selection on wing size, wing FA was found to be significantly lower in the drones collected at the DCA than in those collected at the hives. Our results demonstrate the action of selection during drone mating flights for the first time, showing that developmental stability can influence the mating ability of honeybee drones. We therefore conclude that selection during honeybee drone mating flights may confer some fitness advantages to the queens.     

The effect of growth rate and ageing on colour variation of European pond turtles

Many chelonians have colourful dots, patches and stripes throughout their body that are made up, at least in part, of carotenoids. Therefore, turtles are very suitable models to study the evolution and functionality of carotenoid-based colouration. Recent studies suggested a close link between colouration and immune system in these taxa. However, more research is needed to understand the role of these colourful stripes and patches in turtle visual signalling. The purpose of this study was to explore the relationship between growth rate and colouration in European pond turtles. In particular, we wanted to answer the question of whether there is a trade-off between growth and colour expression. We also aimed to explore the effect of body size and age on colour variation. Turtles from a reintroduction-breeding program were recaptured, weighed and measured over an 8-year period to estimate their growth rates and age. We also measured with a spectrometer the reflectance of colour patches in two different body parts: shell and forelimb. We found that turtles with a faster growth rate had brighter limb stripes independently of their age. On the other hand, shell colouration was related to body size with larger turtles having brighter shell stripes and higher values of carotenoid chroma. Our results suggest that fast-growers may afford to express intense colourful limb stripes likely due to their higher intake of carotenoids that would modulate both growth and colour expression. However, shell colouration was related to body size probably due to ontogenetic differences in the diet, as juveniles are strictly carnivorous while adults are omnivorous. Alternatively, shell colouration might be involved in crypsis as the shell is visually exposed to predators.     

The effect of octopamine on behavioral responses of free-foraging bumblebees to a change in food source profitability

The invertebrate neuromodulator octopamine is known to be involved in bees' associative learning, enhancing the responsiveness of a bee to a conditioned stimulus. In this study, we tested the effect of octopamine on the choice behavior of free-flying bumblebees using a two-phase experiment in an array of artificial flowers. During the first phase of the experiment, the bee was allowed to collect octopamine-laden sugar water from two types of equally rewarding flowers (yellow versus blue). In the second phase, one type of flower was set to be unrewarding. The behavior of the bee (proportion of visits to the unrewarding flowers) over the two phases was fitted to a sigmoid regression model. Our results show that octopamine had no significant effect on the bees' equilibrium choice or on the overall rate of the behavioral change in response to the change in reward. Rather, octopamine significantly affected the time interval between the change in reward status and the initiation of behavioral change in the bee.     

Hunting behaviour and breeding performance of northern goshawks Accipiter gentilis, in relation to resource availability, sex, age and morphology

Animal territories that differ in the availability of food resources will require (all other things being equal) different levels of effort for successful reproduction. As a consequence, breeding performance may become most strongly dependent on factors that affect individual foraging where resources are poor. We investigated potential links between foraging behaviour, reproductive performance and morphology in a goshawk Accipiter gentilis population, which experienced markedly different resource levels in two different parts of the study area (rabbit-rich vs. rabbit-poor areas). Our analyses revealed (1) that rabbit abundance positively affected male reproductive output; (2) that age, size and rabbit abundance (during winter) positively affected different components of female reproductive output; (3) that foraging movements were inversely affected by rabbit abundance for both sexes (for females, this may mainly have reflected poor provisioning by males in the rabbit-poor area); (4) that younger breeders (both in males and females) tended to move over larger distances than older individuals (which may have reflected both a lack of hunting experience and mate searching); and (5) that male body size (wing length) showed some covariation with resource conditions (suggesting possible adaptations to hunting agile avian prey in the rabbit-poor area). Although we are unable to establish firm causal relationships with our observational data set, our results provide an example of how territory quality (here, food abundance) and individual features (here, age and morphology) may combine to shape a predator's foraging behaviour and, ultimately, its breeding performance.     

Rapid matching in Drosophila place learning

The level of conditioned behavior in animals is proportional to the intensity, amount, frequency, or probability of reinforcement. Interestingly, this matching can be dynamic, with performance levels following, for example, a switch in the probability of reinforcement with a short delay. We previously found that conditioned performance levels in Drosophila match reinforcement intensity in a place conditioning paradigm. Whether Drosophila can match conditioned behavior to a change in reinforcement intensity was an open question. In this study, we found that both conditioned behavior and memory levels match reinforcement intensity after a switch, and this rapid matching occurs within 2 min. Thus, fruit flies can dynamically match conditioned behavior and memory levels to a change in reinforcement intensity.     

Social experience influences the development of a central auditory area

Vocal communication develops under social influences that can enhance attention, an important factor in memory formation and perceptual tuning. In songbirds, social conditions can delay sensitive periods of development, overcome learning inhibitions and enable exceptional learning or induce selective learning. However, we do not know how social conditions influence auditory processing in the brain. In the present study, we raised young naive starlings under different social conditions but with the same auditory experience of adult songs, and we compared the effects of these different conditions on the development of the auditory cortex analogue. Several features appeared to be influenced by the social experience, among which the proportion of auditory neuronal sites and the neuronal selectivity. Both physical and social isolation from adult models altered the development of the auditory area in parallel to alterations in vocal development. To our knowledge, this is the first evidence that social deprivation has as much influence on neuronal responsiveness as sensory deprivation.     

A putative octopamine/tyramine receptor mediating appetite in a hungry fly

In the blowfly Phormia regina, experience of simultaneous feeding with d-limonene exposure inhibits proboscis extension reflex (PER) due to decreased tyramine (TA) titer in the brain. To elucidate the molecular mechanism of TA signaling pathway related to the associated feeding behavior, we cloned cDNA encoding the octopamine/TA receptor (PregOAR/TAR). The deduced protein is composed of 607 amino acid residues and has 7 predicted transmembrane domains. Based on homology and phylogenetic analyses, this protein belongs to the OAR/TAR family. The PregOAR/TAR was mainly expressed in head, with low levels of expression in other tissues at adult stages. Gene expression profile is in agreement with a plethora of functions ascribed to TA in various insect tissues. The immunolabeled cell bodies and processes were localized in the medial protocerebrum, outer layer of lobula, antennal lobe, and subesophageal ganglion. These results suggest that decrease of TA level in the brain likely affects neurons expressing PregOAR/TAR, causing mediation of the sensitivity in the sensillum and/or output of motor neurons for PER.     

Reward and non-reward learning of flower colours in the butterfly Byasa alcinous (Lepidoptera: Papilionidae)

Learning plays an important role in food acquisition for a wide range of insects. To increase their foraging efficiency, flower-visiting insects may learn to associate floral cues with the presence (so-called reward learning) or the absence (so-called non-reward learning) of a reward. Reward learning whilst foraging for flowers has been demonstrated in many insect taxa, whilst non-reward learning in flower-visiting insects has been demonstrated only in honeybees, bumblebees and hawkmoths. This study examined both reward and non-reward learning abilities in the butterfly Byasa alcinous whilst foraging among artificial flowers of different colours. This butterfly showed both types of learning, although butterflies of both sexes learned faster via reward learning. In addition, females learned via reward learning faster than males. To the best of our knowledge, these are the first empirical data on the learning speed of both reward and non-reward learning in insects. We discuss the adaptive significance of a lower learning speed for non-reward learning when foraging on flowers.     

Laying date and polygyny as determinants of annual reproductive success in male collared flycatchers (Ficedula albicollis): a long-term study

Annual reproductive success (ARS) is one of the main components of lifetime reproductive success, a reliable measure of individual fitness. Previous studies often dealt with ARS and variables potentially affecting it. Among them, long-term studies that consider multiple factors at the same time are particularly important in understanding the adaptive value of different phenotypes. Here, we used an 18-year dataset to quantify the ARS of male collared flycatchers (Ficedula albicollis) on the basis of recruited offspring. We simultaneously assessed the effect of start of breeding, age, polygyny, body size and the expression of forehead patch (a sexually selected trait). The success of early breeding individuals was appreciably higher than late birds; however, breeding too early was also disadvantaged, and males that bred around the yearly median breeding date had the highest ARS. Polygynous males were more successful in years with good food supply, while in years with low food availability, they did not produce more recruits than monogamous males. The age of males, their forehead patch size and body size did not affect the number of recruits. Our findings support the importance of breeding date and suggest stabilizing selection on it in the long term. We also show that polygyny is not always advantageous for males, and its fitness pay-off may depend on environmental quality.     

A critical number of workers in a honeybee colony triggers investment in reproduction

Social insect colonies, like individual organisms, must decide as they develop how to allocate optimally their resources among survival, growth, and reproduction. Only when colonies reach a certain state do they switch from investing purely in survival and growth to investing also in reproduction. But how do worker bees within a colony detect that their colony has reached the state where it is adaptive to begin investing in reproduction? Previous work has shown that larger honeybee colonies invest more in reproduction (i.e., the production of drones and queens), however, the term ‘larger’ encompasses multiple colony parameters including number of adult workers, size of the nest, amount of brood, and size of the honey stores. These colony parameters were independently increased in this study to test which one(s) would increase a colony’s investment in reproduction via males. This was assayed by measuring the construction of drone comb, the special type of comb in which drones are reared. Only an increase in the number of workers stimulated construction of drone comb. Colonies with over 4,000 workers began building drone comb, independent of the other colony parameters. These results show that attaining a critical number of workers is the key parameter for honeybee colonies to start to shift resources towards reproduction. These findings are relevant to other social systems in which a group’s members must adjust their behavior as a function of the group’s size.