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Manipulating the light/dark cycle: effects on dopamine levels in optic lobes of the honey bee (Apis mellifera) brain.

This study examines the relationship between cyclical variations in optic-lobe dopamine levels and the circadian behavioural rhythmicity exhibited by forager bees. Our results show that changing the light-dark regimen to which bees are exposed has a significant impact not only on forager behaviour, but also on the levels of dopamine that can be detected in the optic lobes of the brain. Consistent with earlier reports, we show that foraging behaviour exhibits properties characteristic of a circadian rhythm. Foraging activity is entrained by daily light cycles to periods close to 24 h, it changes predictably in response to phase shifts in light, and it is able to free-run under constant conditions. Dopamine levels in the optic lobes also undergo cyclical variations, and fluctuations in endogenous dopamine levels are influenced significantly by alterations to the light/dark cycle. However, the time course of these changes is markedly different from changes observed at a behavioural level. No direct correlation could be identified between levels of dopamine in the optic lobes and circadian rhythmic activity of the honey bee.

Animals↗

Pattern vision of the honeybee (Apis mellifera). What is an oriented edge?

Pairs of black patterns on a white background, one rewarded the other not, were presented vertically each in one arm of a Y-maze. During training the locations of the black areas were changed every 5 min to prevent the bees using them as cues, but cues from edges were kept consistent. Bees detect orientation even in a gradient that subtends 36 degrees from black to white (normal to the edge). Orientation cues in short lengths of edge are detected and summed on each side of the fixation point, irrespective of the lay-out of the pattern. Edges at right angles reduce the total orientation cue. The polarity of edges in a sawtooth grating is weakly discriminated, but not the orientation of a fault line where two gratings meet. Edge quality can be discriminated, but is not recognised in unfamiliar orientations. When spot location is excluded as a cue, the orientation of a row of spots or squares which individually provide no net orientation cue is not discriminated. In conclusion, when locations of black areas are shuffled, the bees remember the sum of local orientation cues but not the global pattern, and there is no re-assembly of a pattern based on differently oriented edges. A neuronal model consistent with these results is presented.

Animals↗

Operant conditioning of antennal muscle activity in the honey bee (Apis mellifera L.).

Antennal movements of the honey bee can be conditioned operantly under laboratory conditions. Using this behavioural paradigm we have developed a preparation in which the activity of a single antennal muscle has been operantly conditioned. This muscle, the fast flagellum flexor muscle, is innervated by an identified motoneuron whose action potentials correlate 1:1 with the muscle potentials. The activity of the fast flagellum flexor muscle was recorded extracellularly from the scapus of the antenna. The animal was rewarded with a drop of sucrose solution whenever the muscle activity exceeded a defined reward threshold. The reward threshold was one standard deviation above the mean spontaneous frequency prior to conditioning. After ten conditioning trials, the frequency of the muscle potentials had increased significantly compared to the spontaneous frequency. The conditioned changes of frequency were observed for 30 min after conditioning. No significant changes of the frequency were found in the yoke control group. The firing pattern of the muscle potentials did not change significantly after conditioning or feeding. Fixing the antennal joints reduces or abolishes associative operant conditioning. The conditioned changes of the frequency of muscle potentials in the freely moving antenna are directly comparable to the behavioural changes during operant conditioning.

Animal Structures↗

The effect of genotype on response thresholds to sucrose and foraging behavior of honey bees (Apis mellifera L.).

Honey bee foragers were tested for their proboscis extension response (PER) to water and varying solutions of sucrose. Returning pollen and nectar foragers were collected at the entrance of a colony and were assayed in the laboratory. Pollen foragers had a significantly higher probability of responding to water and to lower concentrations of sucrose. Bees derived from artificially selected high- and low-pollen-hoarding strains were also tested using the proboscis extension assay. Returning foragers were captured and tested for PERs0 to 30% sucrose. Results demonstrated a genotypic effect on PERs of returnining foragers. The PERs of departing high- and low-strain foragers were consistent with those of returning foragers. The PERs were related to nectar and water reward perception of foragers. High strain bees were more likely to return with loads of water and lower concentrations of sucrose than foragers from low pollen strain. Low-strain bees were more likely to return empty. We identified a previously mapped genomic region that contains a variable quantitative trait locus that appears to influence sucrose response thresholds. These studies demonstrate a gene-brain-behavior pathway that can be altered as a consequence of colony-level selection for quantities of stored food.

Animals↗

The effect of genotype, age, sex, and caste on response thresholds to sucrose and foraging behavior of honey bees (Apis mellifera L.).

Bees derived from artificially selected high- and low-pollen-hoarding strains were tested for their proboscis extension reflex response to water and varying sucrose concentrations. High-strain bees had a lower response threshold to sucrose than low-strain bees among pre-foragers, foragers, queens and drones. Pre-foraging low-strain workers showed ontogenetic changes in their response threshold to sucrose which was inversely related to age. High-strain foragers were more likely to return with loads of water compared to low-strain foragers. Whereas low-strain foragers were more likely to return with loads of nectar. Low-strain nectar foragers collected nectar with significantly higher sucrose concentrations than did the high-strain nectar foragers. Alternatively, low-strain foragers were more likely to return empty compared to high-strain foragers. These studies demonstrate how a genotypically varied sensory-physiological process, the perception of sucrose, are associated with a division of labor for foraging.

Aging↗

Modulation of sucrose response thresholds in honey bees (Apis mellifera L.): influence of genotype, feeding, and foraging experience.

The perception of sugar is important to honey bees for making foraging decisions. We measured bees' perception by determining what concentration of sucrose touched to the antennae elicited the proboscis extension response (response threshold). A low response threshold (extension at low concentration) suggests a high perceptual value of sucrose. and vice versa. Perception of sucrose solutions differed between two artificially selected genotypic strains and was modulated by the bees' recent feeding experiences. Bees offered 10%, 30%, or 50% sucrose solutions in small cages overnight, and in large flight-cages or free-flying in the field for several days, had subsequent response thresholds positively correlated to the concentration offered. Empty bees, whether they were nectar, water or pollen foragers, dancers or non-dancers, had a significantly lower threshold than loaded bees. Crop volume affected response thresholds directly and independently of sucrose concentration. We interpret these findings as multiple mechanisms that operate in different time scales, modulating perception of sucrose. Changes occurred in the time scale of evolutionary processes as demonstrated by genotypic differences. Changes with foraging experience occur in hours or minutes while effects of crop filling are instantaneous.

Adaptation, Physiological↗

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.

Animals↗

Free fatty acids digested from pollen and triolein in the honeybee (Apis mellifera carnica Pollmann) midgut.

Honey bees satisfy their lipid requirement by consuming pollen. The free fatty acid content of the midgut was used to quantify fat digestion. Midguts extracted from younger workers of known ages and from foragers were divided into three components: endoperitrophic region (peritrophic membrane with gut contents), extraperitrophic region and intestinal wall. Both the total amount of pollen and the amount of free fatty acids in the endoperitrophic region and in the intestinal wall depend on the bee's age. The amounts increase within the 1st 3 days of a honey bee's life, reach maxima around the age of 8 days and then decrease continuously to the lowest values, measured in forager bees. Forced feeding with triacylglycerol results in significantly higher levels of free fatty acids, especially in the endoperitrophic region, in 8-day-old bees and foragers. This indicates that lipolytic activity depends on age and that the free fatty acid content in 8-day-old bees is primarily limited by the amount and availability of lipids ingested. The results show further that fat digestion depends on the functional status of honey bees, as is the case for pollen consumption, speed of transport of pollen bolus through the alimentary canal and protein digestion.

Animals↗

RNAi-induced phenotypes suggest a novel role for a chemosensory protein CSP5 in the development of embryonic integument in the honeybee (Apis mellifera).

Small chemosensory proteins (CSPs) belong to a conserved, but poorly understood, protein family found in insects and other arthropods. They exhibit both broad and restricted expression patterns during development. In this paper, we used a combination of genome annotation, transcriptional profiling and RNA interference to unravel the functional significance of a honeybee gene (csp5) belonging to the CSP family. We show that csp5 expression resembles the maternal-zygotic pattern that is characterized by the initiation of transcription in the ovary and the replacement of maternal mRNA with embryonic mRNA. Blocking the embryonic expression of csp5 with double-stranded RNA causes abnormalities in all body parts where csp5 is highly expressed. The treated embryos show a "diffuse", often grotesque morphology, and the head skeleton appears to be severely affected. They are 'unable-to-hatch' and cannot progress to the larval stages. Our findings reveal a novel, essential role for this gene family and suggest that csp5 (unable-to-hatch) is an ectodermal gene involved in embryonic integument formation. Our study confirms the utility of an RNAi approach to functional characterization of novel developmental genes uncovered by the honeybee genome project and provides a starting point for further studies on embryonic integument formation in this insect.

Amino Acid Sequence↗

Free amino acids in the haemolymph of honey bee queens (Apis mellifera L.).

In queen honey bees the free amino acid content in the haemolymph clearly depends on the physiological function and social environment of the individual. While in drones and workers the content of free amino acids increases after emergence until it reaches a peak in 5-day-old animals and decreases afterwards, the amino acid content in queens reaches its highest level (>60 nmol/ microl haemolymph) with the onset of egg laying (10 d of age). This level is about 2.5 times more than the highest level found in workers. Queens maintain this high level also when they are older (>30 d) and continue to lay eggs in average colonies. As in drones and workers, in queens the predominant amino acid is proline, which accounts for more than 50% of the total content of free amino acids in egg-laying individuals. When 10-day-old queens are prevented from mating and do not lay eggs, their amino acid content is significantly lower compared to laying queens of the same age. Also the social environment influences the contents of free amino acids in queens. When virgin queens were kept for 6 days with 20 worker bees and sufficient honey and pollen in an incubator, they had significantly lower concentrations of amino acids than virgin queens living for the same period with about 8000 workers in a colony. Most probably, the high amino acid concentration in the haemolymph is the basis for the high protein synthesis activity of laying queens.

Aging↗

Importance of proline and other amino acids during honeybee flight--Apis mellifera carnica POLLMANN).

The levels of proline and other amino acids in the haemolymph and other body parts of honeybee foragers were investigated by HPLC analysis. The concentrations of proline in the blood of glucose-fed or -injected bees finishing their exhaustive tethered flights on a roundabout were significantly reduced compared to bees that were fed and rested for one hour. This indicates some utilization of proline during flight metabolism. The levels of essential amino acids and of the sum of all amino acids except proline remained roughly constant, indicating that the decrease of proline did not result from a changed haemolymph volume. 14C-labelled proline was injected into bees either shortly before starting their flight or before a resting period of equal duration in an incubator at the same temperature. Bees that rested had incorporated more proline into thorax body protein, and less of the labelled substance was unrecovered ("missing") and considered to be respired or less probably defecated. If the entire amount of missing 14C-proline is regarded as exhaled, the oxidative breakdown of proline reached higher levels after flight than in rested bees. This is another hint that proline is utilized during flight. Usually the exhaled amount did not exceed 10 microg proline in half an hour of flight. Although our data indicate involvement of proline in flight metabolism, the amount metabolized is low compared to the utilization of carbohydrates.

Animals↗

Honeybees (Apis mellifera) holding on to memories: response competition causes retroactive interference effects.

Five experiments on honeybees examined how the learning of a second task interferes with what was previously learned. Free flying bees were tested for landmark-based memory in variations on a paradigm of retroactive interference. Bees first learned Task 1, were tested on Task 1 (Test 1), then learned Task 2, and were tested again on Task 1 (Test 2). A 60-min delay (waiting in a box) before Test 2 caused no performance decrements. If the two tasks had conflicting response requirements, (e.g., target right of a green landmark in Task 1 and left of a blue landmark in Task 2), then a strong decrement on Test 2 was found (retroactive interference effect). When response competition was minimised during training or testing, however, the decrement on Test 2 was small or nonexistent. The results implicate response competition as a major contributor to the retroactive interference effect. The honeybee seems to hold on to memories; new memories do not wipe out old ones.

Animals↗

Caste, sex and strain of honey bees (Apis mellifera) affect infestation with tracheal mites (Acarapis woodi).

Worker honey bees from genetic strains selected for being resistant (R) or susceptible (S) to tracheal mites typically show large differences in infestation in field colonies and in bioassays that involve controlled exposure to infested bees. We used bioassays exposing newly emerged individuals to infested workers to compare the propensity for tracheal mites to infest queens, drones and workers from R and S colonies. In tests with queens, newly emerged R and S queens were either simultaneously confined in infested colonies (n = 95 and 87 respectively), or individually caged with groups of 5-20 infested workers (n = 119 and 115 respectively). Mite prevalence (percentage of individuals infested) and abundance (foundress mites per individual) after 4-6 days did not differ between R and S queens. In another test, five newly emerged drones and workers from both an R and an S colony, and a queen of one of the two strains, were caged in each of 38 cages with 20 g of workers infested at 60-96% prevalence. Infestations of the R queens (n = 17) and S queens (n = 19) did not differ significantly, but R workers had half the mite abundance of S workers, while R drones received about a third more migrating mites than S drones. In tests to evaluate possible mechanisms, removal of one mesothoracic leg from R and S workers resulted in 2- to 10-fold increase in mite abundance on the treated side, but excising legs did not affect infestation of the corresponding tracheae in drones. This suggests that differences in infestation between R and S workers, but not drones, are largely determined by their ability to remove mites through autogrooming. If autogrooming is the primary mechanism of colony resistance to tracheal mites, selection for resistance to tracheal mites using infestation of hemizygous drones may be inefficient.

Animals↗

Brood-cell size does not influence the susceptibility of honey bees (Apis mellifera) to infestation by tracheal mites (Acarapis woodi).

Tracheal mites have been associated with the condition in honey bees that devastated colonies in Britain and Ireland in the early 1900s. The first outbreak of this condition, that became known as the 'Isle of Wight' disease, coincided with the period when brood-cell size was increased from about 5.0 mm to about 5.5 mm in width. We undertook an inoculation experiment over a 7-day period to establish if the act of increasing the brood-cell size could have triggered the onset of tracheal mites in honey bees. The standard-sized cells used had a cell width of 5.44 mm and the small-sized cells a width of 5.07 mm. Using callow (newly emerged) bees, from three colonies that had mixed cell sizes, we compared the susceptibility of bees reared in standard-sized cells with that of those raised in small-sized cells. The results indicated similar levels of female mite abundance (0.49 vs. 0.52 mites per bee) and mean fecundity (4.33 vs. 4.22 offspring per female mite), and produced no evidence of any difference in the overall susceptibility between the bees raised in the standard-sized cells versus small-sized brood cells.

Animals↗

The effects of four insect growth-regulating (IGR) insecticides on honeybee (Apis mellifera L.) colony development, queen rearing and drone sperm production.

This study assessed the effects of exposure to IGRs on the long-term development of the honeybee colony, viability of queens and sperm production in drones and integrated the data into a honeybee population model. Colonies treated with diflubenzuron resulted in a short-term reduction in the numbers of adult bees and brood. Colonies treated with fenoxycarb declined during the season earlier and started the season slower. The number of queens that successfully mated and laid eggs was affected in the fenoxycarb treatment group but there were no significant differences in the drone sperm counts between the colonies. An existing honeybee population model was modified to include exposure to IGRs. In the model, fenoxycarb reduced the winter size of the colony, with the greatest effects following a June or an August application. Assuming a 'larvae per nurse bee' ratio of 1.5 for brood rearing capability, the reduction in winter size of a colony following a fenoxycarb application was at its worst about 8%. However, even if only those bees reared within 2 weeks of the IGR being applied are subject to premature ageing, this might significantly reduce the size of over-wintering colonies, and increase the chance of the bee population dwindling and dying in late winter or early spring.

Animals↗

Analyses of avocado (Persea americana) nectar properties and their perception by honey bees (Apis mellifera).

Honey bees are important avocado pollinators. However, due to the low attractiveness of flowers, pollination is often inadequate. Previous work has revealed that avocado honey is relatively unattractive to honey bees when compared with honey from competing flowers. We characterized avocado honey and nectar with respect to their odor, color, and composition of sugars, phenolic compounds, and minerals. Furthermore, we tested how honey bees perceive these parameters, using the proboscis extension response bioassay and preference experiments with free-flying bees. Naïve bees were indifferent to odors of avocado and citrus flowers and honey. Experienced bees, which were collected in the field during the blooming season, responded preferentially to odor of citrus flowers. The unique sugar composition of avocado nectar, which contains almost exclusively sucrose and a low concentration of the rare carbohydrate perseitol, and the dark brown color of avocado honey, had no negative effects on its attractiveness to the bees. Phenolic compounds extracted from avocado honey were attractive to bees and adding them to a solution of sucrose increased its attractiveness. Compared with citrus nectar and nonavocado honey, avocado nectar and honey were rich in a wide range of minerals, including potassium, phosphorus, magnesium, sulfur, iron, and copper. Potassium and phosphorus, the two major minerals, both had a repellent effect on the bees. Possible explanations for the presence of repellent components in avocado nectar are discussed.

Animals↗

Characteristics of neuron activity in the honey bee (Apis mellifera L.) in conditions of kynurenine deficiency.

Neuron activity was studied electrophysiologically in the antennal lobes, mushroom bodies, and cervical connective during ontogenesis (days 1, 3, 7, and 25) in snow(laranija) mutant bees, which suffer kynurenine deficiency, and in wild-type bees. Neuron activity recorded from the cervical connective was found to show the greatest dependence on kynurenine content. Mutation in the structural gene for tryptophan oxygenase, the first and key enzyme in the kynurenine pathway for tryptophan metabolism (KPTM) and which leads to kynurenine deficiency, decreased the frequency of neuron spike activity recorded from the cervical connective. This would appear to be associated with a possible deficiency of glutamic acid; it was independent of the deficiency of kynurenic acid and kynurenine, a point subject to further studies. Clear manifestation of the effect of the mutation requires the presence of two mutant alleles in the bee genome (homozygosity). The effect of the mutation, to inhibit neuron activity in the cerebral ganglion, corresponds to the effect seen previously at the behavioral level. Less clear effects were obtained from recordings of neuron activity from the antennal lobes and mushroom bodies. This may be associated with the complexity of the spectrum of biochemical lesions in different parts of the brain resulting from the mutation.

Action Potentials↗