Behavioral and molecular studies of quantitative differences in hygienic behavior in honeybees.

BACKGROUND: Hygienic behavior (HB) enables honeybees to tolerate parasites, including infection with the parasitic mite Varroa destructor, and it is a well-known example of a quantitative genetic trait. The understanding of the molecular processes underpinning the quantitative differences in this behavior remains limited. RESULTS: We performed gene expression studies in worker bees that displayed quantitative genetic differences in HB. We established a high and low genetic source of HB performance and studied the engagements into HB of single worker bees under the same environmental conditions. We found that the percentage of worker bees that engaged in a hygienic behavioral task tripled in the high versus low HB sources, thus suggesting that genetic differences may mediate differences in stimulated states to perform HB. We found 501 differently expressed genes (DEGs) in the brains of hygienic and non-hygienic performing workers in the high HB source bees, and 342 DEGs in the brains of hygienic performing worker bees, relative to the gene expression in non-hygienic worker bees from the low HB source group. "Cell surface receptor ligand signal transduction" in the high and "negative regulation of cell communication" in the low HB source were overrepresented molecular processes, suggesting that these molecular processes in the brain may play a role in the regulation of quantitative differences in HB. Moreover, only 21 HB-associated DEGs were common between the high and low HB sources. CONCLUSIONS: The better HB colony performance is primarily achieved by a high number of bees engaging in the hygienic tasks that associate with distinct molecular processes in the brain. We propose that different gene products and pathways may mediate the quantitative genetic differences of HB.

Mixing of honeybees with different genotypes affects individual worker behavior and transcription of genes in the neuronal substrate.

Division of labor in social insects has made the evolution of collective traits possible that cannot be achieved by individuals alone. Differences in behavioral responses produce variation in engagement in behavioral tasks, which as a consequence, generates a division of labor. We still have little understanding of the genetic components influencing these behaviors, although several candidate genomic regions and genes influencing individual behavior have been identified. Here, we report that mixing of worker honeybees with different genotypes influences the expression of individual worker behaviors and the transcription of genes in the neuronal substrate. These indirect genetic effects arise in a colony because numerous interactions between workers produce interacting phenotypes and genotypes across organisms. We studied hygienic behavior of honeybee workers, which involves the cleaning of diseased brood cells in the colony. We mixed ∼500 newly emerged honeybee workers with genotypes of preferred Low (L) and High (H) hygienic behaviors. The L/H genotypic mixing affected the behavioral engagement of L worker bees in a hygienic task, the cooperation among workers in uncapping single brood cells, and switching between hygienic tasks. We found no evidence that recruiting and task-related stimuli are the primary source of the indirect genetic effects on behavior. We suggested that behavioral responsiveness of L bees was affected by genotypic mixing and found evidence for changes in the brain in terms of 943 differently expressed genes. The functional categories of cell adhesion, cellular component organization, anatomical structure development, protein localization, developmental growth and cell morphogenesis were overrepresented in this set of 943 genes, suggesting that indirect genetic effects can play a role in modulating and modifying the neuronal substrate. Our results suggest that genotypes of social partners affect the behavioral responsiveness and the neuronal substrate of individual workers, indicating a complex genetic architecture underlying the expression of behavior.

Categorization of visual stimuli in the honeybee Apis mellifera.

Categorization refers to the classification of perceptual input into defined functional groups. We present and discuss evidence suggesting that stimulus categorization can also be found in an invertebrate, the honeybee Apis mellifera, thus underlining the generality across species of this cognitive process. Honeybees show positive transfer of appropriate responding from a trained to a novel set of visual stimuli. Such a transfer was demonstrated for specific isolated features such as symmetry or orientation, but also for assemblies (layouts) of features. Although transfer from training to novel stimuli can be achieved by stimulus generalization of the training stimuli, most of these transfer tests involved clearly distinguishable stimuli for which generalization would be reduced. Though in most cases specific experimental controls such as stimulus balance and discriminability are still required, it seems appropriate to characterize the performance of honeybees as reflecting categorization. Further experiments should address the issue of which categorization theory accounts better for the visual performances of honeybees.

The influence of training length on generalization of visual feature assemblies in honeybees.

Generalization is a fundamental cognitive ability that allows treating similar stimuli as equivalents, and thus responding to them in the same manner. Here, we show that after training free-flying bees with a single, constant pair of patterns made of four quadrants, each displaying different oriented gratings, bees extract the orientation information corresponding to each quadrant and integrate it in a generic layout that preserved the spatial relationship between oriented edges. Our results show that the amount of experience with the training patterns is critical to determine or not generalization to novel stimuli sharing the layout of the rewarded stimulus. Increasing experience results in higher generalization levels reflected in significant responding to novel stimuli. With ongoing training, redundant information seems to be eliminated and reduced to the minimum that is necessary and sufficient to solve the task. Controlling precisely the level of experience of individuals is therefore crucial in experiments on visual recognition.

Honey bees navigate according to a map-like spatial memory.

By using harmonic radar, we report the complete flight paths of displaced bees. Test bees forage at a feeder or are recruited by a waggle dance indicating the feeder. The flights are recorded after the bees are captured when leaving the hive or the feeder and are released at an unexpected release site. A sequence of behavioral routines become apparent: (i) initial straight flights in which they fly the course that they were on when captured (foraging bees) or that they learned during dance communication (recruited bees); (ii) slow search flights with frequent changes of direction in which they attempt to "get their bearings"; and (iii) straight and rapid flights directed either to the hive or first to the feeding station and then to the hive. These straight homing flights start at locations all around the hive and at distances far out of the visual catchment area around the hive or the feeding station. Two essential criteria of a map-like spatial memory are met by these results: bees can set course at any arbitrary location in their familiar area, and they can choose between at least two goals. This finding suggests a rich, map-like organization of spatial memory in navigating honey bees.

Local-feature assembling in visual pattern recognition and generalization in honeybees.

Generalization is a cognitive ability that allows similar stimuli along a given dimension to be treated as equivalents. Insects exhibit high levels of visual generalization. Honeybees trained to recognize complex visual stimuli on the basis of a single feature generalize their choice to novel stimuli sharing that common feature with the trained stimuli. The demonstration of this kind of performance has been limited to the use of a single visual feature, and the possibility that bees link different features in learning a visual pattern has been denied. Here we show that honeybees trained with a series of complex patterns sharing a common layout comprising four edge orientations remember these orientations simultaneously in their appropriate positions, and generalize their response to novel stimuli that preserve the trained layout. Honeybees also generalize their response to patterns with fewer correct orientations, depending on their match with the trained layout. Stimulation of the achromatic L-photoreceptor input is necessary for this task. The mini-brain of the honeybee can thus extract regularities in its environment and establish correspondences among correlated features. It can thus generate a large set of object descriptions from a finite set of elements.