Bias to pollen odors is affected by early exposure and foraging experience.

In many pollinating insects, foraging preferences are adjusted on the basis of floral cues learned at the foraging site. In addition, olfactory experiences gained at early adult stages might also help them to initially choose food sources. To understand pollen search behavior of honeybees, we studied how responses elicited by pollen-based odors are biased in foraging-age workers according to (i) their genetic predisposition to collect pollen, (ii) pollen related information gained during foraging and (iii) different experiences with pollen gained at early adult ages. Bees returning to the hive carrying pollen loads, were strongly biased to unfamiliar pollen bouquets when tested in a food choice device against pure odors. Moreover, pollen foragers' orientation response was specific to the odors emitted by the pollen type they were carrying on their baskets, which suggests that foragers retrieve pollen odor information to recognize rewarding flowers outside the hive. We observed that attraction to pollen odor was mediated by the exposure to a pollen diet during the first week of life. We did not observe the same attraction in foraging-age bees early exposed to an artificial diet that did not contain pollen. Contrary to the specific response observed to cues acquired during foraging, early exposure to single-pollen diets did not bias orientation response towards a specific pollen odor in foraging-age bees (i.e. bees chose equally between the exposed and the novel monofloral pollen odors). Our results show that pollen exposure at early ages together with olfactory experiences gained in a foraging context are both relevant to bias honeybees' pollen search behavior.

Early olfactory experience induces structural changes in the primary olfactory center of an insect brain.

The antennal lobe (AL) is the first olfactory center of the insect brain and is constituted of different functional units, the glomeruli. In the AL, odors are coded as spatiotemporal patterns of glomerular activity. In honeybees, olfactory learning during early adulthood modifies neural activity in the AL on a long-term scale and also enhances later memory retention. By means of behavioral experiments, we first verified that olfactory learning between the fifth and eighth day of adulthood induces better retention performances at a late adult stage than the same experience acquired before or after this period. We checked that the specificity of memory for the odorants used was improved. We then studied whether such early olfactory learning also induces long-term structural changes in the AL consistent with the formation of long-term olfactory memories. We also measured the volume of 15 identified glomeruli in the ALs of 17-day-old honeybees that either experienced an odor associated with sucrose solution between the fifth and eighth day of adulthood or were left untreated. We found that early olfactory experience induces glomerulus-selective increases in volume that were specific to the learned odor. By comparing our volumetric measures with calcium-imaging recordings from a previous study, performed in 17-day-old bees subjected to the same treatment and experimental conditions, we found that glomeruli that showed structural changes after early learning were those that exhibited a significant increase in neural activity. Our results make evident a correlation between structural and functional changes in the AL following early olfactory learning.

Olfactory learning in the stingless bee Tetragonisca angustula (Hymenoptera, Apidae, Meliponini).

Tetragonisca angustula stingless bees are considered as solitary foragers that lack specific communication strategies. In their orientation towards a food source, these social bees use chemical cues left by co-specifics and the information obtained in previous foraging trips by the association of visual stimuli with the food reward. Here, we investigated their ability to learn the association between odors and reward (sugar solution) and the effect on learning of previous encounters with scented food either inside the hive or during foraging. During food choice experiments, when the odor associated with the food was encountered at the feeding site, the bees' choice is biased to the same odor afterwards. The same was not the case when scented food was placed inside the nest. We also performed a differential olfactory conditioning of proboscis extension response with this species for the first time. Inexperienced bees did not show significant discrimination levels. However, when they had had already interacted with scented food inside the hive, they were able to learn the association with a specific odor. Possible olfactory information circulation inside the hive and its use in their foraging strategies is discussed.

Early olfactory experience modifies neural activity in the antennal lobe of a social insect at the adult stage.

In the antennal lobe (AL), the first olfactory centre of the insect brain, odorants are represented as spatiotemporal patterns of glomerular activity. Whether and how such patterns are modified in the long term after precocious olfactory experiences (i.e. in the first days of adulthood) remains unknown. To address this question, we used in vivo optical imaging of calcium activity in the antennal lobe of 17-day-old honeybees which either experienced an odorant associated with sucrose solution 5-8 days after emergence or were left untreated. In both cases, we imaged neural responses to the learned odor and to three novel odors varying in functional group and carbon-chain length. Two different odor concentrations were used. We also measured behavioral responses of 17-day-old honeybees, treated and untreated, to these stimuli. We show that precocious olfactory experience increased general odor-induced activity and the number of activated glomeruli in the adult AL, but also affected qualitative odor representations, which appeared shifted in the neural space of treated animals relative to control animals. Such effects were not limited to the experienced odor, but were generalized to other perceptually similar odors. A similar trend was found in behavioral experiments, in which increased responses to the learned odor extended to perceptually similar odors in treated bees. Our results show that early olfactory experiences have long-lasting effects, reflected in behavioral responses to odorants and concomitant neural activity in the adult olfactory system.

Odor discrimination in classical conditioning of proboscis extension in two stingless bee species in comparison to Africanized honeybees.

Learning in insects has been extensively studied using different experimental approaches. One of them, the proboscis extension response (PER) paradigm, is particularly well suited for quantitative studies of cognitive abilities of honeybees under controlled conditions. The goal of this study was to analyze the capability of three eusocial bee species to be olfactory conditioned in the PER paradigm. We worked with two Brazilian stingless bees species, Melipona quadrifasciata and Scaptotrigona aff. depilis, and with the invasive Africanized honeybee, Apis mellifera. These three species present very different recruitment strategies, which could be related with different odor-learning abilities. We evaluated their gustatory responsiveness and learning capability to discriminate floral odors. Gustatory responsiveness was similar for the three species, although S. aff. depilis workers showed fluctuations along the experimental period. Results for the learning assays revealed that M. quadrifasciata workers can be conditioned to discriminate floral odors in a classical differential conditioning protocol and that this discrimination is maintained 15 min after training. During conditioning, Africanized honeybees presented the highest discrimination, for M. quadrifasciata it was intermediate, and S. aff. depilis bees presented no discrimination. The differences found are discussed considering the putative different learning abilities and procedure effect for each species.

Crop scents affect the occurrence of trophallaxis among forager honeybees.

Previous evidence indicates that the recognition of the nectar delivered by forager honeybees within the colony may have been a primitive method of communication on food resources. Thus, the association between scent and reward that nectar foragers establish while they collect on a given flower species should be retrieved during trophallaxis, i.e., the transfer of liquid food by mouth, and, accordingly, foraging experience could affect the occurrence of these interactions inside the nest. We used experimental arenas to analyze how crop scents carried by donor bees affect trophallaxis among foragers, i.e., donors and receivers, which differ in their foraging experience. Results showed that whenever the foragers had collected unscented sugar solution from a feeder the presence of scents in the solution carried by donors did not affect the occurrence of trophallaxis nor its dynamics. In contrast, whenever the foragers had previous olfactory information, new scents present in the crop of the donors negatively affected the occurrence, but not the dynamics of trophallaxis. Thus, the association learned at the food source seems to be retrieved during trophallaxis, and it is possible that known scents present in the mouthparts of nest-mates may operate as a triggering stimulus to elicit trophallactic behavior within the hive.

Trophallaxis in forager honeybees (Apis mellifera): resource uncertainty enhances begging contacts?

Trophallaxis among adult worker honeybees is the transfer of liquid food by mouth from one individual to another. Within the colony, nectar foragers perform offering contacts (as food-donors) to transfer the contents of their crops to recipient nest-mates and, in addition, they also perform begging contacts (as food-receivers). The biological relevance of these last interactions remains unknown. Previous evidence suggests that begging may be involved in the exchange of information on food resources that occurs naturally between employed foragers and nest-mates. This work was aimed to reveal possible connections between the information obtained while foraging and the begging behavior displayed inside the nest. Experiments were intended to (1) analyze whether chemosensory information obtained while foraging, i.e., odors and sucrose concentrations, affects begging behavior, and (2) determine whether resource uncertainty enhances begging contacts. Results showed that: (1) most begging contacts lasted less than 1 s, a duration which only allows receiving food samples from nest-mates; (2) the diversity of odors and sucrose concentrations at the feeding place enhances the occurrence of begging contacts; and (3) an increased resource uncertainty enhances the forager begging behavior. In addition, results suggest that foragers may direct their begging contacts frequently to other employed nectar foragers.

Assessment of food source profitability in honeybees (Apis mellifera): how does disturbance of foraging activity affect trophallactic behaviour?

When forager honeybees (Apis mellifera) return to the hive after a successful foraging trip, they unload the collected liquid to recipient hive mates through mouth-to-mouth contacts (trophallaxis). The speed at which the liquid is transferred (unloading rate) from donor to recipient is related to the profitability of the recently visited food source. Two main characteristics that define this profitability are the flow of solution delivered by the feeder and the time invested by the forager at the source (visit time). To investigate the effect of visit time on trophallactic behaviour, donor foragers were trained to a rate feeder that could deliver different flows of solution. We dissociated visit time and flow of solution by introducing pauses in the solution's deliverance at different moments of the foraging visit. We analysed whether timing of the non-deliverance period within the visit is important for the forager's assessment of resource profitability. During the subsequent trophallactic encounter with a hive mate, unloading rate was related to the total time invested by the forager at the food source only if the ingestion process had already been started. These results together with previous ones suggest that foragers integrate an overall flow rate of solution of the feeder throughout the entire foraging visit.

Changes in the thoracic temperature of honeybees while receiving nectar from foragers collecting at different reward rates.

Mouth-to-mouth food exchange in eusocial insects (trophallaxis) contributes to the organization of complex social activities. In the case of honeybees, foragers returning from a nectar source transfer the food collected to receiver colony-mates through oral contact. Previous studies have shown that the speed of nectar transfer within each contact (unloading rate) increases when foragers return from feeding sites with higher profitability, i.e. with more concentrated sugar solutions or higher solution flow rates. However, there is no evidence that the nectar unloading rate is actually evaluated by hive-mates during food exchange. To investigate this, trophallaxis between donor bees returning from a feeder with different flow rates of sucrose solution (range 1.0-8.2 microl min(-1) of 50% w/w sucrose solution) and receiver hive-mates was studied by combining behavioural and infrared thermal analysis. The results show that when foraging bees returned from a feeder delivering a higher flow rate they initiated unloading at higher thoracic temperatures and transferred the solution at higher speed. During these food exchanges, the thoraces of receiver bees warmed up faster in proportion to increasing forager temperature and unloading rate. Therefore, whatever the variable actually evaluated by receivers (mostly nectar processors, i.e. bees that handle nectar in the hive) during trophallaxis (unloading rate and/or donor thoracic temperature), they raised their activity level in proportion to that of the foragers. In this way, receiver bees will intensify their nectar processing when nectar foragers return from more profitable sites.

Nectar feeding by the hovering hawk moth Macroglossum stellatarum: intake rate as a function of viscosity and concentration of sucrose solutions.

Although nectar feeding in insects has long been studied, the knowledge of the effect of nectar energy content on the ingestion dynamics separately from the viscosity of the fluid is very limited. To determine the effects of both factors on the feeding behavior of the hovering hawk moth Macroglossum stellatarum, we developed a method to independently manipulate sucrose concentrations and viscosity. The intake rate was analyzed as a function of sucrose concentration, the concentration at constant viscosity (kept constant by adding tylose, an inert polysaccharide), and of the different viscosities of a 30% weight/weight (w/w) sucrose solution (by adding different amounts of tylose). By increasing the concentration, and thus its viscosity, the solution intake rate (in microl s (-1)) decreased beyond a 20% w/w sucrose solution. For a 30% sucrose solution, the intake rate decreased with increasing viscosity. At constant viscosity, the solution intake rate decreased beyond a 30% w/w sucrose solution. However, if we considered the quantity of sucrose ingested per unit time (sucrose intake rate), the same fitted maximum was attained for both series in which the sucrose concentration changed (33.6% w/w). Results suggest that the gustatory input affects the dynamics of fluid ingestion separately from the viscosity.

Trophallaxis in filled-crop honeybees (Apis mellifera L.): food-loading time affects unloading behaviour.

Honeybees ingested 50% w/w (1.8 M) sucrose solution at a rate feeder offering either 16.5, 32.5 or 65 microliters/min. While the time spent ingesting solution at the feeder decreased significantly with increasing flow of solution, bees attained maximum crop loads with this range of flows. Different parameters related to mouth-to-mouth food exchange (trophallaxis) showed important modulations as the offered flow of solution was incremented. Trophallactic transfer rate, i.e. the speed at which liquid food is transferred from donor to recipient bee, was found to increase along with increasing profitability at the rate feeder. In the present case, food source profitability could have been evaluated by foragers either by measuring the time invested in ingesting the solution, or by direct assessment of the flow rate of the feeder. Thus it seems that perception of profitability conditions at the food source suffices for later representation in the hive through trophallactic contacts, independently of crop-filling state.

The interplay between dancing and trophallactic behavior in the honey bee Apis mellifera.

The interplay between the recruitment dance and food-giving trophallactic contacts of returning Apis meellifera foragers was analyzed. Dancing and trophallactic events were recorded for bees returning from a rate feeder that provided 50% weight on weight sucrose solution at a constant flow rate of 5 microl min(-1). Bees that had danced immediately before their trophallactic contact had more recipients per trophallaxis compared with bees that did not dance before. Thus, besides information coded in dancing behavior, dance maneuvers could serve as a stimulus to increase attention of bees located on the dance floor to receive nectar. In addition, the number of bees receiving food during a trophallaxis showed a positive correlation with the probability of dancing immediately after contacting. The time from arrival at the hive to when the first or the subsequent contacts took place presented no correlation with the probability of dancing after trophallaxis. Also, the duration of a trophallaxis was positively correlated with the number of recipients per trophallaxis. These results suggest that returning foragers could receive information during a trophallactic contact with their hive mates that modify thresholds for dancing. Dance maneuvers and trophallactic contacts performed by foraging bees seem to be "mutually" affected.

Nectar feeding by the ant Camponotus mus: intake rate and crop filling as a function of sucrose concentration.

In independent assays, workers of the ant Camponotus mus were conditioned to visit an arena where they found a large drop of sucrose solution of different concentrations, from 5 to 70% weight on weight (w/w). Single ants were allowed to collect the sucrose solution ad libitum, and feeding time, feeding interruptions, crop load, and intake rates were recorded. Feeding time increased exponentially with sucrose concentration, and this relationship was quantitatively described by the increase in viscosity with concentration corresponding to pure sucrose solutions. Ants collecting dilute solutions (5 to 15% w/w) returned to the nest with partial crop loads. Crop filling increased with increasing sucrose concentration, and reached a maximum at 42.6% w/w. Workers collecting highly concentrated solutions (70% w/w) also returned to the nest with a partially-filled crop, as observed for dilute solutions. Nectar intake rate was observed to increase with increasing sucrose concentration in the range 5 to 30% sucrose. It reached a maximum at 30.8%, and declined with increasing sucrose concentration. Results suggest that both sucrose concentration and viscosity of the ingested solution modulate feeding mechanics as well as the worker's decision about the load size to be collected before leaving the source.